Preliminary study of the association between the elimination parameters of phenytoin and phenobarbital.

PubMed

Methaneethorn, Janthima; Panomvana, Duangchit; Vachirayonstien, Thaveechai

2017-09-26

Therapeutic drug monitoring is essential for both phenytoin and phenobarbital therapy given their narrow therapeutic indexes. Nevertheless, the measurement of either phenytoin or phenobarbital concentrations might not be available in some rural hospitals. Information assisting individualized phenytoin and phenobarbital combination therapy is important. This study's objective was to determine the relationship between the maximum rate of metabolism of phenytoin (Vmax) and phenobarbital clearance (CLPB), which can serve as a guide to individualized drug therapy. Data on phenytoin and phenobarbital concentrations of 19 epileptic patients concurrently receiving both drugs were obtained from medical records. Phenytoin and phenobarbital pharmacokinetic parameters were studied at steady-state conditions. The relationship between the elimination parameters of both drugs was determined using simple linear regression. A high correlation coefficient between Vmax and CLPB was found [r=0.744; p<0.001 for Vmax (mg/kg/day) vs. CLPB (L/kg/day)]. Such a relatively strong linear relationship between the elimination parameters of both drugs indicates that Vmax might be predicted from CLPB and vice versa. Regression equations were established for estimating Vmax from CLPB, and vice versa in patients treated with combination of phenytoin and phenobarbital. These proposed equations can be of use in aiding individualized drug therapy.

Rational mutagenesis by engineering disulphide bonds improves Kluyveromyces lactis beta-galactosidase for high-temperature industrial applications

PubMed Central

Rico-Díaz, Agustín; Álvarez-Cao, María-Efigenia; Escuder-Rodríguez, Juan-José; González-Siso, María-Isabel; Cerdán, M. Esperanza; Becerra, Manuel

2017-01-01

Kluyveromyces lactis β-galactosidase (Kl-β-Gal) is one of the most important enzymes in the dairy industry. The poor stability of this enzyme limits its use in the synthesis of galactooligosaccharides (GOS) and other applications requiring high operational temperature. To obtain thermoresistant variants, a rational mutagenesis strategy by introducing disulphide bonds in the interface between the enzyme subunits was used. Two improved mutants, R116C/T270C and R116C/T270C/G818C, had increased half-lives at 45 °C compared to Kl-β-Gal (2.2 and 6.8 fold increases, respectively). Likewise, Tm values of R116C/T270C and R116C/T270C/G818C were 2.4 and 8.5 °C, respectively, higher than Kl-β-Gal Tm. Enrichment in enzymatically active oligomeric forms in these mutant variants also increased their catalytic efficiency, due to the reinforcement of the interface contacts. In this way, using an artificial substrate (p-nitrophenyl-β-D-galactopyranoside), the Vmax values of the mutants were ~1.4 (R116C/T270C) and 2 (R116C/T270C/G818C) fold higher than that of native Kl-β-Gal. Using the natural substrate (lactose) the Vmax for R116C/T270C/G818C almost doubled the Vmax for Kl-β-Gal. Validation of these mutant variants of the enzyme for their use in applications that depend on prolonged incubations at high temperatures was achieved at the laboratory scale by monitoring their catalytic activity in GOS synthesis. PMID:28361909

Permeability, transport, and metabolism of solutes in Caco-2 cell monolayers: a theoretical study.

PubMed

Sun, Huadong; Pang, K Sandy

2008-01-01

We explored the properties of a catenary model that includes the basolateral (B), apical (A), and cellular compartments via simulations under linear and nonlinear conditions to understand the asymmetric observations arising from transporters, enzymes, and permeability in Caco-2 cells. The efflux ratio (EfR; P(app,B-->A)/P(app,A-->B)), obtained from the effective permeability from the A-->B and B-->A direction under linear conditions, was unity for passively permeable drugs whose transport does not involve transporters; the value was unaffected by cellular binding or metabolism, but increased with apical efflux. Metabolism was asymmetric, showing lesser metabolite accrual for the B-->A than A-->B direction because of inherent differences in the volumes for A and B. Moreover, the net flux (total - passive permeation) due to saturable apical efflux, absorption, or metabolism showed nonconformity to simple Michaelis-Menten kinetics against C(D,0), the loading donor concentration. EfR values differed with saturable apical efflux and metabolism (>1), as well as apical absorption (EfRs <1), but approached unity with high passive diffusive clearance (CL(d)) and increasing C(D,0) at a higher degree of saturation of the process. The J(max) (apparent V(max) estimated for the carrier system) and K(m)(') [or the K(m)('') based on a modified equation with the Hill coefficient (beta)] estimates from the Eadie-Hofstee plot revealed spurious correlations with the assigned V(max) and K(m). The sampling time, CL(d), and parameter space of K(m) and V(max) strongly influenced both the correlation and accuracy of estimates. Improved correlation was found for compounds with high CL(d). These observations showed that the catenary model is appropriate in the description of transport and metabolic data in Caco-2 cells.

Rational mutagenesis by engineering disulphide bonds improves Kluyveromyces lactis beta-galactosidase for high-temperature industrial applications.

PubMed

Rico-Díaz, Agustín; Álvarez-Cao, María-Efigenia; Escuder-Rodríguez, Juan-José; González-Siso, María-Isabel; Cerdán, M Esperanza; Becerra, Manuel

2017-03-31

Kluyveromyces lactis β-galactosidase (Kl-β-Gal) is one of the most important enzymes in the dairy industry. The poor stability of this enzyme limits its use in the synthesis of galactooligosaccharides (GOS) and other applications requiring high operational temperature. To obtain thermoresistant variants, a rational mutagenesis strategy by introducing disulphide bonds in the interface between the enzyme subunits was used. Two improved mutants, R116C/T270C and R116C/T270C/G818C, had increased half-lives at 45 °C compared to Kl-β-Gal (2.2 and 6.8 fold increases, respectively). Likewise, Tm values of R116C/T270C and R116C/T270C/G818C were 2.4 and 8.5 °C, respectively, higher than Kl-β-Gal Tm. Enrichment in enzymatically active oligomeric forms in these mutant variants also increased their catalytic efficiency, due to the reinforcement of the interface contacts. In this way, using an artificial substrate (p-nitrophenyl-β-D-galactopyranoside), the Vmax values of the mutants were ~1.4 (R116C/T270C) and 2 (R116C/T270C/G818C) fold higher than that of native Kl-β-Gal. Using the natural substrate (lactose) the Vmax for R116C/T270C/G818C almost doubled the Vmax for Kl-β-Gal. Validation of these mutant variants of the enzyme for their use in applications that depend on prolonged incubations at high temperatures was achieved at the laboratory scale by monitoring their catalytic activity in GOS synthesis.

Doppler Imaging in Aortic Stenosis: The Importance of the Nonapical Imaging Windows to Determine Severity in a Contemporary Cohort.

PubMed

Thaden, Jeremy J; Nkomo, Vuyisile T; Lee, Kwang Je; Oh, Jae K

2015-07-01

Although the highest aortic valve velocity was thought to be obtained from imaging windows other than the apex in about 20% of patients with severe aortic stenosis (AS), its occurrence appears to be increasing as the age of patients has increased with the application of transcatheter aortic valve replacement. The aim of this study was to determine the frequency with which the highest peak jet velocity (Vmax) is found at each imaging window, the degree to which neglecting nonapical imaging windows underestimates AS severity, and factors influencing the location of the optimal imaging window in contemporary patients. Echocardiograms obtained in 100 consecutive patients with severe AS from January 3 to May 23, 2012, in which all imaging windows were interrogated, were retrospectively analyzed. AS severity (aortic valve area and mean gradient) was calculated on the basis of the apical imaging window alone and the imaging window with the highest peak jet velocity. The left ventricular-aortic root angle measured in the parasternal long-axis view as well as clinical variables were correlated with the location of highest peak jet velocity. Vmax was most frequently obtained in the right parasternal window (50%), followed by the apex (39%). Subjects with acute angulation more commonly had Vmax at the right parasternal window (65% vs 43%, P = .05) and less commonly had Vmax at the apical window (19% vs 48%, P = .005), but Vmax was still located outside the apical imaging window in 52% of patients with obtuse aortic root angles. If nonapical windows were neglected, 8% of patients (eight of 100) were misclassified from high-gradient severe AS to low-gradient severe AS, and another 15% (15 of 100) with severe AS (aortic valve area < 1.0 cm(2)) were misclassified as having moderate AS (aortic valve area > 1.0 cm(2)). In this contemporary cohort, Vmax was located outside the apical imaging window in 61% of patients, and neglecting the nonapical imaging windows resulted in the misclassification of AS severity in 23% of patients. Aortic root angulation as measured by two-dimensional echocardiography influences the location of Vmax modestly. Despite increasing time constraints on many echocardiography laboratories, these data confirm that routine Doppler interrogation from multiple imaging windows is critical to accurately determine the severity of AS in contemporary clinical practice. Copyright © 2015 American Society of Echocardiography. Published by Elsevier Inc. All rights reserved.

Three-dimensional quantitative structure-activity relationship studies on UGT1A9-mediated 3-O-glucuronidation of natural flavonols using a pharmacophore-based comparative molecular field analysis model.

PubMed

Wu, Baojian; Morrow, John Kenneth; Singh, Rashim; Zhang, Shuxing; Hu, Ming

2011-02-01

Glucuronidation is often recognized as one of the rate-determining factors that limit the bioavailability of flavonols. Hence, design and synthesis of more bioavailable flavonols would benefit from the establishment of predictive models of glucuronidation using kinetic parameters [e.g., K(m), V(max), intrinsic clearance (CL(int)) = V(max)/K(m)] derived for flavonols. This article aims to construct position (3-OH)-specific comparative molecular field analysis (CoMFA) models to describe UDP-glucuronosyltransferase (UGT) 1A9-mediated glucuronidation of flavonols, which can be used to design poor UGT1A9 substrates. The kinetics of recombinant UGT1A9-mediated 3-O-glucuronidation of 30 flavonols was characterized, and kinetic parameters (K(m), V(max), CL(int)) were obtained. The observed K(m), V(max), and CL(int) values of 3-O-glucuronidation ranged from 0.04 to 0.68 μM, 0.04 to 12.95 nmol/mg/min, and 0.06 to 109.60 ml/mg/min, respectively. To model UGT1A9-mediated glucuronidation, 30 flavonols were split into the training (23 compounds) and test (7 compounds) sets. These flavonols were then aligned by mapping the flavonols to specific common feature pharmacophores, which were used to construct CoMFA models of V(max) and CL(int), respectively. The derived CoMFA models possessed good internal and external consistency and showed statistical significance and substantive predictive abilities (V(max) model: q(2) = 0.738, r(2) = 0.976, r(pred)(2) = 0.735; CL(int) model: q(2) = 0.561, r(2) = 0.938, r(pred)(2) = 0.630). The contour maps derived from CoMFA modeling clearly indicate structural characteristics associated with rapid or slow 3-O-glucuronidation. In conclusion, the approach of coupling CoMFA analysis with a pharmacophore-based structural alignment is viable for constructing a predictive model for regiospecific glucuronidation rates of flavonols by UGT1A9.

Effects of fasting and semistarvation on the kinetics of active and passive sugar absorption across the small intestine in vivo.

PubMed Central

Debnam, E S; Levin, R J

1975-01-01

The effects of dietary restriction on the kinetics of absorption in vivo of glucose, galactose and alpha-methyl glucoside were assessed by electrical and chemical methods in the rat jejunum. 2. The 'apparent Km', maximum absorption or Vmax (mu-mole/10 cm. 15 min) and maximum potential difference (p.d.max) were obtained for the jejunal electrogenic active transfer mechanism from the transfer p.d.s and the chemical absorption data corrected for diffusion using various graphical kinetic plots. 3. Fasting for 3 days greatly decreased the 'apparent Kms', obtained from electrical or chemical data, for all the sugars but had no effect on those for L-valine or L-methionine. Semistarvation caused a less pronounced reduction of the 'apparent Kms' for the sugars. The dietary-induced change in 'apparent Km' for glucose was also observed in the fasted hamster. One interpretation of these changes is that the affinity of the carriers for sugars increases during dietary restriction; the greater the level of restriction the greater the increase. 4. Fasting and semistarvation caused large reductions in the Vmax. These reductions were correlated with a reduced enterocyte population estimated by changes in enterocyte column size. 5. The reduction in the Vmax for galactose was mainly accounted for by the decrease in enterocyte population. In the case of glucose, other factors such as reduced enterocyte metabolism or changes in the carriers must be involved to explain the discrepancy between the large decrease in Vmax and the enterocyte column size. 6. Fasting and semi-starvation had complex, differential actions on the p.d.max for glucose, galactose and alpha-methyl glucoside. These changes did not correlate with those observed in the Vmax measured chemically. 7. A standard diet obtained from two commercial sources was found to differ greatly in its effect on the electrogenic transfer system for alpha-methyl glucoside but had no effect on those for galactose and glucose. PMID:1206572

Human Hepatic Cytochrome P450-Specific Metabolism of the Organophosphorus Pesticides Methyl Parathion and Diazinon

PubMed Central

Tian, Yuan; Knaak, James B.; Kostyniak, Paul J.; Olson, James R.

2012-01-01

Organophosphorus pesticides (OPs) are a public health concern due to their worldwide use and documented human exposures. Phosphorothioate OPs are metabolized by cytochrome P450s (P450s) through either a dearylation reaction to form an inactive metabolite, or through a desulfuration reaction to form an active oxon metabolite, which is a potent cholinesterase inhibitor. This study investigated the rate of desulfuration (activation) and dearylation (detoxification) of methyl parathion and diazinon in human liver microsomes. In addition, recombinant human P450s were used to determine the P450-specific kinetic parameters (Km and Vmax) for each compound for future use in refining human physiologically based pharmacokinetic/pharmacodynamic (PBPK/PD) models of OP exposure. The primary enzymes involved in bioactivation of methyl parathion were CYP2B6 (Km = 1.25 μM; Vmax = 9.78 nmol · min−1 · nmol P450−1), CYP2C19 (Km = 1.03 μM; Vmax = 4.67 nmol · min−1 · nmol P450−1), and CYP1A2 (Km = 1.96 μM; Vmax = 5.14 nmol · min−1 · nmol P450−1), and the bioactivation of diazinon was mediated primarily by CYP1A1 (Km = 3.05 μM; Vmax = 2.35 nmol · min−1 · nmol P450−1), CYP2C19 (Km = 7.74 μM; Vmax = 4.14 nmol · min−1 · nmol P450−1), and CYP2B6 (Km = 14.83 μM; Vmax = 5.44 nmol · min−1 · nmol P450−1). P450-mediated detoxification of methyl parathion only occurred to a limited extent with CYP1A2 (Km = 16.8 μM; Vmax = 1.38 nmol · min−1 · nmol P450−1) and 3A4 (Km = 104 μM; Vmax = 5.15 nmol · min−1 · nmol P450−1), whereas the major enzyme involved in diazinon detoxification was CYP2C19 (Km = 5.04 μM; Vmax = 5.58 nmol · min−1 · nmol P450−1). The OP- and P450-specific kinetic values will be helpful for future use in refining human PBPK/PD models of OP exposure. PMID:21969518

Fast evaluation of enantioselective drug metabolism by electrophoretically mediated microanalysis: application to fluoxetine metabolism by CYP2D6.

PubMed

Asensi-Bernardi, Lucía; Martín-Biosca, Yolanda; Escuder-Gilabert, Laura; Sagrado, Salvador; Medina-Hernández, María José

2013-12-01

In this work, a capillary electrophoretic methodology for the enantioselective in vitro evaluation of drugs metabolism is applied to the evaluation of fluoxetine (FLX) metabolism by cytochrome 2D6 (CYP2D6). This methodology comprises the in-capillary enzymatic reaction and the chiral separation of FLX and its major metabolite, norfluoxetine enantiomers employing highly sulfated β-CD and the partial filling technique. The methodology employed in this work is a fast way to obtain a first approach of the enantioselective in vitro metabolism of racemic drugs, with the additional advantage of an extremely low consumption of enzymes, CDs and all the reagents involved in the process. Michaelis-Menten kinetic parameters (Km and Vmax ) for the metabolism of FLX enantiomers by CYP2D6 have been estimated by nonlinear fitting of experimental data to the Michaelis-Menten equation. Km values have been found to be 30 ± 3 μM for S-FLX and 39 ± 5 μM for R-FLX. Vmax estimations were 28.6 ± 1.2 and 34 ± 2 pmol·min(-1) ·(pmol CYP)(-1) for S- and R-FLX, respectively. Similar results were obtained using a single enantiomer (R-FLX), indicating that the use of the racemate is a good option for obtaining enantioselective estimations. The results obtained show a slight enantioselectivity in favor of R-FLX. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Mode of action of family 10 and 11 endoxylanases on water-unextractable arabinoxylan.

PubMed

Vardakou, Maria; Katapodis, Petros; Samiotaki, Martina; Kekos, Dimitris; Panayotou, George; Christakopoulos, Paul

2003-11-01

Microbial endo-beta-1,4-xylanases (EXs, EC 3.2.1.8) belonging to glycanase families 10 and 11 differ in their action on water-unextractable arabinoxylan (WU-AX). WU-AX was incubated with different levels of a Thermoascus aurantiacus family 10 and a Sporotrichum thermophile family 11 endoxylanases. At 10 g l(-1) arabinoxylan, enzyme concentrations (KE values) needed to obtain half-maximal hydrolysis rates (V(max) values) were 4.4 nM for the xylanase from T. aurantiacus and 7.1 nM for the xylanase from S. thermophile. Determination of Vmax/KE revealed that the family 10 enzyme hydrolysed two times more efficiently WU-AX than the family 11 enzyme. Molecular weights of the products formed were assessed and separation of feruloyl-oligosaccharides was achieved by anion-exchange and size-exclusion chromatography (SEC). The main difference between the feruloylated products by xylanases of family 10 and 11 concerned the length of the products containing feruloyl-arabinosyl substitution. The xylanase from T. aurantiacus liberated from WU-AX a feruloyl arabinoxylodisaccharide (FAX2) as the shortest feruloylated fragment in contrast with the enzyme from S. thermophile, which liberated a feruloyl arabinoxylotrisaccharide (FAX3). These results indicated that different factors govern WU-AX breakdown by the two endoxylanases.

Contractile properties of rat, rhesus monkey, and human type I muscle fibers

NASA Technical Reports Server (NTRS)

Widrick, J. J.; Romatowski, J. G.; Karhanek, M.; Fitts, R. H.

1997-01-01

It is well known that skeletal muscle intrinsic maximal shortening velocity is inversely related to species body mass. However, there is uncertainty regarding the relationship between the contractile properties of muscle fibers obtained from commonly studied laboratory animals and those obtained from humans. In this study we determined the contractile properties of single chemically skinned fibers prepared from rat, rhesus monkey, and human soleus and gastrocnemius muscle samples under identical experimental conditions. All fibers used for analysis expressed type I myosin heavy chain as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Allometric coefficients for type I fibers from each muscle indicated that there was little change in peak tension (force/fiber cross-sectional area) across species. In contrast, both soleus and gastrocnemius type I fiber maximal unloaded shortening velocity (Vo), the y-intercept of the force-velocity relationship (Vmax), peak power per unit fiber length, and peak power normalized for fiber length and cross-sectional area were all inversely related to species body mass. The present allometric coefficients for soleus fiber Vo (-0.18) and Vmax (-0.11) are in good agreement with published values for soleus fibers obtained from common laboratory and domesticated mammals. Taken together, these observations suggest that the Vo of slow fibers from quadrupeds and humans scale similarly and can be described by the same quantitative relationships. These findings have implications in the design and interpretation of experiments, especially those that use small laboratory mammals as a model of human muscle function.

Effect of dietary fatty acids on jejunal and ileal oleic acid uptake by rat brush border membrane vesicles.

PubMed

Prieto, R M; Stremmel, W; Sales, C; Tur, J A

1996-04-18

To test the effect of dietary fatty acids on fatty acid uptake, the influx kinetics of a representative long-chain fatty acid, 3H-oleic acid, in both the jejunum and ileum of rats has been studied using brush border membrane vesicles (BBMV). Animals were fed with semipurified diets containing 5 g fat/100 g diet, as corn oil (control group), safflower oil (unsaturated group) and coconut oil hydrogenated (saturated group). With increasing unbound oleate concentration in the medium, the three dietary groups showed saturable kinetics in both jejunal and ileal BBMV (controls: Vmax = 0.15 +/- 0.01 nmol x mg protein-1 x 5 min-1 and Km = 136 +/- 29.1 nmol for jejunum, and Vmax = 0.23 +/- 0.03 nmol x mg protein-1 x 5 min-1 and Km = 196 +/- 50.3 nmol for ileum; unsaturated: Vmax = 0.28 +/- 0.05 nmol x mg protein-1 x 5 min-1 and Km = 242.7 +/- 91.8 nmol for jejunum, and Vmax = 1.29 +/- 0.06 nmol x mg protein-1 x 5 min-1 and Km = 509.8 +/- 97.5 nmol for ileum; saturated: Vmax = 0.03 +/- 0.01 nmol x mg protein-1 x 5 min-1 and Km = 124.5 +/- 72.6 nmol for jejunum, and Vmax = 0.04 +/- 0.01 nmol x mg protein -1.5 min-1 and Km = 205.6 +/- 85.3 nmol for ileum). These results support the theory that feeding an isocaloric diet containing only unsaturated fatty acids enhanced oleic acid uptake, and feeding an isocaloric diet containing only saturated fatty acids decreased oleic acid uptake. The results obtained in the present work also show the adaptative ability of jejunum and ileum to the type of dietary fat.

Biosynthesis of agmatine in isolated mitochondria and perfused rat liver: studies with 15N-labelled arginine

PubMed Central

2005-01-01

An important but unresolved question is whether mammalian mitochondria metabolize arginine to agmatine by the ADC (arginine decarboxylase) reaction. 15N-labelled arginine was used as a precursor to address this question and to determine the flux through the ADC reaction in isolated mitochondria obtained from rat liver. In addition, liver perfusion system was used to examine a possible action of insulin, glucagon or cAMP on a flux through the ADC reaction. In mitochondria and liver perfusion, 15N-labelled agmatine was generated from external 15N-labelled arginine. The production of 15N-labelled agmatine was time- and dose-dependent. The time-course of [U-15N4]agmatine formation from 2 mM [U-15N4]arginine was best fitted to a one-phase exponential curve with a production rate of approx. 29 pmol·min−1·(mg of protein)−1. Experiments with an increasing concentration (0– 40 mM) of [guanidino-15N2]arginine showed a Michaelis constant Km for arginine of 46 mM and a Vmax of 3.7 nmol·min−1·(mg of protein)−1 for flux through the ADC reaction. Experiments with broken mitochondria showed little changes in Vmax or Km values, suggesting that mitochondrial arginine uptake had little effect on the observed Vmax or Km values. Experiments with liver perfusion demonstrated that over 95% of the effluent agmatine was derived from perfusate [guanidino-15N2]arginine regardless of the experimental condition. However, the output of 15N-labelled agmatine (nmol·min−1·g−1) increased by approx. 2-fold (P<0.05) in perfusions with cAMP. The findings of the present study provide compelling evidence that mitochondrial ADC is present in the rat liver, and suggest that cAMP may stimulate flux through this pathway. PMID:15656789

A determination and comparison of urease activity in feces and fresh manure from pig and cattle in relation to ammonia production and pH changes.

PubMed

Dai, Xiaorong; Karring, Henrik

2014-01-01

Ammonia emission from animal production is a major environmental problem and has impacts on the animal health and working environment inside production houses. Ammonia is formed in manure by the enzymatic degradation of urinary urea and catalyzed by urease that is present in feces. We have determined and compared the urease activity in feces and manure (a urine and feces mixture) from pigs and cattle at 25°C by using Michaelis-Menten kinetics. To obtain accurate estimates of kinetic parameters Vmax and K'm, we used a 5 min reaction time to determine the initial reaction velocities based on total ammoniacal nitrogen (TAN) concentrations. The resulting Vmax value (mmol urea hydrolyzed per kg wet feces per min) was 2.06±0.08 mmol urea/kg/min and 0.80±0.04 mmol urea/kg/min for pig feces and cattle feces, respectively. The K'm values were 32.59±5.65 mmol urea/l and 15.43±2.94 mmol urea/l for pig feces and cattle feces, respectively. Thus, our results reveal that both the Vmax and K'm values of the urease activity for pig feces are more than 2-fold higher than those for cattle feces. The difference in urea hydrolysis rates between animal species is even more significant in fresh manure. The initial velocities of TAN formation are 1.53 mM/min and 0.33 mM/min for pig and cattle manure, respectively. Furthermore, our investigation shows that the maximum urease activity for pig feces occurs at approximately pH 7, and in cattle feces it is closer to pH 8, indicating that the predominant fecal ureolytic bacteria species differ between animal species. We believe that our study contributes to a better understanding of the urea hydrolysis process in manure and provides a basis for more accurate and animal-specific prediction models for urea hydrolysis rates and ammonia concentration in manures and thus can be used to predict ammonia volatilization rates from animal production.

A Determination and Comparison of Urease Activity in Feces and Fresh Manure from Pig and Cattle in Relation to Ammonia Production and pH Changes

PubMed Central

Dai, Xiaorong; Karring, Henrik

2014-01-01

Ammonia emission from animal production is a major environmental problem and has impacts on the animal health and working environment inside production houses. Ammonia is formed in manure by the enzymatic degradation of urinary urea and catalyzed by urease that is present in feces. We have determined and compared the urease activity in feces and manure (a urine and feces mixture) from pigs and cattle at 25°C by using Michaelis-Menten kinetics. To obtain accurate estimates of kinetic parameters Vmax and K'm, we used a 5 min reaction time to determine the initial reaction velocities based on total ammoniacal nitrogen (TAN) concentrations. The resulting Vmax value (mmol urea hydrolyzed per kg wet feces per min) was 2.06±0.08 mmol urea/kg/min and 0.80±0.04 mmol urea/kg/min for pig feces and cattle feces, respectively. The K'm values were 32.59±5.65 mmol urea/l and 15.43±2.94 mmol urea/l for pig feces and cattle feces, respectively. Thus, our results reveal that both the Vmax and K'm values of the urease activity for pig feces are more than 2-fold higher than those for cattle feces. The difference in urea hydrolysis rates between animal species is even more significant in fresh manure. The initial velocities of TAN formation are 1.53 mM/min and 0.33 mM/min for pig and cattle manure, respectively. Furthermore, our investigation shows that the maximum urease activity for pig feces occurs at approximately pH 7, and in cattle feces it is closer to pH 8, indicating that the predominant fecal ureolytic bacteria species differ between animal species. We believe that our study contributes to a better understanding of the urea hydrolysis process in manure and provides a basis for more accurate and animal-specific prediction models for urea hydrolysis rates and ammonia concentration in manures and thus can be used to predict ammonia volatilization rates from animal production. PMID:25397404

Phenytoin intoxication during concurrent diazepam therapy

PubMed Central

Rogers, Howard J.; Haslam, Robert A.; Longstreth, James; Lietman, Paul S.

1977-01-01

Phenytoin elimination is a saturable process obeying Michaelis-Menten kinetics. Plasma phenytoin levels are not related linearly to dose, and small changes in enzyme activity produced by concurrent drug therapy could alter plasma levels. Two cases of phenytoin intoxication associated with simultaneous administration of diazepam are reported. Intravenous phenytoin infusions were given and the apparent Km and Vmax computed from the resulting plasma phenytoin levels. In one case `Km' and `Vmax' were 0.8 μmol/1 and 1.3 μmol/1/hour respectively during concurrent diazepam administration, and 50.3 μmol/1 and 4.4 μmol/1/hour after discontinuation of diazepam. In the second case phenytoin infusion with diazepam gave `Km' and `Vmax' values of 0.012 μmol/1 and 0.95 μmol/1/hour. Without diazepam these were 28.8 μmol/1 and 0.92 μmol/1/hour respectively. PMID:599366

Purification and characterization of a p-coumarate decarboxylase and a vinylphenol reductase from Brettanomyces bruxellensis.

PubMed

Godoy, Liliana; Martínez, Claudio; Carrasco, Nelson; Ganga, María Angélica

2008-09-30

The presence of Brettanomyces bruxellensis has been correlated with an increase of phenolic aromas in wine. The production of these aromas results from the metabolization of cinnamic acids, present in the wine, to their ethyl derivatives. Hence, the participation of two enzymes has been proposed: a p-coumarate decarboxylase (CD) and a vinylphenol reductase (VR). Both enzymes were purified and characterized from B. bruxellensis. In denaturing conditions, the CD enzyme had a molecular mass of 21 kDa, while in native conditions its mass was 41 kDa. The optimal activity was obtained at a temperature of 40 degrees C and a pH of 6.0. For p-coumaric acid, the Km value and Vmax were 1.22+/-0.08 mM and 98+/-0.15 micromol/min mg, respectively. The VR enzyme had a molecular mass of 37 kDa in SDS-PAGE, while in natural conditions its mass was 118 kDa. The Km value was > 3.37+/-2.05 mM and its Vmax was 107.62+/-50.38 micromol/min mg for NADPH used as a cofactor. Both enzymatic activities were stable at pH 3.4, but in the presence of ethanol the CD activity decreased drastically while the VR activity was more stable. This is the first report that shows the presence of a CD and a VR enzyme in B. bruxellensis.

Influence of rete testis fluid deprivation on the kinetic parameters of goat epididymal 5 alpha-reductase.

PubMed

Kelce, W R; Lubis, A M; Braun, W F; Youngquist, R S; Ganjam, V K

1990-01-01

A surgical technique to cannulate the rete testis of the goat was utilized to examine the effects of rete testis fluid (RTF) deprivation on the enzymatic activity of epididymal 5 alpha-reductase. Kinetic techniques were used to determine whether the regional enzymatic effect of RTF deprivation is to decrease the apparent number of 5 alpha-reductase active sites or the catalytic activity of each active site within the epididymal epithelium. Paired comparisons of (Vmax)app and (Km)app values between control and RTF-deprived epididymides indicated that RTF deprivation affected the value of (Vmax)app with no apparent change in the values of (Km)app in caput, corpus, and cauda epididymal regions. We conclude that RTF deprivation in the goat epididymis for 7 days results in a decreased number of apparent 5 alpha-reductase active sites within the epididymal epithelium.

Kinetics of 3H-serotonin uptake by platelets in infantile autism and developmental language disorder (including five pairs of twins)

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

Katsui, T.; Okuda, M.; Usuda, S.

The kinetics of 5-HT uptake by platelets was studied in cases of infantile autism and developmental language disorder (DLD) and normal subjects. Two patients of the autism group were twins, and the seven patients of the DLD group were members of four pairs of twins. The Vmax values (means +/- SD) for autism and DLD were 6.46 +/- .90 pmol 5-HT/10(7) cells/min and 4.85 +/- 1.50 pmol 5-HT/10(7) cells/min, respectively. These values were both significantly higher than that of 2.25 +/- .97 pmole 5-HT/10(7) cells/min for normal children. The Km values of the three groups were not significantly different. Datamore » on the five pairs of twins examined suggested that the elevated Vmax of 5-HT uptake by platelets was determined genetically.« less

Estimation of ground motion for Bhuj (26 January 2001; Mw 7.6 and for future earthquakes in India

USGS Publications Warehouse

Singh, S.K.; Bansal, B.K.; Bhattacharya, S.N.; Pacheco, J.F.; Dattatrayam, R.S.; Ordaz, M.; Suresh, G.; ,; Hough, S.E.

2003-01-01

Only five moderate and large earthquakes (Mw ???5.7) in India-three in the Indian shield region and two in the Himalayan arc region-have given rise to multiple strong ground-motion recordings. Near-source data are available for only two of these events. The Bhuj earthquake (Mw 7.6), which occurred in the shield region, gave rise to useful recordings at distances exceeding 550 km. Because of the scarcity of the data, we use the stochastic method to estimate ground motions. We assume that (1) S waves dominate at R < 100 km and Lg waves at R ??? 100 km, (2) Q = 508f0.48 is valid for the Indian shield as well as the Himalayan arc region, (3) the effective duration is given by fc-1 + 0.05R, where fc is the corner frequency, and R is the hypocentral distance in kilometer, and (4) the acceleration spectra are sharply cut off beyond 35 Hz. We use two finite-source stochastic models. One is an approximate model that reduces to the ??2-source model at distances greater that about twice the source dimension. This model has the advantage that the ground motion is controlled by the familiar stress parameter, ????. In the other finite-source model, which is more reliable for near-source ground-motion estimation, the high-frequency radiation is controlled by the strength factor, sfact, a quantity that is physically related to the maximum slip rate on the fault. We estimate ???? needed to fit the observed Amax and Vmax data of each earthquake (which are mostly in the far field). The corresponding sfact is obtained by requiring that the predicted curves from the two models match each other in the far field up to a distance of about 500 km. The results show: (1) The ???? that explains Amax data for shield events may be a function of depth, increasing from ???50 bars at 10 km to ???400 bars at 36 km. The corresponding sfact values range from 1.0-2.0. The ???? values for the two Himalayan arc events are 75 and 150 bars (sfact = 1.0 and 1.4). (2) The ???? required to explain Vmax data is, roughly, half the corresponding value for Amax, while the same sfact explains both sets of data. (3) The available far-field Amax and Vmax data for the Bhuj mainshock are well explained by ???? = 200 and 100 bars, respectively, or, equivalently, by sfact = 1.4. The predicted Amax and Vmax in the epicentral region of this earthquake are 0.80 to 0.95 g and 40 to 55 cm/sec, respectively.

Effects of deoxygenation on active and passive Ca2+ transport and cytoplasmic Ca2+ buffering in normal human red cells.

PubMed Central

Tiffert, T; Etzion, Z; Bookchin, R M; Lew, V L

1993-01-01

1. The effects of deoxygenation on cytoplasmic Ca2+ buffering, saturated Ca2+ extrusion rate through the Ca2+ pump (Vmax), passive Ca2+ influx and physiological [Ca2+]i level were investigated in human red cells to assess whether or not their Ca2+ metabolism might be altered by deoxygenation in capillaries and venous circulation. 2. The study was performed in fresh human red cells maintained in a tonometer either fully oxygenated or deoxygenated. Cytoplasmic Ca2+ buffering was estimated from the equilibrium distribution of 45Ca2+ induced by the divalent cation ionophore A23187 and the Vmax of the Ca2+ pump was measured either by the Co(2+)-exposure method or following ionophore wash-out. The passive Ca2+ influx and physiological [Ca2+]i were determined in cells preloaded with the Ca2+ chelator benz-2 and resuspended in autologous plasma. 3. Deoxygenation increased the fraction of ionized Ca2+ in cell water by 34-74% and reduced the Vmax of the Ca2+ pump by 18-32%. 4. To elucidate whether or not these effects were secondary to deoxygenation-induced pH shifts, the effects of deoxygenation on cell and medium pH, and of pH on cytoplasmic Ca2+ binding and Ca2+ pump Vmax in oxygenated cells were examined in detail. 5. Deoxygenation generated large alkaline pH shifts that could be explained if the apparent isoelectric point (pI) of haemoglobin increased by 0.2-0.4 pH units in intact cells, consistently higher than the value of 0.15 reported for pure haemoglobin solutions. 6. In oxygenated cells, the fraction of ionized cell calcium, alpha, was little affected by pH within the 7.0-7.7 range. Ca2+ pump Vmax was maximal at a medium pH of about 7.55. Comparison between pH effects elicited by HCl-NaOH additions and by replacing Cl- with gluconate suggested that Vmax was inhibited by both internal acidification and external alkalinization. Since deoxygenation alkalinized cells and medium within a range stimulatory for Vmax, the inhibition observed was not due to pH. 7. There was no significant effect of deoxygenation on passive Ca2+ uptake, or steady-state physiological [Ca2+]i level. 8. The deoxygenation-induced reduction in Ca2+ binding capacity may result from the increased protonation of haemoglobin on deoxygenation and from binding of 2,3-diphosphoglyceric acid (2,3-DPG) and ATP to deoxyhaemoglobin; inhibition of the Ca2+ pump may result from shifts in the [Mg2+]i/[ATP]i ratio away from a near optimal stimulatory value in the oxygenated state. PMID:8229816

Universal Dark Halo Scaling Relation for the Dwarf Spheroidal Satellites

NASA Astrophysics Data System (ADS)

Hayashi, Kohei; Ishiyama, Tomoaki; Ogiya, Go; Chiba, Masashi; Inoue, Shigeki; Mori, Masao

2017-07-01

Motivated by a recently found interesting property of the dark halo surface density within a radius, {r}\\max , giving the maximum circular velocity, {V}\\max , we investigate it for dark halos of the Milky Way’s and Andromeda’s dwarf satellites based on cosmological simulations. We select and analyze the simulated subhalos associated with Milky-Way-sized dark halos and find that the values of their surface densities, {{{Σ }}}{V\\max }, are in good agreement with those for the observed dwarf spheroidal satellites even without employing any fitting procedures. Moreover, all subhalos on the small scales of dwarf satellites are expected to obey the universal relation, irrespective of differences in their orbital evolutions, host halo properties, and observed redshifts. Therefore, we find that the universal scaling relation for dark halos on dwarf galaxy mass scales surely exists and provides us with important clues for understanding fundamental properties of dark halos. We also investigate orbital and dynamical evolutions of subhalos to understand the origin of this universal dark halo relation and find that most subhalos evolve generally along the {r}\\max \\propto {V}\\max sequence, even though these subhalos have undergone different histories of mass assembly and tidal stripping. This sequence, therefore, should be the key feature for understanding the nature of the universality of {{{Σ }}}{V\\max }.

Assessment of Portal Venous and Hepatic Artery Haemodynamic Variation in Non-Alcoholic Fatty Liver Disease (NAFLD) Patients.

PubMed

Balasubramanian, Padhmini; Boopathy, Vinoth; Govindasamy, Ezhumalai; Venkatesh, Basavaiya Prabhu

2016-08-01

Non-Alcoholic Fatty Liver Disease (NAFLD) has various spectrums of liver diseases like isolated fatty liver, steatohepatitis and cirrhosis usually progressing in a linear fashion. In this process they are known to cause certain haemodynamic changes in the portal flow and hepatic artery flow. The aim of the study was to study these haemodynamic changes in patients with NAFLD and to correlate it with the disease severity. Ninety patients diagnosed to have NAFLD based on ultrasound abdomen (30 each in grade1, grade2 and grade3 NAFLD) and 30 controls (Normal liver on ultrasound abdomen) were subjected to portal vein and hepatic artery Doppler study. Peak maximum velocity (Vmax), Peak minimum velocity (Vmin), Mean flow velocity (MFV), and Vein pulsality index (VPI) of the portal vein and hepatic artery resistivity index (HARI) of the hepatic artery were the doppler parameters which were assessed. Liver span was also assessed both for the fatty liver and controls. The mean Vmax, Vmin, MFV and VPI of the portal vein in patients with NAFLD was 12.23±1.74cm/sec, 9.31±1.45cm/sec, 10.76±1.48cm/sec, and 0.24±0.04 as compared to 14.05±2.43cm/sec, 10.01±2.27cm/sec, 12.23±2.47cm/sec, 0.3±0.08 in controls respectively. All these differences were statistically significant except for Vmin. The Mean HARI in patients with fatty liver was 0.65±0.06 when compared to controls of 0.75±0.06 (p=0.001). HARI (r-value of -0.517) had a better negative correlation followed by VPI (r-value of -0.44) and Vmax (r-value of -0.293) with the severity of NAFLD. MFV had a very weak negative correlation (r-value of -0.182) with the severity of NAFLD. The Vmax, MFV, VPI and HARI were significantly less when compared to controls suggesting a reduced portal flow and an increased hepatic arterial flow in patients with NAFLD. Among the parameters, HARI correlated better with the severity of NAFLD followed by VPI.

Anti-lipase and antioxidant properties of 30 medicinal plants used in Oaxaca, México.

PubMed

Villa-Ruano, Nemesio; Zurita-Vásquez, Guilibaldo G; Pacheco-Hernández, Yesenia; Betancourt-Jiménez, Martha G; Cruz-Durán, Ramiro; Duque-Bautista, Horacio

2013-01-01

We report the results of in vitro anti-lipase and antioxidant assays using crude ethanolic extracts from 30 plants grown in Oaxaca, México. Anti-lipase tests were performed by using porcine pancreatic lipase (PPL) [EC 3.1.1.3] from Affymetrix/USB. The extracts of Solanum erianthum, Salvia microphylla, Brungmansia suaveolens and Cuphea aequipetala showed up to 60% PPL inhibition. The effect of these extracts on the kinetic parameters of PPL (Km= 0.36 mM, and Vmax=0.085 mM min -1) revealed that the alcoholic preparations of S. erianthum and C. aequipetala engendered a non-competitive inhibition (Vmax=0.055 mM min -1; Vmax= 0.053 mM min -1), whereas those of S. microphylla and B. suaveolens produced a mixed inhibition (Km= 0.567 mM, Vmax=0.051 mM min _1; Km=0.643 mM, Vmax= 0.042 mM min ¹). In addition to these findings, seven extracts from different plants were able to inhibit PPL in the range of 30-50%. Antioxidant tests against 2,2-Diphenyl-1-picryl hydrazyl (DPPH) confirmed that Arctostaphylos pungens, Gnaphalium roseum, Crotalaria pumila, Cuphea aequipetala, Rhus chondroloma, and Satureja laevigata possess relevant antioxidant activity (IC(5)0=50-80 μg mL¹). The general composition of the most effective ethanolic extracts was obtained in order to confirm their known chemistry reported by previous works. Comprehensive chemical analysis of the ethanolic extracts and their poisoning effects suggests that S. microphylla, C. aequipetala and A. pungens could be considered as the best sources with both desired properties.

Cellulose binding domain assisted immobilization of lipase (GSlip-CBD) onto cellulosic nanogel: characterization and application in organic medium.

PubMed

Kumar, Ashok; Zhang, Shaowei; Wu, Gaobing; Wu, Cheng Chao; Chen, JunPeng; Baskaran, R; Liu, Ziduo

2015-12-01

A cbd gene was cloned into the C-terminal region of a lip gene from Geobacillus stearothermophilus. The native lipase (43.5 kDa) and CBD-Lip fusion protein (60.2 kDa) were purified to homogeneity by SDS-PAGE. A highly stable cellulosic nanogel was prepared by controlled hydrolysis of microcrystalline cellulose onto which the CBD-lip fusion protein was immobilized through bio-affinity based binding. The nanogel-bound lipase showed optimum activity at 55 °C, and it remains stable and active at pH 10-10.5. Furthermore, the immobilized lipase showed an over two-fold increase of relative activity in the presence of DMSO, isopropanol, isoamyl alcohol and n-butanol, but a mild activity decrease at a low concentration of methanol and ethanol. The immobilized biocatalyst retained ~50% activity after eight repetitive hydrolytic cycles. Enzyme kinetic studies of the immobilized lipase showed a 1.24 fold increase in Vmax and 5.25 fold increase in kcat towards p-NPP hydrolysis. Additionally, the nanogel bound lipase was tested to synthesize a biodiesel ester, ethyl oleate in DMSO. Kinetic analysis showed the km 100.5 ± 4.3 mmol and Vmax 0.19 ± 0.015 mmolmin(-1) at varied oleic acid concentration. Also, the values of km and Vmax at varying concentration of ethanol were observed to be 95.9 ± 13.9 mmol and 0.22 ± 0.013 mmolmin(-1) respectively. The maximum yield of ethyl oleate 111.2 ± 1.24 mM was obtained under optimized reaction conditions in organic medium. These results suggest that this immobilized biocatalyst can be used as an efficient tool for the biotransformation reactions on an industrial scale. Copyright © 2015 Elsevier B.V. All rights reserved.

Validation of a Video Analysis Software Package for Quantifying Movement Velocity in Resistance Exercises.

PubMed

Sañudo, Borja; Rueda, David; Pozo-Cruz, Borja Del; de Hoyo, Moisés; Carrasco, Luis

2016-10-01

Sañudo, B, Rueda, D, del Pozo-Cruz, B, de Hoyo, M, and Carrasco, L. Validation of a video analysis software package for quantifying movement velocity in resistance exercises. J Strength Cond Res 30(10): 2934-2941, 2016-The aim of this study was to establish the validity of a video analysis software package in measuring mean propulsive velocity (MPV) and the maximal velocity during bench press. Twenty-one healthy males (21 ± 1 year) with weight training experience were recruited, and the MPV and the maximal velocity of the concentric phase (Vmax) were compared with a linear position transducer system during a standard bench press exercise. Participants performed a 1 repetition maximum test using the supine bench press exercise. The testing procedures involved the simultaneous assessment of bench press propulsive velocity using 2 kinematic (linear position transducer and semi-automated tracking software) systems. High Pearson's correlation coefficients for MPV and Vmax between both devices (r = 0.473 to 0.993) were observed. The intraclass correlation coefficients for barbell velocity data and the kinematic data obtained from video analysis were high (>0.79). In addition, the low coefficients of variation indicate that measurements had low variability. Finally, Bland-Altman plots with the limits of agreement of the MPV and Vmax with different loads showed a negative trend, which indicated that the video analysis had higher values than the linear transducer. In conclusion, this study has demonstrated that the software used for the video analysis was an easy to use and cost-effective tool with a very high degree of concurrent validity. This software can be used to evaluate changes in velocity of training load in resistance training, which may be important for the prescription and monitoring of training programmes.

Effects of CYP2C19 Variants on Fluoxetine Metabolism in vitro.

PubMed

Fang, Ping; He, Jia-Yang; Han, Ai-Xia; Lan, Tian; Dai, Da-Peng; Cai, Jian-Ping; Hu, Guo-Xin

2017-01-01

CYP2C19 is an important member of the cytochrome P450 enzyme superfamily. We recently identified 31 CYP2C19 alleles in the Han Chinese population. The aim of this study was to assess the catalytic activities of these allelic isoforms and their effects on the metabolism of fluoxetine in vitro. The wild-type and 30 CYP2C19 variants were expressed in insect cells and each variant was characterized using fluoxetine as the substrate. Reactions were performed at 37°C with 20-1,000 µmol/L substrate for 30 min. By using ultra-high performance liquid chromatography-mass spectrometry to detect the products, the kinetic parameters Km, Vmax, and intrinsic clearance (Vmax/Km) of norfluoxetine were determined. Among the CYP2C19 variants tested, T130M showed similar intrinsic clearance (Vmax/Km) values with CYP2C19*1, while the intrinsic clearance values of other variants were significantly decreased (from 9.56 to 77.77%). In addition, CYP2C19*3 and *35FS could not be detected because they have no detectable enzyme activity. In China, the assessment of CYP2C19 variants in vitro offers valuable information relevant to the personalized medicine for CYP2C19-metabolized drug. © 2017 S. Karger AG, Basel.

Temperature Sensitivities of Extracellular Enzyme Vmax and Km Across Thermal Environments

NASA Astrophysics Data System (ADS)

Allison, S. D.; Romero-Olivares, A.; Lu, Y.; Taylor, J.; Treseder, K. K.

2017-12-01

The magnitude and direction of carbon cycle feedbacks under climate warming remain uncertain due to insufficient knowledge about the temperature sensitivity of microbial processes in soil. Enzymatic rates could increase at higher temperatures, but this response is determined by multiple parameters that may change over time if soil microbes adapt to warming. We used the Michaelis-Menten relationship, the Arrhenius relationship, and biochemical transition state theory to construct hypotheses about the responses of extracellular enzyme Vmax and Km to temperature. Based on the Arrhenius relationship, we hypothesized that Vmax and Km would show positive temperature sensitivities. For enzymes from warmer environments, we expected to find lower Vmax, Km, and Km temperature sensitivity but higher Vmax temperature sensitivity. We tested these hypotheses with enzymes from isolates of the filamentous fungus Neurospora discreta collected around the globe and from decomposing leaf litter in a warming experiment in Alaskan boreal forest. Vmax and Km of most Neurospora extracellular enzymes were temperature sensitive with average Vmax Q10 ranging from 1.48 to 2.25 and Km Q10 ranging from 0.71 to 2.80. For both Vmax and Km, there was a tendency for the parameter to correlate negatively with its temperature sensitivity, a pattern predicted by biochemical theory. Also in agreement with theory, Vmax and Km were positively correlated for some enzymes. In contrast, there was little support for biochemical theory when comparing Vmax and Km across thermal environments. There was no relationship between temperature sensitivity of Vmax or Km and mean annual temperature of the isolation site for Neurospora strains. There was some evidence for greater Vmax under experimental warming in Alaskan litter, but the temperature sensitivities of Vmax and Km did not vary with warming as expected. We conclude that relationships among Vmax, Km, and temperature are largely consistent with biochemical theory, and our enzyme data should be useful for parameterizing trait-based models of microbial processes. However, theoretical predictions about adaptation to thermal environment were not supported by our data, suggesting that covarying edaphic and ecological factors may play a dominant role in soil enzyme responses to climate warming.

Catalytical Properties of Free and Immobilized Aspergillus niger Tannase.

PubMed

Flores-Maltos, Abril; Rodríguez-Durán, Luis V; Renovato, Jacqueline; Contreras, Juan C; Rodríguez, Raúl; Aguilar, Cristóbal N

2011-01-01

A fungal tannase was produced, recovered, and immobilized by entrapment in calcium alginate beads. Catalytical properties of the immobilized enzyme were compared with those of the free one. Tannase was produced intracellularly by the xerophilic fungus Aspergillus niger GH1 in a submerged fermentation system. Enzyme was recovered by cell disruption and the crude extract was partially purified. The catalytical properties of free and immobilized tannase were evaluated using tannic acid and methyl gallate as substrates. K(M) and V(max) values for free enzyme were very similar for both substrates. But, after immobilization, K(M) and V(max) values increased drastically using tannic acid as substrate. These results indicated that immobilized tannase is a better biocatalyst than free enzyme for applications on liquid systems with high tannin content, such as bioremediation of tannery or olive-mill wastewater.

pH regulation in barnacle muscle fibers: dependence on extracellular sodium and bicarbonate.

PubMed

Boron, W F; McCormick, W C; Roos, A

1981-01-01

Intracellular pH (pHi) regulation was studied in barnacle muscle fibers with pH-sensitive microelectrodes. The cells were acid loaded, and the subsequent recovery of pHi was monitored. The rate of recovery was reduced by one-third when external Na+ ([Na+]o) was replaced by Li+, but recovery was completely abolished when Na+ was replaced by choline or N-methyl-D-glucamine. In other experiments, varying amounts of Na+ were replaced by choline, and the acid extrusion rate, derived from the recovery rate of pHi, was calculated at a single value of pHi, 6.80. The dependence of the acid extrusion rate on [Na+]o could be described by Michaelis-Menten kinetics; at pHo (extracellular) = 8.0 and [HCO3-]o (extracellular) = 10 mM, the apparent Km and Vmax were 59 mM and 1.3 mmol x l(-1) x min-1. When [HCO3-]o was reduced to 2.5 mM at the same pHo, Km did not change significantly, but Vmax was substantially reduced. On the other hand, when pHo was reduced to 7.4 at constant [HCO3-]o, Vmax changed only slightly, but Km increased substantially. In similar experiments, we examined the dependence of the acid extrusion rate on [HCO3-]o. At pHo = 8.0 and [Na+]o = 440 mM, the apparent Km and Vmax were 4.1 mM and 2.1 mmol x 1-1 x min-1. When pHo was reduced to 7.4, Vmax was not altered, but Km substantially increased. The kinetic data are discussed in terms of the role of pHo, [Na+]o, and [HCO3-]o in the pHi-regulating system.

Stereospecificity of mushroom tyrosinase immobilized on a chiral and a nonchiral support.

PubMed

Marín-Zamora, María Elisa; Rojas-Melgarejo, Francisco; García-Canovas, Francisco; García-Ruiz, Pedro Antonio

2007-05-30

Mushroom tyrosinase was immobilized from an extract onto glass beads covered with the cross-linked totally cinnamoylated derivates of d-sorbitol (sorbitol cinnamate) and glycerine (glycerine cinnamate). The enzyme was immobilized onto the support by direct adsorption, and the quantity of immobilized tyrosinase was higher for sorbitol cinnamate, the support with the higher number of esterified hydroxyls per unit of monosacharide, than for glycerine cinnamate. The results obtained from the stereospecificity study of the monophenolase and diphenolase activity of immobilized mushroom tyrosinase are reported. The enantiomers L-tyrosine, DL-tyrosine, D-tyrosine, L-dopa, DL-dopa, D-dopa, L-alpha-methyldopa, DL-alpha-methyldopa, L-isoprenaline, DL-isoprenaline, L-adrenaline, DL-adrenaline, L-noradrenaline, and D-noradrenaline were assayed with tyrosinase immobilized on a chiral support (sorbitol cinnamate), whereas L-tyrosine, DL-tyrosine, D-tyrosine, L-dopa, DL-dopa, D-dopa, L-alpha-methyldopa, and DL-alpha-methyldopa were assayed with tyrosinase immobilized on a nonchiral support (glycerine cinnamate). The same Vmax(app) values for each series of enantiomers were obtained. However, the Km(app) values were different, the l isomers showing lower values than the dl isomers, whereas the highest Km(app) value was obtained with d isomers. No difference was observed in the stereospecificity of tyrosinase immobilized on a chiral (sorbitol cinnamate) or nonchiral (glycerine cinnamate) support.

Pyruvate and ketone-body transport across the mitochondrial membrane. Exchange properties, pH-dependence and mechanism of the carrier.

PubMed

Halestrap, A P

1978-06-15

The effects of exchangeable ions and pH on the efflux of pyruvate from preloaded mitochondria are reported. Efflux obeys first-order kinetics, and the stimulation of efflux by exchangeable ions such as acetoacetate and lactate obeys Michaelis--Menten kinetics. The apparent Km value +/- S.E. for acetoacetate was 0.56 +/- 0.14 mM (n = 5) and that for lactate 12.3 +/- 2.3 mM (n = 6). The Vmax. values +/- S.E. at 0 degrees C were 16.2 +/- 2.0 and 21.9 +/- 2.7 nmol/min per mg of protein. The exchange of a variety of other substituted monocarboxylates was also studied. Efflux was also stimulated by increasing the external pH. The data gave a pK for the transport process of 8.35 and a Vmax. of 3.31 +/- 0.14 nmol/min per mg. The similarity of the Vmax. values for various exchangeable ions but the difference of this from the Vmax. in the absence of exchangeable ions may indicate that transport of pyruvate occurs with H+ and not in exchange for an OH- ion. The inhibition of transport by alpha-cyano-4-hydroxycinnamate took several seconds to reach completion at 0 degrees C. It is proposed that inhibition occurs by binding to the substrate site and subsequent reaction with an -SH group on the inside of the membrane. The inhibitor can be displaced by substrates that can also enter the mitochondria independently of the carrier and so compete with the inhibitor for the substrate-binding site on the inside of the membrane. A mechanism for transport is proposed that invokes a transition state of pyruvate involving addition of an -SH group to the 2-carbon of pyruvate. Evidence is presented that suggests that ketone bodies may cross the mitochondrial membrane either on the carrier or by free diffusion. The physiological involvement of the carrier in ketone-body metabolism is discussed. The role of ketone bodies and pH in the physiological regulation of pyruvate transport is considered.

Characterization of protease from bacillus sp. on medium containing FeCl3 exposed to magnetic field 0.2 mt

NASA Astrophysics Data System (ADS)

Sumardi; Agustrina, Rochmah; Nugroho Ekowati, Christina; Selvie Pasaribu, Yovita

2018-03-01

This purpose of this research is to determine the character of the protease enzymes from Bacillus sp. on media content of FeCl3 exposed to 0.2 mT magnetic field. The data obtained were analyzed descriptively. The result showed that protease enzyme without Fe resulted in the highest activity at pH 8, temperature. 30°C with the addition of activator Mn2+, and Vmax of 0.28 U/ml, and Km of 4.60 U/ml. The protease enzyme on media without magnetic field exposure and containing Fe yielded the highest activity at pH 8, temperature 30°C with the addition of activator Mn2+, and Vmax of 0.33 U/ml, and Km of 5.64 U/ml. The protease enzyme on medium with magnetic field exposure and use Fe as inductors have the highest activity at pH 9, the temperature of 55° C with the addition of activator Mn2+, and Vmax of 0.35 U/ml, and Km 10.04 U/ml.

Tunnel transport and interlayer excitons in bilayer fractional quantum Hall systems

NASA Astrophysics Data System (ADS)

Zhang, Yuhe; Jain, J. K.; Eisenstein, J. P.

2017-05-01

In a bilayer system consisting of a composite-fermion (CF) Fermi sea in each layer, the tunnel current is exponentially suppressed at zero bias, followed by a strong peak at a finite-bias voltage Vmax. This behavior, which is qualitatively different from that observed for the electron Fermi sea, provides fundamental insight into the strongly correlated non-Fermi-liquid nature of the CF Fermi sea and, in particular, offers a window into the short-distance high-energy physics of this highly nontrivial state. We identify the exciton responsible for the peak current and provide a quantitative account of the value of Vmax. The excitonic attraction is shown to be quantitatively significant, and its variation accounts for the increase of Vmax with the application of an in-plane magnetic field. We also estimate the critical Zeeman energy where transition occurs from a fully spin-polarized composite-fermion Fermi sea to a partially spin-polarized one, carefully incorporating corrections due to finite width and Landau level mixing, and find it to be in satisfactory agreement with the Zeeman energy where a qualitative change has been observed for the onset bias voltage [J. P. Eisenstein et al., Phys. Rev. B 94, 125409 (2016), 10.1103/PhysRevB.94.125409]. For fractional quantum Hall states, we predict a substantial discontinuous jump in Vmax when the system undergoes a transition from a fully spin-polarized state to a spin singlet or a partially spin-polarized state.

Leaf chlorophyll constraint on model simulated gross primary productivity in agricultural systems

NASA Astrophysics Data System (ADS)

Houborg, Rasmus; McCabe, Matthew F.; Cescatti, Alessandro; Gitelson, Anatoly A.

2015-12-01

Leaf chlorophyll content (Chll) may serve as an observational proxy for the maximum rate of carboxylation (Vmax), which describes leaf photosynthetic capacity and represents the single most important control on modeled leaf photosynthesis within most Terrestrial Biosphere Models (TBMs). The parameterization of Vmax is associated with great uncertainty as it can vary significantly between plants and in response to changes in leaf nitrogen (N) availability, plant phenology and environmental conditions. Houborg et al. (2013) outlined a semi-mechanistic relationship between Vmax25 (Vmax normalized to 25 °C) and Chll based on inter-linkages between Vmax25, Rubisco enzyme kinetics, N and Chll. Here, these relationships are parameterized for a wider range of important agricultural crops and embedded within the leaf photosynthesis-conductance scheme of the Community Land Model (CLM), bypassing the questionable use of temporally invariant and broadly defined plant functional type (PFT) specific Vmax25 values. In this study, the new Chll constrained version of CLM is refined with an updated parameterization scheme for specific application to soybean and maize. The benefit of using in-situ measured and satellite retrieved Chll for constraining model simulations of Gross Primary Productivity (GPP) is evaluated over fields in central Nebraska, U.S.A between 2001 and 2005. Landsat-based Chll time-series records derived from the Regularized Canopy Reflectance model (REGFLEC) are used as forcing to the CLM. Validation of simulated GPP against 15 site-years of flux tower observations demonstrate the utility of Chll as a model constraint, with the coefficient of efficiency increasing from 0.91 to 0.94 and from 0.87 to 0.91 for maize and soybean, respectively. Model performances particularly improve during the late reproductive and senescence stage, where the largest temporal variations in Chll (averaging 35-55 μg cm-2 for maize and 20-35 μg cm-2 for soybean) are observed. While prolonged periods of vegetation stress did not occur over the studied fields, given the usefulness of Chll as an indicator of plant health, enhanced GPP predictabilities should be expected in fields exposed to longer periods of moisture and nutrient stress. While the results support the use of Chll as an observational proxy for Vmax25, future work needs to be directed towards improving the Chll retrieval accuracy from space observations and developing consistent and physically realistic modeling schemes that can be parameterized with acceptable accuracy over spatial and temporal domains.

Current status of the thiol redox model for the regulation of hexose transport by insulin.

PubMed

Czech, M P

1976-12-01

Data obtained over the last two years pertinent to the thiol redox model for the modulation of hexose transport activity by insulin is summarized. The model proposes that activation of hexose transport in fat cells involves sulfhydryl oxidation to the disulfide form in a key protein component of the fat cell surface membrane. Theoretically, the rapid activation of transport by insulin may involve either the conversion of inactive membrane carriers to the active form as originally proposed, or the conversion of a low Vmax transport system to a high Vmax form. The present experiments showed that the percent inhibition of insulin-activated transport rates by submaximal levels of cytochalasin B was decreased compared to its effects on basal transport. Treatment of fat cells with N-ethylmaleimide inhibited cytochalasin B action but not transport activity. When insulin or the oxidant vitamin K5 was added to cells 5 minutes before the N-ethylmaleimide, the elevated transport activity was also resistant to the sulfhydryl reagent, but cytochalasin B retained its potent inhibitory effect on transport. The data demonstrate that unique properties characterize basal versus insulin-activated transport activity with respect to the sensitivity of cytochalasin B action to sulfhydryl blockade in isolated fat cells. The data are consistent with the concept that activation of transport activity reflects the conversion of a reduced (sulfhydryl) system characterized by a low Vmax to an oxidized (disulfide), high Vmax transport system.

Inhibition, by 2-oxo acids that accumulate in maple-syrup-urine disease, of lactate, pyruvate, and 3-hydroxybutyrate transport across the blood-brain barrier.

PubMed

Cremer, J E; Teal, H M; Cunningham, V J

1982-09-01

Data are presented in support of the transport of (-)-D-3-hydroxybutyrate across the blood-brain barrier (BBB) being a carrier-mediated process. The kinetic parameters in 21-day-old pentobarbital-anaesthetized rats were Vmax 2.0 mumol.g-1.min-1, Km 29 mM, and KD 0.024 ml.g-1.min-1. The value for Vmax was the same as that for L-lactate and pyruvate transport in animals of the same age. The transport of all three substrates was sensitive to inhibition by low concentrations of either 2-oxo-3-methylbutanoate or 2-oxo-4-methylpentanoate, the 2-oxo acids that can accumulate in patients with maple-syrup-urine disease. The Ki values for the 2-oxo acids were severalfold lower than the respective Km values. 2-Oxo-3-phenylpropionate was a poor inhibitor. The relative affinities of the various monocarboxylic acids for the transport system of the BBB distinguished it from similar systems described in brain, heart, and liver mitochondria; human erythrocytes; and Ehrlich ascites-tumour cells.

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

Attia, R.M.; Gamal, R.F.

The enzymatic properties of cellulase Cx (B-14-glucan 4-glucano hydrolase, 3.2.1.4) were studied. The enzyme was obtained from a local isolate of Aspergillus niger R-1237. The reaction followed first order kinetics. The apparent temperature optimum fell at about 60 degrees Centigrade. The enthalpy of activiation of the ES-complex was calculated as about 2600 cal/mole. The Arrhenius equation is valid, and the energy of activation of the forward reaction (E) was calculated to be 12600 cal/mole. The standard free energy change (delta G) and the standard entropy change (delta G) were found to be - 102.7 cal/mole and plus 39.3 cal/mole/degree atmore » 50 degrees Centigrade. The values of thermodynamic quantities at other temperatures ranging from 30 degrees to 60 degrees Centigrade were also studied. The effect of temperatures on the two parameters, i.e. Vmax and Km values were discussed.« less

Effect of salt acclimatization on 3 beta-hydroxysteroid dehydrogenase/isomerase activity in the interrenal of Bufo arenarum.

PubMed

Pozzi, Andrea G; Lantos, Carlos P; Ceballos, Nora R

2002-03-01

In amphibians, aldosterone (Aldo) is particularly important in the regulation of Na(+) exchange by skin and urinary bladder. In previous works we studied a key enzyme in Aldo biosynthesis, the 3 beta-hydroxysteroid dehydrogenase/isomerase (3 beta HSD/I), in the interrenals of Bufo arenarum. In those works a dual localization of the 3 beta HSD/I in both microsomes and mitochondria was described. The mitochondrial, but not the microsomal, enzyme prefers the immediate Aldo precursor, 3 beta-analogue of aldosterone, as substrate. In this order, the enzyme 3 beta HSD/I would be not only a key enzyme for the synthesis of Aldo but additionally, due to its microsomal and mitochondrial localization, a possible target for the regulation of Aldo biosynthesis. With this rationale in mind, we have used in vivo and in vitro approaches to study Aldo regulation. In the present investigation the levels of Aldo were determined in plasma of winter (W) and summer (S) toads subjected to different saline concentrations (0.125 and 0.15 M) or kept on wet land. Saline hyperosmotically treated toads had significantly lower levels than isoosmotically treated toads. These results are consistent with the response in mammals, in which salt loading provokes a reduction in Aldo secretion. In W toads, plasmatic corticosterone (B) concentration was higher than Aldo concentration, whereas in S toads, B/Aldo ratio approached unity. The reduction of Aldo levels after saline dehydration was due to a decline in its biosynthesis. K(m) and V(max) values for 3 beta HSD/I were calculated for mitochondrial and microsomal fractions obtained from animals acclimated to 0.15 M NaCl or kept on land. As previously described, V(max) differs between W and S toads. However, only mitochondrial V(max) changed as a consequence of saline adaptation, suggesting that the mitochondrial enzyme could be involved in the regulation of Aldo biosynthesis.

Evaluation of possible reasons for the low phenylalanine ammonia lyase activity in cellulose nitrate membrane microcapsules.

PubMed

Habibi-Moini, S; D'mello, A P

2001-03-14

Microencapsulated phenylalanine ammonia lyase (PAL) exhibits a marked reduction in activity compared to the activity of the free enzyme in pH 8.5 Tris buffer. The purpose of this investigation was to evaluate the contribution of incomplete entrapment, the internal environment of cellulose nitrate membrane microcapsules, the diffusional barrier of the membrane and the microcapsulation process to the low activity of encapsulated PAL. A solution of PAL and 10% w/v hemoglobin was incorporated into cellulose nitrate membrane microcapsules. Hemoglobin incorporation was used as a surrogate marker of PAL entrapment. Using 14C hemoglobin, the encapsulation efficiency was determined to be 70% and suggested that incomplete entrapment might partially account for the low activity of encapsulated PAL. The effect of the internal environment of the microcapsule (10% hemoglobin solution) on PAL activity was evaluated by comparing enzyme activity in 10% w/v hemoglobin solution and pH 8.5 Tris buffer. Similar K(M) and V(max) values of PAL in the two media indicated that the internal environment of the microcapsule did not contribute to the reduction in activity of the encapsulated enzyme. The contribution of a membrane diffusional barrier was determined by breaking the putative barrier and measuring PAL activity in intact and broken microcapsules. Similar activity of PAL in these two conditions is evidence for the lack of a diffusional barrier. The effect of the microencapsulation process on PAL activity was evaluated by comparing K(M) and V(max) of free and encapsulated PAL. Similar K(M) values in these two media suggested that the process did not affect the conformation of PAL. However, encapsulated PAL had a 50% lower V(max) value compared to free PAL, which showed that the microencapsulation process deactivated a substantial proportion of the enzyme.

[Contrast enhanced power Doppler and color Doppler ultrasound in breast masses: Efficiency in diagnosis and contributions to differential diagnosis].

PubMed

Algül, Ali; Balci, Pinar; Seçil, Mustafa; Canda, Tülay

2003-06-01

To compare ability of detection of vascular structures by utilizing ultrasonographic contrast agent (Levovist) prior to and following power Doppler ultrasound (PDUS) and colour Doppler ultrasound (CDUS) and to determine useful parameters in the differentiation of malignant and benign breast masses by means of verified data. Vascularisation characteristics of 38 breast masses (22 malignant, 16 benign) which were confirmed by mammography and B-mode sonography were evaluated by both CDUS and PDUS following and prior to intravenous contrast application. In addition, Vmax and RI values of vascular structures were calculated by Doppler spectral evaluation. Malignant lesions showed more vascularity than benign lesions both with and without contrast enhancement. With both methods, by utilizing contrast agent, central, penetrating and tortuous vascular structures became more significant in malignant lesions when compared with benign lesions. PDUS was able to detect vascular structures better than CDUS; however, the difference was not statistically significant. Presence of peripheral vascularity was not useful in differentiating malignant from benign lesions. Vmax and RI values were higher in malignant lesions and the difference was statistically significant. In both methods, Vmax > 15 cm/sec and RI > 0.80 (CDUS), and RI > 0.70 (PDUS) were accepted as malignancy parameters. Vascular patterns of breast masses as determined with PDUS and CDUS with contrast enhancement and Doppler spectral examinations enabled differentiation of malignant and benign breast lesions. Thus, it is possible to decrease the number of unnecessary surgical interventions.

Adaptation of skeletal muscle energy metabolism to repeated hypoxic-normoxic exposures and drug treatment.

PubMed

Pastoris, O; Dossena, M; Gorini, A; Vercesi, L; Benzi, G

1985-03-01

Muscular glycolytic fuels, intermediates and end-products (glycogen, glucose, glucose-6-phosphate, pyruvate, lactate), Krebs cycle intermediates (citrate, alpha-ketoglutarate, succinate, malate), related free amino acids (glutamate, alanine), ammonia, energy store (creatine phosphate), energy mediators (ATP, ADP, AMP) and energy charge potential were evaluated. Furthermore the maximum rate (Vmax) of the following muscular enzyme activities was evaluated in the crude extract and/or mitochondrial fraction: for the anaerobic glycolytic pathway: hexokinase, phosphofructokinase, pyruvate kinase, lactate dehydrogenase; for the tricarboxylic acid cycle: citrate synthase, malate dehydrogenase; for the electron transfer chain: total NADH cytochrome c reductase, cytochrome oxidase. The rat gastrocnemius muscles were analyzed in normoxia and after repeated, alternate hypoxic and normoxic exposures (12 hours of hypoxia daily; for 5 days). Naftidrofuryl was administered daily at three different doses: 10, 15 and 22.5 mg/kg i.m., 30 min before the beginning of the experimental hypoxia. The biochemical adaptation to intermittent normobaric hypoxic-normoxic exposures was characterized by the decrease of the muscular contents of creatine phosphate, citrate, alpha-ketoglutarate and glutamate. This adaptation occurred in absence of significant changes in the Vmax of the muscle enzymes tested. By naftidrofuryl treatment, in gastrocnemius muscle from hypoxic rats both alpha-ketoglutarate and creatine phosphate contents maintained normal values, while glutamate concentration remained reduced to subnormal values. With the exception of hexokinase, naftidrofuryl treatment did not modify the Vmax of marker enzymes related to energy transduction.

Purification and Characterization of Glucose 6-Phosphate Dehydrogenase, 6-Phosphogluconate Dehydrogenase, and Glutathione Reductase from Rat Heart and Inhibition Effects of Furosemide, Digoxin, and Dopamine on the Enzymes Activities.

PubMed

Adem, Sevki; Ciftci, Mehmet

2016-06-01

The present study was aimed to investigate characterization and purification of glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, and glutathione reductase from rat heart and the inhibitory effect of three drugs. The purification of the enzymes was performed using 2',5'-ADP sepharose 4B affinity material. The subunit and the natural molecular weights were analyzed by SDS-PAGE and gel filtration. Biochemical characteristics such as the optimum temperature, pH, stable pH, and salt concentration were examined for each enzyme. Types of product inhibition and Ki values with Km and Vmax values of the substrates and coenzymes were determined. According to the obtained Ki and IC50 values, furosemide, digoxin, and dopamine showed inhibitory effect on the enzyme activities at low millimolar concentrations in vitro conditions. Dopamine inhibited the activity of these enzymes as competitive, whereas furosemide and digoxin inhibited the activity of the enzyme as noncompetitive. © 2016 Wiley Periodicals, Inc.

Comparison of peak flow velocity through the left ventricular outflow tract and effective orifice area indexed to body surface area in Golden Retriever puppies to predict development of subaortic stenosis in adult dogs.

PubMed

Javard, Romain; Bélanger, Marie-Claude; Côté, Etienne; Beauchamp, Guy; Pibarot, Philippe

2014-12-15

To evaluate the usefulness of Doppler-derived peak flow velocity through the left ventricular outflow tract (LVOT Vmax) and effective orifice area indexed to body surface area (EOAi) in puppies to predict development of subaortic stenosis (SAS) in the same dogs as adults. Prospective, longitudinal, observational study. 38 Golden Retrievers. Cardiac auscultation and echocardiography were performed on 2- to 6-month-old puppies, then repeated at 12 to 18 months. Subaortic stenosis was diagnosed when LVOT Vmax was ≥ 2.3 m/s in adult dogs with left basilar systolic murmurs. All puppies with EOAi < 1.46 cm(2)/m(2) had SAS as adults. All adults with EOAi < 1.29 cm(2)/m(2) had SAS. An LVOT Vmax > 2.3 m/s in puppyhood was 63% sensitive and 100% specific for SAS in adulthood. In puppies, LVOT Vmax was more strongly associated with a future diagnosis of SAS (area under the curve [AUC], 0.89) than was EOAi (AUC, 0.80). In puppies, the combination of LVOT Vmax and EOAi yielded slightly higher sensitivity (69%) and specificity (100%) for adult SAS than did LVOT Vmax alone. In unaffected and affected dogs, LVOT Vmax increased significantly from puppyhood to adulthood but EOAi did not. In Golden Retriever puppies, LVOT Vmax > 2.3 m/s and EOAi < 1.46 cm(2)/m(2) were both associated with a diagnosis of SAS at adulthood. The combination of these 2 criteria may result in higher sensitivity for SAS screening. Unlike LVOT Vmax, EOAi did not change during growth in either unaffected Golden Retrievers or those with SAS.

Inhibition of monoamine oxidase A and B activities by imidazol(ine)/guanidine drugs, nature of the interaction and distinction from I2-imidazoline receptors in rat liver

PubMed Central

Ozaita, Andrés; Olmos, Gabriel; Assumpció Boronat, M; Miguel Lizcano, José; Unzeta, Mercedes; García-Sevilla, Jesús A

1997-01-01

I2-Imidazoline sites ([3H]-idazoxan binding) have been identified on monoamine oxidase (MAO) and proposed to modulate the activity of the enzyme through an allosteric inhibitory mechanism (Tesson et al., 1995). The main aim of this study was to assess the inhibitory effects and nature of the inhibition of imidazol(ine)/guanidine drugs on rat liver MAO-A and MAO-B isoforms and to compare their inhibitory potencies with their affinities for the sites labelled by [3H]-clonidine in the same tissue. Competition for [3H]-clonidine binding in rat liver mitochondrial fractions by imidazol(ine)/guanidine compounds revealed that the pharmacological profile of the interaction (2 - styryl - 2 - imidazoline, LSL 61112>idazoxan>2 - benzofuranyl - 2 - imidazoline, 2-BFI=cirazoline>guanabenz>oxymetazoline>>clonidine) was typical of that for I2-sites. Clonidine inhibited rat liver MAO-A and MAO-B activities with very low potency (IC50s: 700 μM and 6 mM, respectively) and displayed the typical pattern of competitive enzyme inhibition (Lineweaver-Burk plots: increased Km and unchanged Vmax values). Other imidazol(ine)/guanidine drugs also were weak MAO inhibitors with the exception of guanabenz, 2-BFI and cirazoline on MAO-A (IC50s: 4–11 μM) and 2-benzofuranyl-2-imidazol (LSL 60101) on MAO-B (IC50: 16 μM). Idazoxan was a full inhibitor, although with rather low potency, on both MAO-A and MAO-B isoenzymes (IC50s: 280 μM and 624 μM, respectively). Kinetic analyses of MAO-A inhibition by these drugs revealed that the interactions were competitive. For the same drugs acting on MAO-B the interactions were of the mixed type inhibition (increased Km and decreased Vmax values), although the greater inhibitory effects on the apparent value of Vmax/Km than on the Vmax value indicated that the competitive element of the MAO-B inhibition predominated. Competition for [3H]-Ro 41-1049 binding to MAO-A or [3H]-Ro 19-6327 binding to MAO-B in rat liver mitochondrial fractions by imidazol(ine)/guanidine compounds revealed that the drug inhibition constants (Ki values) were similar to the IC50 values displayed for the inhibition of MAO-A or MAO-B activities. In fact, very good correlations were obtained when the affinities of drugs at MAO-A or MAO-B catalytic sites were correlated with their potencies in inhibiting MAO-A (r=0.92) or MAO-B (r=0.99) activity. This further suggested a direct drug interaction with the catalytic sites of MAO-A and MAO-B isoforms. No significant correlations were found when the potencies of imidazol(ine)/guanidine drugs at the high affinity site (pKiH, nanomolar range) or the low-affinity site (pKiL, micromolar range) of I2-imidazoline receptors labelled with [3H]-clonidine were correlated with the pIC50 values of the same drugs for inhibition of MAO-A or MAO-B activity. These discrepancies indicated that I2-imidazoline receptors are not directly related to the site of action of these drugs on MAO activity in rat liver mitochondrial fractions. Although these studies cannot exclude the presence of additional binding sites on MAO that do not affect the activity of the enzyme, they would suggest that I2-imidazoline receptors represent molecular species that are distinct from MAO. PMID:9222546

Dynamical relationship between wind speed magnitude and meridional temperature contrast: Application to an interannual oscillation in Venusian middle atmosphere GCM

NASA Astrophysics Data System (ADS)

Yamamoto, Masaru; Takahashi, Masaaki

2018-03-01

We derive simple dynamical relationships between wind speed magnitude and meridional temperature contrast. The relationship explains scatter plot distributions of time series of three variables (maximum zonal wind speed UMAX, meridional wind speed VMAX, and equator-pole temperature contrast dTMAX), which are obtained from a Venus general circulation model with equatorial Kelvin-wave forcing. Along with VMAX and dTMAX, UMAX likely increases with the phase velocity and amplitude of a forced wave. In the scatter diagram of UMAX versus dTMAX, points are plotted along a linear equation obtained from a thermal-wind relationship in the cloud layer. In the scatter diagram of VMAX versus UMAX, the apparent slope is somewhat steep in the high UMAX regime, compared with the low UMAX regime. The scatter plot distributions are qualitatively consistent with a quadratic equation obtained from a diagnostic equation of the stream function above the cloud top. The plotted points in the scatter diagrams form a linear cluster for weak wave forcing, whereas they form a small cluster for strong wave forcing. An interannual oscillation of the general circulation forming the linear cluster in the scatter diagram is apparent in the experiment of weak 5.5-day wave forcing. Although a pair of equatorial Kelvin and high-latitude Rossby waves with a same period (Kelvin-Rossby wave) produces equatorward heat and momentum fluxes in the region below 60 km, the equatorial wave does not contribute to the long-period oscillation. The interannual fluctuation of the high-latitude jet core leading to the time variation of UMAX is produced by growth and decay of a polar mixed Rossby-gravity wave with a 14-day period.

Echinococcus granulosus (Cestoda): uptake of L-amino acids by secondary hydatid cysts.

PubMed

Jeffs, S A; Arme, C

1988-02-01

The uptake of cycloleucine, L-proline, L-alanine and L-threonine by secondary hydatid cysts of Echinococcus granulosus (U.K. horse strain 3-8 mm in diameter, derived from Balb/c mice infected 300-400 days previously) occurs by passive diffusion into the cyst wall (laminated layer plus germinal layer) and by mediated mechanisms into the fluid-filled interior. The maximal concentrations of these compounds are achieved after incubation for 2 h in vitro and approach those in vivo. Kt and Vmax values describing the uptake of these compounds are given. The flux rates for these compounds are extremely slow compared to those obtained with the protoscolex. A rationale for standardizing the experimental method for uptake studies with hydatid cysts is described.

A simple theory of motor protein kinetics and energetics. II.

PubMed

Qian, H

2000-01-10

A three-state stochastic model of motor protein [Qian, Biophys. Chem. 67 (1997) pp. 263-267] is further developed to illustrate the relationship between the external load on an individual motor protein in aqueous solution with various ATP concentrations and its steady-state velocity. A wide variety of dynamic motor behavior are obtained from this simple model. For the particular case of free-load translocation being the most unfavorable step within the hydrolysis cycle, the load-velocity curve is quasi-linear, V/Vmax = (cF/Fmax-c)/(1-c), in contrast to the hyperbolic relationship proposed by A.V. Hill for macroscopic muscle. Significant deviation from the linearity is expected when the velocity is less than 10% of its maximal (free-load) value--a situation under which the processivity of motor diminishes and experimental observations are less certain. We then investigate the dependence of load-velocity curve on ATP (ADP) concentration. It is shown that the free load Vmax exhibits a Michaelis-Menten like behavior, and the isometric Fmax increases linearly with ln([ATP]/[ADP]). However, the quasi-linear region is independent of the ATP concentration, yielding an apparently ATP-independent maximal force below the true isometric force. Finally, the heat production as a function of ATP concentration and external load are calculated. In simple terms and solved with elementary algebra, the present model provides an integrated picture of biochemical kinetics and mechanical energetics of motor proteins.

Explicit solution of integrated 1 - exp equation for predicting accumulation and decline of concentrations for drugs obeying nonlinear saturation kinetics.

PubMed

Keller, Frieder; Hartmann, Bertram; Czock, David

2009-12-01

To describe nonlinear, saturable pharmacokinetics, the Michaelis-Menten equation is frequently used. However, the Michaelis-Menten equation has no integrated solution for concentrations but only for the time factor. Application of the Lambert W function was proposed recently to obtain an integrated solution of the Michaelis-Menten equation. As an alternative to the Michaelis-Menten equation, a 1 - exp equation has been used to describe saturable kinetics, with the advantage that the integrated 1 - exp equation has an explicit solution for concentrations. We used the integrated 1 - exp equation to predict the accumulation kinetics and the nonlinear concentration decline for a proposed fictive drug. In agreement with the recently proposed method, we found that for the integrated 1 - exp equation no steady state is obtained if the maximum rate of change in concentrations (Vmax) within interval (Tau) is less than the difference between peak and trough concentrations (Vmax x Tau < C peak - C trough).

Functional characterisation of a tropine-forming reductase gene from Brugmansia arborea, a woody plant species producing tropane alkaloids.

PubMed

Qiang, Wei; Xia, Ke; Zhang, Qiaozhuo; Zeng, Junlan; Huang, Yuanshe; Yang, Chunxian; Chen, Min; Liu, Xiaoqiang; Lan, Xiaozhong; Liao, Zhihua

2016-07-01

Brugmansia arborea is a woody plant species that produces tropane alkaloids (TAs). The gene encoding tropine-forming reductase or tropinone reductase I (BaTRI) in this plant species was functionally characterised. The full-length cDNA of BaTRI encoded a 272-amino-acid polypeptide that was highly similar to tropinone reductase I from TAs-producing herbal plant species. The purified 29kDa recombinant BaTRI exhibited maximum reduction activity at pH 6.8-8.0 when tropinone was used as substrate; it also exhibited maximum oxidation activity at pH 9.6 when tropine was used as substrate. The Km, Vmax and Kcat values of BaTRI for tropinone were 2.65mM, 88.3nkatmg(-1) and 2.93S(-1), respectively, at pH 6.4; the Km, Vmax and Kcat values of TRI from Datura stramonium (DsTRI) for tropinone were respectively 4.18mM, 81.20nkatmg(-1) and 2.40S(-1) at pH 6.4. At pH 6.4, 6.8 and 7.0, BaTRI had a significantly higher activity than DsTRI. Analogues of tropinone, 4-methylcyclohexanone and 3-quinuclidinone hydrochloride, were also used to investigate the enzymatic kinetics of BaTRI. The Km, Vmax and Kcat values of BaTRI for tropine were 0.56mM, 171.62nkat.mg(-1) and 5.69S(-1), respectively, at pH 9.6; the Km, Vmax and Kcat values of DsTRI for tropine were 0.34mM, 111.90nkatmg(-1) and 3.30S(-1), respectively, at pH 9.6. The tissue profiles of BaTRI differed from those in TAs-producing herbal plant species. BaTRI was expressed in all examined organs but was most abundant in secondary roots. Finally, tropane alkaloids, including hyoscyamine, anisodamine and scopolamine, were detected in various organs of B. arborea by HPLC. Interestingly, scopolamine constituted most of the tropane alkaloids content in B. arborea, which suggests that B. arborea is a scopolamine-rich plant species. The scopolamine content was much higher in the leaves and stems than in other organs. The gene expression and TAs accumulation suggest that the biosynthesis of hyoscyamine, especially scopolamine, occurred not only in the roots but also in the aerial parts of B. arborea. Copyright © 2016 Elsevier Ltd. All rights reserved.

Studies on long chain cis- and trans-acyl-CoA esters and Acyl-CoA dehydrogenase from rat heart mitochondria.

PubMed

Korsrud, G O; Conacher, H B; Jarvis, G A; Beare-Rogers, J L

1977-02-01

The beta-oxidation of long chain fatty acids was investigated in a preparation of rat heart mitochondria. The acyl-CoA esters of the cis and trans isomers of delta9-hexadecenoic, delta9-octadecenoic, delta11-eicosenoic, and delta13-docosenoic acids were prepared. Rates of the acyl-CoA reaction were determined with an extract from rat heart mitochondria. The apparent Michaelis constant (Km) and maximum velocity (Vmax) were calculated for each substrate. In general, apparent Vmax values decreased with increasing chain length of the monoenoic substrates. Reduced activity of acyl-CoA dehydrogenase with long chain acyl-CoA esters could have contributed to accumulation of lipids in hearts of rats fed diets containing long chain fatty acids.

Enzymatic synthesis of dithiolopyrrolone antibiotics using cell-free extract of Saccharothrix algeriensis NRRL B-24137 and biochemical characterization of two pyrrothine N-acyltransferases in this extract.

PubMed

Saker, Safwan; Almousa Almaksour, Ziade; Chorin, Anne-Claire; Lebrihi, Ahmed; Mathieu, Florence

2014-01-01

Saccharothrix algeriensis NRRL B-24137 produces naturally different dithiolopyrrolone derivatives. The enzymatic activity of pyrrothine N-acyltransferase was determined to be responsible for the transfer of an acyl group from acyl-CoA to pyrrothine core. This activity was also reported to be responsible for the diversity of the dithiolopyrrolone derivatives. Based on this fact, nine dithiolopyrrolone derivatives were produced in vitro via the crude extract of Sa. algeriensis. Three of them have never been obtained before by natural fermentation: acetoacetyl-pyrrothine, hydroxybutyryl-pyrrothine, and dimethyl thiolutin (holomycin). Two acyltransferase activities, acetyltransferase and benzoyltransferase catalyzing the incorporation of linear and cyclic acyl groups to the pyrrothine core, respectively, were biochemically characterized in this crude extract. The first one is responsible for formation of acetyl-pyrrothine and the second for benzoyl-pyrrothine. Both enzymes were sensitive to temperature changes: For example, the loss of acetyltransferase and benzoyltransferase activity was 53% and 80% respectively after pre-incubation of crude extract for 60 min at 20°C. The two enzymes were more active in neutral and basal media (pH 7-10) than in the acidic one (pH 3-6). The optimum temperature and pH of acetyltransferase were 40°C and 7, with a Km value of 7.9 μM and a Vmax of 0.63 μM/min when acetyl-CoA was used as limited substrate. Benzoyltransferase had a temperature and a pH optimum at 55°C and 9, a Km value of 14.7 μM, and a Vmax of 0.67 μM/min when benzoyl- CoA was used as limited substrate.

Hopantenate interference on the adaptation of muscular energy metabolism to intermittent hypoxia.

PubMed

Pastoris, O; Vercesi, L; Mazzocchi, A; Dossena, M; Benzi, G

1986-06-01

In rat gastrocnemius muscle, the concentrations of glycolytic fuels, intermediates and end-products; Krebs cycle intermediates and related free amino acids; ammonia; energy store and mediators; and the energy charge potential were evaluated in normoxia or after repeated, alternate hypoxic and normoxic exposures (12 hr of hypoxia daily; for 5 days) with or without treatment with hopantenate (HOPA). Furthermore, in the crude extract and/or mitochondrial fraction the maximum rate (Vmax) of some muscular enzymes related to the anaerobic glycolytic pathway; the tricarboxylic acid cycle; and the electron transfer chain were evaluated. Hopantenate was administered daily at the dose of 250 mg.kg-1 i.p., for 5 days, 30 min before the beginning of the experimental normobaric hypoxia. The biochemical adaptation to intermittent normobaric hypoxic-normoxic exposures was characterized by the decrease of the muscular concentrations of citrate, alpha-ketoglutarate and glutamate, in absence of changes in the Vmax of the muscle enzymes related to energy transduction. In gastrocnemius muscle from hypoxic rats, by HOPA treatment, both citrate and alpha-ketoglutarate maintained normal values, aspartate decreased, while glutamate remained reduced to subnormal values. In the muscle from hypoxic animals, by hopantenate treatment the Vmax of the mitochondrial enzymes tested (citrate synthase, malate dehydrogenase, total NADH cytochrome c reductase, cytochrome oxidase) decreased in comparison with both hypoxic and normoxic untreated animals. This behaviour could be tentatively related to a mitochondrial sparing action concomitant with an intervention of the glutamate group of amino acids, even if the results do not allow a clear interpretation of the mechanism of HOPA action.

Variable-period surface-wave magnitudes: A rapid and robust estimator of seismic moments

USGS Publications Warehouse

Bonner, J.; Herrmann, R.; Benz, H.

2010-01-01

We demonstrate that surface-wave magnitudes (Ms), measured at local, regional, and teleseismic distances, can be used as a rapid and robust estimator of seismic moment magnitude (Mw). We used the Russell (2006) variable-period surface-wave magnitude formula, henceforth called Ms(VMAX), to estimate the Ms for 165 North American events with 3.2

Allosteric modulation of semicarbazide-sensitive amine oxidase activities in vitro by imidazoline receptor ligands

PubMed Central

Holt, Andrew; Wieland, Barbara; Baker, Glen B

2004-01-01

Evidence indicates that imidazoline I2 binding sites (I2BSs) are present on monoamine oxidase (MAO) and on soluble (plasma) semicarbazide-sensitive amine oxidase enzymes. The binding site on MAO has been described as a modulatory site, although no effects on activity are thought to have been observed as a result of ligands binding to these sites. We examined the effects in vitro of several imidazoline binding site ligands on activities of bovine plasma amine oxidase (BPAO) and porcine kidney diamine oxidase (PKDAO) in a spectrophotometric protocol. While both enzymes were inhibited at high concentrations of all ligands, clonidine, cirazoline and oxymetazoline were seen, at lower concentrations, to increase activity of BPAO versus benzylamine, but not of PKDAO versus putrescine. This effect was substrate dependent, with mixed or biphasic inhibition of spermidine, methylamine, p-tyramine and β-phenylethylamine oxidation observed at cirazoline concentrations that increased benzylamine oxidation. With benzylamine as substrate, clonidine decreased KM (EC50 8.82 μM, Emax 75.1% of control) and increased Vmax (EC50 164.6 μM, Emax 154.1% of control). Cirazoline decreased Vmax (EC50 2.15 μM, Emax 91.4% of control), then decreased KM (EC50 5.63 μM, Emax 42.6% of control) and increased Vmax (EC50 49.0 μM, Emax 114.4% of decreased Vmax value). Data for clonidine fitted a mathematical model for two-site nonessential activation plus linear intersecting noncompetitive inhibition. Data for cirazoline were consistent with involvement of a fourth site. These results reveal an ability of imidazoline ligands to modulate BPAO kinetics allosterically. The derived mechanism may have functional significance with respect to modulation of MAO by I2BS ligands. PMID:15451775

Another baryon miracle? Testing solutions to the `missing dwarfs' problem

NASA Astrophysics Data System (ADS)

Trujillo-Gomez, Sebastian; Schneider, Aurel; Papastergis, Emmanouil; Reed, Darren S.; Lake, George

2018-04-01

The dearth of dwarf galaxies in the local Universe is hard to reconcile with the large number of low-mass haloes expected within the concordance Λ cold dark matter (ΛCDM) paradigm. In this paper, we perform a systematic evaluation of the uncertainties affecting the measurement of dark matter halo abundance using galaxy kinematics. Using a large sample of dwarf galaxies with spatially resolved kinematics, we derive a correction to obtain the abundance of galaxies as a function of maximum circular velocity - a direct probe of halo mass - from the line-of-sight velocity function in the Local Volume. This method provides a direct means of comparing the predictions of theoretical models and simulations (including non-standard cosmologies and novel galaxy formation physics) to the observational constraints. The new `galactic Vmax' function is steeper than the line-of-sight velocity function but still shallower than the theoretical CDM expectation, implying that unaccounted baryonic physics may be necessary to reduce the predicted abundance of galaxies. Using the galactic Vmax function, we investigate the theoretical effects of feedback-powered outflows and photoevaporation of gas due to reionization. At the 3σ confidence level, we find that feedback and reionization are not effective enough to reconcile the disagreement. In the case of maximum baryonic effects, the theoretical prediction still deviates significantly from the observations for Vmax < 60 km s-1. CDM predicts at least 1.8 times more galaxies with Vmax = 50 km s-1 and 2.5 times more than observed at 30 km s-1. Recent hydrodynamic simulations seem to resolve the discrepancy but disagree with the properties of observed galaxies with spatially resolved kinematics. This abundance problem might point to the need to modify cosmological predictions at small scales.

High-intensity interval training increases in vivo oxidative capacity with no effect on P(i)→ATP rate in resting human muscle.

PubMed

Larsen, Ryan G; Befroy, Douglas E; Kent-Braun, Jane A

2013-03-01

Mitochondrial ATP production is vital for meeting cellular energy demand at rest and during periods of high ATP turnover. We hypothesized that high-intensity interval training (HIT) would increase ATP flux in resting muscle (VPi→ATP) in response to a single bout of exercise, whereas changes in the capacity for oxidative ATP production (Vmax) would require repeated bouts. Eight untrained men (27 ± 4 yr; peak oxygen uptake = 36 ± 4 ml·kg(-1)·min(-1)) performed six sessions of HIT (4-6 × 30-s bouts of all-out cycling with 4-min recovery). After standardized meals and a 10-h fast, VPi→ATP and Vmax of the vastus lateralis muscle were measured using phosphorus magnetic resonance spectroscopy at 4 Tesla. Measurements were obtained at baseline, 15 h after the first training session, and 15 h after completion of the sixth session. VPi→ATP was determined from the unidirectional flux between Pi and ATP, using the saturation transfer technique. The rate of phosphocreatine recovery (kPCr) following a maximal contraction was used to calculate Vmax. While kPCr and Vmax were unchanged after a single session of HIT, completion of six training sessions resulted in a ∼14% increase in muscle oxidative capacity (P ≤ 0.004). In contrast, neither a single nor six training sessions altered VPi→ATP (P = 0.74). This novel analysis of resting and maximal high-energy phosphate kinetics in vivo in response to HIT provides evidence that distinct aspects of human skeletal muscle metabolism respond differently to this type of training.

Influence of carbon source and inoculum type on anaerobic biomass adhesion on polyurethane foam in reactors fed with acid mine drainage.

PubMed

Rodriguez, Renata P; Zaiat, Marcelo

2011-04-01

This paper analyzes the influence of carbon source and inoculum origin on the dynamics of biomass adhesion to an inert support in anaerobic reactors fed with acid mine drainage. Formic acid, lactic acid and ethanol were used as carbon sources. Two different inocula were evaluated: one taken from an UASB reactor and other from the sediment of a uranium mine. The values of average colonization rates and the maximum biomass concentration (C(max)) were inversely proportional to the number of carbon atoms in each substrate. The highest C(max) value (0.35 g TVS g(-1) foam) was observed with formic acid and anaerobic sludge as inoculum. Maximum colonization rates (v(max)) were strongly influenced by the type of inoculum when ethanol and lactic acid were used. For both carbon sources, the use of mine sediment as inoculum resulted in a v(max) of 0.013 g TVS g(-1) foam day(-1), whereas 0.024 g TVS g(-1) foam day(-1) was achieved with anaerobic sludge. Copyright © 2011 Elsevier Ltd. All rights reserved.

Temporal and spatial variations in kinetics of alkaline phosphatase in sediments of a shallow Chinese eutrophic lake (Lake Donghu).

PubMed

Yiyong, Zhou; Jianqiu, Li; Min, Zhang

2002-04-01

Monthly sediment and interstitial water samples were collected in a shallow Chinese freshwater lake (Lake Donghu) from three areas to determine if alkaline phosphatase activity (APA) plays an important role, in phosphorus cycling in sediment. The seasonal variability in the kinetics of APA and other relevant parameters were investigated from 1995-1996. The phosphatase hydrolyzable phosphorus (PHP) fluctuated seasonally in interstitial water, peaking in the spring. A synchronous pattern was observed in chlorophyll a contents in surface water in general. The orthophosphate (o-P) concentrations in the interstitial water increased during the spring. An expected negative relationship between PHP and Vmax of APA is not evident in interstitial water. The most striking feature of the two variables is their co-occurring, which can be explained in terms of an induction mechanism. It is argued that phosphatase activity mainly contributes to the driving force of o-P regeneration from PHP in interstitial water, supporting the development of phytoplankton biomass in spring. The Vmax values in sediment increased during the summer, in conjunction with lower Km values in interstitial water that suggest a higher affinity for the substrate. The accumulation of organic matter in the sediment could be traced back to the breakdown of the algal spring bloom, which may stimulate APA with higher kinetic efficiency, by a combination of the higher Vmax in sediments plus lower Km values in interstitial water, in summer. In summary, a focus on phosphatase and its substrate in annual scale may provide a useful framework for the development of novel P cycling, possible explanations for the absence of a clear relationship between PHP and APA were PHP released from the sediment which induced APA, and the presence of kinetically higher APA both in sediment and interstitial water which permitted summer mineralization of organic matter derived from the spring bloom to occur. The study highlighted the need for distinguishing functionally distinct extracellular enzymes between the sediment and interstitial water of lakes.

Identification of functional amino acid residues involved in polyamine and agmatine transport by human organic cation transporter 2.

PubMed

Higashi, Kyohei; Imamura, Masataka; Fudo, Satoshi; Uemura, Takeshi; Saiki, Ryotaro; Hoshino, Tyuji; Toida, Toshihiko; Kashiwagi, Keiko; Igarashi, Kazuei

2014-01-01

Polyamine (putrescine, spermidine and spermine) and agmatine uptake by the human organic cation transporter 2 (hOCT2) was studied using HEK293 cells transfected with pCMV6-XL4/hOCT2. The Km values for putrescine and spermidine were 7.50 and 6.76 mM, and the Vmax values were 4.71 and 2.34 nmol/min/mg protein, respectively. Spermine uptake by hOCT2 was not observed at pH 7.4, although it inhibited both putrescine and spermidine uptake. Agmatine was also taken up by hOCT2, with Km value: 3.27 mM and a Vmax value of 3.14 nmol/min/mg protein. Amino acid residues involved in putrescine, agmatine and spermidine uptake by hOCT2 were Asp427, Glu448, Glu456, Asp475, and Glu516. In addition, Glu524 and Glu530 were involved in putrescine and spermidine uptake activity, and Glu528 and Glu540 were weakly involved in putrescine uptake activity. Furthermore, Asp551 was also involved in the recognition of spermidine. These results indicate that the recognition sites for putrescine, agmatine and spermidine on hOCT2 strongly overlap, consistent with the observation that the three amines are transported with similar affinity and velocity. A model of spermidine binding to hOCT2 was constructed based on the functional amino acid residues.

Hymenolepis diminuta (Cestoda): uptake of cycloleucine by metacestodes.

PubMed

Jeffs, S A; Arme, C

1985-01-01

Cycloleucine uptake by metacestodes of H. diminuta of various ages was investigated. Absorption occurs by active mediated transport, mean Kt = 0.28 mM. Vmax values are age-related, and can be correlated to developmental changes. Cycloleucine uptake in the metacestode is very similar to that in the adult worm and the implications of this are discussed.

Direct Detection of Stereospecific Soman Hydrolysis by Wild-Type Human Serum Paraoxonase

DTIC Science & Technology

2007-01-01

was used as Tc = (Ao/VmaA)(1 - (C/Co)( Kmc /KwA)(VmaxA/Vmac)) the carrier gas at a linear velocity of 45 crns-1. The oven + (B0/VmaxB)(1 - (C/Co)( Kmc ...KmB)(VmaxB/Vm.c)) temperature was held initially at 80 ’C for 14 min, pro- + (Co/Vmac)(1 - (C/Co)( Kmc / Kmc )(Vm.c/Vmac)) grammed from 80 to 90 ’C at 5...oC’min-1, and held at + (DO/VmaD)(1 - (C/Co)(K.C/KmD)(Vm.D1/V.aC)) 90 ’C for 3 min. Split injections of 1 tiL volume were made ( Kmc /VmaxC) Log(C/Co

Influence of cimetidine and diethyldithiocarbamate on the metabolism of halothane and methoxyflurane in vitro.

PubMed

Loesch, J; Siegers, C P; Younes, M

1987-06-01

The metabolism of halothane and methoxyflurane was measured in vitro by the vial equilibration method using the S-9-fraction from rat liver as source of enzymes. Kinetic values were measured for halothane: Vmax = 11.6 nmol/g.min, KM = 19.6 mumol/l and methoxyflurane: Vmax = 12.0 nmol/g.min, KM = 17.5 mumol/l. Dithiocarb showed strong inhibitory activity on halothane and methoxyflurane metabolism; inhibition constants were calculated as Ki = 0.051 mmol/l and Ki = 0.004 mmol/l, respectively. Cimetidine inhibited the metabolism of both anesthetics to a lesser extent. Inhibition constants were calculated as Ki = 16.2 mmol/l and Ki = 8.2 mmol/l for halothane and methoxyflurane, respectively. The observed inhibitory properties of dithiocarb and cimetidine on the metabolism of halothane and methoxyflurane may be of interest in connection with the problem of toxic liver and kidney injury after anesthesia with these agents.

Force interaction and 3D pole movement in double poling.

PubMed

Stöggl, T; Holmberg, H-C

2011-12-01

The aim of this study was to analyze double poling using combined kinetic and 3D kinematic analysis at high skiing speeds as regards pole force components, pole angles and pole behavior during the poling and swing phase. The hypothesis was that a horizontal pole force is more predictive for maximal skiing speed (V(max)) than the resultant pole force. Sixteen elite skiers performed a double-poling V(max) test while treadmill roller skiing. Pole forces and 3D kinematics of pole movement at a speed of 30 km/h were analyzed and related to V(max). The duration of the "preparation phase" showed the strongest relationship with V(max) (r=0.87, P<0.001). Faster skiers generated longer cycle lengths with longer swing and poling times, had less inclined pole angles at pole plant and a later peak pole force. Horizontal pole forces were not more highly related to V(max) compared with the resultant pole force. Impact force was not related to V(max). At high skiing speeds, skiers should aim to combine high pole forces with appropriate timing of pole forces and appropriate pole and body positions during the swing and poling phase. The emphasis in training should be on the development of specific strength capacities for pole force production and the utilization of these capacities in double-poling training sessions. © 2011 John Wiley & Sons A/S.

The topology of large-scale structure. III - Analysis of observations

NASA Astrophysics Data System (ADS)

Gott, J. Richard, III; Miller, John; Thuan, Trinh X.; Schneider, Stephen E.; Weinberg, David H.; Gammie, Charles; Polk, Kevin; Vogeley, Michael; Jeffrey, Scott; Bhavsar, Suketu P.; Melott, Adrian L.; Giovanelli, Riccardo; Hayes, Martha P.; Tully, R. Brent; Hamilton, Andrew J. S.

1989-05-01

A recently developed algorithm for quantitatively measuring the topology of large-scale structures in the universe was applied to a number of important observational data sets. The data sets included an Abell (1958) cluster sample out to Vmax = 22,600 km/sec, the Giovanelli and Haynes (1985) sample out to Vmax = 11,800 km/sec, the CfA sample out to Vmax = 5000 km/sec, the Thuan and Schneider (1988) dwarf sample out to Vmax = 3000 km/sec, and the Tully (1987) sample out to Vmax = 3000 km/sec. It was found that, when the topology is studied on smoothing scales significantly larger than the correlation length (i.e., smoothing length, lambda, not below 1200 km/sec), the topology is spongelike and is consistent with the standard model in which the structure seen today has grown from small fluctuations caused by random noise in the early universe. When the topology is studied on the scale of lambda of about 600 km/sec, a small shift is observed in the genus curve in the direction of a 'meatball' topology.

The topology of large-scale structure. III - Analysis of observations. [in universe

NASA Technical Reports Server (NTRS)

Gott, J. Richard, III; Weinberg, David H.; Miller, John; Thuan, Trinh X.; Schneider, Stephen E.

1989-01-01

A recently developed algorithm for quantitatively measuring the topology of large-scale structures in the universe was applied to a number of important observational data sets. The data sets included an Abell (1958) cluster sample out to Vmax = 22,600 km/sec, the Giovanelli and Haynes (1985) sample out to Vmax = 11,800 km/sec, the CfA sample out to Vmax = 5000 km/sec, the Thuan and Schneider (1988) dwarf sample out to Vmax = 3000 km/sec, and the Tully (1987) sample out to Vmax = 3000 km/sec. It was found that, when the topology is studied on smoothing scales significantly larger than the correlation length (i.e., smoothing length, lambda, not below 1200 km/sec), the topology is spongelike and is consistent with the standard model in which the structure seen today has grown from small fluctuations caused by random noise in the early universe. When the topology is studied on the scale of lambda of about 600 km/sec, a small shift is observed in the genus curve in the direction of a 'meatball' topology.

Control of speed during the double poling technique performed by elite cross-country skiers.

PubMed

Lindinger, Stefan Josef; Stöggl, Thomas; Müller, Erich; Holmberg, Hans-Christer

2009-01-01

Double poling (DP) as a main technique in cross-country skiing has developed substantially over the last 15 yr. The purpose of the present study was to analyze the question, "How do modern elite skiers control DP speed?" Twelve male elite cross-country skiers roller skied using DP at 9, 15, 21, and 27 km.h(-1) and maximum velocity (V(max)). Cycle characteristics, pole and plantar forces, and elbow, hip, and knee joint angles were analyzed. Both poling frequency and cycle length increased up to 27 km.h (-1)(P < 0.05), with a further increase in poling frequency at V(max) (P < 0.05). Peak pole force, rate of force development, and rearfoot plantar force increased with submaximal velocities (V(sm)), whereas poling time and time-to-peak pole force gradually shortened (P < 0.05). Changes in elbow joint kinematics during the poling phase were characterized by a decreased angle minimum and an increased flexion and extension ranges of motion as well as angular velocities across V(sm) (P < 0.05), with no further changes at V(max). Hip and knee joint kinematics adapted across V(sm) by 1) decreasing angles at pole plant and angle minima during the poling phase, 2) increasing the ranges of motion and angular velocities during the flexion phases occurring around pole plant, and 3) increasing extension ranges of motion and angular velocities during the recovery phase (all P values <0.05), with no further changes at V(max). Elite skiers control DP speed by increasing both poling frequency and cycle length; the latter is achieved by increased pole force despite reduced poling time. Adaptation to higher speeds was assisted by an increased range of motion, smaller angle minima, and higher angular velocities in the elbow, the hip, and the knee joints.

In vivo and in vitro kinetics of ethylene oxide metabolism in rats and mice.

PubMed

Brown, C D; Wong, B A; Fennell, T R

1996-01-01

Ethylene oxide (EO) is a direct-acting mutagen and animal carcinogen used as an industrial intermediate and sterilant with a high potential for human exposure. Kinetics of EO metabolism in rodents can be used to develop human EO dosimetry models. This study examined the kinetics of EO metabolism in vivo and in vitro in male and female F-344 rats and B6C3F1 mice. In vivo studies measured blood and tissue EO levels during and 2-20 min following whole-body inhalation exposure (4 hr, 100 or 330 ppm EO). At 100 ppm EO, the half-life of elimination (t1/2) in rats was 13.8 +/- 0.3 (mean +/- SD) and 10.8 +/- 2.4 min for males and females, respectively, compared to a t1/2 in mice of 3.12 +/- 0.2 and 2.4 +/- 0.2 min in males and females, respectively. On exposure to 330 ppm EO, the t1/2 in mice increased approx twofold, while no change in t1/2 was observed in rats. In vitro kinetic parameters (Vmax and KM) of EO metabolism were determined using tissue cytosol and microsomes. EO metabolism in vitro occurred primarily via cytosolic glutathione S-transferase-mediated EO-GSH conjugation (cGST-EO), with highest activity in the liver. Liver cGST-EO activity (Vmax) was 258 +/- 86.9 and 287 +/- 49.0 nmol/mg protein/min (mean +/- SD) in male and female mice, respectively, compared to 52.7 +/- 10.8 and 29.3 +/- 4.9 in male and female rats, respectively. In rats, but not mice, there was a statistically significant (p < 0.05) gender difference in the Vmax for liver cGST. The KM for liver cGST-EO was approximately 10 mM in both species. The higher Vmax values observed in mice are consistent with the more rapid elimination of EO observed for this species in vivo compared to rats.

Morphological and metabolic adjustments in the small intestine to energy demands of growth, storage, and fasting in the first annual cycle of a hibernating lizard (Tupinambis merianae).

PubMed

do Nascimento, Lucas Francisco R; da Silveira, Lilian Cristina; Nisembaum, Laura Gabriela; Colquhoun, Alison; Abe, Agusto S; Mandarim-de-Lacerda, Carlos Alberto; de Souza, Silvia Cristina R

2016-05-01

Seasonal plasticity in the small intestine of neonatal tegu lizards was investigated using morphometry and analysis of enzymes involved in supplying energy to the intestinal tissue. In the autumn, the intestinal mass (Mi) was 1.0% of body mass and the scaling exponent b=0.92 indicated that Mi was larger in smaller neonates. During arousal from dormancy Mi was 23% smaller; later in spring, Mi increased 60% in relation to the autumn and the exponent b=0.14 indicated that the recovery was disproportionate in smaller tegus. During the autumn, the intestinal villi were greatly elongated; by midwinter, the Hv, SvEp, and VvEp were smaller than during the autumn (59%, 54%, 29%) and were restored to autumn levels during spring. In the active tegus, the maximum activity (Vmax) of enzymes indicated that the enterocytes can obtain energy from different sources, and possess gluconeogenic capacity. During winter, the Vmax of CS, HOAD, GDH, PEPCK was 40-50% lower in relation to the autumn and spring, while the Vmax of HK, PK, LDH, AST was unchanged. The hypoglycemia and the mucosal atrophy/ischemia during winter would prevent the enterocytes from using glucose, whereas they could slowly oxidize fatty acids released from body stores and amino acids from the tissue proteolysis to satisfy their needs of energy. Contrastingly, starvation during spring caused severe mass loss (50%); the tissue protein and the VvEp and VvLP did not change while the thickness of the muscular layer increased 51%, which suggested different effects along the length of the organ. In addition, the Vmax of the glycolytic enzymes was lower, indicating that a regulatory mechanism would spare blood glucose for vital organs during unanticipated food restriction. Copyright © 2016 Elsevier Inc. All rights reserved.

Identification of Functional Amino Acid Residues Involved in Polyamine and Agmatine Transport by Human Organic Cation Transporter 2

PubMed Central

Higashi, Kyohei; Imamura, Masataka; Fudo, Satoshi; Uemura, Takeshi; Saiki, Ryotaro; Hoshino, Tyuji; Toida, Toshihiko; Kashiwagi, Keiko; Igarashi, Kazuei

2014-01-01

Polyamine (putrescine, spermidine and spermine) and agmatine uptake by the human organic cation transporter 2 (hOCT2) was studied using HEK293 cells transfected with pCMV6-XL4/hOCT2. The Km values for putrescine and spermidine were 7.50 and 6.76 mM, and the Vmax values were 4.71 and 2.34 nmol/min/mg protein, respectively. Spermine uptake by hOCT2 was not observed at pH 7.4, although it inhibited both putrescine and spermidine uptake. Agmatine was also taken up by hOCT2, with Km value: 3.27 mM and a Vmax value of 3.14 nmol/min/mg protein. Amino acid residues involved in putrescine, agmatine and spermidine uptake by hOCT2 were Asp427, Glu448, Glu456, Asp475, and Glu516. In addition, Glu524 and Glu530 were involved in putrescine and spermidine uptake activity, and Glu528 and Glu540 were weakly involved in putrescine uptake activity. Furthermore, Asp551 was also involved in the recognition of spermidine. These results indicate that the recognition sites for putrescine, agmatine and spermidine on hOCT2 strongly overlap, consistent with the observation that the three amines are transported with similar affinity and velocity. A model of spermidine binding to hOCT2 was constructed based on the functional amino acid residues. PMID:25019617

Breakdown and Limit of Continuum Diffusion Velocity for Binary Gas Mixtures from Direct Simulation

NASA Astrophysics Data System (ADS)

Martin, Robert Scott; Najmabadi, Farrokh

2011-05-01

This work investigates the breakdown of the continuum relations for diffusion velocity in inert binary gas mixtures. Values of the relative diffusion velocities for components of a gas mixture may be calculated using of Chapman-Enskog theory and occur not only due to concentration gradients, but also pressure and temperature gradients in the flow as described by Hirschfelder. Because Chapman-Enskog theory employs a linear perturbation around equilibrium, it is expected to break down when the velocity distribution deviates significantly from equilibrium. This breakdown of the overall flow has long been an area of interest in rarefied gas dynamics. By comparing the continuum values to results from Bird's DS2V Monte Carlo code, we propose a new limit on the continuum approach specific to binary gases. To remove the confounding influence of an inconsistent molecular model, we also present the application of the variable hard sphere (VSS) model used in DS2V to the continuum diffusion velocity calculation. Fitting sample asymptotic curves to the breakdown, a limit, Vmax, that is a fraction of an analytically derived limit resulting from the kinetic temperature of the mixture is proposed. With an expected deviation of only 2% between the physical values and continuum calculations within ±Vmax/4, we suggest this as a conservative estimate on the range of applicability for the continuum theory.

Rachiplusia nu larva as a biofactory to achieve high level expression of horseradish peroxidase.

PubMed

Romero, Lucía Virginia; Targovnik, Alexandra Marisa; Wolman, Federico Javier; Cascone, Osvaldo; Miranda, María Victoria

2011-05-01

A process based on orally-infected Rachiplusia nu larvae as biological factories for expression and one-step purification of horseradish peroxidase isozyme C (HRP-C) is described. The process allows obtaining high levels of pure HRP-C by membrane chromatography purification. The introduction of the partial polyhedrin homology sequence element in the target gene increased HRP-C expression level by 2.8-fold whereas it increased 1.8-fold when the larvae were reared at 27 °C instead of at 24 °C, summing up a 4.6-fold overall increase in the expression level. Additionally, HRP-C purification by membrane chromatography at a high flow rate greatly increase D the productivity without affecting the resolution. The V(max) and K(m) values of the recombinant HRP-C were similar to those of the HRP from Armoracia rusticana roots. © Springer Science+Business Media B.V. 2011

Enhancing Activity and Stability of Uricase from Lactobacillus plantarum by Zeolite immobilization

NASA Astrophysics Data System (ADS)

Iswantini, D.; Nurhidayat, N.; Sarah

2017-03-01

Lactobacillus plantarum has been known be able to produce uricase for uric acid biosensor. Durability and stability of L. plantarum in generating uricase enzyme was low. Hence, we tried to enhance its durability and stability by immobilizing it onto activated 250 mg zeolite at room temperature using 100 μL L.plantarum suspension and 2.87 mM uric acid, while Michaelis-Menten constant (KM) and Vmax were obtained at 6.7431 mM and 0.9171 µA consecutively, and the linearity range was 0.1-3.3 mM (R2 = 0.9667). Limit of detection (LOD) and limit of quantification (LOQ) value of the measurement were 0.4827 mM and 1.6092 mM respectively. Biosensor stability treatment was carried out in two different treatments, using the same electrode and using disposable electrode. The disposable electrode stability showed better result based on repeated measurements, but stability was still need improvement.

Designing a multistage supply chain in cross-stage reverse logistics environments: application of particle swarm optimization algorithms.

PubMed

Chiang, Tzu-An; Che, Z H; Cui, Zhihua

2014-01-01

This study designed a cross-stage reverse logistics course for defective products so that damaged products generated in downstream partners can be directly returned to upstream partners throughout the stages of a supply chain for rework and maintenance. To solve this reverse supply chain design problem, an optimal cross-stage reverse logistics mathematical model was developed. In addition, we developed a genetic algorithm (GA) and three particle swarm optimization (PSO) algorithms: the inertia weight method (PSOA_IWM), V(Max) method (PSOA_VMM), and constriction factor method (PSOA_CFM), which we employed to find solutions to support this mathematical model. Finally, a real case and five simulative cases with different scopes were used to compare the execution times, convergence times, and objective function values of the four algorithms used to validate the model proposed in this study. Regarding system execution time, the GA consumed more time than the other three PSOs did. Regarding objective function value, the GA, PSOA_IWM, and PSOA_CFM could obtain a lower convergence value than PSOA_VMM could. Finally, PSOA_IWM demonstrated a faster convergence speed than PSOA_VMM, PSOA_CFM, and the GA did.

Cycling of Dissolved Organic Phosphorus and Alkaline Phosphatase Activity in Euphotic Zone of the Western North Pacific

NASA Astrophysics Data System (ADS)

Suzumura, M.

2010-12-01

Phosphorus is an essential nutrient for marine organisms. In oligotrophic environments, concentrations of dissolved inorganic phosphate (SRP), the most bioavailable form of phosphorus, are low and have been hypothesized to constrain the primary productivity. Evidence has been found that dissolved organic phosphorus (DOP) supports a significant fraction of primary production through hydrolytic remineralization of DOP to SRP by alkaline phosphatase (APA). In this study, DOP biogeochemistry was investigated at three locations of the open-ocean environment in the Kuroshio region and at a semi-eutrophic coastal site of the western North Pacific. Concentrations of SRP, DOP and hydrolyzable ester-P were measured in the euphotic zone. Kinetic parameters of APA were determined using a fluorogenic substrate, including potential maximum velocity (Vmax), apparent Michaelis-Menten half-saturation constant (Km), and turnover time (TA) of APA hydrolyzable DOP. SRP concentrations were quite low (≤ 10 nM) in the surface seawater and rapidly increased below the chlorophyll a maximum layer (CML). DOP concentration ranged from 29 to 223 nM. Above the CML, DOP composed a major fraction accounting for 60-100% of dissolved total P. A significant linear relationship was found between the concentrations of SRP and hydrolyzable ester-P (R2 = 0.83, P < 0.01). This suggests active utilization of ester-P under phosphate-depleted conditions. In the Kuroshio region, Vmax of APA exhibited the highest value at the surface water (0 m) and decreased rapidly with depth, while at the coastal site the peak value was found at CML. TA of hydrolyzable DOP was quite variable among the locations and increased with depth especially below CML. The estimated values of in situ hydrolysis rate were much lower (2-34%) than the potential Vmax which was determined with the addition of an excess amount of the substrate. The results suggest that marine microbes can efficiently and rapidly utilize hydrolyzable DOP under phosphate-depleted conditions and that there is still room in the in situ APA activity. Utilization of DOP, however, is likely regulated by the ambient concentrations of hydrolyzable ester-P lower than the apparent Km.

New Tool to Control and Monitor Weighted Vest Training Load for Sprinting and Jumping in Soccer.

PubMed

Carlos-Vivas, Jorge; Freitas, Tomás T; Cuesta, Miguel; Perez-Gomez, Jorge; De Hoyo, Moisés; Alcaraz, Pedro E

2018-04-26

Carlos-Vivas, J, Freitas, TT, Cuesta, M, Perez-Gomez, J, De Hoyo, M, and Alcaraz, PE. New tool to control and monitor weighted vest training load for sprinting and jumping in soccer. J Strength Cond Res XX(X): 000-000, 2018-The purpose of this study was to develop 2 regression equations that accurately describe the relationship between weighted vest loads and performance indicators in sprinting (i.e., maximum velocity, Vmax) and jumping (i.e., maximum height, Hmax). Also, this study aimed to investigate the effects of increasing the load on spatio-temporal variables and power development in soccer players and to determine the "optimal load" for sprinting and jumping. Twenty-five semiprofessional soccer players performed the sprint test, whereas a total of 46 completed the vertical jump test. Two different regression equations were developed for calculating the load for each exercise. The following equations were obtained: % body mass (BM) = -2.0762·%Vmax + 207.99 for the sprint and % BM = -0.7156·%Hmax + 71.588 for the vertical jump. For both sprinting and jumping, when the load increased, Vmax and Hmax decreased. The "optimal load" for resisted training using weighted vest was unclear for sprinting and close to BM for vertical jump. This study presents a new tool to individualize the training load for resisted sprinting and jumping using weighted vest in soccer players and to develop the whole force-velocity spectrum according to the objectives of the different periods of the season.

The Core of Allosteric Motion in Thermus caldophilus l-Lactate Dehydrogenase*

PubMed Central

Ikehara, Yoko; Arai, Kazuhito; Furukawa, Nayuta; Ohno, Tadashi; Miyake, Tatsuya; Fushinobu, Shinya; Nakajima, Masahiro; Miyanaga, Akimasa; Taguchi, Hayao

2014-01-01

For Thermus caldophilus l-lactate dehydrogenase (TcLDH), fructose 1,6-bisphosphate (FBP) reduced the pyruvate S0.5 value 103-fold and increased the Vmax value 4-fold at 30 °C and pH 7.0, indicating that TcLDH has a much more T state-sided allosteric equilibrium than Thermus thermophilus l-lactate dehydrogenase, which has only two amino acid replacements, A154G and H179Y. The inactive (T) and active (R) state structures of TcLDH were determined at 1.8 and 2.0 Å resolution, respectively. The structures indicated that two mobile regions, MR1 (positions 172–185) and MR2 (positions 211–221), form a compact core for allosteric motion, and His179 of MR1 forms constitutive hydrogen bonds with MR2. The Q4(R) mutation, which comprises the L67E, H68D, E178K, and A235R replacements, increased Vmax 4-fold but reduced pyruvate S0.5 only 5-fold in the reaction without FBP. In contrast, the P2 mutation, comprising the R173Q and R216L replacements, did not markedly increase Vmax, but 102-reduced pyruvate S0.5, and additively increased the FBP-independent activity of the Q4(R) enzyme. The two types of mutation consistently increased the thermal stability of the enzyme. The MR1-MR2 area is a positively charged cluster, and its center approaches another positively charged cluster (N domain cluster) across the Q-axis subunit interface by 5 Å, when the enzyme undergoes the T to R transition. Structural and kinetic analyses thus revealed the simple and unique allosteric machinery of TcLDH, where the MR1-MR2 area pivotally moves during the allosteric motion and mediates the allosteric equilibrium through electrostatic repulsion within the protein molecule. PMID:25258319

Kinetics of Ethylene and Ethylene Oxide in Subcellular Fractions of Lungs and Livers of Male B6C3F1 Mice and Male Fischer 344 Rats and of Human Livers

PubMed Central

Csanády, György András; Kessler, Winfried; Klein, Dominik; Pankratz, Helmut; Pütz, Christian; Richter, Nadine; Filser, Johannes Georg

2011-01-01

Ethylene (ET) is metabolized in mammals to the carcinogenic ethylene oxide (EO). Although both gases are of high industrial relevance, only limited data exist on the toxicokinetics of ET in mice and of EO in humans. Metabolism of ET is related to cytochrome P450-dependent mono-oxygenase (CYP) and of EO to epoxide hydrolase (EH) and glutathione S-transferase (GST). Kinetics of ET metabolism to EO and of elimination of EO were investigated in headspace vessels containing incubations of subcellular fractions of mouse, rat, or human liver or of mouse or rat lung. CYP-associated metabolism of ET and GST-related metabolism of EO were found in microsomes and cytosol, respectively, of each species. EH-related metabolism of EO was not detectable in hepatic microsomes of rats and mice but obeyed saturation kinetics in hepatic microsomes of humans. In ET-exposed liver microsomes, metabolism of ET to EO followed Michaelis-Menten-like kinetics. Mean values of Vmax [nmol/(min·mg protein)] and of the apparent Michaelis constant (Km [mmol/l ET in microsomal suspension]) were 0.567 and 0.0093 (mouse), 0.401 and 0.031 (rat), and 0.219 and 0.013 (human). In lung microsomes, Vmax values were 0.073 (mouse) and 0.055 (rat). During ET exposure, the rate of EO production decreased rapidly. By modeling a suicide inhibition mechanism, rate constants for CYP-mediated catalysis and CYP inactivation were estimated. In liver cytosol, mean GST activities to EO expressed as Vmax/Km [μl/(min·mg protein)] were 27.90 (mouse), 5.30 (rat), and 1.14 (human). The parameters are most relevant for reducing uncertainties in the risk assessment of ET and EO. PMID:21785163

Kinetics and spatial distribution of enzymes of carbon, nitrogen and phosphorus cycles in earthworm biopores

NASA Astrophysics Data System (ADS)

Hoang Thi Thu, Duyen; Razavi, Bahar S.

2016-04-01

Earthworms boost microbial activities and consequently form hotspots in soil. The distribution of enzyme activities inside the earthworm biopores is completely unknown. For the first time, we analyzed enzyme kinetics and visualized enzyme distribution inside and outside biopores by in situ soil zymography. Kinetic parameters (Vmax and Km) of 6 enzymes β-glucosidase (GLU), cellobiohydrolase (CBH), xylanase (XYL), chitinase (NAG), leucine aminopeptidase (LAP) and acid phosphatase (APT) were determined in biopores formed by Lumbricus terrestris L.. The spatial distributions of GLU, NAG and APT become visible via zymograms in comparison between earthworm-inhabited and earthworm-free soil. Zymography showed heterogeneous distribution of hotspots in the rhizosphere and biopores. The hotspot areas were 2.4 to 14 times larger in the biopores than in soil without earthworms. The significantly higher Vmax values for GLU, CBH, XYL, NAG and APT in biopores confirmed the stimulation of enzyme activities by earthworms. For CBH, XYL and NAG, the 2- to 3-fold higher Km values in biopores indicated different enzyme systems with lower substrate affinity compared to control soil. The positive effects of earthworms on Vmax were cancelled by the Km increase for CBH, XYL and NAG at a substrate concentration below 20 μmol g-1 soil. The change of enzyme systems reflected a shift in dominant microbial populations toward species with lower affinity to holo-celluloses and to N-acetylglucosamine, and with higher affinity to proteins as compared to the biopores-free soil. We conclude that earthworm biopores are microbial hotspots with much higher and dense distribution of enzyme activities compared to bulk soil. 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. Blagodatskaya, E., Kuzyakov, Y., 2013. Review paper: Active microorganisms in soil: Critical review of estimation criteria and approaches. Soil Biology & Biochemistry 67, 192-211.

A study of the temporal effect of alcohol on human erythrocyte sodium-lithium countertransport in relation to membrane cholesterol and phospholipids.

PubMed

Adebayo, G I; Gaffney, P; Feely, J

1996-01-01

The effect of a single dose of alcohol (0.8 g/kg), given with "diet coke," on erythrocyte sodium-lithium countertransport (SLC) in relation to membrane cholesterol and phospholipids was assessed over 24 h in 10 healthy volunteers. Baseline passive lithium efflux (0.168 +/- 0.008 mmol l-1 Cell H-1) was increased 1 h (0.202 +/- 0.014 mmol l-1 cell h-1; p < 0.030), and 4 h (0.200 +/- 0.014 mmol l-1 cell h-1; p < 0.020), but similar to that at 24 h postalcohol (0.173 +/- 0.011 mmol l-1 cell h-1). These changes were not associated with any change in intracellular lithium. Control SLC VMAX of 0.387 +/- 0.054 mmol l-1 cell h-1 fell at 1 h (0.328 +/- 0.050 mmol l-1 cell h-1; p = 0.0012) and 4 h (0.312 +/- 0.048 mmol l-1 cell h-1; p < 0.0005). Its value 24 h postalcohol (0.371 +/- 0.047 mmol l-1 cell h-1) was comparable to that at baseline. There was no significant change in the affinity of the transporter for external sodium throughout the experimental period, suggesting that the reduction in VMAX 1 and 4 h after alcohol ingestion resulted from a noncompetitive inhibition. Intracellular sodium 4 h after alcohol was lower than at baseline, but returned to the control value within 24 h. In a control group (n = 5), pretreatment with "diet coke" alone did not alter any of the measured parameters. It is concluded that alcohol pretreatment increases passive lithium efflux and decreases SLC Vmax. Both effects are evident up to at least 4 h postdosing, but recover within 24 h in the absence of further alcohol intake.

Sympathomimetic effects of MIBG: comparison with tyramine.

PubMed

Graefe, K H; Bossle, F; Wölfel, R; Burger, A; Souladaki, M; Bier, D; Dutschka, K; Farahati, J; Bönisch, H

1999-08-01

Because nothing is known about whether metaiodobenzylguanidine (MIBG) has tyramine-like actions, the sympathomimetic effects of MIBG were determined in the isolated rabbit heart and compared with those of tyramine. Spontaneously beating rabbit hearts were perfused with Tyrode's solution (Langendorff technique; 37 degrees C; 26 mL/min), and the heart rate as well as the norepinephrine and dopamine overflow into the perfusate was measured before and after doses of MIBG or tyramine (0.03-10 micromol) given as bolus injections (100 microL) into the aortic cannula. Km and Vmax values for the neuronal uptake (uptake1) of 125I-MIBG and 14C-tyramine were obtained in human neuroblastoma (SK-N-SH) cells. The Ki of MIBG for inhibition of the 3H-catecholamine uptake mediated by the vesicular monoamine transporter was determined in membrane vesicles obtained from bovine chromaffin granules and compared with the previously reported Ki value for tyramine determined under identical experimental conditions. By producing increases in heart rate and norepinephrine overflow, both compounds had dose-dependent sympathomimetic effects in the rabbit heart. MIBG was much less effective than tyramine in increasing heart rate (maximum effect 59 versus 156 beats/min) and norepinephrine overflow (maximum effect 35 versus 218 pmol/g). Tyramine also caused increases in dopamine overflow, whereas MIBG was a poor dopamine releaser. At a dose of 10 micromol, the increase in heart rate lasted more than 60 min after MIBG and about 20 min after tyramine injection. Accordingly, the norepinephrine overflow caused by 10 micromol MIBG and tyramine declined with half-lives of 57.8 and 2.2 min, respectively. The effects of both drugs were drastically reduced in hearts exposed to 2 micromol/L desipramine. The kinetic parameters characterizing the saturation of neuronal uptake by 125I-MIBG and 14C-tyramine were similar for the two compounds: Km values of MIBG and tyramine were 1.6 and 1.7 micromol/L, respectively, and Vmax values of MIBG and tyramine were 43 and 37 pmol/mg protein/min, respectively. However, in inhibiting the vesicular 3H-catecholamine uptake, MIBG was eight times less potent than tyramine. MIBG is much less effective than tyramine as an indirect sympathomimetic agent. This is probably a result of its relatively low affinity for the vesicular monoamine transporter and explains the relatively poor ability of the drug to mobilize norepinephrine stored in synaptic vesicles. The long duration of MIBG action results primarily from the drug not being metabolized by monoamine oxidase. The sympathomimetic effects of MIBG described here are not likely to come into play in patients given diagnostic or common therapeutic doses of radioiodinated MIBG.

Kinetics of sulfate and hydrogen uptake by the thermophilic sulfate-reducing bacteria thermodesulfobacterium sp. Strain JSP and thermodesulfovibrio sp. Strain R1Ha3

PubMed

Sonne-Hansen; Westermann; Ahring

1999-03-01

Half-saturation constants (Km), maximum uptake rates (Vmax), and threshold concentrations for sulfate and hydrogen were determined for two thermophilic sulfate-reducing bacteria (SRB) in an incubation system without headspace. Km values determined for the thermophilic SRB were similar to the constants described for mesophilic SRB isolated from environments with low sulfate concentrations.

Kinetics of Sulfate and Hydrogen Uptake by the Thermophilic Sulfate-Reducing Bacteria Thermodesulfobacterium sp. Strain JSP and Thermodesulfovibrio sp. Strain R1Ha3

PubMed Central

Sonne-Hansen, Jacob; Westermann, Peter; Ahring, Birgitte K.

1999-01-01

Half-saturation constants (Km), maximum uptake rates (Vmax), and threshold concentrations for sulfate and hydrogen were determined for two thermophilic sulfate-reducing bacteria (SRB) in an incubation system without headspace. Km values determined for the thermophilic SRB were similar to the constants described for mesophilic SRB isolated from environments with low sulfate concentrations. PMID:10049897

United States Air Force Graduate Student Summer Support Program (1987). Program Technical Report. Volume 2

DTIC Science & Technology

1987-12-01

Metabolism (VMAX) Using Quantitative Structure- Activity Relationships (QSAR) 17 Directed Motion Doppler Shift Effects on Mitric Oxide (0,0) Gamma Band...chemiluminescence values were observed at characteristic times after adding glucose to the disks. We also produced virus-sized nanoparticles (Glucose...These nanoparticles were able to penetrate a .2 um filter,and they retained their enzymatic activity for weeks. They produced 20-fold greater

Role of 3D Ultrasound and Doppler in Differentiating Clinically Suspected Cases of Leiomyoma and Adenomyosis of Uterus

PubMed Central

Sharma, Kaveri; Venkatesh, B.P; Barman, Partho; Roy, Sumit Kumar; Jayagurunathan, Usha; Sellamuthu, Eswaramoorthy; Moidu, Fazil

2015-01-01

Introduction Adenomyosis and Leiomyoma are common disorders affecting females in their reproductive age. They mimic each other in clinical presentation. Due to similarities in clinical symptoms and signs, missing one diagnosis in favour of the other is not very uncommon. Accurate diagnosis of these two conditions is important for their management. In this study we evaluated role of 3D Ultrasound and Doppler in differentiating clinically suspected cases of leiomyoma and adenomyosis of uterus. Materials and Methods A total of 100 patients with symptoms of abnormal uterine bleeding (with or without dysmenorrhoea), lump abdomen, chronic pelvic pain or dysparaunia who were clinically diagnosed as leiomyoma of uterus and/or adenomyosis were enrolled in to the study. These patients underwent transvaginal sonography (TVS), trans abdominal sonography (TAS) along with color and spectral Doppler sonography. Scanning was done in follicular phase of the menstrual cycle to avoid bias due high vascularity of endometrium in secretory phase. The morphology of the lesion, its vascularity, and Pulsality Index (PI), Resistive Index (RI) and Vmax (maximum velocity) were measured. Only those patients who were chosen for operative treatment were included in the study. Radiological diagnosis was then correlated with intra-operative and histopathological diagnosis. Results On imaging, while using morphological criteria and Doppler for diagnosing leiomyoma, it was found that “peripheral vascularity” was seen in 52 (89%) cases, which was the highest. Similarly while diagnosing adenomyosis it was, the criteria “central vascularity” was seen in 28 cases (93%) and “ill defined junctional zone in 3D ultrasound” was seen in 26 cases (86%), which was also observed to be highest. With the cut off values taken for PI,RI and Vmax, diagnosis of leiomyoma was found to be 93.4% sensitive, 95.6% specific and with a positive predictive value of 97.6% and negative predictive value of 88.6%. Diagnosis of adenomyosis showed a sensitivity of 95.6%, specificity of 93.4% and a positive predictive value of 88.6% and negative predictive value of 97.6%. Imaging dignosed the co-existence of both the conditions correctly in 8 (66%) cases. Conclusion The parameters of blood flow impedance (that is PI, RI, and Vmax) of arteries within or around the uterine lesions revealed a consistent and significant difference between leiomyoma and adenomyosis. So apart from morphological criteria used in 3D TAS and TVS, aid of color Doppler can more accurately differentiate and diagnose these conditions. PMID:26023602

Active uptake of substance P carboxy-terminal heptapeptide (5-11) into rat brain and rabbit spinal cord slices.

PubMed

Nakata, Y; Kusaka, Y; Yajima, H; Segawa, T

1981-12-01

We previously reported that nerve terminals and glial cells lack an active uptake system capable of terminating transmitter action of substance P (SP). In the present study, we demonstrated the existence of an active uptake system for SP carboxy-terminal heptapeptide, (5-11)SP. When the slices from either rat brain or rabbit spinal cord were incubated with [3H](5-11)SP, the uptake of (5-11)SP into slices was observed. The uptake system has the properties of an active transport mechanism: it is dependent on temperature and sensitive to hypoosmotic treatment and is inhibited by ouabain and dinitrophenol (DNP). In the brain, (5-11)SP was accumulated by means of a high-affinity and a low-affinity uptake system. The Km and the Vmax values for the high-affinity system were 4.20 x 10(-8) M and 7.59 fmol/10 mg wet weight/min, respectively, whereas these values for the low-affinity system were 1.00 x 10(-6) M and 100 fmol/10 mg wet weight/min, respectively. In the spinal cord, there was only one uptake system, with a Km value of 2.16 x 10(-7) M and Vmax value of 26.2 fmol/10 mg wet weight/min. These results suggest that when SP is released from nerve terminals, it is hydrolysed into (5-11)SP before or after acting as a neurotransmitter, which is in turn accumulated into nerve terminals. Therefore, the uptake system may represent a possible mechanism for the inactivation of SP.

Models of disuse - A comparison of hindlimb suspension and immobilization

NASA Technical Reports Server (NTRS)

Fitts, R. H.; Metzger, J. M.; Riley, D. A.; Unsworth, B. R.

1986-01-01

The effects of 1 and 2 weeks of hindlimb suspension (HS) on the contractile properties of fast- and slow-twitch skeletal muscles of male Sprague Dawley rats are studied and compared with hindlimb immobilization (HI) data. The optimal length and contractile properties of the slow-twitch soleus, fast-twitch extensor digitorum longus, and the vastus lateralis are measured. It is observed that HS and HI affect slow-twitch muscles; isometric twitch duration in the slow-twitch soleus is decreased. Soleus muscle mass and peak tetanic tension declines with disuse. A major difference in the influence of HS and HI on the maximal speed of soleus muscle shortening, V(max) is detected; HS produced a twofold increase in V(max) compared to control data and HI had no significant effect on V(max). The relation between V(max) and myosin concentration is analyzed. The data reveal that HS modifies slow-twitch muscle yielding hybrid fibers with elevated shortening velocities and this change may be dependent on the elimination of load-bearing contractions.

Kinetic analysis of dihydroxyacetone production from crude glycerol by immobilized cells of Gluconobacter oxydans MTCC 904.

PubMed

Dikshit, Pritam Kumar; Moholkar, Vijayanand S

2016-09-01

The present study has investigated kinetic features of bioconversion of biodiesel-derived crude glycerol to dihydroxyacetone with immobilized Gluconobacter oxydans cells using modified Haldane substrate-inhibition model. The results have been compared against free cells and pure glycerol. Relative variations in the kinetic parameters KS, KI, Vmax, n and X reveal that immobilized G. oxydans cells (on PU foam substrate) with crude glycerol as substrate give higher order of inhibition (n) and lower maximum reaction velocities (Vmax). These results are essentially implications of substrate transport restrictions across immobilization matrix, which causes retention of substrate in the matrix and reduction in fractional available substrate (X) for the cells. This causes reduction in both KS (substrate concentration at Vmax/2) and KI (inhibition constant) as compared to free cells. For immobilized cells, substrate concentration (Smax) corresponding to Vmax is practically same for both pure and crude glycerol as substrate. Copyright © 2016 Elsevier Ltd. All rights reserved.

A K-band Frequency Agile Microstrip Bandpass Filter using a Thin Film HTS/Ferroelectric/dielectric Multilayer Configuration

NASA Technical Reports Server (NTRS)

Subramanyam, Guru; VanKeuls, Fred; Miranda, Felix A.

1998-01-01

We report on YBa2Cu3O(7-delta) (YBCO) thin film/SrTiO3 (STO) thin film K-band tunable bandpass filters on LaAlO3 (LAO) dielectric substrates. The 2 pole filter has a center frequency of 19 GHz and a 4% bandwidth. Tunability is achieved through the non-linear dc electric field dependence of the relative dielectric constant of STO(epsilon(sub rSTO). A large tunability ((Delta)f/f(sub 0) = (f(sub Vmax) - f(sub 0)/f(sub 0), where f(sub 0) is the center frequency of the filter at no bias and f(sub Vmax) is the center frequency of the filter at the maximum applied bias) of greater than 10% was obtained in YBCO/STO/LAO microstrip bandpass filters operating below 77 K. A center frequency shift of 2.3 GHz (i.e., a tunability factor of approximately 15%) was obtained at a 400 V bipolar dc bias, and 30 K, with minimal degradation in the insertion loss of the filter. This paper addresses design, fabrication and testing of tunable filters based on STO ferroelectric thin films. The performance of the YBCO/STO/LAO filters is compared to that of gold/STO/LAO counterparts.

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

PubMed Central

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

2013-01-01

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

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

PubMed

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

2013-09-01

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

The effects of a supportive knee brace on leg performance in healthy subjects.

PubMed

Veldhuizen, J W; Koene, F M; Oostvogel, H J; von Thiel, T P; Verstappen, F T

1991-12-01

Eight healthy volunteers were fitted with a supportive knee brace (Push Brace 'Heavy') to one knee for a duration of four weeks wherein they were tested before, during and after the application to establish the effect of bracing on performance. The tests consisted of isokinetic strength measurement of knee flexion and extension, 60 meter dash, vertical jump height and a progressive horizontal treadmill test until exhaustion (Vmax) with determination of oxygen uptake, heart rate and plasma lactate concentration. Wearing the brace for one day, the performance indicators showed a decline compared with the test before application (base values). Sprint time was 4% longer (p less than 0.01) and Vmax 6% slower (p less than 0.01). Peak torque of knee flexion at 60 and 240 deg.sec-1 was 6% (p less than 0.05) respectively 9% (p less than 0.05) less. Peak extension torque at 60 deg.sec-1 was 9% less (p less than 0.05). While wearing the brace for four weeks, the test performances were practically identical to their base values. After removal of the brace, all test parameters were statistically similar to the base values. Heart rate at submaximal exercise levels was even lower (p less than 0.05). In conclusion, performance in sports with test-like exercise patterns is not affected by the brace tested. Bracing does not "weaken the knee" as it is widely believed in sports practice.

Influence of temperature on muscle recruitment and muscle function in vivo.

PubMed

Rome, L C

1990-08-01

Temperature has a large influence on the maximum velocity of shortening (Vmax) and maximum power output of muscle (Q10 = 1.5-3). In some animals, maximum performance and maximum sustainable performance show large temperature sensitivities, because these parameters are dependent solely on mechanical power output of the muscles. The mechanics of locomotion (sarcomere length excursions and muscle-shortening velocities, V) at a given speed, however, are precisely the same at all temperatures. Animals compensate for the diminished power output of their muscles at low temperatures by compressing their recruitment order into a narrower range of locomotor speeds, that is, recruiting more muscle fibers and faster fiber types at a given speed. By examining V/Vmax, I calculate that fish at 10 degrees C must recruit 1.53-fold greater fiber cross section than at 20 degrees C. V/Vmax also appears to be an important design constraint in muscle. It sets the lowest V and the highest V over which a muscle can be used effectively. Because the Vmax of carp slow red muscle has a Q10 of 1.6 between 10 and 20 degrees C, the slow aerobic fibers can be used over a 1.6-fold greater range of swim speeds at the warmer temperature. In some species of fish, Vmax can be increased during thermal acclimation, enabling animals to swim at higher speeds.

Characterization of Two Distinct Glycosyl Hydrolase Family 78 α-l-Rhamnosidases from Pediococcus acidilactici▿†

PubMed Central

Michlmayr, Herbert; Brandes, Walter; Eder, Reinhard; Schümann, Christina; del Hierro, Andrés M.; Kulbe, Klaus D.

2011-01-01

α-l-Rhamnosidases play an important role in the hydrolysis of glycosylated aroma compounds (especially terpenes) from wine. Although several authors have demonstrated the enological importance of fungal rhamnosidases, the information on bacterial enzymes in this context is still limited. In order to fill this important gap, two putative rhamnosidase genes (ram and ram2) from Pediococcus acidilactici DSM 20284 were heterologously expressed, and the respective gene products were characterized. In combination with a bacterial β-glucosidase, both enzymes released the monoterpenes linalool and cis-linalool oxide from a muscat wine extract under ideal conditions. Additionally, Ram could release significant amounts of geraniol and citronellol/nerol. Nevertheless, the potential enological value of these enzymes is limited by the strong negative effects of acidity and ethanol on the activities of Ram and Ram2. Therefore, a direct application in winemaking seems unlikely. Although both enzymes are members of the same glycosyl hydrolase family (GH 78), our results clearly suggest the distinct functionalities of Ram and Ram2, probably representing two subclasses within GH 78: Ram could efficiently hydrolyze only the synthetic substrate p-nitrophenyl-α-l-rhamnopyranoside (Vmax = 243 U mg−1). In contrast, Ram2 displayed considerable specificity toward hesperidin (Vmax = 34 U mg−1) and, especially, rutinose (Vmax = 1,200 U mg−1), a disaccharide composed of glucose and rhamnose. Both enzymes were unable to hydrolyze the flavanone glycoside naringin. Interestingly, both enzymes displayed indications of positive substrate cooperativity. This study presents detailed kinetic data on two novel rhamnosidases, which could be relevant for the further study of bacterial glycosidases. PMID:21784921

Metabolism of ethylbenzene by human liver microsomes and recombinant human cytochrome P450s (CYP).

PubMed

Sams, Craig; Loizou, George D; Cocker, John; Lennard, Martin S

2004-03-07

The enzyme kinetics of the initial hydroxylation of ethylbenzene to form 1-phenylethanol were determined in human liver microsomes. The individual cytochrome P450 (CYP) forms catalysing this reaction were identified using selective inhibitors and recombinant preparations of hepatic CYPs. Production of 1-phenylethanol in hepatic microsomes exhibited biphasic kinetics with a high affinity, low Km, component (mean Km = 8 microM; V(max) = 689 pmol/min/mg protein; n = 6 livers) and a low affinity, high Km, component (Km = 391 microM; V(max) = 3039 pmol/min/mg protein; n = 6). The high-affinity component was inhibited 79%-95% (mean 86%) by diethyldithiocarbamate, and recombinant CYP2E1 was shown to metabolise ethylbenzene with low Km (35 microM), but also low (max) (7 pmol/min/pmol P450), indicating that this isoform catalysed the high-affinity component. Recombinant CYP1A2 and CYP2B6 exhibited high V(max) (88 and 71 pmol/min/pmol P450, respectively) and high Km (502 and 219 microM, respectively), suggesting their involvement in catalysing the low-affinity component. This study has demonstrated that CYP2E1 is the major enzyme responsible for high-affinity side chain hydroxylation of ethylbenzene in human liver microsomes. Activity of this enzyme in the population is highly variable due to induction or inhibition by physiological factors, chemicals in the diet or some pharmaceuticals. This variability can be incorporated into the risk assessment process to improve the setting of occupational exposure limits and guidance values for biological monitoring.

THE IMPACT OF SCALING FACTOR VARIABILITY ON RISK-RELEVANT TOXICOKINETIC OUTCOMES IN CHILDREN: A CASE STUDY USING BROMODICHLOROMETHANE (BDCM)

EPA Science Inventory

Biotransformation rates (Vmax) extrapolated from in vitro data are used increasingly in human physiologically based pharmacokinetic (PBPK) models. Extrapolation of Vmax from in vitro data requires use of scaling factors, including mg of microsomal protein/g liver (MPPGL), nmol of...

Peripheral Airway Smooth Muscle, but Not the Trachealis, Is Hypercontractile in an Equine Model of Asthma.

PubMed

Matusovsky, Oleg S; Kachmar, Linda; Ijpma, Gijs; Bates, Genevieve; Zitouni, Nedjma; Benedetti, Andrea; Lavoie, Jean-Pierre; Lauzon, Anne-Marie

2016-05-01

Heaves is a naturally occurring equine disease that shares many similarities with human asthma, including reversible antigen-induced bronchoconstriction, airway inflammation, and remodeling. The purpose of this study was to determine whether the trachealis muscle is mechanically representative of the peripheral airway smooth muscle (ASM) in an equine model of asthma. Tracheal and peripheral ASM of heaves-affected horses under exacerbation, or under clinical remission of the disease, and control horses were dissected and freed of epithelium to measure unloaded shortening velocity (Vmax), stress (force/cross-sectional area), methacholine effective concentration at which 50% of the maximum response is obtained, and stiffness. Myofibrillar Mg(2+)-ATPase activity, actomyosin in vitro motility, and contractile protein expression were also measured. Horses with heaves had significantly greater Vmax and Mg(2+)-ATPase activity in peripheral airway but not in tracheal smooth muscle. In addition, a significant correlation was found between Vmax and the time elapsed since the end of the corticosteroid treatment for the peripheral airways in horses with heaves. Maximal stress and stiffness were greater in the peripheral airways of the horses under remission compared with controls and the horses under exacerbation, potentially due to remodeling. Actomyosin in vitro motility was not different between controls and horses with heaves. These data demonstrate that peripheral ASM is mechanically and biochemically altered in heaves, whereas the trachealis behaves as in control horses. It is therefore conceivable that the trachealis muscle may not be representative of the peripheral ASM in human asthma either, but this will require further investigation.

Adenosine transport systems on dissociated brain cells from mouse, guinea-pig, and rat

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

Johnston, M.E.; Geiger, J.D.

1990-09-01

The kinetics and sodium dependence of adenosine transport were determined using an inhibitor-stop method on dissociated cell body preparations obtained from mouse, guinea-pig and rat brain. Transport affinity (KT) values for the high affinity adenosine transport systems KT(H) were significantly different between these three species; mean +/- SEM values were 0.34 +/- 0.1 in mouse, 0.9 +/- 0.2 in rat, and 1.5 +/- 0.5 microM in guinea-pig. The KT values for the low affinity transport system KT(L) were not different between the three species. Brain cells from rat displayed a significantly greater maximal capacity to accumulate (3H)adenosine (Vmax) than didmore » mouse or guinea-pig for the high affinity system, or than did mouse for the low affinity system. When sodium chloride was replaced in the transport medium with choline chloride, the KT(H) values for guinea-pig and rat were both increased by approximately 100%; only in rat did the change reach statistical significance. The sodium-dependence of adenosine transport in mouse brain was clearly absent. The differences between KT(H) values in mouse and those in guinea-pig or rat were accentuated in the absence of sodium. The differences in kinetic values, ionic requirements, and pharmacological characteristics between adenosine transporters in CNS tissues of mouse, guinea-pig and rat may help account for some of the variability noted among species in terms of their physiological responses to adenosine.« less

Benzydamine N-oxidation as an index reaction reflecting FMO activity in human liver microsomes and impact of FMO3 polymorphisms on enzyme activity

PubMed Central

Störmer, Elke; Roots, Ivar; Brockmöller, Jürgen

2000-01-01

Aims The role of flavin containing monooxygenases (FMO) on the disposition of many drugs has been insufficiently explored. In vitro and in vivo tests are required to study FMO activity in humans. Benzydamine (BZD) N-oxidation was evaluated as an index reaction for FMO as was the impact of genetic polymorphisms of FMO3 on activity. Methods BZD was incubated with human liver microsomes (HLM) and recombinant enzymes. Human liver samples were genotyped using PCR-RFLP. Results BZD N-oxide formation rates in HLM followed Michaelis-Menten kinetics (mean Km = 64.0 μm, mean Vmax = 6.9 nmol mg−1 protein min−1; n = 35). N-benzylimidazole, a nonspecific CYP inhibitor, and various CYP isoform selective inhibitors did not affect BZD N-oxidation. In contrast, formation of BZD N-oxide was almost abolished by heat treatment of microsomes in the absence of NADPH and strongly inhibited by methimazole, a competitive FMO inhibitor. Recombinant FMO3 and FMO1 (which is not expressed in human liver), but not FMO5, showed BZD N-oxidase activity. Respective Km values for FMO3 and FMO1 were 40.4 μm and 23.6 μm, and respective Vmax values for FMO3 and FMO1 were 29.1 and 40.8 nmol mg−1 protein min−1. Human liver samples (n = 35) were analysed for six known FMO3 polymorphisms. The variants I66M, P135L and E305X were not detected. Samples homozygous for the K158 variant showed significantly reduced vmax values (median 2.7 nmol mg−1 protein min−1) compared to the carriers of at least one wild type allele (median 6.2 nmol mg−1 protein min−1) (P<0.05, Mann–Whitney- U-test). The V257M and E308G substitutions had no effect on enzyme activity. Conclusions BZD N-oxidation in human liver is mainly catalysed by FMO3 and enzyme activity is affected by FMO3 genotype. BZD may be used as a model substrate for human liver FMO3 activity in vitro and may be further developed as an in vivo probe reflecting FMO3 activity. PMID:11136294

A molecular dynamics study on the role of attractive and repulsive forces in internal energy, internal pressure and structure of dense fluids

NASA Astrophysics Data System (ADS)

Goharshadi, Elaheh K.; Morsali, Ali; Mansoori, G. Ali

2007-01-01

Isotherms of experimental data of internal pressure of dense fluids versus molar volume, Vm are shown to have each a maximum point at a Vmax below the critical molar volume. In this study, we investigated the role of attractive and repulsive intermolecular energies on this behavior using a molecular dynamics simulation technique. In the simulation, we choose the Lennard-Jones (LJ) intermolecular potential energy function. The LJ potential is known to be an effective potential representing a statistical average of the true pair and many-body interactions in simple molecular systems. The LJ potential function is divided into attractive and repulsive parts. MD calculations have produced internal energy, potential energy, transitional kinetic energy, and radial distribution function (RDF) for argon at 180 K and 450 K using LJ potential, LJ repulsive, and LJ attractive parts. It is shown that the LJ potential function is well capable of predicting the inflection point in the internal energy-molar volume curve as well as maximum point in the internal pressure-molar volume curve. It is also shown that at molar volumes higher than Vmax, the attractive forces have strong influence on determination of internal energy and internal pressure. At volumes lower than Vmax, neither repulsive nor attractive forces are dominating. Also, the coincidence between RDFs resulting from LJ potential and repulsive parts of LJ potential improves as molar volume approaches Vmax from high molar volumes. The coincidence becomes complete at Vmax ⩾ V.

In vitro metabolism of phenytoin in 36 CYP2C9 variants found in the Chinese population.

PubMed

Chen, Lian-Guo; Wang, Zhe; Zhu, Yuan; Xiong, Jian-Hua; Sun, Li-Rong; Dai, Da-Peng; Cai, Jian-Ping; Hu, Guo-Xin

2016-06-25

Cytochrome P450 2C9 (CYP2C9) is an important member of the cytochrome P450 enzyme superfamily, with 57 CYP2C9 allelic variants being previously reported. Recently, we identified 22 novel alleles (*36 -*56 and N418T) in the Han Chinese population. This study aims to assess the catalytic activities of wild-type (CYP2C9*1) and 36 CYP2C9 allelic variants found in the Chinese population toward phenytoin (PHT) in vitro. Insect microsomes expressing CYP2C9*1 and 36 CYP2C9 variants were incubated with 1-200 μM phenytoin for 30 min at 37 °C. Then, these products were extracted and the signal detection was performed by HPLC-MS/MS. The intrinsic clearance (Vmax/Km) values of all variants, with the exception of CYP2C9*2, CYP2C9*11, CYP2C9*23, CYP2C9*29, CYP2C9*34, CYP2C9*38, CYP2C9*44, CYP2C9*46 and CYP2C9*48, were significantly different from CYP2C9*1. CYP2C9*27, *40, *41, *47, *49, *51, *53, *54, *56 and N418T variant exhibited markedly larger values than CYP2C9*1 (>152.8%), whereas 17 variants exhibited smaller values (from 48.6% to 99.9%) due to larger Km and/or smaller Vmax values than CYP2C9*1. The findings suggest that more attention should be paid on subjects carrying these infrequent CYP2C9 alleles when administering phenytoin in clinic. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Screening and Characterization of Polygalacturonase as Potential Enzyme for Keprok Garut Orange (Citrus nobilis var. chrysocarpa) Juice Clarification

NASA Astrophysics Data System (ADS)

Widowati, E.; Utami, R.; Kalistyatika, K.

2017-11-01

Use of thermostable enzyme from bacilli for industrial application is significant. This research aimed to isolate thermophilic pectinolytic bacteria from orange peel and vegetable waste which produced thermostable polygalacturonase, to investigate the polygalacturonase ability in clarifying keprok Garut orange juice, and to characterize polygalacturonase based on pH optimum, temperature optimum, enzyme stability, enzyme kinetics KM, and Vmax. Obtained, 14 isolates that further selected to 4 best isolates based on highest polygalacturonase activity and keprok Garut orange juice clarification ability. Four selected enzyme isolates were AR 2, AR 4, KK 4, and KK 5 had ability to increase juice transmittance, decrease juice viscosity and also reduce total soluble solid. Furthermore 4 selected isolates were partially purified by ammonium sulphate precipitation and dialysis method. Four partially purified enzymes were known that enzyme character of AR 2 optimum at pH 6; AR 4 optimum at pH 5.5; KK 4 optimum at pH 6; and KK 5 optimum at pH 4.5. Four enzymes were optimum at temperature 60°C thus stable at temperature 50-60°C, this characteristic indicate that enzymes were thermostable. AR 2 showed active activity stable at pH 4-7; AR 4 showed active activity stable at pH 6-7; KK 4 showed active activity stable at pH 4-6; however KK 5 stable at pH 4-5. Enzyme AR 2 and KK 4 was getting inactive at pH 11, thus AR 4 and KK 5 inactive at pH 12. KM value of AR 2, AR 4, KK 4, and KK 5 was 0.0959; 0.0974; 0.0966; and 0.178 mg/ml respectively. Vmax of AR 2, AR 4, KK 4, and KK 5 was 0.0203; 0.0202; 0.0185; and 0.0229 U/ml respectively. Enzyme AR 2 was the most compatible enzyme to be applied in keprok Garut orange juice clarification for it had the lowest KM value.

Comparison of Oxidation Kinetics of Nitrite-Oxidizing Bacteria: Nitrite Availability as a Key Factor in Niche Differentiation

PubMed Central

Nowka, Boris; Daims, Holger

2014-01-01

Nitrification has an immense impact on nitrogen cycling in natural ecosystems and in wastewater treatment plants. Mathematical models function as tools to capture the complexity of these biological systems, but kinetic parameters especially of nitrite-oxidizing bacteria (NOB) are lacking because of a limited number of pure cultures until recently. In this study, we compared the nitrite oxidation kinetics of six pure cultures and one enrichment culture representing three genera of NOB (Nitrobacter, Nitrospira, Nitrotoga). With half-saturation constants (Km) between 9 and 27 μM nitrite, Nitrospira bacteria are adapted to live under significant substrate limitation. Nitrobacter showed a wide range of lower substrate affinities, with Km values between 49 and 544 μM nitrite. However, the advantage of Nitrobacter emerged under excess nitrite supply, sustaining high maximum specific activities (Vmax) of 64 to 164 μmol nitrite/mg protein/h, contrary to the lower activities of Nitrospira of 18 to 48 μmol nitrite/mg protein/h. The Vmax (26 μmol nitrite/mg protein/h) and Km (58 μM nitrite) of “Candidatus Nitrotoga arctica” measured at a low temperature of 17°C suggest that Nitrotoga can advantageously compete with other NOB, especially in cold habitats. The kinetic parameters determined represent improved basis values for nitrifying models and will support predictions of community structure and nitrification rates in natural and engineered ecosystems. PMID:25398863

An extended heterogeneous car-following model accounting for anticipation driving behavior and mixed maximum speeds

NASA Astrophysics Data System (ADS)

Sun, Fengxin; Wang, Jufeng; Cheng, Rongjun; Ge, Hongxia

2018-02-01

The optimal driving speeds of the different vehicles may be different for the same headway. In the optimal velocity function of the optimal velocity (OV) model, the maximum speed vmax is an important parameter determining the optimal driving speed. A vehicle with higher maximum speed is more willing to drive faster than that with lower maximum speed in similar situation. By incorporating the anticipation driving behavior of relative velocity and mixed maximum speeds of different percentages into optimal velocity function, an extended heterogeneous car-following model is presented in this paper. The analytical linear stable condition for this extended heterogeneous traffic model is obtained by using linear stability theory. Numerical simulations are carried out to explore the complex phenomenon resulted from the cooperation between anticipation driving behavior and heterogeneous maximum speeds in the optimal velocity function. The analytical and numerical results all demonstrate that strengthening driver's anticipation effect can improve the stability of heterogeneous traffic flow, and increasing the lowest value in the mixed maximum speeds will result in more instability, but increasing the value or proportion of the part already having higher maximum speed will cause different stabilities at high or low traffic densities.

Production, isolation, and purification of L-asparaginase from Pseudomonas aeruginosa 50071 using solid-state fermentation.

PubMed

El-Bessoumy, Ashraf A; Sarhan, Mohamed; Mansour, Jehan

2004-07-31

The L-asparaginase (E. C. 3. 5. 1. 1) enzyme was purified to homogeneity from Pseudomonas aeruginosa 50071 cells that were grown on solid-state fermentation. Different purification steps (including ammonium sulfate fractionation followed by separation on Sephadex G-100 gel filtration and CM-Sephadex C50) were applied to the crude culture filtrate to obtain a pure enzyme preparation. The enzyme was purified 106-fold and showed a final specific activity of 1900 IU/mg with a 43% yield. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of the purified enzyme revealed it was one peptide chain with M(r) of 160 kDa. A Lineweaver-Burk analysis showed a K(m) value of 0.147 mM and V(max) of 35.7 IU. The enzyme showed maximum activity at pH 9 when incubated at 37 degrees C for 30 min. The amino acid composition of the purified enzyme was also determined.

Enhanced cellulase producing mutants developed from heterokaryotic Aspergillus strain.

PubMed

Kaur, Baljit; Oberoi, H S; Chadha, B S

2014-03-01

A heterokaryon 28, derived through protoplast fusion between Aspergillus nidulans and Aspergillus tubingensis (Dal8), was subjected cyclic mutagenesis followed by selection on increasing levels of 2-deoxy glucose (2-DG) as selection marker. The derived deregulated cellulase hyper producing mutant '64', when compared to fusant 28, produced 9.83, 7.8, 3.2, 4.2 and 19.74 folds higher endoglucanase, β-glucosidase, cellobiohydrolase, FPase and xylanase, respectively, under shake cultures. The sequence analysis of PCR amplified β-glucosidase gene from wild and mutant showed nucleotide deletion/substitution. The mutants showed highly catalytic efficient β-glucosidase as evident from low Km and high Vmax values. The expression profiling through zymogram analysis also indicated towards over-expression of cellulases. The up/down regulated expressed proteins observed through SDS-PAGE were identified by Peptide mass fingerprinting The cellulase produced by mutants in conjunction with cellulase free xylanase derived from Thermomyces lanuginosus was used for efficient utilization of alkali treated rice straw for obtaining xylo-oligosaccharides and ethanol. Copyright © 2014 Elsevier Ltd. All rights reserved.

Responses of soil hydrolase kinetics to nitrogen and phosphorus additions in Chinese fir plantations of subtropical China

NASA Astrophysics Data System (ADS)

Zhang, X.; Zhang, C.; Yang, Y.; Wang, H.; Chen, F.; Fu, X.; Fang, X.; Sun, X.

2016-12-01

Nitrogen (N) deposition and low soil phosphorus (P) content aggravate the P limitation in subtropical forest soils. However, the responses of soil organic matter related hydrolyase kinetics to N and P additions in subtropical plantations are still not clear. We tested the hypothesis that P application can improve the potential maximum activities of soil carbon (C) and N related hydrolayase but substrate demand (Km) may tradeoff the catalytic efficiency of the enzymes. Thirty 20m×20m plots were established in November 2011 and six different treatments were randomly distributed with five replicates in the Chinese fir plantations in subtropical China. The ongoing treatments are control (CK, no N and P application), low N addition (N1:50 kg N ha-1 yr-1), high N addition (N2: 100 kg N ha-1 yr-1), P addition (P: 50 kg P ha-1 yr-1), low N andP addition (N1P: 50 kg N ha-1 yr-1 and 50 kg P ha-1 yr-1) and high N and P addition (N2P: 100 kg N ha-1yr-1and 50 kg P ha-1 yr-1). Soil enzyme kinetic parameters for b-1,4-glucosidase (βG), β-1,4-N-acetylglucosaminidase (NAG), and acid phosphatase (aP) were measured in November 2015. The substrate affinities (Km) of βG and NAG were not affected by N or /and P additions. However, the substrate affinities of aP were decreased by N additions (N1, N2) with higher Km values than the other treatments. N additions (N1, N2) or higher N combined P additions (N2P) increased Vmax and catalytic efficiencies for βG, while with P addition treatments (N1P, N2P, and P) decreased Vmax and catalytic efficiencies for aP. The effects of N combined P treatments (N1P and N2P) on kinetic parameters (Vmax, Km) and catalytic efficiencies for AP were similar to P treatment, indicating that P had stronger effects on organic phosphorus hydrolysis than N in the research site. The N additions (N1 and N2) did not affect the catalytic efficiencies for NAG despite of their positive responses to Vmax for NAG compared with CK. The catalytic efficiencies of aP and NAG were negatively correlated with soil TP and available P contents, and both the enzyme kinetics for aP exhibited strong negative correlations with TP and available P contents. However, the Vmax for BG and NAG were positively correlated with SOC contents, but were negatively correlated with soil pH.

Accurate mass and velocity functions of dark matter haloes

NASA Astrophysics Data System (ADS)

Comparat, Johan; Prada, Francisco; Yepes, Gustavo; Klypin, Anatoly

2017-08-01

N-body cosmological simulations are an essential tool to understand the observed distribution of galaxies. We use the MultiDark simulation suite, run with the Planck cosmological parameters, to revisit the mass and velocity functions. At redshift z = 0, the simulations cover four orders of magnitude in halo mass from ˜1011M⊙ with 8783 874 distinct haloes and 532 533 subhaloes. The total volume used is ˜515 Gpc3, more than eight times larger than in previous studies. We measure and model the halo mass function, its covariance matrix w.r.t halo mass and the large-scale halo bias. With the formalism of the excursion-set mass function, we explicit the tight interconnection between the covariance matrix, bias and halo mass function. We obtain a very accurate (<2 per cent level) model of the distinct halo mass function. We also model the subhalo mass function and its relation to the distinct halo mass function. The set of models obtained provides a complete and precise framework for the description of haloes in the concordance Planck cosmology. Finally, we provide precise analytical fits of the Vmax maximum velocity function up to redshift z < 2.3 to push for the development of halo occupation distribution using Vmax. The data and the analysis code are made publicly available in the Skies and Universes data base.

Properties and mutation studies of a bacteriophage-derived chimeric recombinant staphylolytic protein P128

PubMed Central

Saravanan, Sanjeev Rajagopalan; Paul, Vivek Daniel; George, Shilpa; Sundarrajan, Sudarson; Kumar, Nirmal; Hebbur, Madhavi; Kumar, Naveen; Veena, Ananda; Maheshwari, Uma; Appaiah, Chemira Biddappa; Chidambaran, Muralidharan; Bhat, Anuradha Gopal; Hariharan, Sukumar; Padmanabhan, Sriram

2013-01-01

P128 is a chimeric anti-staphylococcal protein having a catalytic domain from a Staphylococcus bacteriophage K tail associated structural protein and a cell wall targeting domain from the Staphylococcus bacteriocin-lysostaphin. In this study, we disclose additional properties of P128 and compared the same with lysostaphin. While lysostaphin was found to get inactivated by heat and was inactive on its parent strain S. simulans biovar staphylolyticus, P128 was thermostable and was lytic towards S. simulans biovar staphylolyticus demonstrating a difference in their mechanism of action. Selected mutation studies of the catalytic domain of P128 showed that arginine and cysteine, at 40th and 76th positions respectively, are critical for the staphylolytic activity of P128, although these amino acids are not conserved residues. In comparison to native P128, only the R40S mutant (P301) was catalytically active on zymogram gel and had a similar secondary structure, as assessed by circular dichroism analysis and in silico modeling with similar cell binding properties. Mutation of the arginine residue at 40th position of the P128 molecule caused dramatic reduction in the Vmax (∆OD600 [mg/min]) value (nearly 270 fold) and the recombinant lysostaphin also showed lesser Vmax value (nearly 1.5 fold) in comparison to the unmodified P128 protein. The kinetic parameters such as apparent Km (Km APP) and apparent Kcat (KcatAPP) of the native P128 protein also showed significant differences in comparison to the values observed for P301 and lysostaphin. PMID:24251076

Determinant Factors of the Squat Jump in Sprinting and Jumping Athletes

PubMed Central

González-Badillo, Juan José; Jiménez-Reyes, Pedro; Ramírez-Lechuga, Jorge

2017-01-01

Abstract The aim of this study was to assess the relationship between strength variables and maximum velocity (Vmax) in the squat jump (SJ) in sprinting and jumping athletes. Thirty-two sprinting and jumping athletes of national level (25.4 ± 4.5 years; 79.4 ± 6.9 kg and 180.4 ± 6.0 cm) participated in the study. Vmax in the SJ showed significant relationships with peak force 1 (PF1) (r = 0.82, p ≤ 0.001), peak force 2 (PF2) (r = 0.68, p ≤ 0.001), PF2 by controlling for PF1 (r = 0.30, non-significant), the maximum rate of force development at peak force 1 (RFDmax1) (r = 0.62, p ≤ 0.001), mean RFD 1 (RFDmean1) (r = 0.48, p ≤ 0.01), mean RFD 2 (RFDmean2) (r = 0.70, p ≤ 0.001), force at RFDmax1 (r = 0.36, p ≤ 0.05), force at RFDmax2 (r = 0.83, p ≤ 0.001) and force at RFDmax2 by controlling for PF1 (r = 0.40, p ≤ 0.05). However, Vmax in the SJ was associated negatively with the ratio PF2/PF1 (r = -0.54, p ≤ 0.01), time at peak force 2 (Tp2) (r = -0.64, p ≤ 0.001) and maximum rate of force development at peak force 2 (RFDmax2) (r = -0.71, p ≤ 0.001). These findings indicate that the peak force achieved at the beginning of the movement (PF1) is the main predictor of performance in jumping, although the RFDmax values and the ratio PF2/PF1 are also variables to be taken into account when analyzing the determinant factors of vertical jumping. PMID:28828074

Substrate preferences and catalytic parameters determined by structural characteristics of sterol 14alpha-demethylase (CYP51) from Leishmania infantum.

PubMed

Hargrove, Tatiana Y; Wawrzak, Zdzislaw; Liu, Jialin; Nes, W David; Waterman, Michael R; Lepesheva, Galina I

2011-07-29

Leishmaniasis is a major health problem that affects populations of ∼90 countries worldwide, with no vaccine and only a few moderately effective drugs. Here we report the structure/function characterization of sterol 14α-demethylase (CYP51) from Leishmania infantum. The enzyme catalyzes removal of the 14α-methyl group from sterol precursors. The reaction is essential for membrane biogenesis and therefore has great potential to become a target for antileishmanial chemotherapy. Although L. infantum CYP51 prefers C4-monomethylated sterol substrates such as C4-norlanosterol and obtusifoliol (V(max) of ∼10 and 8 min(-1), respectively), it is also found to 14α-demethylate C4-dimethylated lanosterol (V(max) = 0.9 min(-1)) and C4-desmethylated 14α-methylzymosterol (V(max) = 1.9 min(-1)). Binding parameters with six sterols were tested, with K(d) values ranging from 0.25 to 1.4 μM. Thus, L. infantum CYP51 is the first example of a plant-like sterol 14α-demethylase, where requirements toward the composition of the C4 atom substituents are not strict, indicative of possible branching in the postsqualene portion of sterol biosynthesis in the parasite. Comparative analysis of three CYP51 substrate binding cavities (Trypanosoma brucei, Trypanosoma cruzi, and L. infantum) suggests that substrate preferences of plant- and fungal-like protozoan CYP51s largely depend on the differences in the enzyme active site topology. These minor structural differences are also likely to underlie CYP51 catalytic rates and drug susceptibility and can be used to design potent and specific inhibitors.

Incorporating Love- and Rayleigh-wave magnitudes, unequal earthquake and explosion variance assumptions and interstation complexity for improved event screening

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

Anderson, Dale N; Bonner, Jessie L; Stroujkova, Anastasia

Our objective is to improve seismic event screening using the properties of surface waves, We are accomplishing this through (1) the development of a Love-wave magnitude formula that is complementary to the Russell (2006) formula for Rayleigh waves and (2) quantifying differences in complexities and magnitude variances for earthquake and explosion-generated surface waves. We have applied the M{sub s} (VMAX) analysis (Bonner et al., 2006) using both Love and Rayleigh waves to events in the Middle East and Korean Peninsula, For the Middle East dataset consisting of approximately 100 events, the Love M{sub s} (VMAX) is greater than the Rayleighmore » M{sub s} (VMAX) estimated for individual stations for the majority of the events and azimuths, with the exception of the measurements for the smaller events from European stations to the northeast. It is unclear whether these smaller events suffer from magnitude bias for the Love waves or whether the paths, which include the Caspian and Mediterranean, have variable attenuation for Love and Rayleigh waves. For the Korean Peninsula, we have estimated Rayleigh- and Love-wave magnitudes for 31 earthquakes and two nuclear explosions, including the 25 May 2009 event. For 25 of the earthquakes, the network-averaged Love-wave magnitude is larger than the Rayleigh-wave estimate. For the 2009 nuclear explosion, the Love-wave M{sub s} (VMAX) was 3.1 while the Rayleigh-wave magnitude was 3.6. We are also utilizing the potential of observed variances in M{sub s} estimates that differ significantly in earthquake and explosion populations. We have considered two possible methods for incorporating unequal variances into the discrimination problem and compared the performance of various approaches on a population of 73 western United States earthquakes and 131 Nevada Test Site explosions. The approach proposes replacing the M{sub s} component by M{sub s} + a* {sigma}, where {sigma} denotes the interstation standard deviation obtained from the stations in the sample that produced the M{sub s} value. We replace the usual linear discriminant a* M{sub s}+b*{sub m{sub b}} with a* M{sub s}+b*{sub m{sub b}} + C*{sigma}. In the second approach, we estimate the optimum hybrid linear-quadratic discriminant function resulting from the unequal variance assumption. We observed slight improvement for the discriminant functions resulting from the theoretical interpretations of the unequal variance function. We have also studied the complexity of the ''magnitude spectra'' at each station. Our hypothesis is that explosion spectra should have fewer focal mechanism-produced complexities in the magnitude spectra than earthquakes. We have developed an intrastation ''complexity'' metric {Delta}M{sub s}, where {Delta}M{sub s} = M{sub s}(i)-M{sub s}(i+1) at periods, i, which are between 9 and 25 seconds. The complexity by itself has discriminating power but does not add substantially to the conditional hybrid discriminant that incorporates the differing spreads of the earthquake and explosion standard deviations.« less

Addressing the too big to fail problem with baryon physics and sterile neutrino dark matter

NASA Astrophysics Data System (ADS)

Lovell, Mark R.; Gonzalez-Perez, Violeta; Bose, Sownak; Boyarsky, Alexey; Cole, Shaun; Frenk, Carlos S.; Ruchayskiy, Oleg

2017-07-01

N-body dark matter simulations of structure formation in the Λ cold dark matter (ΛCDM) model predict a population of subhaloes within Galactic haloes that have higher central densities than inferred for the Milky Way satellites, a tension known as the 'too big to fail' problem. Proposed solutions include baryonic effects, a smaller mass for the Milky Way halo and warm dark matter (WDM). We test these possibilities using a semi-analytic model of galaxy formation to generate luminosity functions for Milky Way halo-analogue satellite populations, the results of which are then coupled to the Jiang & van den Bosch model of subhalo stripping to predict the subhalo Vmax functions for the 10 brightest satellites. We find that selecting the brightest satellites (as opposed to the most massive) and modelling the expulsion of gas by supernovae at early times increases the likelihood of generating the observed Milky Way satellite Vmax function. The preferred halo mass is 6 × 1011 M⊙, which has a 14 per cent probability to host a Vmax function like that of the Milky Way satellites. We conclude that the Milky Way satellite Vmax function is compatible with a CDM cosmology, as previously found by Sawala et al. using hydrodynamic simulations. Sterile neutrino-WDM models achieve a higher degree of agreement with the observations, with a maximum 50 per cent chance of generating the observed Milky Way satellite Vmax function. However, more work is required to check that the semi-analytic stripping model is calibrated correctly for each sterile neutrino cosmology.

A spectral model for signal elements isolated from zebrafish photopic electroretinogram

PubMed Central

Nelson, Ralph; Singla, Nirmish

2009-01-01

The zebrafish photopic ERG sums isolatable elements. In each element red, blue, green and UV (r, g, b, u) cone signals combine in a way that reflects retinal organization. ERG responses to monochromatic stimuli of different wavelengths and irradiances were recorded on a white, rod suppressing background using superfused eyecups. Onset elements were isolated with glutamatergic blockers and response subtractions. CNQX blocked ionotropic (AMPA/kainate) glutamate receptors; L-AP4 or CPPG blocked metabotropic (mGluR6) glutamate receptors; TBOA blocked glutamate transporters; and L-Aspartate inactivated all glutamatergic mechanisms. Seven elements emerged: photopic PIII, the L-Aspartate-isolated cone response; b1, a CNQX-sensitive early b-wave element of inner retinal origin; PII, a photopic, CNQX-insensitive, composite b-wave element from ON bipolar cells; PIIm, an L-AP4/CPPG-sensitive, CNQX-insensitive metabotropic sub-element of PII; PIInm, an L-AP4/CPPG/CNQX-insensitive, non-metabotropic sub-element of PII; a1nm, a TBOA-sensitive, CNQX/L-AP4/CPPG-insensitive, non-metabotropic, post-photoreceptor a-wave element; and a2, a CNQX-sensitive a-wave element linked to OFF bipolar cells. The first five elements were fit with a spectral model that demonstrates independence of cone color pathways. From this Vmax and half-saturation values (k) for the contributing r- g- b- and u-cone signals were calculated. Two signal patterns emerged. For PIII or PIInm the Vmax order was Vr > Vg ≫ Vb ≈ Vu. For b1, PII, and PIIm the Vmax order was Vr ≈ Vb > Vg > Vu. In either pattern u-cone amplitude (Vu) was smallest, but u-cone sensitivity (ku362) was greatest, some 10-30 times greater than r-cone (kr570). The spectra of b1/PII/PIIm elements peaked near b-cone and u-cone absorbance maxima regardless of criteria, but the spectra of PIII/PIInm elements shifted from b- towards r-cone absorbance maxima as criterion levels increased. The greatest gains in Vmax relative to PIII occurred for the b- and u-cone signals in the b1/PII/PIIm b-wave elements. This suggests a high-gain, prolific metabotropic circuitry for b- and u-cone bipolar cells. PMID:19723365

Dependence of renal (Na+ + k+)-adenosine triphosphatase activity on thyroid status.

PubMed

Lo, S C; August, T R; Liberman, U A; Edelman, I S

1976-12-25

In thyroidectomized rats, a single injection of L-2,,5,2'-triiodothyronine (T3) (50mug/100 g body weight) elicited at 45% increase in (Na+ + k+)-dependent adenosine triphosphatase (NaK-ATPase) activity of the membrane-rich fraction of renal cortex at the optimal time of response, 48 h after injection. Three successive doses of T3 (50 mug/100 g body weight), given on alternate days, increased NaK-ATPase by 67% in the renal cortex but had no significant effect on the outer medulla or the papilla. Moreover, T3 had no effect on Mg2+-dependent adenosine trisphatase (MgATPase) in cortex, cedulla, or papilla. Three doses of T3 (50 mug/100 g body weight) given on alternate days to thyroidectomized rats elecited a 134, 79, and 46% increase in Vmax for ATP, Na4, and K+, respectively. There were no changes in the Km for ATP or the K1/2 values for Na+ and K+. Two methods were used to estimate the effect of T3 on the number of NaK-ATPase units (assumed to represent the number of Na+ pump sites); rat renal plasma membrane fractions were incubated with [gamma-32P]ATP, Mg2+, and Na+; the 32P-labeled membrane protein yeild was quantitatively dependent on Na+ and was hydrolyzed on addition of K+. There was a linear correlation between the specific activity of NaK-ATPase (Vmax) and the amount of phosphorylated intermediate formed, in renal cortical membrane fractions from thyroidectomized rats given T3 or the diluent. There was also a linear correlation between the specific activity of NaK-ATPase (Vmax) and the amount of [3H]ouabain specifically bound (Na+-, Mg2+-, APT-dependent) to the NaK-ATPase preparation. Injection of T3 resulted in a 70% increase in NaK-ATPase activity, a 79% increase in formation of the phosphorylated intermediate, and a 65% increase in the [H]ouabain specifically bound to the NaK-ATPase system. The T3-dependent increases in Vmax for ATP, Na+, and K+ and the proportionate increases in the phosphorylated intermediate and in the amount of [3H]ouabain bound indicate that T3 increases the number of NaK-ATPase units and that this increase accounts for the increase in NaK-ATPase activity.

Actinobacterial Nitrate Reducers and Proteobacterial Denitrifiers Are Abundant in N2O-Metabolizing Palsa Peat

PubMed Central

Palmer, Katharina

2012-01-01

Palsa peats are characterized by elevated, circular frost heaves (peat soil on top of a permanently frozen ice lens) and are strong to moderate sources or even temporary sinks for the greenhouse gas nitrous oxide (N2O). Palsa peats are predicted to react sensitively to global warming. The acidic palsa peat Skalluvaara (approximate pH 4.4) is located in the discontinuous permafrost zone in northwestern Finnish Lapland. In situ N2O fluxes were spatially variable, ranging from 0.01 to −0.02 μmol of N2O m−2 h−1. Fertilization with nitrate stimulated in situ N2O emissions and N2O production in anoxic microcosms without apparent delay. N2O was subsequently consumed in microcosms. Maximal reaction velocities (vmax) of nitrate-dependent denitrification approximated 3 and 1 nmol of N2O per h per gram (dry weight [gDW]) in soil from 0 to 20 cm and below 20 cm of depth, respectively. vmax values of nitrite-dependent denitrification were 2- to 5-fold higher than the vmax nitrate-dependent denitrification, and vmax of N2O consumption was 1- to 6-fold higher than that of nitrite-dependent denitrification, highlighting a high N2O consumption potential. Up to 12 species-level operational taxonomic units (OTUs) of narG, nirK and nirS, and nosZ were retrieved. Detected OTUs suggested the presence of diverse uncultured soil denitrifiers and dissimilatory nitrate reducers, hitherto undetected species, as well as Actino-, Alpha-, and Betaproteobacteria. Copy numbers of nirS always outnumbered those of nirK by 2 orders of magnitude. Copy numbers of nirS tended to be higher, while copy numbers of narG and nosZ tended to be lower in 0- to 20-cm soil than in soil below 20 cm. The collective data suggest that (i) the source and sink functions of palsa peat soils for N2O are associated with denitrification, (ii) actinobacterial nitrate reducers and nirS-type and nosZ-harboring proteobacterial denitrifiers are important players, and (iii) acidic soils like palsa peats represent reservoirs of diverse acid-tolerant denitrifiers associated with N2O fluxes. PMID:22660709

Novel transporters from Kluyveromyces marxianus and Pichia guilliermondii expressed in Saccharomyces cerevisiae enable growth on L-arabinose and D-xylose.

PubMed

Knoshaug, Eric P; Vidgren, Virve; Magalhães, Frederico; Jarvis, Eric E; Franden, Mary Ann; Zhang, Min; Singh, Arjun

2015-10-01

Genes encoding L-arabinose transporters in Kluyveromyces marxianus and Pichia guilliermondii were identified by functional complementation of Saccharomyces cerevisiae whose growth on L-arabinose was dependent on a functioning L-arabinose transporter, or by screening a differential display library, respectively. These transporters also transport D-xylose and were designated KmAXT1 (arabinose-xylose transporter) and PgAXT1, respectively. Transport assays using L-arabinose showed that KmAxt1p has K(m) 263 mM and V(max) 57 nM/mg/min, and PgAxt1p has K(m) 0.13 mM and V(max) 18 nM/mg/min. Glucose, galactose and xylose significantly inhibit L-arabinose transport by both transporters. Transport assays using D-xylose showed that KmAxt1p has K(m) 27 mM and V(max) 3.8 nM/mg/min, and PgAxt1p has K(m) 65 mM and V(max) 8.7 nM/mg/min. Neither transporter is capable of recovering growth on glucose or galactose in a S. cerevisiae strain deleted for hexose and galactose transporters. Transport kinetics of S. cerevisiae Gal2p showed K(m) 371 mM and V(max) 341 nM/mg/min for L-arabinose, and K(m) 25 mM and V(max) 76 nM/mg/min for galactose. Due to the ability of Gal2p and these two newly characterized transporters to transport both L-arabinose and D-xylose, one scenario for the complete usage of biomass-derived pentose sugars would require only the low-affinity, high-throughput transporter Gal2p and one additional high-affinity general pentose transporter, rather than dedicated D-xylose or L-arabinose transporters. Additionally, alignment of these transporters with other characterized pentose transporters provides potential targets for substrate recognition engineering. Copyright © 2015 John Wiley & Sons, Ltd.

Low-Concentration Kinetics of Atmospheric CH4 Oxidation in Soil and Mechanism of NH4+ Inhibition

PubMed Central

Gulledge, Jay; Schimel, Joshua P.

1998-01-01

NH4+ inhibition kinetics for CH4 oxidation were examined at near-atmospheric CH4 concentrations in three upland forest soils. Whether NH4+-independent salt effects could be neutralized by adding nonammoniacal salts to control samples in lieu of deionized water was also investigated. Because the levels of exchangeable endogenous NH4+ were very low in the three soils, desorption of endogenous NH4+ was not a significant factor in this study. The Km(app) values for water-treated controls were 9.8, 22, and 57 nM for temperate pine, temperate hardwood, and birch taiga soils, respectively. At CH4 concentrations of ≤15 μl liter−1, oxidation followed first-order kinetics in the fine-textured taiga soil, whereas the coarse-textured temperate soils exhibited Michaelis-Menten kinetics. Compared to water controls, the Km(app) values in the temperate soils increased in the presence of NH4+ salts, whereas the Vmax(app) values decreased substantially, indicating that there was a mixture of competitive and noncompetitive inhibition mechanisms for whole NH4+ salts. Compared to the corresponding K+ salt controls, the Km(app) values for NH4+ salts increased substantially, whereas the Vmax(app) values remained virtually unchanged, indicating that NH4+ acted by competitive inhibition. Nonammoniacal salts caused inhibition to increase with increasing CH4 concentrations in all three soils. In the birch taiga soil, this trend occurred with both NH4+ and K+ salts, and the slope of the increase was not affected by the addition of NH4+. Hence, the increase in inhibition resulted from an NH4+-independent mechanism. These results show that NH4+ inhibition of atmospheric CH4 oxidation resulted from enzymatic substrate competition and that additional inhibition that was not competitive resulted from a general salt effect that was independent of NH4+. PMID:9797279

Examining the effect of galaxy evolution on the stellar-halo mass relation in the EAGLE simulation

NASA Astrophysics Data System (ADS)

Kulier, Andrea; Padilla, Nelson; Schaye, Joop; Crain, Robert; Schaller, Matthieu; Bower, Richard; Theuns, Tom; Paillas, Enrique

2018-01-01

The EAGLE hydrodynamical simulation was used in Matthee et al. 2016 to examine the scatter in the stellar mass-halo mass relation of central galaxies, finding that the stellar mass (M*) correlates well with the maximum circular velocity (Vmax) of the host halo, but with a substantial scatter that does not correlate significantly with other host halo properties. Here we further examine the scatter in the stellar mass-halo mass relation of central galaxies in EAGLE, its correlation with other properties, and its origin. We find that at fixed Vmax, galaxies with lower concentration have younger stellar populations, as expected from the relationship between concentration and halo assembly time. However, at fixed Vmax and halo concentration, galaxies with larger M* have younger stellar ages, so that combining the two effects, galaxies with younger stellar ages at fixed halo mass have higher stellar masses. The host halos of galaxies with larger M* at fixed Vmax and concentration also contain more gas than those with smaller stellar masses at z = 0.1, i.e. the baryon fraction of the halos is larger. There is an even stronger correlation between the scatter in M* at z = 0.1 and the scatter in the baryon fraction of the galaxy's progenitors at z ~ 1, such that the latter sets ~50% of the scatter in M* at z = 0.1. We conclude that most of the scatter between Vmax and M* at z = 0.1 is set at earlier redshifts by the scatter in the baryon fraction of halos, which in turn is primarily the result of differences in feedback strength within halos.

Lethal effect of electric fields on isolated ventricular myocytes.

PubMed

de Oliveira, Pedro Xavier; Bassani, Rosana Almada; Bassani, José Wilson Magalhães

2008-11-01

Defibrillator-type shocks may cause electric and contractile dysfunction. In this study, we determined the relationship between probability of lethal injury and electric field intensity (E in isolated rat ventricular myocytes, with emphasis on field orientation and stimulus waveform. This relationship was sigmoidal with irreversible injury for E > 50 V/cm . During both threshold and lethal stimulation, cells were twofold more sensitive to the field when it was applied longitudinally (versus transversally) to the cell major axis. For a given E, the estimated maximum variation of transmembrane potential (Delta V(max)) was greater for longitudinal stimuli, which might account for the greater sensitivity to the field. Cell death, however, occurred at lower maximum Delta V(max) values for transversal shocks. This might be explained by a less steep spatial decay of transmembrane potential predicted for transversal stimulation, which would possibly result in occurrence of electroporation in a larger membrane area. For the same stimulus duration, cells were less sensitive to field-induced injury when shocks were biphasic (versus monophasic). Ours results indicate that, although significant myocyte death may occur in the E range expected during clinical defibrillation, biphasic shocks are less likely to produce irreversible cell injury.

Calcium pump kinetics determined in single erythrocyte ghosts by microphotolysis and confocal imaging.

PubMed

Kubitscheck, U; Pratsch, L; Passow, H; Peters, R

1995-07-01

The activity of the plasma membrane calcium pump was measured in single cells. Human red blood cell ghosts were loaded with a fluorescent calcium indicator and either caged calcium and ATP (protocol A) or caged ATP and calcium (protocol B). In a suitably modified laser scanning microscope either calcium or ATP were released by a short UV light pulse. The time-dependent fluorescence intensity of the calcium indicator was then followed in single ghosts by repetitive confocal imaging. The fluorescence intensity was converted into calcium concentration, which in turn was used to derive the kinetic parameters of the calcium pump, the Michaelis-Menten constant Km, and the maximal transport rate vmax. Km and vmax values derived in this manner were 24 +/- 14 microM and 1.0 +/- 0.6 microM/(ghost s) for protocol A, and 4 +/- 3 microM and 1.0 +/- 0.6 microM/(ghost s) for protocol B, respectively. The difference between A and B is presumably caused by calmodulin, which is inactive in the experiments with protocol A. The possibilities to extend the new method to living nucleus-containing cells transiently transfected with mutants of the plasma membrane calcium pump are discussed.

The first evidence of cholinesterases in skin mucus of carps and its applicability as biomarker of organophosphate exposure.

PubMed

Nigam, Ashwini Kumar; Srivastava, Nidhi; Rai, Amita Kumari; Kumari, Usha; Mittal, Ajay Kumar; Mittal, Swati

2014-05-01

The presence of cholinesterase (ChE) activity in skin mucus of three carps, Cirrhinus mrigala, Labeo rohita, and Catla catla and its applicability as biomarker of the organophosphorus insecticide exposure were investigated. Biochemical characterization, using specific substrates and inhibitors, indicated that measured esterase activity in skin mucus was mainly owing to ChEs. Significant difference in the proportion of acetylcholinesterase and butyrylcholinesterase activities was observed in skin mucus of three carps. Enzyme kinetic analysis, using the substrate acetylthiocholine iodide revealed significantly high Vmax value in C. catla compared to that in L. rohita and C. mrigala. In contrast, Vmax value using the substrate butyrylthiocholine iodide was significantly high in C. mrigala than in L. rohita and C. catla. In vitro treatment of skin mucus of three carps, with the organophosphorus insecticide Nuvan®, showed strong inhibition of ChE activities. In vivo experiments conducted using C. mrigala and exposing the fish to the sublethal test concentrations (5 and 15 mg/L) of the insecticide also revealed significant inhibition of ChE activity in mucus. In C. mrigala, exposed to the sublethal test concentrations of the insecticide for 4 days and then kept for recovery for 16 days, mucus ChE activity recovered to the control level. Thus, ChE activity in skin mucus could be considered a good biomarker of the organophosphorus insecticide exposure to fish and a useful tool in monitoring environmental toxicity. Copyright © 2012 Wiley Periodicals, Inc., a Wiley company.

Competition between roots and microorganisms for nitrogen: mechanisms and ecological relevance

NASA Astrophysics Data System (ADS)

Kuzyakov, Yakov; Xu, Xingliang

2014-05-01

Demand of all living organisms on the same nutrients forms the basis for interspecific competition between plants and microorganisms in soils. This competition is especially strong in the rhizosphere. To evaluate competitive and mutualistic interactions between plants and microorganisms and to analyse ecological consequences of these interactions, we analysed 424 data pairs from 41 15N-labelling studies that investigated 15N redistribution between roots and microorganisms. Calculated Michaelis-Menten kinetics based on Km (Michaelis constant) and Vmax (maximum uptake capacity) values from 77 studies on the uptake of nitrate, ammonia, and amino acids by roots and microorganisms clearly showed that, shortly after nitrogen (N) mobilization from soil organic matter and litter, microorganisms take up most N. Lower Km values of microorganisms suggest that they are especially efficient at low N concentrations, but can also acquire more N at higher N concentrations (Vmax) compared with roots. Because of the unidirectional flow of nutrients from soil to roots, plants are the winners for N acquisition in the long run. Therefore, despite strong competition between roots and microorganisms for N, a temporal niche differentiation reflecting their generation times leads to mutualistic relationships in the rhizosphere. This temporal niche differentiation is highly relevant ecologically because it: protects ecosystems from N losses by leaching during periods of slow or no root uptake; continuously provides roots with available N according to plant demand; and contributes to the evolutionary development of mutualistic interactions between roots and microorganisms.

Identification, purification and characterization of furfural transforming enzymes from Clostridium beijerinckii NCIMB 8052.

PubMed

Zhang, Yan; Ujor, Victor; Wick, Macdonald; Ezeji, Thaddeus Chukwuemeka

2015-06-01

Generation of microbial inhibitory compounds such as furfural and 5-hydroxymethylfurfural (HMF) is a formidable roadblock to fermentation of lignocellulose-derived sugars to butanol. Bioabatement offers a cost effective strategy to circumvent this challenge. Although Clostridium beijerinckii NCIMB 8052 can transform 2-3 g/L of furfural and HMF to their less toxic alcohols, higher concentrations present in biomass hydrolysates are intractable to microbial transformation. To delineate the mechanism by which C. beijerinckii detoxifies furfural and HMF, an aldo/keto reductase (AKR) and a short-chain dehydrogenase/reductase (SDR) found to be over-expressed in furfural-challenged cultures of C. beijerinckii were cloned and over-expressed in Escherichia coli Rosetta-gami™ B(DE3)pLysS, and purified by histidine tag-assisted immobilized metal affinity chromatography. Protein gel analysis showed that the molecular weights of purified AKR and SDR are close to the predicted values of 37 kDa and 27 kDa, respectively. While AKR has apparent Km and Vmax values of 32.4 mM and 254.2 mM s(-1) respectively, using furfural as substrate, SDR showed lower Km (26.4 mM) and Vmax (22.6 mM s(-1)) values on the same substrate. However, AKR showed 7.1-fold higher specific activity on furfural than SDR. Further, both AKR and SDR were found to be active on HMF, benzaldehyde, and butyraldehyde. Both enzymes require NADPH as a cofactor for aldehydes reduction. Based on these results, it is proposed that AKR and SDR are involved in the biotransformation of furfural and HMF by C. beijerinckii. Copyright © 2015 Elsevier Ltd. All rights reserved.

Transport mechanism of L-[14C]glutamate in cortical slices and synaptosomes of rabbits exposed to brain ischemia and reperfusion.

PubMed

Solyakov, L; Dobrota, D; Drany, O; Vachova, M; Machac, S; Mezesova, V; Bachurin, S; Lombardi, V

1995-01-01

Changes in the functioning of the glutamatergic system in rabbit brain were studied after partial brain ischemia and reperfusion. In vitro studies were conducted relating to the release of L-[14C]glutamate from cortical brain slices, L-[14C]glutamate uptake in synaptosomes, and 45Ca uptake in synaptosomes. It was found that basal release of L-[14C]glutamate from rabbit brain cortical slices after 30 min of partial ischemia and 1 d of reperfusion was essentially without change compared to the control values. After 3 d of reperfusion, there was an increase in basal release of L-[14C]glutamate from rabbit brain cortical slices. K+ stimulated release of L-[14C]glutamate in normal Krebs-Ringer medium was essentially the same in the control group and in the experimental group after 30 min of ischemia. The K+ stimulated release of L-[14C]glutamate independent of calcium was increased to 145% after 30 min of ischemia and 1 d of reperfusion. The decreased Km value at the glutamate transporter may have contributed to this difference. Kinetic parameters of the L-[14C]glutamate uptake (Km and Vmax) in synaptosomes from rabbit brain were significantly lower after 30 min of ischemia. The authors discovered that during the reperfusion period, Vmax was almost the same as in the control group. The activity of the Na+/Ca2+ exchanger in synaptosomes of rat brain was about 70% of the control values after 30 min of ischemia and 72 h of reperfusion. According to our results, increased L-[14C]glutamate release after 30 min of ischemia appears to be the result of higher intracellular calcium concentration and possibly also of a higher uptake of glutamate.

Endurance performance and nocturnal HRV indices.

PubMed

Nummela, A; Hynynen, E; Kaikkonen, P; Rusko, H

2010-03-01

The effects of endurance training on endurance performance characteristics and cardiac autonomic modulation during night sleep were investigated. Twenty-four sedentary subjects trained over four weeks two hours per week at an average running intensity of 76+/-4% of their heart rate reserve. The R to R ECG-intervals were recorded and heart rate variability indices including high frequency power (HFP) were calculated for the nights following the training days every week. The subjects were divided into responders and non-responders according to the improvements in the maximal velocity of the incremental treadmill test (v(max)). The responders improved their v(max) by 10.9+/-46 % (p < 0.001) while no changes were observed in the non-responders (1.6+/-3.0%), although there were no differences in any training load variables between the groups. In the responders nocturnal HFP was significantly higher during the fourth training week compared to the first training week (p=0.036). Furthermore, a significant correlation was observed between the change in v(max) and the change in nocturnal HFP (r=0.482, p=0.042). It was concluded that after similar training, an increase in cardiac vagal modulation was related to improved v(max) in the sedentary subjects. Georg Thieme Verlag KG Stuttgart.New York.

Optimal experimental design for assessment of enzyme kinetics in a drug discovery screening environment.

PubMed

Sjögren, Erik; Nyberg, Joakim; Magnusson, Mats O; Lennernäs, Hans; Hooker, Andrew; Bredberg, Ulf

2011-05-01

A penalized expectation of determinant (ED)-optimal design with a discrete parameter distribution was used to find an optimal experimental design for assessment of enzyme kinetics in a screening environment. A data set for enzyme kinetic data (V(max) and K(m)) was collected from previously reported studies, and every V(max)/K(m) pair (n = 76) was taken to represent a unique drug compound. The design was restricted to 15 samples, an incubation time of up to 40 min, and starting concentrations (C(0)) for the incubation between 0.01 and 100 μM. The optimization was performed by finding the sample times and C(0) returning the lowest uncertainty (S.E.) of the model parameter estimates. Individual optimal designs, one general optimal design and one, for laboratory practice suitable, pragmatic optimal design (OD) were obtained. In addition, a standard design (STD-D), representing a commonly applied approach for metabolic stability investigations, was constructed. Simulations were performed for OD and STD-D by using the Michaelis-Menten (MM) equation, and enzyme kinetic parameters were estimated with both MM and a monoexponential decay. OD generated a better result (relative standard error) for 99% of the compounds and an equal or better result [(root mean square error (RMSE)] for 78% of the compounds in estimation of metabolic intrinsic clearance. Furthermore, high-quality estimates (RMSE < 30%) of both V(max) and K(m) could be obtained for a considerable number (26%) of the investigated compounds by using the suggested OD. The results presented in this study demonstrate that the output could generally be improved compared with that obtained from the standard approaches used today.

[Alkaline-adapted beta-mannanase of Bacillus pumilus: gene heterologous expression and enzyme characterization].

PubMed

Tang, Jiajie; Guo, Su; Wang, Wei; Wei, Wei; Wei, Dongzhi

2015-11-04

We expressed a novel alkaline-adapted beta-mannanase gene and characterized the enzyme for potential industrial applications. We obtained a mannanase gene (named man(B)) from Bacillus pumilus Nsic2 and expressed the gene man(B) in Escherichia coli and Bacillus subtilis. Furthermore, we characterized the enzyme. The gene man(B) had an open reading frame of 1104 bp that encoded a polypeptide of 367-amino-acid beta-mannanase (Man(B)). The protein sequence showed the highest identity with the beta-mannanase from B. pumilus CCAM080065. We expressed the gene man(B) in E. coli BL21 (DE3) with the enzyme activity of 11021.3 U/mL. Compared with other mannanases, Man(B) showed higher stability under alkaline conditions and was stable at pH6.0 -9.0. The specific activity of purified Man(B) was 4191 ± 107 U/mg. The K(m) and V(max) values of purified Man(B) were 35.7 mg/mL and 14.9 μmol/(mL x min), respectively. Meanwhile, we achieved recombinant protein secretion expression in B. subtilis WB800N. We achieved heterologous expression of the gene man(B) and characterized its enzyme. The alkaline-adapted Man(B) showed potential value in industrial applications due to its pH stability.

Highly Efficient Synthesis of Fructooligosaccharides by Extracellular Fructooligosaccharide-Producing Enzymes and Immobilized Cells of Aspergillus aculeatus M105 and Purification and Biochemical Characterization of a Fructosyltransferase from the Fungus.

PubMed

Huang, Mei-Ping; Wu, Min; Xu, Qiang-Sheng; Mo, De-Jiao; Feng, Jia-Xun

2016-08-24

In this work, Aspergillus aculeatus M105 was obtained to produce high extracellular fructooligosaccharide-producing enzyme activity. The maximum yields of fructooligosaccharides produced by its extracellular enzymes and immobilized cells were 67.54 and 65.47% (w/w), respectively. A fructosyltransferase (FTase), AaFT32A, was purified from M105. The optimal pH and temperature of AaFT32A were pH 5.0-6.0 and 65 °C, respectively. The Km, Vmax, and kcat values for the transfructosylating activity of AaFT32A were 2267 mM, 1347 μmol/min/mg protein, and 1550.2 s(-1), respectively, and those values for the hydrolytic activity of AaFT32A were 6.10 mM, 32.44 μmol/min/mg protein, and 37.3 s(-1), respectively. The sequence of AaFT32A deduced from the cloned gene shared 99.4% identity with a FTase from Aspergillus japonicus CB05 and a fructofuranosidase from Aspergillus niger and 96.5% identity with a FTase (Aspacl_37092) from A. aculeatus ATCC 16872. The fungal strain and its FTase may have potential applications in the prebiotics industry.

Single transporter for sulfate, selenate, and selenite in Escherichia coli K-12.

PubMed Central

Lindblow-Kull, C; Kull, F J; Shrift, A

1985-01-01

A Michaelis-Menten kinetic analysis of the transport of sulfate, selenate, and selenite into Escherichia coli K-12 showed that the three dianions were transported by the same carrier. Km values, used as a measure of the affinity of each ligand for the carrier, showed that sulfate was bound 5 times more tightly than selenate and 37 times more tightly than selenite. The specificity ratio, Vmax/Km, also indicated that sulfate was the preferred ligand. There was little difference in the ratios for selenate and selenite. PMID:3897189

Nicotinamide riboside, an unusual, non-typical, substrate of purified purine-nucleoside phosphorylases.

PubMed

Wielgus-Kutrowska, B; Kulikowska, E; Wierzchowski, J; Bzowska, A; Shugar, D

1997-01-15

Nicotinamide 1-beta-D-riboside (Nir), the cationic, reducible moiety of the coenzyme NAD+, has been confirmed as an unusual substrate for purified purine-nucleoside phosphorylase (PNP) from a mammalian source (calf spleen). It is also a substrate of the enzyme from Escherichia coli. The Km values at pH 7, 1.48 mM and 0.62 mM, respectively, were 1-2 orders of magnitude higher than for the natural substrate inosine, but the Vmax values were comparable, 96% and 35% that for Ino. The pseudo first-order rate constants, Vmax/Km, were 1.1% and 2.5% for the calf spleen and E. coli enzymes. The aglycon, nicotinamide, was neither a substrate nor an inhibitor of PNP. Nir was a weak inhibitor of inosine phosphorolysis catalyzed by both enzymes, with Ki values close to the Km for its phosphorolysis, consistent with simple competitive inhibition; this was further confirmed by Dixon plots. Phosphorolysis of the fluorescent positively charged substrate 7-methylguanosine was also inhibited in a competitive manner by both Ino and Nir. Phosphorolysis of Nir by both enzymes was inhibited competitively by several specific inhibitors of calf spleen and E. coli PNP, with Ki values similar to those for inhibition of other natural substrates. The pH dependence of the kinetic constants for the phosphorolysis of Nir and of a variety of other substrates, was extensively investigated, particularly in the alkaline pH range, where Nir exhibited abnormally high substrate activity relative to the reduced reaction rates of both enzymes towards other anionic or neutral substrates. The overall results are discussed in relation to present concepts regarding binding and phosphorolysis of substrates by PNP based on crystallographic data of enzyme-inhibitor complexes, and current studies on enzymatic and nonenzymatic mechanisms of the cleavage of the Nir glycosidic bond.

Enzymes loaded chitosan/coconut fibre/zinc oxide nanoparticles strip for polyamine determination.

PubMed

Hooda, Vinita; Archita

2018-01-15

Most often, the immobilized enzyme based quantification is an attractive alternative to other chromatographic, electrochemical and mass spectrometry based methods due to its specificity and simplicity. In the present study, polyamine oxidase specific for spermine and spermidine and diamine oxidase specific for putrescine, were co-immobilized onto a novel chitosan/coconut fibre/zinc oxide nanoparticles (CS/CF/nZnO) hybrid support to yield a polyamine sensing strip. The strip worked optimally at pH 7.0, temperature 25°C and 6min of incubation time. Pretty good values for kinetic constants Km app (6.60mM), Vmax (17.69μmol/min mg protein) and kcat app (1987.64s -1 ) as well as for thermal (<50 % activity retained at 40°C), storage (half life-40days) and operational stabilities (<90 % activity retained after 20 reuses) were obtained. The strip was employed for polyamine determination in some of the locally grown fruit and vegetables and the results were found to be comparable, reliable and reproducible. Copyright © 2017 Elsevier Ltd. All rights reserved.

Demethylation of the pesticide methoxychlor in liver and intestine from untreated, methoxychlor-treated, and 3-methylcholanthrene-treated channel catfish (Ictalurus punctatus): evidence for roles of CYP1 and CYP3A family isozymes.

PubMed

Stuchal, Leah D; Kleinow, Kevin M; Stegeman, John J; James, Margaret O

2006-06-01

Exposure to the organochlorine pesticide methoxychlor (MXC) is associated with endocrine disruption in several species through biotransformation to mono-desmethyl-MXC (OH-MXC) and bis-desmethyl-MXC (HPTE), which interact with estrogen receptors. The biotransformation of [14C]methoxychlor was examined in channel catfish (Ictalurus punctatus), a freshwater species found in the southern United States. Hepatic microsomes formed OH-MXC and HPTE, assessed by comigration with authentic standards. The Km for OH-MXC formation by control liver microsomes was 3.8 +/- 1.3 microM (mean +/- S.D., n = 4), and Vmax was 131 +/- 53 pmol/min/mg protein. These values were similar to those of catfish pretreated with 2 mg/kg methoxychlor i.p. for 6 days (Km 3.3 +/- 0.8 microM and Vmax 99 +/- 17 pmol/min/mg) but less (p < 0.05) than the kinetic parameters for catfish treated with 3-methylcholanthrene (3-MC), which had Km of 6.0 +/- 1.1 microM and Vmax of 246 +/- 6 pmol/min/mg protein. Liver microsomes from 3-MC-treated fish produced significantly more of the secondary metabolite and more potent estrogen, HPTE. Intestinal microsomes formed OH-MXC at lower rates than liver. Methoxychlor pretreatment significantly reduced intestinal metabolite formation from 32 +/- 4 to 15 +/- 6 pmol/min/mg (mean +/- S.D., n = 4), whereas 3-MC treatment significantly increased OH-MXC production to 72 +/- 22 pmol/min/mg. Ketoconazole, clotrimazole, and alpha-naphthoflavone all decreased the production of OH-MXC in liver microsomes, whereas alpha-naphthoflavone stimulated HPTE formation, suggesting that CYP1 and CYP3 family isozymes demethylated methoxychlor. The results suggest that the formation of estrogenic metabolites from methoxychlor would be more rapid in catfish coexposed to CYP1 inducers.

Predicting vertical jump height from bar velocity.

PubMed

García-Ramos, Amador; Štirn, Igor; Padial, Paulino; Argüelles-Cienfuegos, Javier; De la Fuente, Blanca; Strojnik, Vojko; Feriche, Belén

2015-06-01

The objective of the study was to assess the use of maximum (Vmax) and final propulsive phase (FPV) bar velocity to predict jump height in the weighted jump squat. FPV was defined as the velocity reached just before bar acceleration was lower than gravity (-9.81 m·s(-2)). Vertical jump height was calculated from the take-off velocity (Vtake-off) provided by a force platform. Thirty swimmers belonging to the National Slovenian swimming team performed a jump squat incremental loading test, lifting 25%, 50%, 75% and 100% of body weight in a Smith machine. Jump performance was simultaneously monitored using an AMTI portable force platform and a linear velocity transducer attached to the barbell. Simple linear regression was used to estimate jump height from the Vmax and FPV recorded by the linear velocity transducer. Vmax (y = 16.577x - 16.384) was able to explain 93% of jump height variance with a standard error of the estimate of 1.47 cm. FPV (y = 12.828x - 6.504) was able to explain 91% of jump height variance with a standard error of the estimate of 1.66 cm. Despite that both variables resulted to be good predictors, heteroscedasticity in the differences between FPV and Vtake-off was observed (r(2) = 0.307), while the differences between Vmax and Vtake-off were homogenously distributed (r(2) = 0.071). These results suggest that Vmax is a valid tool for estimating vertical jump height in a loaded jump squat test performed in a Smith machine. Key pointsVertical jump height in the loaded jump squat can be estimated with acceptable precision from the maximum bar velocity recorded by a linear velocity transducer.The relationship between the point at which bar acceleration is less than -9.81 m·s(-2) and the real take-off is affected by the velocity of movement.Mean propulsive velocity recorded by a linear velocity transducer does not appear to be optimal to monitor ballistic exercise performance.

Predicting Vertical Jump Height from Bar Velocity

PubMed Central

García-Ramos, Amador; Štirn, Igor; Padial, Paulino; Argüelles-Cienfuegos, Javier; De la Fuente, Blanca; Strojnik, Vojko; Feriche, Belén

2015-01-01

The objective of the study was to assess the use of maximum (Vmax) and final propulsive phase (FPV) bar velocity to predict jump height in the weighted jump squat. FPV was defined as the velocity reached just before bar acceleration was lower than gravity (-9.81 m·s-2). Vertical jump height was calculated from the take-off velocity (Vtake-off) provided by a force platform. Thirty swimmers belonging to the National Slovenian swimming team performed a jump squat incremental loading test, lifting 25%, 50%, 75% and 100% of body weight in a Smith machine. Jump performance was simultaneously monitored using an AMTI portable force platform and a linear velocity transducer attached to the barbell. Simple linear regression was used to estimate jump height from the Vmax and FPV recorded by the linear velocity transducer. Vmax (y = 16.577x - 16.384) was able to explain 93% of jump height variance with a standard error of the estimate of 1.47 cm. FPV (y = 12.828x - 6.504) was able to explain 91% of jump height variance with a standard error of the estimate of 1.66 cm. Despite that both variables resulted to be good predictors, heteroscedasticity in the differences between FPV and Vtake-off was observed (r2 = 0.307), while the differences between Vmax and Vtake-off were homogenously distributed (r2 = 0.071). These results suggest that Vmax is a valid tool for estimating vertical jump height in a loaded jump squat test performed in a Smith machine. Key points Vertical jump height in the loaded jump squat can be estimated with acceptable precision from the maximum bar velocity recorded by a linear velocity transducer. The relationship between the point at which bar acceleration is less than -9.81 m·s-2 and the real take-off is affected by the velocity of movement. Mean propulsive velocity recorded by a linear velocity transducer does not appear to be optimal to monitor ballistic exercise performance. PMID:25983572

Ketopantoyl-lactone reductase from Candida parapsilosis: purification and characterization as a conjugated polyketone reductase.

PubMed

Hata, H; Shimizu, S; Hattori, S; Yamada, H

1989-02-24

Ketopantoyl-lactone reductase (2-dehydropantoyl-lactone reductase, EC 1.1.1.168) was purified and crystallized from cells of Candida parapsilosis IFO 0708. The enzyme was found to be homogeneous on ultracentrifugation, high-performance gel-permeation liquid chromatography and SDS-polyacrylamide gel electrophoresis. The relative molecular mass of the native and SDS-treated enzyme is approximately 40,000. The isoelectric point of the enzyme is 6.3. The enzyme was found to catalyze specifically the reduction of a variety of natural and unnatural polyketones and quinones other than ketopantoyl lactone in the presence of NADPH. Isatin and 5-methylisatin are rapidly reduced by the enzyme, the Km and Vmax values for isatin being 14 microM and 306 mumol/min per mg protein, respectively. Ketopantoyl lactone is also a good substrate (Km = 333 microM and Vmax = 481 mumol/min per mg protein). Reverse reaction was not detected with pantoyl lactone and NADP+. The enzyme is inhibited by quercetin, several polyketones and SH-reagents. 3,4-Dihydroxy-3-cyclobutene-1,2-dione, cyclohexenediol-1,2,3,4-tetraone and parabanic acid are uncompetitive inhibitors for the enzyme, the Ki values being 1.4, 0.2 and 3140 microM, respectively, with isatin as substrate. Comparison of the enzyme with the conjugated polyketone reductase of Mucor ambiguus (S. Shimizu, H. Hattori, H. Hata and H. Yamada (1988) Eur. J. Biochem. 174, 37-44) and ketopantoyl-lactone reductase of Saccharomyces cerevisiae suggested that ketopantoyl-lactone reductase is a kind of conjugated polyketone reductase.

Competition between roots and microorganisms for nitrogen: mechanisms and ecological relevance.

PubMed

Kuzyakov, Yakov; Xu, Xingliang

2013-05-01

Demand of all living organisms on the same nutrients forms the basis for interspecific competition between plants and microorganisms in soils. This competition is especially strong in the rhizosphere. To evaluate competitive and mutualistic interactions between plants and microorganisms and to analyse ecological consequences of these interactions, we analysed 424 data pairs from 41 (15)N-labelling studies that investigated (15)N redistribution between roots and microorganisms. Calculated Michaelis-Menten kinetics based on K(m) (Michaelis constant) and V(max) (maximum uptake capacity) values from 77 studies on the uptake of nitrate, ammonia, and amino acids by roots and microorganisms clearly showed that, shortly after nitrogen (N) mobilization from soil organic matter and litter, microorganisms take up most N. Lower K(m) values of microorganisms suggest that they are especially efficient at low N concentrations, but can also acquire more N at higher N concentrations (V(max)) compared with roots. Because of the unidirectional flow of nutrients from soil to roots, plants are the winners for N acquisition in the long run. Therefore, despite strong competition between roots and microorganisms for N, a temporal niche differentiation reflecting their generation times leads to mutualistic relationships in the rhizosphere. This temporal niche differentiation is highly relevant ecologically because it: protects ecosystems from N losses by leaching during periods of slow or no root uptake; continuously provides roots with available N according to plant demand; and contributes to the evolutionary development of mutualistic interactions between roots and microorganisms. © 2013 The Authors. New Phytologist © 2013 New Phytologist Trust.

Kinetic characterization of arginine deiminase and carbamate kinase from Streptococcus pyogenes M49.

PubMed

Hering, Silvio; Sieg, Antje; Kreikemeyer, Bernd; Fiedler, Tomas

2013-09-01

Streptococcus pyogenes (group A Streptococcus, GAS) is an important human pathogen causing mild superficial infections of skin and mucous membranes, but also life-threatening systemic diseases. S. pyogenes and other prokaryotic organisms use the arginine deiminase system (ADS) for survival in acidic environments. In this study, the arginine deiminase (AD), and carbamate kinase (CK) from S. pyogenes M49 strain 591 were heterologously expressed in Escherichia coli DH5α, purified, and kinetically characterized. AD and CK from S. pyogenes M49 share high amino acid sequence similarity with the respective enzymes from Lactococcus lactis subsp. lactis IL1403 (45.6% and 53.5% identical amino acids) and Enterococcus faecalis V583 (66.8% and 66.8% identical amino acids). We found that the arginine deiminase of S. pyogenes is not allosterically regulated by the intermediates and products of the arginine degradation (e.g., ATP, citrulline, carbamoyl phosphate). The Km and Vmax values for arginine were 1.13±0.12mM (mean±SD) and 1.51±0.07μmol/min/mg protein. The carbamate kinase is inhibited by ATP but unaffected by arginine and citrulline. The Km and Vmax values for ADP were 0.72±0.08mM and 1.10±0.10μmol/min/mg protein and the Km for carbamoyl phosphate was 0.65±0.07mM. The optimum pH and temperature for both enzymes were 6.5 and 37°C, respectively. Copyright © 2013 Elsevier Inc. All rights reserved.

Effect of saposins on acid sphingomyelinase.

PubMed Central

Tayama, M; Soeda, S; Kishimoto, Y; Martin, B M; Callahan, J W; Hiraiwa, M; O'Brien, J S

1993-01-01

The effect of saposins (A, B, C and D) on acid sphingomyelinase activity was determined using a crude human kidney sphingomyelinase preparation and a purified sphingomyelinase preparation from human placenta. Saposin D stimulated the activity of the crude enzyme by increasing its apparent Km and Vmax. values for sphingomyelin hydrolysis. Unlike the crude enzyme, the activity of the purified enzyme was strongly inhibited by saposin D as well as other saposins. Saposin D decreased the apparent Km and Vmax values of purified sphingomyelinase activity. The effects of saposin D on the activity of different sphingomyelinase preparations appear to depend on Triton X-100, which is present in the crude enzyme but not in the purified enzyme. When the detergent was removed from the crude preparation, the effect of saposin D changed from being stimulatory to inhibitory. Conversely, when the detergent is added to the purified enzyme, the effect of saposin D on sphingomyelinase activity changed from being inhibitory to stimulatory. While other saposins were inhibitory or had no effect on sphingomyelinase activity in the above assay system, not only saposin D but also saposins A and C exhibited a stimulatory effect upon purified sphingomyelinase activity when the substrate, sphingomyelin, was added in the form of liposomes without detergent. Saposin B was not only inhibitory in the liposome system, but also reduced the stimulatory effect of saposins A, C and D. These observations indicate that the stimulatory effect of saposins A, C and D on acid sphingomyelinase activity is greatly influenced by the physical environment of the enzyme and suggest that similar effects by saposins may be exerted in lysosomal membranes. PMID:8452527

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.

Enzymatic hydrolysis of short-chain lecithin/long-chain phospholipid unilamellar vesicles: sensitivity of phospholipases to matrix phase state.

PubMed

Gabriel, N E; Agman, N V; Roberts, M F

1987-11-17

Short-chain lecithin/long-chain phospholipid unilamellar vesicles (SLUVs), unlike pure long-chain lecithin vesicles, are excellent substrates for water-soluble phospholipases. Hemolysis assays show that greater than 99.5% of the short-chain lecithin is partitioned in the bilayer. In these binary component vesicles, the short-chain species is the preferred substrate, while the long-chain phospholipid can be treated as an inhibitor (phospholipase C) or poor substrate (phospholipase A2). For phospholipase C Bacillus cereus, apparent Km and Vmax values show that bilayer-solubilized diheptanoylphosphatidylcholine (diheptanoyl-PC) is nearly as good a substrate as pure micellar diheptanoyl-PC, although the extent of short-chain lecithin hydrolysis depends on the phase state of the long-chain lipid. For phospholipase A2 Naja naja naja, both Km and Vmax values show a greater range: in a gel-state matrix, diheptanoyl-PC is hydrolyzed with micellelike kinetic parameters; in a liquid-crystalline matrix, the short-chain lecithin becomes comparable to the long-chain component. Both enzymes also show an anomalous increase in specific activity toward diheptanoyl-PC around the phase transition temperature of the long-chain phospholipid. Since the short-chain lecithin does not exhibit a phase transition, this must reflect fluctuations in head-group area or vertical motions of the short-chain lecithin caused by surrounding long-chain lecithin molecules. These results are discussed in terms of a specific model for SLUV hydrolysis and a general explanation for the "interfacial activation" observed with water-soluble phospholipases.

Cytochrome P450 isoenzymes involved in rat liver microsomal metabolism of californine and protopine.

PubMed

Paul, Liane D; Springer, Dietmar; Staack, Roland F; Kraemer, Thomas; Maurer, Hans H

2004-02-06

Studies are described on the cytochrome P450 (CYP) isoenzyme dependence of the main metabolic steps of the Eschscholtzia californica alkaloids californine and protopine using rat liver microsomes. Preparations of E. californica are in use as phytopharmaceuticals and as herbal drugs of abuse. CYP isoenzyme dependences were studied using specific chemical inhibitors for CYP1A2, CYP2D1, and CYP3A2 (alpha-naphthoflavone, quinine, and ketoconazole, respectively). CYP2C11 was inhibited by specific antibodies for lack of specific chemical inhibitors. Californine N-demethylation was mainly catalyzed by CYP3A2 and to a minor extent by CYP1A2 and CYP2D1, but not by CYP2C11. CYP2D1 and CYP2C11 were shown to be mainly involved in demethylenation of both, californine and protopine, while CYP1A2 and CYP3A2 showed only minor contribution. Kinetic parameters of the reactions were established. K(m) and V(max) values for the californine N-demethylation were 4.5+/-4.7 microM and 22.9+/-13.7 min/mg protein (high affinity) and 161.3+/-16.7 microM and 311.8+/-39.4 min/mg protein (low affinity), respectively. Californine demethylenation and protopine demethylenation showed substrate inhibition and K(m) and V(max) values were 5.0+/-0.5 and 7.1+/-0.6 microM and 83.3+/-2.6 and 160.7+/-4.0 min/mg protein, respectively.

Lion (Panthera leo) and caracal (Caracal caracal) type IIx single muscle fibre force and power exceed that of trained humans.

PubMed

Kohn, Tertius A; Noakes, Timothy D

2013-03-15

This study investigated for the first time maximum force production, shortening velocity (Vmax) and power output in permeabilised single muscle fibres at 12°C from lion, Panthera leo (Linnaeus 1758), and caracal, Caracal caracal (Schreber 1776), and compared the values with those from human cyclists. Additionally, the use and validation of previously frozen tissue for contractile experiments is reported. Only type IIx muscle fibres were identified in the caracal sample, whereas type IIx and only two type I fibres were found in the lion sample. Only pure type I and IIa, and hybrid type IIax fibres were identified in the human samples - there were no pure type IIx fibres. Nevertheless, compared with all the human fibre types, the lion and caracal fibres were smaller (P<0.01) in cross-sectional area (human: 6194±230 μm(2), lion: 3008±151 μm(2), caracal: 2583±221 μm(2)). On average, the felid type IIx fibres produced significantly greater force (191-211 kN m(-2)) and ~3 times more power (29.0-30.3 kN m(-2) fibre lengths s(-1)) than the human IIax fibres (100-150 kN m(-2), 4-11 kN m(-2) fibre lengths s(-1)). Vmax values of the lion type IIx fibres were also higher than those of human type IIax fibres. The findings suggest that the same fibre type may differ substantially between species and potential explanations are discussed.

Characteristics of butanol metabolism in alcohol dehydrogenase-deficient deermice.

PubMed Central

Alderman, J A; Kato, S; Lieber, C S

1989-01-01

Deermice lacking the low-Km alcohol dehydrogenase eliminated butan-1-ol, a substrate for microsomal oxidation but not for catalase, at 117 mumol/min per kg body wt. Microsomal fractions and hepatocytes metabolized butan-1-ol also (Vmax. = 6.7 nmol/min per nmol of cytochrome P-450, Km = 0.85 mM; Vmax. = 5.3 nmol/min per 10(6) cells, Km = 0.71 mM respectively). These results are consistent with alcohol oxidation by the microsomal system in these deermice. PMID:2930472

Temperature sensitivity and enzymatic mechanisms of soil organic matter decomposition along an altitudinal gradient on Mount Kilimanjaro

NASA Astrophysics Data System (ADS)

Blagodatskaya, Evgenia; Blagodatsky, Sergey; Khomyakov, Nikita; Myachina, Olga; Kuzyakov, Yakov

2016-02-01

Short-term acceleration of soil organic matter decomposition by increasing temperature conflicts with the thermal adaptation observed in long-term studies. Here we used the altitudinal gradient on Mt. Kilimanjaro to demonstrate the mechanisms of thermal adaptation of extra- and intracellular enzymes that hydrolyze cellulose, chitin and phytate and oxidize monomers (14C-glucose) in warm- and cold-climate soils. We revealed that no response of decomposition rate to temperature occurs because of a cancelling effect consisting in an increase in half-saturation constants (Km), which counteracts the increase in maximal reaction rates (Vmax with temperature). We used the parameters of enzyme kinetics to predict thresholds of substrate concentration (Scrit) below which decomposition rates will be insensitive to global warming. Increasing values of Scrit, and hence stronger canceling effects with increasing altitude on Mt. Kilimanjaro, explained the thermal adaptation of polymer decomposition. The reduction of the temperature sensitivity of Vmax along the altitudinal gradient contributed to thermal adaptation of both polymer and monomer degradation. Extrapolating the altitudinal gradient to the large-scale latitudinal gradient, these results show that the soils of cold climates with stronger and more frequent temperature variation are less sensitive to global warming than soils adapted to high temperatures.

Methylmercury-cholinesterase interactions in rats.

PubMed Central

Hastings, F L; Lucier, G W; Klein, R

1975-01-01

The interaction of methylmercury hydroxide (MMH) and cholinesterases was studied in male and female rats. MMH administered subcutaneously in doses of 10 mg/kg for 2 days reduced the level of plasma cholinesterase (ButChE) by 68% in females and 47% in males while brain acetylcholinesterase (AChE) was unaffected. Normal females had higher but more variable ButChE levels than normal males. In a time-course experiment, a single dose of MMH (10 mg/kg) reduced ButChE levels when mercury levels reached 22 mug/ml in the blood. A 10% reduction in brain AChE was observed at 72 hours; however, mercury reached a concentration of only 2.0 mug/g in brain tissue. The determination of the Michaelis constant Km and maximum velocity value Vmax for butyrylcholine and ButChE in control and MMH-treated (1 mg/kg) animals indicated that MMH reduced Vmax only. Since no loss in ButChE activity occurred when MMH and control plasma were incubated in vitro, MMH is not a direct inhibitor of ButChE. Because only the inactive monomeric form of ButChE contains free sulfhydryl groups, it is postulated that MMH combines covalently with the sulfur, preventing formation of active enzyme. By analogy, it is believed this is also the case with AChE. PMID:1227853

Relationships between maximal anaerobic power of the arms and legs and javelin performance.

PubMed

Bouhlel, E; Chelly, M S; Tabka, Z; Shephard, R

2007-06-01

The aim of this study was to examine relationships between maximal anaerobic power, as measured by leg and arm force-velocity tests, estimates of local muscle volume and javelin performance. Ten trained national level male javelin throwers (mean age 19.6+/- 2 years) participated in this study. Maximal anaerobic power, maximal force and maximal velocity were measured during leg (Wmax-L) and arm (Wmax-A) force-velocity tests, performed on appropriately modified forms of Monark cycle ergometer. Estimates of leg and arm muscle volume were made using a standard anthropometric kit. Maximal force of the leg (Fmax-L) was significantly correlated with estimated leg muscle volume (r=0.71, P<0.05). Wmax-L and Wmax-A were both significantly correlated with javelin performance (r=0.76, P<0.01; r=0.71, P <0.05, respectively). Maximal velocity of the leg (Vmax-L) was also significantly correlated with throwing performance (r=0.83; P<0.001). Wmax of both legs and arms were significantly correlated with javelin performance, the closest correlation being for Wmax-L; this emphasizes the importance of the leg muscles in this sport. Fmax-L and Vmax-L were related to muscle volume and to javelin performance, respectively. Force-velocity testing may have value in regulating conditioning and rehabilitation in sports involving throwing.

Spectrophotometric determination of pyrrole-like substances in urine of rat and man: an assay for the evaluation of 2,5-hexanedione formed from n-hexane.

PubMed

Kessler, W; Heilmaier, H; Kreuzer, P; Shen, J H; Filser, M; Filser, J G

1990-01-01

Male Wistar rats exposed to atmospheric n-hexane excreted in their urine substances which gave rise to absorption spectra like those of pyrroles after the reaction with Ehrlich's reagent. A simple spectrophotometric assay was developed to determine these "pyrrole-like substances" in urine. Their excretion kinetics were evaluated by exposing rats for 8 h to atmospheric n-hexane concentrations between 50 and 3000 ppm. The dose-response curve revealed saturation kinetics according to Michaelis-Menten, Vmax being 1.12 [delta E526.ml urine/8 h n-hexane exposure] and "Km", the atmospheric n-hexane concentration at Vmax/2, being 250 ppm. The excretion of pyrrole-like substances closely correlated with that of 2,5-hexanedione measured by Fedtke and Bolt (1987). Pyrrole-like substances were also found in the urine of a male volunteer. When exposing the person for 3 h to atmospheric n-hexane at a concentration of 146 ppm (equivalent to 55 ppm/8 h) the excreted amount was twice the background value. Due to the sensitivity of this assay it is possible to determine pyrrole-like substances in urine according to the present German MAK or US TLV conditions for n-hexane (50 ppm/8 h).

Kinetic behaviour of calf intestinal alkaline phosphatase with pNPP.

PubMed

Chaudhuri, Gouri; Chatterjee, Saswata; Venu-Babu, P; Ramasamy, K; Thilagaraj, W Richard

2013-02-01

The hydrolysis of p-nitrophenyl phosphate (pNPP) by calf intestinal alkaline phosphatase (CIAP) was investigated with respect to kinetic parameters such as V(max), K(m) and K(cat) under varying pH, buffers, substrate concentration, temperature and period of incubation. Highest activity was obtained with Tris-HCl at pH 11, while in the case of glycine-NaOH buffer the peak activity was recorded at pH 9.5. The enzyme showed the following kinetic characteristics with pNPP in 50 mM Tris-HCl at pH 11 and 100 mM glycine-NaOH at pH 9.5 at an incubation temperature of 37 degrees C: V(max), 3.12 and 1.6 micromoles min(-1) unit(-1); K(m), 7.6 x 10(-4) M and 4 x 10(-4) M; and K(cat), 82.98 s(-1) and 42.55 s(-1), respectively. CIAP displayed a high temperature optimum of 45 degrees C at pH 11. The kinetic behaviour of the enzyme under different parameters suggested that the enzyme might undergo subtle conformational changes in response to the buffers displaying unique characteristics. Bioprecipitation of Cu2+ from 50 ppm of CuCl2 solution was studied where 64.3% of precipitation was obtained. P(i) generated from CIAP-mediated hydrolysis of pNPP was found to bind with copper and precipitated as copper-phosphate. Thus, CIAP could be used as a test candidate in bioremediation of heavy metals from industrial wastes through generation of metal-phosphate complexes.

omega-Amino acid:pyruvate transaminase from Alcaligenes denitrificans Y2k-2: a new catalyst for kinetic resolution of beta-amino acids and amines.

PubMed

Yun, Hyungdon; Lim, Seongyop; Cho, Byung-Kwan; Kim, Byung-Gee

2004-04-01

Alcaligenes denitrificans Y2k-2 was obtained by selective enrichment followed by screening from soil samples, which showed omega-amino acid:pyruvate transaminase activity, to kinetically resolve aliphatic beta-amino acid, and the corresponding structural gene (aptA) was cloned. The gene was functionally expressed in Escherichia coli BL21 by using an isopropyl-beta-D-thiogalactopyranoside (IPTG)-inducible pET expression system (9.6 U/mg), and the recombinant AptA was purified to show a specific activity of 77.2 U/mg for L-beta-amino-n-butyric acid (L-beta-ABA). The enzyme converts various beta-amino acids and amines to the corresponding beta-keto acids and ketones by using pyruvate as an amine acceptor. The apparent K(m) and V(max) for L-beta-ABA were 56 mM and 500 U/mg, respectively, in the presence of 10 mM pyruvate. In the presence of 10 mM L-beta-ABA, the apparent K(m) and V(max) for pyruvate were 11 mM and 370 U/mg, respectively. The enzyme exhibits high stereoselectivity (E > 80) in the kinetic resolution of 50 mM D,L-beta-ABA, producing optically pure D-beta-ABA (99% enantiomeric excess) with 53% conversion.

ω-Amino Acid:Pyruvate Transaminase from Alcaligenes denitrificans Y2k-2: a New Catalyst for Kinetic Resolution of β-Amino Acids and Amines

PubMed Central

Yun, Hyungdon; Lim, Seongyop; Cho, Byung-Kwan; Kim, Byung-Gee

2004-01-01

Alcaligenes denitrificans Y2k-2 was obtained by selective enrichment followed by screening from soil samples, which showed ω-amino acid:pyruvate transaminase activity, to kinetically resolve aliphatic β-amino acid, and the corresponding structural gene (aptA) was cloned. The gene was functionally expressed in Escherichia coli BL21 by using an isopropyl-β-d-thiogalactopyranoside (IPTG)-inducible pET expression system (9.6 U/mg), and the recombinant AptA was purified to show a specific activity of 77.2 U/mg for l-β-amino-n-butyric acid (l-β-ABA). The enzyme converts various β-amino acids and amines to the corresponding β-keto acids and ketones by using pyruvate as an amine acceptor. The apparent Km and Vmax for l-β-ABA were 56 mM and 500 U/mg, respectively, in the presence of 10 mM pyruvate. In the presence of 10 mM l-β-ABA, the apparent Km and Vmax for pyruvate were 11 mM and 370 U/mg, respectively. The enzyme exhibits high stereoselectivity (E > 80) in the kinetic resolution of 50 mM d,l-β-ABA, producing optically pure d-β-ABA (99% enantiomeric excess) with 53% conversion. PMID:15066855

Universality and robustness of revivals in the transverse field XY model

NASA Astrophysics Data System (ADS)

Häppölä, Juho; Halász, Gábor B.; Hamma, Alioscia

2012-03-01

We study the structure of the revivals in an integrable quantum many-body system, the transverse field XY spin chain, after a quantum quench. The time evolutions of the Loschmidt echo, the magnetization, and the single-spin entanglement entropy are calculated. We find that the revival times for all of these observables are given by integer multiples of Trev≃L/vmax, where L is the linear size of the system and vmax is the maximal group velocity of quasiparticles. This revival structure is universal in the sense that it does not depend on the initial state and the size of the quench. Applying nonintegrable perturbations to the XY model, we observe that the revivals are robust against such perturbations: they are still visible at time scales much larger than the quasiparticle lifetime. We therefore propose a generic connection between the revival structure and the locality of the dynamics, where the quasiparticle speed vmax generalizes into the Lieb-Robinson speed vLR.

[Temperature sensitivity of soil organic carbon mineralization and β-glucosidase enzymekinetics in the northern temperate forests at different altitudes, China].

PubMed

Fan, Jin-juan; Li, Dan-dan; Zhang, Xin-yu; He, Nian-peng; Bu, Jin-feng; Wang, Qing; Sun, Xiao-min; Wen, Xue-fa

2016-01-01

Soil samples, which were collected from three typical forests, i.e., Betula ermanii forest, coniferous mixed broad-leaved forest, and Pinus koraiensis forest, at different altitudes along the southern slope of Laotuding Mountain of Changbai Mountain range in Liaoning Province of China, were incubated over a temperature gradient in laboratory. Soil organic carbon mineralization rates (Cmin), soil β-1,4-glucosidase (βG) kinetics and their temperature sensitivity (Q₁₀) were measured. The results showed that both altitude and temperature had significant effects on Cmin · Cmin increased with temperature and was highest in the B. ermanii forest. The temperature sensitivity of Cmin [Q₁₀(Cmin)] ranked in order of B. ermanii forest > P. koraiensis forest > coniferous mixed broad-leaved forest, but did not differ significantly among the three forests. Both the maximum activity (Vmax) and the Michaelis constant (Km) of the βG responded positively to temperature for all the forests. The temperature sensitivity of Vmax [Q₁₀(Vmax)] ranged from 1.78 to 1.90, and the temperature sensitivity of Km [Q₁₀(Km)] ranged from 1.79 to 2.00. The Q₁₀(Vmax)/Q10(Km) ratios were significantly greater in the B. ermanii soil than in the other two forest soils, suggesting that the βG kinetics-dependent impacts of the global warming or temperature increase on the decomposition of soil organic carbon were temperature sensitive for the forests at the higher altitudes.

Crataegus extract prolongs action potential duration in guinea-pig papillary muscle.

PubMed

Müller, A; Linke, W; Zhao, Y; Klaus, W

1996-11-01

Crataegus extract is used in cardiology for the treatment of moderate heart failure (NYHA II). Recently it was shown that Crataegus extract prolongs the refractory period in isolated perfused guinea pig hearts. In order to find out what mechanism is responsible for this prolongation of refractory period, we investigated the effects of Crataegus extract (LI 132) on the action potential of guinea pig papillary muscle with the help of conventional microelectrode techniques. Crataegus extract, when put in a concentration (10 mg/l) capable of inducing an inotropic effect of about 20%, significantly increased action potential duration at all investigated levels of repolarisation. Maximum prolongation was 8.5±2.3 ms, 12.5±2.6 ms and 11.7±2.9 ms at 20%, 50% and 90% repolarisation, respectively (control APD(90): 172±4 ms). Experiments on the time course of recovery of the maximum upstroke velocity (V(max)) of the action potential revealed that Crataegus extract increased the time constant of recovery of V(max) from 8.80±2.33 ms to 22.60±5.77 ms, indicating a weak Class I-like antiarrhythmic action. In addition, we observed a small reduction in V(max). In summary, our results show that Crataegus extract prolongs action potential duration and delays recovery of V(max). We, therefore, suggest that Crataegus extract possesses certain antiarrhythmic properties. Copyright © 1996 Gustav Fischer Verlag · Stuttgart · Jena · New York. Published by Elsevier GmbH.. All rights reserved.

Calcium alginate gel as encapsulation matrix for coimmobilized enzyme systems.

PubMed

Blandino, A; Macías, M; Cantero, D

2003-07-01

Encapsulation within calcium alginate gel capsules was used to produce a coimmobilized enzyme system. Glucose oxidase (GOD) and catalase (CAT) were chosen as model enzymes. The same values of Vmax and Km app for the GOD encapsulated system and for the GOD-CAT coencapsulated system were calculated. When gel beads and capsules were compared, the same catalyst deactivation sequence for the two enzymes was observed. However, when capsules were employed as immobilization support, GOD efficiencies were higher than for the gel beads. These results were explained in terms of the structure of the capsules.

Morphine-induced kinetic alterations of choline acetyltransferase of the rat caudate nucleus

PubMed Central

Datta, K.; Wajda, I. J.

1972-01-01

1. In order to explain the decrease of choline acetyltransferase (2.3.1.6.) activity observed in the caudate nucleus of morphine-treated rats, partially purified preparations of the enzyme were used in kinetic studies, with choline as substrate. 2. The apparent Michaelis constant for the enzyme obtained from normal rats was found to be 0·9 mM choline; this value doubled when the animals were killed one hour after a single injection of morphine (30 mg/kg). When the rats were injected daily for 4 or 15 days, and killed one hour after the last injection, the apparent Km value was 2·1 mM in each case. Prolonged daily treatment with morphine, followed by 48 h withdrawal, or by administration of 4 mg/kg of naloxone (given half an hour after the last injection of morphine) resulted in apparent Km values of 1·3-1·5 mM of choline, suggesting a gradual return to the lower, normal substrate requirement. Vmax changes were insignificant. 3. The effect of morphine added in vitro to different enzyme preparations was also studied. The Km values of 0·9 mM, in the enzyme isolated from normal rats, increased to 2·0 after incubation in vitro with 12·5 mM morphine. Similar increases were found in enzymes obtained from rats 48 h after the withdrawal of morphine or from rats injected with naloxone after prolonged morphine treatment. The high apparent Km values, found in enzyme obtained from animals killed one hour after the last dose of morphine, did not change upon incubation with 12·5 mM morphine. A similar pattern of Km changes was noticed after incubation with 25 mM acetylcholine. 4. An increase of 32% in acetylcholine (ACh) level was found in the caudate nucleus one hour after subcutaneous injection of 30 mg/kg of morphine. Return to normal values was observed when morphine was administered daily. After two to three weeks of daily treatment and subsequent withdrawal from morphine for 48 h, the levels of ACh were normal. If the daily treated rats were given naloxone within half an hour of the last injection of morphine, and killed 30 min later, the levels of ACh remained normal. 5. Fifty per cent inhibition of enzyme activity was observed upon in vitro incubation with 75 mM acetylcholine, or with 25 mM morphine. The same degree of inhibition was noticed when the enzyme was obtained from normal or from morphine-treated rats. PMID:5041452

Assessment and kinetics of soil phosphatase in Brazilian Savanna systems.

PubMed

Ferreira, Adão S; Espíndola, Suéllen P; Campos, Maria Rita C

2016-05-31

The activity and kinetics of soil phosphatases are important indicators to evaluate soil quality in specific sites such as the Cerrado (Brazilian Savanna). This study aimed to determine the activity and kinetic parameters of soil phosphatase in Cerrado systems. Soil phosphatase activity was assessed in samples of native Cerrado (NC), no-tillage (NT), conventional tillage (CT) and pasture with Brachiaria brizantha (PBb) and evaluated with acetate buffer (AB), tris-HCl buffer (TB), modified universal buffer (MUB) and low MUB. The Michaelis-Menten equation and Eadie-Hofstee model were applied to obtain the kinetic parameters of soil phosphatase using different concentrations of p-nitrophenol phosphate (p-NPP). MUB showed the lowest soil phosphatase activity in all soils whereas AB in NC and NT presented the highest. Low MUB decreased interferences in the assessment of soil phosphatase activity when compared to MUB, suggesting that organic acids interfere on the soil phosphatase activity. In NC and NT, soil phosphatase activity performed with TB was similar to AB and low MUB. Km values from the Michaels-Menten equation were higher in NC than in NT, which indicate a lower affinity of phosphatase activity for the substrate in NC. Vmax values were also higher in NC than in NT. The Eadie-Hofstee model suggests that NC had more phosphatase isoforms than NT. The study showed that buffer type is of fundamental importance when assessing soil phosphatase activity in Cerrado soils.

Production of Recombinant Trichoderma reesei Cellobiohydrolase II in a New Expression System Based on Wickerhamomyces anomalus

PubMed Central

Díaz-Rincón, Dennis J.; Duque, Ivonne; Osorio, Erika; Rodríguez-López, Alexander; Espejo-Mojica, Angela; Parra-Giraldo, Claudia M.

2017-01-01

Cellulase is a family of at least three groups of enzymes that participate in the sequential hydrolysis of cellulose. Recombinant expression of cellulases might allow reducing their production times and increasing the low proteins concentrations obtained with filamentous fungi. In this study, we describe the production of Trichoderma reesei cellobiohydrolase II (CBHII) in a native strain of Wickerhamomyces anomalus. Recombinant CBHII was expressed in W. anomalus 54-A reaching enzyme activity values of up to 14.5 U L−1. The enzyme extract showed optimum pH and temperature of 5.0–6.0 and 40°C, respectively. Enzyme kinetic parameters (KM of 2.73 mM and Vmax of 23.1 µM min−1) were between the ranges of values reported for other CBHII enzymes. Finally, the results showed that an enzymatic extract of W. anomalus 54-A carrying the recombinant T. reesei CBHII allows production of reducing sugars similar to that of a crude extract from cellulolytic fungi. These results show the first report on the use of W. anomalus as a host to produce recombinant proteins. In addition, recombinant T. reesei CBHII enzyme could potentially be used in the degradation of lignocellulosic residues to produce bioethanol, based on its pH and temperature activity profile. PMID:28951785

Echinococcus granulosus: absorption of cycloleucine and alpha-aminoisobutyric acid by protoscoleces.

PubMed

Jeffs, S A; Arme, C

1986-02-01

Protoscoleces of Echinococcus granulosus absorb the amino acids cycloleucine and alpha-aminoisobutyric acid (AIB) by a combination of mediated uptake and diffusion. After correcting for the latter, values for Kt and Vmax of 0.124 mM and 0.947 nmoles/mg protein/2 min for cycloleucine were calculated; corresponding values for AIB were 0.039 mM and 0.139 nmoles/mg protein/2 min. Both amino acids were accumulated against a concentration gradient and a comparison of Kt and Ki values determined in mutual inhibition experiments suggested that both cycloleucine and AIB share a common uptake locus (loci). Cycloleucine uptake was pH-dependent and could be inhibited by a variety of other amino acids. Neither D- nor L-proline inhibited cycloleucine absorption but D-methionine, D-alanine, D-leucine, D-valine and D-serine were much more effective inhibitors than their L-counterparts.

The activity of pyruvate carrier in a reconstituted system: substrate specificity and inhibitor sensitivity.

PubMed

Nałecz, K A; Kamińska, J; Nałecz, M J; Azzi, A

1992-08-15

The pyruvate carrier, of molecular mass 34 kDa, was purified from mitochondria isolated from rat liver, rat brain, and bovine heart, by affinity chromatography on immobilized 2-cyano-4-hydroxycinnamate. Its activity after reconstitution in phosphatidylcholine vesicles was measured either as uptake of [1-14C]pyruvate or as exchange with different 2-oxoacids. All preparations exhibited similar apparent Km values for pyruvate, but somewhat different V(max) values. The ability to exchange different anions of physiological significance, including branched-chain 2-oxoacids, confirmed the known substrate specificity described for the pyruvate carrier in mitochondria. The sensitivity of pyruvate transport toward phenylglyoxal suggested an important role of arginyl residues in the transport activity, while a role of lysyl and histidyl residues was not confirmed.

Cloning, Expression, and Purification of Choline Dehydrogenase from the Moderate Halophile Halomonas elongata

PubMed Central

Gadda, Giovanni; McAllister-Wilkins, Elien Elizabeth

2003-01-01

Choline dehydrogenase (EC 1.1.99.1) catalyzes the four-electron oxidation of choline to glycine-betaine via a betaine-aldehyde intermediate. Such a reaction is of considerable interest for biotechnological applications in that transgenic plants engineered with bacterial glycine-betaine-synthesizing enzymes have been shown to have enhanced tolerance towards various environmental stresses, such as hypersalinity, freezing, and high temperatures. To date, choline dehydrogenase has been poorly characterized in its biochemical and kinetic properties, mainly because its purification has been hampered by instability of the enzyme in vitro. In the present report, we cloned and expressed in Escherichia coli the betA gene from the moderate halophile Halomonas elongata which codes for a hypothetical choline dehydrogenase. The recombinant enzyme was purified to more than 70% homogeneity as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and by treatment with 30 to 50% saturation of ammonium sulfate followed by column chromatography using DEAE-Sepharose. The purified enzyme showed similar substrate specificities with either choline or betaine-aldehyde as the substrate, as indicated by the apparent V/K values (where V is the maximal velocity and K is the Michaelis constant) of 0.9 and 0.6 μmol of O2 min−1 mg−1 mM−1 at pH 7 and 25°C, respectively. With 1 mM phenazine methosulfate as the primary electron acceptor, the apparent Vmax values for choline and betaine-aldehyde were 10.9 and 5.7 μmol of O2 min−1 mg−1, respectively. These Vmax values decreased four- to sevenfold when molecular oxygen was used as the electron acceptor. Altogether, the kinetic data are consistent with the conclusion that H. elongata betA codes for a choline dehydrogenase that can also act as an oxidase when electron acceptors other than molecular oxygen are not available. PMID:12676692

Decreased cardiac SERCA2 expression, SR Ca uptake, and contractile function in hypothyroidism are attenuated in SERCA2 overexpressing transgenic rats.

PubMed

Vetter, Roland; Rehfeld, Uwe; Reissfelder, Christoph; Fechner, Henry; Seppet, Enn; Kreutz, Reinhold

2011-03-01

The sarco/endoplasmic reticulum (SR) Ca(2+)-ATPase SERCA2a has a key role in controlling cardiac contraction and relaxation. In hypothyroidism, decreased expression of the thyroid hormone (TH)-responsive SERCA2 gene contributes to slowed SR Ca(2+) reuptake and relaxation. We investigated whether cardiac expression of a TH-insensitive SERCA2a cDNA minigene can rescue SR Ca(2+) handling and contractile function in female SERCA2a-transgenic rats (TG) with experimental hypothyroidism. Wild-type rats (WT) and TG were rendered hypothyroid by 6-N-propyl-2-thiouracil treatment for 6 wk; control rats received no treatment. In vivo measured left ventricular (LV) hemodynamic parameters were compared with SERCA2a expression and function in LV tissue. Hypothyroidism decreased LV peak systolic pressure, dP/dt(max), and dP/dt(min) in both WT and TG. However, loss of function was less in TG. Thus slowed relaxation in hypothyroidism was found to be 1.5-fold faster in TG compared with WT (P < 0.05). In parallel, a 1.4-fold higher V(max) value of homogenate SR Ca(2+) uptake was observed in hypothyroid TG (P < 0.05 vs. hypothyroid WT), and the hypothyroidism-caused decline of LV SERCA2a mRNA expression in TG by -24% was markedly less than the decrease of -49% in WT (P < 0.05). A linear relationship was observed between the SERCA2a/PLB mRNA ratio values and the V(max) values of SR Ca(2+) uptake when the respective data of all experimental groups were plotted together (r = 0.90). The data show that expression of the TH-insensitive SERCA2a minigene compensates for loss of expressional activity of the TH-responsive native SERCA2a gene in the female hypothyroid rat heart. However, SR Ca(2+) uptake and in vivo heart function were only partially rescued.

Are there differences in the catalytic activity per unit enzyme of recombinantly expressed and human liver microsomal cytochrome P450 2C9? A systematic investigation into inter-system extrapolation factors.

PubMed

Crewe, H K; Barter, Z E; Yeo, K Rowland; Rostami-Hodjegan, A

2011-09-01

The 'relative activity factor' (RAF) compares the activity per unit of microsomal protein in recombinantly expressed cytochrome P450 enzymes (rhCYP) and human liver without separating the potential sources of variation (i.e. abundance of enzyme per mg of protein or variation of activity per unit enzyme). The dimensionless 'inter-system extrapolation factor' (ISEF) dissects differences in activity from those in CYP abundance. Detailed protocols for the determination of this scalar, which is used in population in vitro-in vivo extrapolation (IVIVE), are currently lacking. The present study determined an ISEF for CYP2C9 and, for the first time, systematically evaluated the effects of probe substrate, cytochrome b5 and methods for assessing the intrinsic clearance (CL(int) ). Values of ISEF for S-warfarin, tolbutamide and diclofenac were 0.75 ± 0.18, 0.57 ± 0.07 and 0.37 ± 0.07, respectively, using CL(int) values derived from the kinetic values V(max) and K(m) of metabolite formation in rhCYP2C9 + reductase + b5 BD Supersomes™. The ISEF values obtained using rhCYP2C9 + reductase BD Supersomes™ were more variable, with values of 7.16 ± 1.25, 0.89 ± 0.52 and 0.50 ± 0.05 for S-warfarin, tolbutamide and diclofenac, respectively. Although the ISEF values obtained from rhCYP2C9 + reductase + b5 for the three probe substrates were statistically different (p < 0.001), the use of the mean value of 0.54 resulted in predicted oral clearance values for all three substrates within 1.4 fold of the observed literature values. For consistency in the relative activity across substrates, use of a b5 expressing recombinant system, with the intrinsic clearance calculated from full kinetic data is recommended for generation of the CYP2C9 ISEF. Furthermore, as ISEFs have been found to be sensitive to differences in accessory proteins, rhCYP system specific ISEFs are recommended. Copyright © 2011 John Wiley & Sons, Ltd.

Optimal Protective Hypothermia in Arrested Mammalian Hearts

PubMed Central

Villet, Outi M.; Ge, Ming; Sekhar, Laigam N.; Corson, Marshall A.; Tylee, Tracy S.; Fan, Lu-Ping; Yao, Lin; Zhu, Chun; Olson, Aaron K.; Buroker, Norman E.; Xu, Cheng-Su; Anderson, David L.; Soh, Yong-Kian; Wang, Elise; Chen, Shi-Han; Portman, Michael A.

2015-01-01

Many therapeutic hypothermia recommendations have been reported, but the information supporting them is sparse, and reveals a need for the data of target therapeutic hypothermia (TTH) from well-controlled experiments. The core temperature ≤35°C is considered as hypothermia, and 29°C is a cooling injury threshold in pig heart in vivo. Thus, an optimal protective hypothermia (OPH) should be in the range 29–35°C. This study was conducted with a pig cardiopulmonary bypass preparation to decrease the core temperature to 29–35°C range at 20 minutes before and 60 minutes during heart arrest. The left ventricular (LV) developed pressure, maximum of the first derivative of LV (dP/dtmax), cardiac power, heart rate, cardiac output, and myocardial velocity (Vmax) were recorded continuously via an LV pressure catheter and an aortic flow probe. At 20 minutes of off-pump during reperfusion after 60 minutes arrest, 17 hypothermic hearts showed that the recovery of Vmax and dP/dtmax established sigmoid curves that consisted of two plateaus: a good recovery plateau at 29–30.5°C, the function recovered to baseline level (BL) (Vmax=118.4%±3.9% of BL, LV dP/dtmax=120.7%±3.1% of BL, n=6); another poor recovery plateau at 34–35°C (Vmax=60.2%±2.8% of BL, LV dP/dtmax=28.0%±5.9% of BL, p<0.05, n=6; ), which are similar to the four normothermia arrest (37°C) hearts (Vmax=55.9%±4.8% of BL, LV dP/dtmax=24.5%±2.1% of BL, n=4). The 32–32.5°C arrest hearts showed moderate recovery (n=5). A point of inflection (around 30.5–31°C) existed at the edge of a good recovery plateau followed by a steep slope. The point presented an OPH that should be the TTH. The results are concordant with data in the mammalian hearts, suggesting that the TTH should be initiated to cool core temperature at 31°C. PMID:25514569

Methane and Trichloroethylene Degradation by Methylosinus trichosporium OB3b Expressing Particulate Methane Monooxygenase

PubMed Central

Lontoh, Sonny; Semrau, Jeremy D.

1998-01-01

Whole-cell assays of methane and trichloroethylene (TCE) consumption have been performed on Methylosinus trichosporium OB3b expressing particulate methane monooxygenase (pMMO). From these assays it is apparent that varying the growth concentration of copper causes a change in the kinetics of methane and TCE degradation. For M. trichosporium OB3b, increasing the copper growth concentration from 2.5 to 20 μM caused the maximal degradation rate of methane (Vmax) to decrease from 300 to 82 nmol of methane/min/mg of protein. The methane concentration at half the maximal degradation rate (Ks) also decreased from 62 to 8.3 μM. The pseudo-first-order rate constant for methane, Vmax/Ks, doubled from 4.9 × 10−3 to 9.9 × 10−3 liters/min/mg of protein, however, as the growth concentration of copper increased from 2.5 to 20 μM. TCE degradation by M. trichosporium OB3b was also examined with varying copper and formate concentrations. M. trichosporium OB3b grown with 2.5 μM copper was unable to degrade TCE in both the absence and presence of an exogenous source of reducing equivalents in the form of formate. Cells grown with 20 μM copper, however, were able to degrade TCE regardless of whether formate was provided. Without formate the Vmax for TCE was 2.5 nmol/min/mg of protein, while providing formate increased the Vmax to 4.1 nmol/min/mg of protein. The affinity for TCE also increased with increasing copper, as seen by a change in Ks from 36 to 7.9 μM. Vmax/Ks for TCE degradation by pMMO also increased from 6.9 × 10−5 to 5.2 × 10−4 liters/min/mg of protein with the addition of formate. From these whole-cell studies it is apparent that the amount of copper available is critical in determining the oxidation of substrates in methanotrophs that are expressing only pMMO. PMID:16349516

Electrophysiological effects of haloperidol on isolated rabbit Purkinje fibers and guinea pigs papillary muscles under normal and simulated ischemia.

PubMed

Yan, Dong; Cheng, Lu-feng; Song, Hong-Yan; Turdi, Subat; Kerram, Parhat

2007-08-01

Overdoses of haloperidol are associated with major ventricular arrhythmias, cardiac conduction block, and sudden death. The aim of this experiment was to study the effect of haloperidol on the action potentials in cardiac Purkinje fibers and papillary muscles under normal and simulated ischemia conditions in rabbits and guinea pigs. Using the standard intracellular microelectrode technique, we examined the effects of haloperidol on the action potential parameters [action potential amplitude (APA), phase 0 maximum upstroke velocity (V(max)), action potential amplitude at 90% of repolarization (APD(90)), and effective refractory period (ERP)] in rabbit cardiac Purkinje fibers and guinea pig cardiac papillary cells, in which both tissues were under simulated ischemic conditions. Under ischemic conditions, different concentrations of haloperidol depressed APA and prolonged APD(90) in a concentration-dependent manner in rabbit Purkinje fibers. Haloperidol (3 micromol/L) significantly depressed APA and prolonged APD(90), and from 1 micromol/L, haloperidol showed significant depression on V(max); ERP was not significantly affected. In guinea pig cardiac papillary muscles, the thresholds of significant reduction in APA, V(max), EPR, and APD(90) were 10, 0.3, 1, and 1 mumol/L, respectively, for haloperidol. Compared with cardiac conductive tissues, papillary muscles were more sensitive to ischemic conditions. Under ischemia, haloperidol prolonged ERP and APD(90) in a concentration-dependent manner and precipitated the decrease in V(max) induced by ischemia. The shortening of ERP and APD(90) in papillary muscle action potentials may be inhibited by haloperidol.

Antioxidant and Inhibitory Effects of Saponin Extracts from Dianthus basuticus Burtt Davy on Key Enzymes Implicated in Type 2 Diabetes In vitro.

PubMed

Nafiu, Mikhail Olugbemiro; Ashafa, Anofi Omotayo Tom

2017-01-01

Dianthus basuticus is a plant of South African origin with various acclaimed pharmaceutical potentials. This study explored the antioxidant and antidiabetic activities of saponin extract from D. basuticus in vitro . Antioxidant activity of saponin was evaluated by 2,2-diphenyl-1-picrylhydrazyl (DPPH) and nitric oxide (*NO)-free radical scavenging activity while antidiabetic potentials were measured by the α-amylase and α-glucosidase inhibitory activities of the saponin extract. The results showed that the saponin extract, compared with quercetin, displayed better DPPH (IC 50 = 6.95 mg/ml) and NO (IC 50 = 3.31 mg/ml) radical scavenging capabilities. Similarly, the saponin extracts elicited stronger α-glucosidase (IC 50 = 3.80 mg/ml) and moderate α-amylase (IC 50 = 4.18 mg/ml) inhibitory activities as compared to acarbose. Saponin exhibited a competitive mode of inhibition on α-amylase with same maximum velocity (Vmax) of 0.0093 mM/min for saponin compared with control 0.0095 mM/min and different the Michaelis constant (Km) values of 2.6 × 10 -6 mM and 2.1 × 10 -5 mM, respectively, while for α-glucosidase, the inhibition was uncompetitive, Vmax of 0.027 mM/min compared with control 0.039 mM/min and Km values of 1.02 × 10 -6 mM and 1.38 × 10 -6 mM, respectively. The gas chromatography-mass spectrometric analysis revealed the presence of bioactive like β- and α-amyrin, 3-O-methyl-D-glucose, methyl commate, and olean-12-en-3-beta-ol. Overall, the data suggested that the saponin extract from D. basuticus has potentials as natural antioxidants and antidiabetics. Saponin extract from Dianthus basuticus displayed promising antidiabetic and antioxidant activitySaponin competitively and uncompetitively inhibited a-amylase and a-glucosidase, respectivelyThe stronger inhibition of α-glucosidase and moderate inhibition of α-amylase by saponin extract from D. basuticus is promising good antidiabetes compared with existing drugs with associated side effects. Abbreviations used: DPPH: 2,2-diphenyl-1-picrylhydrazyl, Km: The Michaelis constant, Vmax: Maximum velocity, ROS: Reactive oxygen species, NIDDM: Non-insulin-dependent diabetes mellitus, UFS: University of the Free State, GC-MS: Gas chromatography-mass spectrometric, MS: Mass spectrometry, NIST: National Institute of Standards and Technology, DNS: 3,5-dinitrosalicylic acid, NO: Nitric oxide, RNS: Reactive nitrogen species, PNPG: p-Nitrophenyl-α-D-glucopyranoside.

Antioxidant and Inhibitory Effects of Saponin Extracts from Dianthus basuticus Burtt Davy on Key Enzymes Implicated in Type 2 Diabetes In vitro

PubMed Central

Nafiu, Mikhail Olugbemiro; Ashafa, Anofi Omotayo Tom

2017-01-01

Context: Dianthus basuticus is a plant of South African origin with various acclaimed pharmaceutical potentials. Aims: This study explored the antioxidant and antidiabetic activities of saponin extract from D. basuticus in vitro. Materials and Methods: Antioxidant activity of saponin was evaluated by 2,2-diphenyl-1-picrylhydrazyl (DPPH) and nitric oxide (*NO)-free radical scavenging activity while antidiabetic potentials were measured by the α-amylase and α-glucosidase inhibitory activities of the saponin extract. Results: The results showed that the saponin extract, compared with quercetin, displayed better DPPH (IC50 = 6.95 mg/ml) and NO (IC50 = 3.31 mg/ml) radical scavenging capabilities. Similarly, the saponin extracts elicited stronger α-glucosidase (IC50 = 3.80 mg/ml) and moderate α-amylase (IC50 = 4.18 mg/ml) inhibitory activities as compared to acarbose. Saponin exhibited a competitive mode of inhibition on α-amylase with same maximum velocity (Vmax) of 0.0093 mM/min for saponin compared with control 0.0095 mM/min and different the Michaelis constant (Km) values of 2.6 × 10-6 mM and 2.1 × 10-5 mM, respectively, while for α-glucosidase, the inhibition was uncompetitive, Vmax of 0.027 mM/min compared with control 0.039 mM/min and Km values of 1.02 × 10-6 mM and 1.38 × 10-6 mM, respectively. The gas chromatography-mass spectrometric analysis revealed the presence of bioactive like β- and α-amyrin, 3-O-methyl-D-glucose, methyl commate, and olean-12-en-3-beta-ol. Conclusion: Overall, the data suggested that the saponin extract from D. basuticus has potentials as natural antioxidants and antidiabetics. SUMMARY Saponin extract from Dianthus basuticus displayed promising antidiabetic and antioxidant activitySaponin competitively and uncompetitively inhibited a-amylase and a-glucosidase, respectivelyThe stronger inhibition of α-glucosidase and moderate inhibition of α-amylase by saponin extract from D. basuticus is promising good antidiabetes compared with existing drugs with associated side effects. Abbreviations used: DPPH: 2,2-diphenyl-1-picrylhydrazyl, Km: The Michaelis constant, Vmax: Maximum velocity, ROS: Reactive oxygen species, NIDDM: Non-insulin-dependent diabetes mellitus, UFS: University of the Free State, GC-MS: Gas chromatography-mass spectrometric, MS: Mass spectrometry, NIST: National Institute of Standards and Technology, DNS: 3,5-dinitrosalicylic acid, NO: Nitric oxide, RNS: Reactive nitrogen species, PNPG: p-Nitrophenyl-α-D-glucopyranoside. PMID:29200716

Ethanol intake and sup 3 H-serotonin uptake I: A study in Fawn-Hooded rats

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

Daoust, M.; Compagnon, P.; Legrand, E.

1991-01-01

Ethanol intake and synaptosomal {sup 3}H-serotonin uptake were studied in male Fawn-Hooded and Sprague-Dawley rats. Fawn-Hooded rats consumed more alcohol and more water than Sprague-Dawley rats. Plasma alcohol levels of Sprague-Dawley rats were not detectable but were about 5 mg/dl in Fawn-Hooded rats. Ethanol intake increased the Vmax of serotonin uptake in Fawn-Hooded rats in hippocampus and cortex, but not in thalamus. In Fawn-Hooded rats, serotonin uptake (Vmax) was higher than in Sprague-Dawley rats cortex. Ethanol intake reduced the Vmax of serotonin uptake in Fawn-Hooded rats in hippocampus and cortex. In cortex, the carrier affinity for serotonin was increased inmore » alcoholized Fawn-Hooded rats. These results indicate that synaptosomal {sup 3}H-serotonin uptake is affected by ethanol intake. In Fawn-Hooded rats, high ethanol consumption is associated with high serotonin uptake. In rats presenting high serotonin uptake, alcoholization reduces {sup 3}H-serotonin internalization in synaptosomes, indicating a specific sensitivity to alcohol intake of serotonin uptake system.« less

Nonlinear pharmacokinetics of visnagin in rats after intravenous bolus administration.

PubMed

Haug, Karin G; Weber, Benjamin; Hochhaus, Guenther; Butterweck, Veronika

2012-01-23

Ammi visnaga L. (syn. Khella, Apiaceae) preparations have traditionally been used in the Middle East for the treatment of kidney stone disease. Visnagin, a furanocoumarin derivative, is one of the main compounds of Ammi visnaga with potential effects on kidney stone prevention. To date, no information is available about the pharmacokinetic (PK) properties of visnagin. It was the aim of the study to characterize the PK properties of visnagin after intravenous (i.v.) bolus administration in rats and to develop an adequate model for the description of the observed data, including model parameter estimates. Therefore, three doses of visnagin (1.25, 2.5, and 5mg/kg) solubilized in 25% Captisol® were administered by i.v. bolus injection to male Sprague-Dawley rats. Plasma samples were extracted and subsequently analyzed using a validated LC-MS/MS method. Both non-compartmental and compartmental PK analyses were performed. A stepwise model building approach was applied including nonlinear mixed effect modeling for final model selection and to obtain final model estimates in NONMEM VI. The average areas under the curve (AUC(0-last)) after doses of 1.25, 2.5, and 5mg/kg were 1.03, 3.61, and 12.6 mg *h/l, respectively. The shape of the plasma concentration-time profiles and the observed disproportionate increase in AUC(0-last) with increasing dose suggested nonlinearity in the elimination of visnagin. A two-compartment Michaelis-Menten model provided the best fit with following typical values of the parameter estimates: 2.09 mg/(l*h) (V(max)), 0.08 mg/l (K(M)), 0.175 l (V(C)), 1.0 h⁻¹ (k₁₂), and 1.22 h⁻¹ (k₂₁). Associated inter-subject variability estimates (% CV) for V(max), K(M) and V(C) were 21.8, 70.9, and 9.2, respectively. Intra-subject variability (constant CV error model) was estimated to be 7.0%. The results suggest the involvement of a saturable process in the elimination of visnagin, possibly an enzyme or transporter system. Copyright © 2011 Elsevier B.V. All rights reserved.

Kinetic and crystallographic studies of Escherichia coli UDP-N-acetylmuramate:L-alanine ligase.

PubMed Central

Emanuele, J. J.; Jin, H.; Jacobson, B. L.; Chang, C. Y.; Einspahr, H. M.; Villafranca, J. J.

1996-01-01

Uridine diphosphate-N-acetylmuramate:L-alanine ligase (EC 6.3.2.8, UNAM:L-Ala ligase or MurC gene product) catalyzes the ATP-dependent ligation of the first amino acid to the sugar moiety of the peptidoglycan precursor. This is an essential step in cell wall biosynthesis for both gram-positive and gram-negative bacteria. Optimal assay conditions for initial velocity studies have been established. Steady-state assays were carried out to determine the effect of various parameters on enzyme activity. Factors studies included: cation specificity, ionic strength, buffer composition and pH. At 37 degrees C and pH 8.0, kcat was equal to 980 +/- 40 min-1, while K(m) values for ATP, UNAM, and L-alanine were, 130 +/- 10, 44 +/- 3, and 48 +/- 6 microM, respectively. Of the metals tested only Mn, Mg, and Co were able to support activity. Sodium chloride, potassium chloride, ammonium chloride, and ammonium sulfate had no effect on activity up to 75 mM levels. The enzyme, in appropriate buffer, was stable enough to be assayed over the pH range of 5.6 to 10.1. pH profiles of Vmax/K(m) for the three substrates and of Vmax were obtained. Crystallization experiments with the enzyme produced two crystal forms. One of these has been characterized by X-ray diffraction as monoclinic, space group C2, with cell dimensions a = 189.6, b = 92.1, c = 75.2 A, beta = 105 degrees, and two 54 kDa molecules per asymmetric unit. It was discovered that the enzyme will hydrolyze ATP in the absence of L-alanine. This L-alanine independent activity is dependent upon the concentrations of both ATP and UNAM; kcat for this activity is less than 4% of the biosynthetic activity measured in the presence of saturating levels of L-alanine. Numerous L-alanine analogs tested were shown to stimulate ATP hydrolysis. A number of these L-alanine analogs produced novel products as accessed by HPLC and mass spectral analysis. All of the L-alanine analogs tested as inhibitors were competitive versus L-alanine. PMID:8976565

Isolation and properties of AMP deaminase from jumbo squid (Dosidicus gigas) mantle muscle from the Gulf of California, Mexico.

PubMed

Marquez-Rios, E; Pacheco-Aguilar, R; Castillo-Yañez, F J; Figueroa-Soto, C G; Ezquerra-Brauer, J M; Gollas-Galvan, T

2008-09-01

Adenosine monophosphate (AMP) deaminase was purified from jumbo squid mantle muscle by chromatography in cellulose phosphate, Q-Fast and 5'-AMP sepharose. Specific activity of 2.5U/mg protein, 4.5% recovery and 133.68 purification fold were obtained at the end of the experiment. SDS-PAGE showed a single band with 87kDa molecular mass, native PAGE proved a band of 178kDa, whereas gel filtration detected a 180kDa protein, suggesting the homodimeric nature of this enzyme, in which subunits are not linked by covalent forces. Isoelectric focusing of this enzyme showed a pI of 5.76, which agrees with pI values of AMP deaminase from other invertebrate organisms. AMP deaminase presented a kinetic sigmoidal plot with Vmax of 1.16μM/min/mg, Km of 13mM, Kcat of 3.48μM.s(-1) and a Kcat/Km of 267 (mol/L)(-1).s(-1). The apparent relative low catalytic activity of jumbo squid muscle AMP deaminase in the absence of positive effectors is similar to that reported for homologous enzymes in other invertebrate organisms. Copyright © 2008 Elsevier Ltd. All rights reserved.

Optimization studies on production of a salt-tolerant protease from Pseudomonas aeruginosa strain BC1 and its application on tannery saline wastewater treatment

PubMed Central

Sivaprakasam, Senthilkumar; Dhandapani, Balaji; Mahadevan, Surianarayanan

2011-01-01

Treatment and safe disposal of tannery saline wastewater, a primary effluent stream that is generated by soaking salt-laden hides and skin is one of the major problems faced by the leather manufacturing industries. Conventional treatment methods like solar evaporation ponds and land composting are not eco-friendly as they deteriorate the ground water quality. Though, this waste stream is comprised of high concentration of dissolved proteins the presence of high salinity (1–6 % NaCl by wt) makes it non-biodegradable. Enzymatic treatment is one of the positive alternatives for management of such kind of waste streams. A novel salt-tolerant alkaline protease obtained from P.aeruginosa (isolated from tannery saline wastewater) was used for enzymatic degradation studies. The effect of various physical factors including pH, temperature, incubation time, protein source and salinity on the activity of identified protease were investigated. Kinetic parameters (Km , Vmax) were calculated for the identified alkaline protease at varying substrate concentrations. Tannery saline wastewater treated with identified salt tolerant protease showed 75 % protein removal at 6 h duration and 2 % (v/v) protease addition was found to be the optimum dosage value. PMID:24031785

Soy Pulp Extract Inhibits Angiotensin I-Converting Enzyme (ACE) Activity In Vitro: Evidence for Its Potential Hypertension-Improving Action.

PubMed

Nishibori, Naoyoshi; Kishibuchi, Reina; Morita, Kyoji

2017-05-04

Soy pulp, called "okara" in Japanese, is known as a by-product of the production of bean curd (tofu), and expected to contain a variety of biologically active substances derived from soybean. However, the biological activities of okara ingredients have not yet been fully understood, and the effectiveness of okara as a functional food seems necessary to be further evaluated. Then the effect of okara extract on angiotensin I-converting enzyme (ACE) activity was examined in vitro, and the extract was shown to cause the inhibition of ACE activity in a manner depending on its concentration. Kinetic analysis indicated that this enzyme inhibition was accompanied by an increase in the Km value without any change in Vmax. Further studies suggested that putative inhibitory substances contained in the extract might be heat stable and dialyzable, and recovered mostly in the peptide fraction obtained by a spin-column separation and a high performance liquid chromatography (HPLC) fractionation. Therefore, the extract was speculated to contain small-size peptides responsible for the inhibitory effect of okara extract on ACE activity, and could be expected to improve the hypertensive conditions by reducing the production of hypertensive peptide.

A Search for Low-Luminosity BL Lacertae Objects

NASA Astrophysics Data System (ADS)

Rector, Travis A.; Stocke, John T.; Perlman, Eric S.

1999-05-01

Many properties of BL Lacs have become explicable in terms of the ``relativistic beaming'' hypothesis, whereby BL Lacs are FR 1 radio galaxies viewed nearly along the jet axis. However, a possible problem with this model is that a transition population between beamed BL Lacs and unbeamed FR 1 galaxies has not been detected. A transition population of ``low-luminosity BL Lacs'' was predicted to exist in abundance in X-ray-selected samples such as the Einstein Extended Medium Sensitivity Survey (EMSS) by Browne & Marcha. However, these BL Lacs may have been misidentified as clusters of galaxies. We have conducted a search for such objects in the EMSS with the ROSAT High-Resolution Imager (HRI) here we present ROSAT HRI images, optical spectra, and VLA radio maps for a small number of BL Lacs that were previously misidentified in the EMSS catalog as clusters of galaxies. While these objects are slightly lower in luminosity than other EMSS BL Lacs, their properties are too similar to the other BL Lacs in the EMSS sample to ``bridge the gap'' between BL Lacs and FR 1 radio galaxies. Also, the number of new BL Lacs found is too low to alter significantly the X-ray luminosity function or value for the X-ray-selected EMSS BL Lac sample. Thus, these observations do not explain fully the discrepancy between the X-ray- and radio-selected BL Lac samples.

Power output and force-velocity relationship of red and white muscle fibres from the Pacific blue marlin (Makaira nigricans).

PubMed

Johnston, I A; Salamonski, J

1984-07-01

Single white fibres and small bundles (two to three) of red fibres were isolated from the trunk muscle of Pacific Blue Marlin (50-121 kg body weight). Fibres were chemically skinned with 1% Brij. Maximum Ca2+-activated force production (Po) was 57 kN m-2 for red fibres and 176 kN m-2 for white fibres at 25 degrees C. The force-velocity (P-V) characteristics of these fibres were determined at 15 and 25 degrees C. Points below 0.6 Po on the P-V curve could be fitted to a linear form of Hill's equation. The degree of curvature of the P-V curve was similar at 15 and 25 degrees C (Hill's constant a/Po = 0.24 and 0.12 for red and white fibres respectively). Extrapolated maximum contraction velocities (Vmax) were 2.5 muscle lengths s-1 (Lo S-1) (red fibres) and 5.3 Lo S-1 (white fibres) at 25 degrees C. Q10(15-25 degrees C) values for Vmax were 1.4 and 1.3 for red and white fibres respectively. Maximum power output had a similar low temperature dependence and amounted to 13 W kg-1 for red and 57 W kg-1 for white muscle at 25 degrees C. The results are briefly discussed in relation to the locomotion and ecology of marlin.

A Method for Multiplexed Measurement of Mitochondrial Pyruvate Carrier Activity*

PubMed Central

Gray, Lawrence R.; Rauckhorst, Adam J.; Taylor, Eric B.

2016-01-01

The discovery that the MPC1 and MPC2 genes encode the protein components of the mitochondrial pyruvate carrier (MPC) has invigorated studies of mitochondrial pyruvate transport and its regulation in normal and disease states. Indeed, recent reports have demonstrated MPC involvement in the control of cell fate in cancer and gluconeogenesis in models of type 2 diabetes. Biochemical measurements of MPC activity are foundational for understanding the role of pyruvate transport in health and disease. We developed a 96-well scaled method of [14C]pyruvate uptake that markedly decreases sample requirements and increases throughput relative to previous techniques. This method was applied to determine the mouse liver MPC Km (28.0 ± 3.9 μm) and Vmax (1.08 ± 0.05 nmol/min/mg), which have not previously been reported. Km and Vmax of the rat liver MPC were found to be 71.2 ± 17 μm and 1.42 ± 0.14 nmol/min/mg, respectively. Additionally, we performed parallel pyruvate uptake and oxidation experiments with the same biological samples and show differential results in response to fasting, demonstrating the continued importance of a direct MPC activity assay. We expect this method will be of value for understanding the contribution of the MPC activity to health and disease states where pyruvate metabolism is expected to play a prominent role. PMID:26823462

Disk galaxy scaling relations at intermediate redshifts. I. The Tully-Fisher and velocity-size relations

NASA Astrophysics Data System (ADS)

Böhm, Asmus; Ziegler, Bodo L.

2016-07-01

Aims: Galaxy scaling relations such as the Tully-Fisher relation (between the maximum rotation velocity Vmax and luminosity) and the velocity-size relation (between Vmax and the disk scale length) are powerful tools to quantify the evolution of disk galaxies with cosmic time. Methods: We took spatially resolved slit spectra of 261 field disk galaxies at redshifts up to z ≈ 1 using the FORS instruments of the ESO Very Large Telescope. The targets were selected from the FORS Deep Field and William Herschel Deep Field. Our spectroscopy was complemented with HST/ACS imaging in the F814W filter. We analyzed the ionized gas kinematics by extracting rotation curves from the two-dimensional spectra. Taking into account all geometrical, observational, and instrumental effects, these rotation curves were used to derive the intrinsic Vmax. Results: Neglecting galaxies with disturbed kinematics or insufficient spatial rotation curve extent, Vmax was reliably determined for 124 galaxies covering redshifts 0.05 < z < 0.97. This is one of the largest kinematic samples of distant disk galaxies to date. We compared this data set to the local B-band Tully-Fisher relation and the local velocity-size relation. The scatter in both scaling relations is a factor of ~2 larger at z ≈ 0.5 than at z ≈ 0. The deviations of individual distant galaxies from the local Tully-Fisher relation are systematic in the sense that the galaxies are increasingly overluminous toward higher redshifts, corresponding to an overluminosity ΔMB = -(1.2 ± 0.5) mag at z = 1. This luminosity evolution at given Vmax is probably driven by younger stellar populations of distant galaxies with respect to their local counterparts, potentially combined with modest changes in dark matter mass fractions. The analysis of the velocity-size relation reveals that disk galaxies of a given Vmax have grown in size by a factor of ~1.5 over the past ~8 Gyr, most likely through accretion of cold gas and/or small satellites. From scrutinizing the combined evolution in luminosity and size, we find that the galaxies that show the strongest evolution toward smaller sizes at z ≈ 1 are not those that feature the strongest evolution in luminosity, and vice versa. Based on observations with the European Southern Observatory Very Large Telescope (ESO-VLT), observing run IDs 65.O-0049, 66.A-0547, 68.A-0013, 69.B-0278B, 70.B-0251A and 081.B-0107A.The full Table 1 is only available at the CDS via anonymous ftp to http://cdsarc.u-strasbg.fr (http://130.79.128.5) or via http://cdsarc.u-strasbg.fr/viz-bin/qcat?J/A+A/592/A64

Autotrophic, hydrogen-oxidizing, denitrifying bacteria in groundwater, potential agents for bioremediation of nitrate contamination

USGS Publications Warehouse

Smith, R.L.; Ceazan, M.L.; Brooks, M.H.

1994-01-01

Addition of hydrogen or formate significantly enhanced the rate of consumption of nitrate in slurried core samples obtained from an active zone of denitrification in a nitrate-contaminated sand and gravel aquifer (Cape Cod, Mass.). Hydrogen uptake by the core material was immediate and rapid, with an apparent K(m) of 0.45 to 0.60 ??M and a V(max) of 18.7 nmol cm-3 h-1 at 30??C. Nine strains of hydrogen-oxidizing denitrifying bacteria were subsequently isolated from the aquifer. Eight of the strains grew autotrophically on hydrogen with either oxygen or nitrate as the electron acceptor. One strain grew mixotrophically. All of the isolates were capable of heterotrophic growth, but none were similar to Paracoccus denitrificans, a well-characterized hydrogen-oxidizing denitrifier. The kinetics for hydrogen uptake during denitrification were determined for each isolate with substrate depletion progress curves; the K(m)s ranged from 0.30 to 3.32 ??M, with V(max)s of 1.85 to 13.29 fmol cell-1 h-1. Because these organisms appear to be common constituents of the in situ population of the aquifer, produce innocuous end products, and could be manipulated to sequentially consume oxygen and then nitrate when both were present, these results suggest that these organisms may have significant potential for in situ bioremediation of nitrate contamination in groundwater.

Effects of genetic polymorphisms on the OCT1 and OCT2-mediated uptake of ranitidine.

PubMed

Meyer, Marleen Julia; Seitz, Tina; Brockmöller, Jürgen; Tzvetkov, Mladen Vassilev

2017-01-01

Ranitidine (Zantac®) is a H2-receptor antagonist commonly used for the treatment of acid-related gastrointestinal diseases. Ranitidine was reported to be a substrate of the organic cation transporters OCT1 and OCT2. The hepatic transporter OCT1 is highly genetically variable. Twelve major alleles confer partial or complete loss of OCT1 activity. The effects of these polymorphisms are highly substrate-specific and therefore difficult to predict. The renal transporter OCT2 has a common polymorphism, Ala270Ser, which was reported to affect OCT2 activity. In this study we analyzed the effects of genetic polymorphisms in OCT1 and OCT2 on the uptake of ranitidine and on its potency to inhibit uptake of other drugs. We characterized ranitidine uptake using HEK293 and CHO cells stably transfected to overexpress wild type OCT1, OCT2, or their naturally occurring allelic variants. Ranitidine was transported by wild-type OCT1 with a Km of 62.9 μM and a vmax of 1125 pmol/min/mg protein. Alleles OCT1*5, *6, *12, and *13 completely lacked ranitidine uptake. Alleles OCT1*2, *3, *4, and *10 had vmax values decreased by more than 50%. In contrast, OCT1*8 showed an increase of vmax by 25%. The effects of OCT1 alleles on ranitidine uptake strongly correlated with the effects on morphine uptake suggesting common interaction mechanisms of both drugs with OCT1. Ranitidine inhibited the OCT1-mediated uptake of metformin and morphine at clinically relevant concentrations. The inhibitory potency for morphine uptake was affected by the OCT1*2 allele. OCT2 showed only a limited uptake of ranitidine that was not significantly affected by the Ala270Ser polymorphism. We confirmed ranitidine as an OCT1 substrate and demonstrated that common genetic polymorphisms in OCT1 strongly affect ranitidine uptake and modulate ranitidine's potential to cause drug-drug interactions. The effects of the frequent OCT1 polymorphisms on ranitidine pharmacokinetics in humans remain to be analyzed.

Effects of genetic polymorphisms on the OCT1 and OCT2-mediated uptake of ranitidine

PubMed Central

Meyer, Marleen Julia; Seitz, Tina; Brockmöller, Jürgen

2017-01-01

Background Ranitidine (Zantac®) is a H2-receptor antagonist commonly used for the treatment of acid-related gastrointestinal diseases. Ranitidine was reported to be a substrate of the organic cation transporters OCT1 and OCT2. The hepatic transporter OCT1 is highly genetically variable. Twelve major alleles confer partial or complete loss of OCT1 activity. The effects of these polymorphisms are highly substrate-specific and therefore difficult to predict. The renal transporter OCT2 has a common polymorphism, Ala270Ser, which was reported to affect OCT2 activity. Aim In this study we analyzed the effects of genetic polymorphisms in OCT1 and OCT2 on the uptake of ranitidine and on its potency to inhibit uptake of other drugs. Methods and results We characterized ranitidine uptake using HEK293 and CHO cells stably transfected to overexpress wild type OCT1, OCT2, or their naturally occurring allelic variants. Ranitidine was transported by wild-type OCT1 with a Km of 62.9 μM and a vmax of 1125 pmol/min/mg protein. Alleles OCT1*5, *6, *12, and *13 completely lacked ranitidine uptake. Alleles OCT1*2, *3, *4, and *10 had vmax values decreased by more than 50%. In contrast, OCT1*8 showed an increase of vmax by 25%. The effects of OCT1 alleles on ranitidine uptake strongly correlated with the effects on morphine uptake suggesting common interaction mechanisms of both drugs with OCT1. Ranitidine inhibited the OCT1-mediated uptake of metformin and morphine at clinically relevant concentrations. The inhibitory potency for morphine uptake was affected by the OCT1*2 allele. OCT2 showed only a limited uptake of ranitidine that was not significantly affected by the Ala270Ser polymorphism. Conclusions We confirmed ranitidine as an OCT1 substrate and demonstrated that common genetic polymorphisms in OCT1 strongly affect ranitidine uptake and modulate ranitidine’s potential to cause drug-drug interactions. The effects of the frequent OCT1 polymorphisms on ranitidine pharmacokinetics in humans remain to be analyzed. PMID:29236753

Biochemical characterization of the bifunctional enzyme dihydrofolate reductase-thymidylate synthase from Leishmania (Viannia) and its evaluation as a drug target.

PubMed

Osorio, Edison; Aguilera, Carolina; Naranjo, Nelson; Marín, Marcel; Muskus, Carlos

2013-01-01

Dihydrofolate reductase (DHFR) has been used successfully as a drug target in the area of anti-bacterial, anti-cancer and anti-malarial therapy. Although this bifunctional enzyme is also a potential drug target for treatment of leishmaniasis, there have been no reports on its efficacy against Leishmania (Viannia) species. The gene encoding the bifunctional DHFR and thymidylate synthase (TS) of Le. (V.) braziliensis was isolated and expressed in E. coli. The enzyme was purified and characterized. The inhibitory effects of antifolates and four aporphine alkaloids on its activity were evaluated. The full-length gene consists of a 1560-bp open reading frame encoding a 58 kDa translated peptide containing DHFR and TS domains linked together in a single polypeptide chain. The recombinant DHFR-TS enzyme revealed Km and Vmax values of 55.35 ± 4.02 µ M (mean ± SE) and 0.02 ± 5.34 x 10 -4 µ M/min respectively for dihydrofolic acid (H₂F). The Le. braziliensis rDHFR-TS have Ki values for antimicrobial antifolates in the µM range. Methotrexate (MTX) was a more-potent inhibitor of enzymatic activity (Ki = 22.0 µM) than trimethoprim (Ki = 33 µM) and pyrimethamine (Ki = 68 µM). These Ki values are significantly lower than those obtained for the aporphine alkaloids. The results of the study show the inhibitory effect of antifolate drugs on enzymatic activity, indicating that Le. braziliensis rDHFR-TS could be a model to studying antifolate compounds as potential antiprotozoal drugs.

Differential responses of targeted lung redox enzymes to rat exposure to 60 or 85% oxygen

PubMed Central

Gan, Zhuohui; Roerig, David L.; Clough, Anne V.

2011-01-01

Rat exposure to 60% O2 (hyper-60) or 85% O2 (hyper-85) for 7 days confers susceptibility or tolerance, respectively, of the otherwise lethal effects of exposure to 100% O2. The objective of this study was to determine whether activities of the antioxidant cytosolic enzyme NAD(P)H:quinone oxidoreductase 1 (NQO1) and mitochondrial complex III are differentially altered in hyper-60 and hyper-85 lungs. Duroquinone (DQ), an NQO1 substrate, or its hydroquinone (DQH2), a complex III substrate, was infused into the arterial inflow of isolated, perfused lungs, and the venous efflux rates of DQH2 and DQ were measured. Based on inhibitor effects and kinetic modeling, capacities of NQO1-mediated DQ reduction (Vmax1) and complex III-mediated DQH2 oxidation (Vmax2) increased by ∼140 and ∼180% in hyper-85 lungs, respectively, compared with rates in lungs of rats exposed to room air (normoxic). In hyper-60 lungs, Vmax1 increased by ∼80%, with no effect on Vmax2. Additional studies revealed that mitochondrial complex I activity in hyper-60 and hyper-85 lung tissue homogenates was ∼50% lower than in normoxic lung homogenates, whereas mitochondrial complex IV activity was ∼90% higher in only hyper-85 lung tissue homogenates. Thus NQO1 activity increased in both hyper-60 and hyper-85 lungs, whereas complex III activity increased in hyper-85 lungs only. This increase, along with the increase in complex IV activity, may counter the effects the depression in complex I activity might have on tissue mitochondrial function and/or reactive oxygen species production and may be important to the tolerance of 100% O2 observed in hyper-85 rats. PMID:21551015

Differential responses of targeted lung redox enzymes to rat exposure to 60 or 85% oxygen.

PubMed

Gan, Zhuohui; Roerig, David L; Clough, Anne V; Audi, Said H

2011-07-01

Rat exposure to 60% O(2) (hyper-60) or 85% O(2) (hyper-85) for 7 days confers susceptibility or tolerance, respectively, of the otherwise lethal effects of exposure to 100% O(2). The objective of this study was to determine whether activities of the antioxidant cytosolic enzyme NAD(P)H:quinone oxidoreductase 1 (NQO1) and mitochondrial complex III are differentially altered in hyper-60 and hyper-85 lungs. Duroquinone (DQ), an NQO1 substrate, or its hydroquinone (DQH(2)), a complex III substrate, was infused into the arterial inflow of isolated, perfused lungs, and the venous efflux rates of DQH(2) and DQ were measured. Based on inhibitor effects and kinetic modeling, capacities of NQO1-mediated DQ reduction (V(max1)) and complex III-mediated DQH(2) oxidation (V(max2)) increased by ∼140 and ∼180% in hyper-85 lungs, respectively, compared with rates in lungs of rats exposed to room air (normoxic). In hyper-60 lungs, V(max1) increased by ∼80%, with no effect on V(max2). Additional studies revealed that mitochondrial complex I activity in hyper-60 and hyper-85 lung tissue homogenates was ∼50% lower than in normoxic lung homogenates, whereas mitochondrial complex IV activity was ∼90% higher in only hyper-85 lung tissue homogenates. Thus NQO1 activity increased in both hyper-60 and hyper-85 lungs, whereas complex III activity increased in hyper-85 lungs only. This increase, along with the increase in complex IV activity, may counter the effects the depression in complex I activity might have on tissue mitochondrial function and/or reactive oxygen species production and may be important to the tolerance of 100% O(2) observed in hyper-85 rats.

Is ADH1C genotype relevant for the cardioprotective effect of alcohol?

PubMed

Høiseth, Gudrun; Magnus, Per; Knudsen, Gun Peggy; Jansen, Mona Dverdal; Næss, Oyvind; Tambs, Kristian; Mørland, Jørg

2013-03-01

The cardioprotective effect of ethanol has been suggested to be linked to one of the ethanol metabolizing enzymes (ADH1C), which constitutes a high V(max) and a low V(max) variant. This has been demonstrated in some studies, while others have not been able to replicate the findings. The aim of the present study was to investigate the relation between the different ADH1C genotypes, death from coronary heart disease (CHD) and alcohol in a material larger than the previously published studies. Eight hundred CHD deaths as well as 1303 controls were genotyped for the high V(max) (γ1) and the low V(max) (γ2) ADH1C variant. Information of alcohol use was available for all subjects. Multiple logistic regression analyses was used to study if the decreased risk of death from CHD in alcohol consuming subjects was more pronounced in subjects homozygous for the γ2 allele (γ2γ2 subjects) compared to γ1γ1 and γ1γ2 subjects. The odds ratio (OR) for death from CHD in alcohol consumers compared to abstainers was similar in the genotype groups, i.e., 0.62 (95% CI: 0.43-0.88) in γ1γ1 subjects and 0.62 (95% CI: 0.42-0.91) in γ2γ2 subjects. Also when stratifying the results by gender and when dividing alcohol consumers into different alcohol consumption groups, there was no difference in the OR between the different genotype groups. This study, which included the largest study group published so far, failed to find any link between the ADH1C genotype and the cardioprotective effects of alcohol. Copyright © 2013 Elsevier Inc. All rights reserved.

Saccharomyces cerevisiae FLO1 Gene Demonstrates Genetic Linkage to Increased Fermentation Rate at Low Temperatures

PubMed Central

Deed, Rebecca C.; Fedrizzi, Bruno; Gardner, Richard C.

2017-01-01

Low fermentation temperatures are of importance to food and beverage industries working with Saccharomyces cerevisiae. Therefore, the identification of genes demonstrating a positive impact on fermentation kinetics is of significant interest. A set of 121 mapped F1 progeny, derived from a cross between haploid strains BY4716 (a derivative of the laboratory yeast S288C) and wine yeast RM11-1a, were fermented in New Zealand Sauvignon Blanc grape juice at 12.5°. Analyses of five key fermentation kinetic parameters among the F1 progeny identified a quantitative trait locus (QTL) on chromosome I with a significant degree of linkage to maximal fermentation rate (Vmax) at low temperature. Independent deletions of two candidate genes within the region, FLO1 and SWH1, were constructed in the parental strains (with S288C representing BY4716). Fermentation of wild-type and deletion strains at 12.5 and 25° confirmed that the genetic linkage to Vmax corresponds to the S288C version of the FLO1 allele, as the absence of this allele reduced Vmax by ∼50% at 12.5°, but not at 25°. Reciprocal hemizygosity analysis (RHA) between S288C and RM11-1a FLO1 alleles did not confirm the prediction that the S288C version of FLO1 was promoting more rapid fermentation in the opposing strain background, suggesting that the positive effect on Vmax derived from S288C FLO1 may only provide an advantage in haploids, or is dependent on strain-specific cis or trans effects. This research adds to the growing body of evidence demonstrating the role of FLO1 in providing stress tolerance to S. cerevisiae during fermentation. PMID:28143947

Decline in spirometric variables in grain workers from start of employment: differential effect of duration of follow up.

PubMed Central

Zejda, J E; Pahwa, P; Dosman, J A

1992-01-01

Prospective study of 164 young men from the start of employment in grain elevators showed that of those seen at the initial evaluation of respiratory state only 30% were available for a complete four year follow up. The drop out of subjects could represent a health related selection leading to the underestimation of respiratory effects of exposure to grain dust as assessed in the survivor group. This hypothesis was examined by comparisons of longitudinal changes in lung function in four groups defined by the duration of follow up involving the initial examination and periodic evaluations after one, two, and four years of work. Sixty four men were tested only on the initial examination (group I), 18 underwent two (group II), 31 underwent three (group III), and 51 (group IV) all four examinations. The groups had similar mean ages (range: 19.4-20.1 years), mean duration of previous exposure to grain dust (range: 8-13 weeks), smoking habits, lung function, and prevalences of respiratory symptoms evaluated on the initial occasion. The average decline in lung function over the first year was associated with duration of follow up. The annual decline in FVC (ml) was 58 in group II, 41 in group III and -55 (increase) in group IV; the decline in FEV1 (ml) was 224, 130, and 70 respectively. The differences for the annual declines of FEV1, FEF25-759 Vmax509 and Vmax25 were significant between groups II and IV, and the FEF25-759 Vmax509 and Vmax25 differed significantly between groups II and III. The results show that the restriction of analysis to the survivors may underestimate the relation between work and respiratory impairment. PMID:1515349

Cholinesterase assay for monitoring the kinetics of the JD6. 5 organophosphorus acid anhydrase in detoxification of diisopropylfluorophosphate. Final report, January-June 1988

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

Yeh, H.R.; Cheng, T.C.; DeFrank, J.J.

1992-06-01

In the present studies, cholinesterase was used for monitoring the enzymatic activities of the JD6.5 organophosphorus acid anhydrase. The kinetic data indicated that: (1) the first order of kinetic constants (k) and Vmax values of the enzymatic reactions increased as the concentrations of the enzyme increased; (2) while the half-life (tl/2) of diisopropylfluorophosphate (DFP) hydrolysis decreased as the enzyme concentrations increased; (3) the minimum time required for hydrolysis of 9mM of DFP was 3 min at the concentrations of the enzyme present; Km values of DFP were found to be in range of 5mM; and (4) both MnCl2 and NaClmore » were found to be required for the optimal activity of the enzyme.« less

Risk factors for left ventricular hypertrophy: role of Na(+)-Li+ countertransport.

PubMed

Neves, P L; Faisca, M; Gomes, V; Cacodcar, S; Bernardo, I; Anunciada, A I; Viegas, E; Martins, H; da Silva, A M

1996-06-01

Left ventricular hypertrophy (LVH) is associated with an increase in cardiovascular death in essential hypertension (EH). The factors involved in LVH are multiple and complex. We looked for risk factors of LVH in a group of 28 nonobese patients with EH (mean age = 45.3 years). We analyzed the activity of several erythrocyte ion transports (Vmax of NaLi countertransport, NaKCl cotransport and NaK-pump, and the Na-leak Kp Na), the intracellular Na and the insulin sensitivity index. All these parameters were used as independent variables whereas the left ventricular mass index (LVMI) was used as the dependent variable. Variables showing a significant univariate correlation (age, time of EH, mean blood pressure and Vmax of NaLi countertransport) were introduced in a stepwise multiple regression model. Only age (P = 0.014), time of EH (P = 0.038) and Vmax of NaLi countertransport (P = 0.032) were independently associated with LVMI (R2 = 0.581, P = 0.0001). The NaLi CT, an operating mode of the NaH exchanger that facilitates cellular growth, may be a marker of LVH, and consequently a marker of increased cardiovascular risk.

Small intestinal sulphoxidation of albendazole.

PubMed

Villaverde, C; Alvarez, A I; Redondo, P; Voces, J; Del Estal, J L; Prieto, J G

1995-05-01

1. The in vitro sulphoxidation of Albendazole (ABZ) by rat intestinal microsomes has been examined. The results revealed intestinal sulphoxidation of ABZ by intestinal microsomes in a NADPH-dependent enzymatic system. The kinetic constants for sulphoxidase activity were Vmax = 46 pmol/min/mg protein and Michaelis constant Km = 6.8 microM. 2. The possible effect of inducers (Arochlor 1254 and ABZ pretreatment) and inhibitors (erythromycin, methimazole, carbon monoxide and fenbendazole), was also studied. In rat pretreated with Arochlor 1254, Vmax was 52 pmol/min/mg protein, whereas oral administration of ABZ increased the intestinal sulphoxidation of the drug, Vmax being 103 pmol/min/mg protein. 3. Erythromycin did not change the enzymatic bioconversion of ABZ, but methimazole and carbon monoxide inhibited the enzyme activity by approximately 60 and 30% respectively. Fenbendazole (a structural analogue of ABZ) was a competitive inhibitor of the sulphoxidation process, characterized by a Ki or 69 microM. 4. These data demonstrate that the intestinal enzymes contributing to the initial sulphoxidation of ABZ may be similar to the hepatic enzymes involved in the biotransformation process by the P450 and FMO systems, a conclusion that needs to be further established.

Inositol phosphates influence the membrane bound Ca/sup 2 +//Mg/sup 2 +/ stimulated ATPase from human erythrocyte membranes

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

Kester, M.; Ekholm, J.; Kumar, R.

1986-03-01

The modulation by exogenous inositol phosphates of the membrane Ca/sup 2 +//Mg/sup 2 +/ ATPase from saponin/EGTA lysed human erythrocytes was determined in a buffer (pH 7.6) containing histidine, 80 mM, MgCl/sub 2/, 3.3 mM, NaCl, 74 mM, KCl, 30 mM, Na/sub 2/ATP, 2.3 mM, ouabain, 0.83 mM, with variable amounts of CaCl/sub 2/ and EGTA. The ATPase assay was linear with time at 44/sup 0/C. The inositol phosphates were commercially obtained and were also prepared from /sup 32/P labeled rabbit platelet inositol phospholipids. Inositol triphosphate (IP/sub 3/) elevated the Ca/sup 2 +//Mg/sup 2 +/ ATPase activity over basal levelsmore » in a dose, time, and calcium dependent manner and were increased up to 85% of control values. Activities for the Na/sup +//K/sup +/-ATPase and a Mg/sup 2 +/ ATPase were not effected by IP/sub 3/. Ca/sup 2 +//Mg/sup 2 +/APTase activity with IP/sub 2/ or IP/sub 3/ could be synergistically elevated with calmodulin addition. The activation of the ATPase with IP/sub 3/ was calcium dependent in a range from .001 to .02 mM. The apparent Km and Vmax values were determined for IP/sub 3/ stimulated Ca/sup 2 +//Mg/sup 2 +/ ATPase.« less

Purification and Characterization of Thermostable and Detergent-Stable α-Amylase from Anoxybacillus sp. AH1

PubMed Central

Bekler, Fatma Matpan; Pirinççioğlu, Hemşe; Güven, Reyhan Gül; Güven, Kemal

2016-01-01

Summary A thermostable and detergent-stable α-amylase from a newly isolated Anoxybacillus sp. AH1 was purified and characterized. Maximum enzyme production (1874.8 U/mL) was obtained at 24 h of incubation. The amylase was purified by using Sephadex G-75 gel filtration, after which an 18-fold increase in specific activity and a yield of 9% were achieved. The molecular mass of the purified enzyme was estimated at 85 kDa by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). The optimum pH and temperature values of the enzyme were 7.0 and 60 °C, respectively. The enzyme was highly stable in the presence of 30% glycerol, retaining 85% of its original activity at 60 °C within 120 min. Km and vmax values were 0.102 µmol and 0.929 µmol/min, respectively, using Lineweaver-Burk plot. The enzyme activity was increased by various detergents, but it was significantly inhibited in the presence of urea. Mg2+ and Ca2+ also significantly activated α-amylase, while Zn2+, Cu2+ and metal ion chelators ethylenediaminetetraacetic acid (EDTA) and 1,10-phenanthroline (phen) greatly inhibited the enzyme activity. α-Amylase activity was enhanced by β-mercaptoethanol (β-ME) and dithiothreitol (DTT) to a great extent, but inhibited by p-chloromercuribenzoic acid (PCMB). Iodoacetamide (IAA) and N-ethylmaleimide (NEM) had a slight, whereas phenylmethylsulfonyl fluoride (PMSF) had a strong inhibitory effect on the amylase activity. PMID:27904395

[Enzymatic characteristics of peroxidase from Chrysanthemum morifolium cv. Bo-ju].

PubMed

Zhu, Yu-Yun; Lyu, Xin-Lin; Li, Xiang-Wei; Zhang, Dong; Dong, Li-Hua; Zhu, Jing-Jing; Wang, Zhi-Min; Zhang, Jin-Zhen

2018-04-01

The enzymatic browning is one of the main reasons for affecting the quality of medicinal flowers. In the process of chrysanthemum harvesting and processing, improper treatment will lead to the browning and severely impact the appearance and quality of chrysanthemum. Peroxidase enzyme is one of the oxidoreductases that cause enzymatic browning of fresh chrysanthemum. The enzymatic characteristics of peroxidase (POD) in chrysanthemum were studied in this paper. In this experiment, the effects of different reaction substrates and their concentrations, PH value of buffer and reaction temperatures on the activity of POD enzyme were investigated. The results showed that the optimal substrate of POD was guaiacol, and the optimal concentration of POD was 50 mmol·L⁻¹. The optimal pH value and reaction temperature were 4.4 and 30-35 °C, respectively. Michaelis-Menten equation was obtained to express the kinetics of enzyme-catalyzed reaction of POD, Km=0.193 mol·L⁻¹, Vmax=0.329 D·min⁻¹. In addition, the results of POD enzyme thermal stability test showed that the POD enzyme activity was inhibited when being treated at 80 °C for 4 min or at 100 °C for 2 min. The above results were of practical significance to reveal the enzymatic browning mechanism, control the enzymatic browning and improve the quality of chrysanthemum, and can also provide the basis for the harvesting and processing of medicinal materials containing polyphenols. Copyright© by the Chinese Pharmaceutical Association.

Cloning, sequencing, and expression of the gene encoding cyclic 2, 3-diphosphoglycerate synthetase, the key enzyme of cyclic 2, 3-diphosphoglycerate metabolism in Methanothermus fervidus.

PubMed

Matussek, K; Moritz, P; Brunner, N; Eckerskorn, C; Hensel, R

1998-11-01

Cyclic 2,3-diphosphoglycerate synthetase (cDPGS) catalyzes the synthesis of cyclic 2,3-diphosphoglycerate (cDPG) by formation of an intramolecular phosphoanhydride bond in 2,3-diphosphoglycerate. cDPG is known to be accumulated to high intracellular concentrations (>300 mM) as a putative thermoadapter in some hyperthermophilic methanogens. For the first time, we have purified active cDPGS from a methanogen, the hyperthermophilic archaeon Methanothermus fervidus, sequenced the coding gene, and expressed it in Escherichia coli. cDPGS purification resulted in enzyme preparations containing two isoforms differing in their electrophoretic mobility under denaturing conditions. Since both polypeptides showed the same N-terminal amino acid sequence and Southern analyses indicate the presence of only one gene coding for cDPGS in M. fervidus, the two polypeptides originate from the same gene but differ by a not yet identified modification. The native cDPGS represents a dimer with an apparent molecular mass of 112 kDa and catalyzes the reversible formation of the intramolecular phosphoanhydride bond at the expense of ATP. The enzyme shows a clear preference for the synthetic reaction: the substrate affinity and the Vmax of the synthetic reaction are a factor of 8 to 10 higher than the corresponding values for the reverse reaction. Comparison with the kinetic properties of the electrophoretically homogeneous, apparently unmodified recombinant enzyme from E. coli revealed a twofold-higher Vmax of the enzyme from M. fervidus in the synthesizing direction.

Effect of bio-column composed of aged refuse on methane abatement--a novel configuration of biological oxidation in refuse landfill.

PubMed

Han, Dan; Zhao, Youcai; Xue, Binjie; Chai, Xiaoli

2010-01-01

An experimental bio-column composed of aged refuse was installed around the exhaust pipe as a new way to mitigate methane in refuse landfill. One of the objectives of this work was to assess the effect of aged refuse thickness in bio-column on reducing CH4 emissions. Over the study period, methane oxidation was observed at various thicknesses, 5 cm (small size), 10 cm (middle size) and 15 cm (large size), representing one to three times of pipeline diameters. The middle and large size both showed over 90% methane conversion, and the highest methane conversion rate of above 95% occurred in the middle-size column cell. Michaelis-Menten equation addressed the methanotrophs diffusion in different layers of the bio-columns. Maximum methanotrophic activity (Vmax) measured at the three thicknesses ranged from 6.4 x 10(-3) to 15.6 x 10(-3) units, and the half-saturation value (K(M)) ranged from 0.85% to 1.67%. Both the highest Vmax and K(M) were observed at the middle-size of the bio-column, as well as the largest methanotrophs population, suggesting a significant efficiency of methane mitigation happened in the optimum zone with greatest affinity and methanotrophic bacteria activities. Therefore, bio-column is a potential style for methane abatement in landfill, and the aged refuse both naturally formed and artificially placed in the column plays a critical role in CH4 emission.

Cytochrome P-450 isoforms involved in carboxylic acid ester cleavage of Hantzsch pyridine ester of pranidipine.

PubMed

Kudo, S; Okumura, H; Miyamoto, G; Ishizaki, T

1999-02-01

Cytochrome P-450 (CYP) isoforms responsible for the cleavage of Hantzsch pyridine ester at the 3-position of pranidipine were studied in vitro using cDNA-expressed human CYP enzymes. CYP1A1, 1A2, 2D6, and 3A4 cleaved the ester with a catalytic activity of 5.5, 0. 93, 13.1, and 22.4 nmol/30 min/nmol P-450, respectively. CYP2A6, 2B6, 2C8, 2C9, 2C19, and 2E1 were not involved in the de-esterification. The Km and Vmax values for the de-esterification were 11.8 microM and 0.47 nmol/min/nmol P-450 in the CYP2D6-catalyzed reaction and 8. 7 microM and 0.84 nmol/min/nmol P-450 in the CYP3A4-catalyzed reaction. The intrinsic clearance (Vmax/Km) of the de-esterification by CYP3A4 was 2-fold greater than that by CYP2D6. Quinidine almost completely inhibited the CYP2D6-mediated de-esterification at the concentration of 1 x 10(-6) M. Ketoconazole and troleandomycin inhibited the CYP3A4-mediated reaction in a dose-related manner. The results indicate that although the multiple CYP isoforms can catalyze the de-esterification, CYP3A4 and 2D6 are the major isoforms.

Hydrolysis of triolein in phospholipid vesicles and microemulsions by a purified rat liver acid lipase.

PubMed

Burrier, R E; Brecher, P

1983-10-10

An acid lipase was purified from rat liver lysosomes. Lipase purification involved affinity chromatography, gel filtration, and stabilization of the purified preparation using ethylene glycol and Triton X-100. A molecular weight of 67,000-69,000 was determined independently using density gradient centrifugation, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and gel filtration. To study enzyme action, model substrates were prepared by incorporating radiolabeled triolein into either unilamellar vesicles or microemulsions. Substrates were prepared by cosonicating aqueous dispersions of lecithin and triolein. Formation of vesicles or emulsions depended on the relative amount of each lipid and on sonication conditions. Vesicles were prepared at molar ratios between 70:1 and 26:1 (lecithin:triolein) and the microemulsion preparation at a molar ratio of 1:1. The substrate particles were of similar size (220-250 A) as determined by Bio-Gel A-15m chromatography. Hydrolysis of triolein contained in vesicles or emulsions was similar with respect to pH, temperature, and reaction products. Kinetic studies on vesicles with increasing triolein content showed progressively greater Vmax values (0-0.6 mumol/min/mg), and Vmax for the emulsion was 3.1 mumol/min/mg. Addition of human very low or low density lipoprotein produced a dose-dependent inhibition with both substrates. The results show that synthetically prepared microemulsions are stable and effective substrates for the acid lipase and indicate that surface-oriented triolein is hydrolyzed in both preparations.

A designed bifunctional laccase/β-1,3-1,4-glucanase enzyme shows synergistic sugar release from milled sugarcane bagasse.

PubMed

Furtado, G P; Ribeiro, L F; Lourenzoni, M R; Ward, R J

2013-01-01

A bifunctional enzyme has been created by fusing two Bacillus subtilis enzymes: the β-1,3-1,4-glucanase (BglS, EC 3.2.1.73) that hydrolyzes plant cell wall β-glucans and the copper-dependent oxidase laccase (CotA, EC 1.10.3.2) that catalyzes the oxidation of aromatic compounds with simultaneous reduction of oxygen to water. The chimeric laccase/β-1,3-1,4-glucanase was created by insertion fusion of the bglS and cotA genes, and expressed in Escherichia coli. The affinity-purified recombinant chimeric enzyme showed both laccase and glucanase activities, with a maximum laccase activity at pH 4.5 and 75°C that showed a V(max) 30% higher than observed for the parental laccase. The maximum glucanase activity in the chimeric enzyme was at pH 6.0 and 50°C, with a slight reduction in V(max) by ∼10% compared with the parental glucanase. A decreased K(M) resulted in an overall increase in the K(cat)/K(M) value for the glucanase activity of the chimeric enzyme. The hydrolytic activity of the chimera was 20% higher against natural milled sugarcane bagasse as compared with equimolar mixtures of the separate parental enzymes. Molecular dynamics simulations indicated the approximation of the two catalytic domains in the chimeric enzyme, and the formation of an inter-domain interface may underlie the improved catalytic function.

Microbial Group Specific Uptake Kinetics of Inorganic Phosphate and Adenosine-5′-Triphosphate (ATP) in the North Pacific Subtropical Gyre

PubMed Central

Björkman, Karin; Duhamel, Solange; Karl, David M.

2012-01-01

We investigated the concentration dependent uptake of inorganic phosphate (Pi) and adenosine-5′-triphosphate (ATP) in microbial populations in the North Pacific Subtropical Gyre (NPSG). We used radiotracers to measure substrate uptake into whole water communities, differentiated microbial size classes, and two flow sorted groups; Prochlorococcus (PRO) and non-pigmented bacteria (NPB). The Pi concentrations, uptake rates, and Pi pool turnover times (Tt) were (mean, ±SD); 54.9 ± 35.0 nmol L−1 (n = 22), 4.8 ± 1.9 nmol L−1 day−1 (n = 19), and 14.7 ± 10.2 days (n = 19), respectively. Pi uptake into >2 μm cells was on average 12 ± 7% (n = 15) of the total uptake. The kinetic response to Pi (10–500 nmol L−1) was small, indicating that the microorganisms were close to their maximum uptake velocity (Vmax). Vmax averaged 8.0 ± 3.6 nmol L−1 day−1 (n = 19) in the >0.2 μm group, with half saturation constants (Km) of 40 ± 28 nmol L−1 (n = 19). PRO had three times the cell specific Pi uptake rate of NPB, at ambient concentrations, but when adjusted to cells L−1 the rates were similar, and these two groups were equally competitive for Pi. The Tt of γ-P-ATP in the >0.2 μm group were shorter than for the Pi pool (4.4 ± 1.0 days; n = 6), but this difference diminished in the larger size classes. The kinetic response to ATP was large in the >0.2 μm class with Vmax exceeding the rates at ambient concentrations (mean 62 ± 27 times; n = 6) with a mean Vmax for γ-P-ATP of 2.8 ± 1.0 nmol L−1 day−1, and Km at 11.5 ± 5.4 nmol L−1 (n = 6). The NPB contribution to γ-P-ATP uptake was high (95 ± 3%, n = 4) at ambient concentrations but decreased to ∼50% at the highest ATP amendment. PRO had Km values 5–10 times greater than NPB. The above indicates that PRO and NPB were in close competition in terms of Pi acquisition, whereas P uptake from ATP could be attributed to NPB. This apparent resource partitioning may be a niche separating strategy and an important factor in the successful co-existence within the oligotrophic upper ocean of the NPSG. PMID:22701449

Islet transplantation under the kidney capsule fully corrects the impaired skeletal muscle glucose transport system of streptozocin diabetic rats.

PubMed Central

Napoli, R; Davalli, A M; Hirshman, M F; Weitgasser, R; Weir, G C; Horton, E S

1996-01-01

Chronic insulin therapy improves but does not restore impaired insulin-mediated muscle glucose uptake in human diabetes or muscle glucose uptake, transport, and transporter translocation in streptozocin diabetic rats. To determine whether this inability is due to inadequate insulin replacement, we studied fasted streptozocin-induced diabetic Lewis rats either untreated or after islet transplantation under the kidney capsule. Plasma glucose was increased in untreated diabetics and normalized by the islet transplantation (110 +/- 5, 452 +/- 9, and 102 +/- 3 mg/dl in controls, untreated diabetics, and transplanted diabetics, respectively). Plasma membrane and intracellular microsomal membrane vesicles were prepared from hindlimb skeletal muscle of basal and maximally insulin-stimulated rats. Islet transplantation normalized plasma membrane carrier-mediated glucose transport Vmax, plasma membrane glucose transporter content, and insulin-induced transporter translocation. There were no differences in transporter intrinsic activity (Vmax/Ro) among the three groups. Microsomal membrane GLUT4 content was reduced by 30% in untreated diabetic rats and normal in transplanted diabetics, whereas the insulin-induced changes in microsomal membrane GLUT4 content were quantitatively similar in the three groups. There were no differences in plasma membrane GLUT1 among the groups and between basal and insulin stimulated states. Microsomal membrane GLUT1 content was increased 60% in untreated diabetics and normalized by the transplantation. In conclusion, an adequate insulin delivery in the peripheral circulation, obtained by islet transplantation, fully restores the muscle glucose transport system to normal in streptozocin diabetic rats. PMID:8617870

Screening for stroke in sickle cell anemia: comparison of transcranial Doppler imaging and nonimaging US techniques.

PubMed

Neish, Ariane S; Blews, David E; Simms, Catherine A; Merritt, Robert K; Spinks, Alice J

2002-03-01

To determine whether criteria for screening patients with sickle cell anemia for stroke established with a nonimaging transcranial Doppler ultrasonographic (US) technique are applicable to studies performed with a transcranial Doppler US imaging technique. One hundred sixty-eight examinations in 66 children were performed for sickle cell stroke screening. Children were examined with nonimaging and imaging transcranial Doppler US techniques on the same day, for a total of 84 paired examinations. The time-averaged maximum mean velocity (V(mean)) and resistive index (RI) were calculated in the middle cerebral arteries, bifurcations of the distal internal carotid arteries, distal internal carotid arteries, anterior cerebral arteries, posterior cerebral arteries, and basilar arteries. The maximum systolic velocity (V(max)) was evaluated in the distal internal carotid arteries and middle cerebral arteries. V(mean), V(max), and RI measurements were subjected to repeated-measures multivariate analysis of covariance, and the Pearson product moment correlation was used for middle cerebral artery velocity, age, and hemoglobin. V(mean) measurements obtained with nonimaging and imaging techniques varied substantially for the bifurcation of the distal internal carotid artery, the posterior cerebral artery, and the basilar artery. Substantial differences were found in RIs for every vessel. Examination time was shorter with the nonimaging technique. V(mean) measurements in the middle cerebral artery, distal internal carotid artery, and anterior cerebral artery did not vary substantially between nonimaging and imaging transcranial Doppler US. RI data did not yield comparable measurements.

The Star Formation Demographics of Galaxies in the Local Volume

NASA Astrophysics Data System (ADS)

Lee, Janice C.; Kennicutt, Robert C.; Funes, S. J., José G.; Sakai, Shoko; Akiyama, Sanae

2007-12-01

We examine the connections between the current global star formation activity, luminosity, dynamical mass, and morphology of galaxies in the Local Volume, using Hα data from the 11 Mpc Hα and Ultraviolet Galaxy Survey (11HUGS). Taking the equivalent width (EW) of the Hα emission line as a tracer of the specific star formation rate, we analyze the distribution of galaxies in the MB-EW and rotational velocity (Vmax)-EW planes. Star-forming galaxies show two characteristic transitions in these planes. A narrowing of the galaxy locus occurs at MB~-15 and Vmax~50 km s-1, where the scatter in the logarithmic EWs drops by a factor of 2 as the luminosities/masses increase, and galaxy morphologies shift from predominately irregular to late-type spiral. Another transition occurs at MB~-19 and Vmax~120 km s-1, above which the sequence turns off toward lower EWs and becomes mostly populated by intermediate- and early-type bulge-prominent spirals. Between these two transitions, the mean logarithmic EW appears to remain constant at 30 Å. We comment on how these features reflect established empirical relationships, and provide clues for identifying the large-scale physical processes that both drive and regulate star formation, with emphasis on the low-mass galaxies which dominate our approximately volume-limited sample.

Kinetics of Papain: An Introductory Biochemistry Laboratory Experiment

NASA Astrophysics Data System (ADS)

Cornely, Kathleen; Crespo, Eric; Earley, Michael; Kloter, Rachel; Levesque, Aime; Pickering, Mary

1999-05-01

Enzyme kinetics experiments are popular in the undergraduate laboratory. These experiments have pedagogic value because they reinforce the concepts of Michaelis-Menten kinetics covered in the lecture portion of the course and give students the experience of calculating kinetic constants from data they themselves have generated. In this experiment, we investigate the kinetics of the thiol protease papain. The source of the papain is commercially available papaya latex. A specific substrate, Na-benzoyl-arginine-p-nitroanilide (BAPNA), is used, which takes advantage of the fact that papain interacts with a phenylalanine residue two amino acids away from the peptide bond cleaved. Upon hydrolysis by papain, a bright yellow product is released, p-nitroaniline. This allows the reaction to be monitored spectrophotometrically by measuring the rate of formation of the p-nitroaniline product as a function of the increase in absorbance of the solution at the lmax of p-nitroaniline (400 nm) over time at various substrate concentrations. These data are used to plot a Lineweaver-Burk plot from which the vmax and KM are obtained. If time permits, students carry out additional investigations in which e of p-nitroaniline is measured, the enzyme solution protein concentration is measured, the enzyme purity is evaluated by SDS-PAGE, and a pH-rate profile is constructed from experimental data.

Production of epoxide hydrolases in batch fermentations of Botryosphaeria rhodina.

PubMed

Melzer, Guido; Junne, Stefan; Wohlgemuth, Roland; Hempel, Dietmar C; Götz, Peter

2008-06-01

The filamentous fungus Botryosphaeria rhodina (ATCC 9055) was investigated related to its ability for epoxide hydrolase (EH) production. Epoxide hydrolase activity is located at two different sites of the cells. The larger part is present in the cytosol (70%), while the smaller part is associated to membranes (30%). In media optimization experiments, an activity of 3.5 U/gDW for aromatic epoxide hydrolysis of para-nitro-styrene oxide (pNSO) could be obtained. Activity increased by 30% when pNSO was added to the culture during exponential growth. An increase of enzyme activity up to 6 U/gDW was achieved during batch-fermentations in a bioreactor with 2.7 l working volume. Evaluation of fermentations with 30 l working volume revealed a relation of oxygen uptake rate to EH expression. Oxygen limitation resulted in a decreased EH activity. Parameter estimation by the linearization method of Hanes yielded Km values of 2.54 and 1.00 mM for the substrates S-pNSO and R-pNSO, respectively. vmax was 3.4 times higher when using R-pNSO. A protein purification strategy leading to a 47-fold increase in specific activity (940 U/mgProtein) was developed as a first step to investigate molecular and structural characteristics of the EH.

Purification and some kinetic properties of catalase from parsley (Petroselinum hortense Hoffm., Apiaceae) leaves.

PubMed

Oztürk, Lokman; Bülbül, Metin; Elmastas, Mahfuz; Ciftçi, Mehmet

2007-01-01

In this study, catalase (CAT: EC 1.11.1.6) was purified from parsley (Petroselinum hortense) leaves; analysis of the kinetic behavior and some properties of the enzyme were investigated. The purification consisted of three steps, including preparation of homogenate, ammonium sulfate fractionation, and fractionation by DEAE-Sephadex A50 ion exchange chromatography. The enzyme was obtained with a yield of 9.5% and had a specific activity of 1126 U (mg proteins)(-1). The overall purification was about 5.83-fold. A temperature of 4 degrees C was maintained during the purification process. Enzyme activity was spectrophotometrically measured at 240 nm. In order to control the purification of the enzyme, SDS-polyacrylamide gel electrophoresis was carried out in 4% and 10% acryl amide for stacking and running gel, respectively. SDS-polyacrylamide gel electrophoresis showed a single band for the enzyme. The molecular weight was found to be 183.29 kDa by Sephadex G-200 gel filtration chromatography. The stable pH, optimum pH, and ionic strength were determined for phosphate and Tris-HCl buffer systems. In addition, K(M) and V(max) values for H(2)O(2), at optimum pH and 25 degrees C, were determined by means of Lineweaver-Burk plots.

Absolute and Relative Training Load and Its Relation to Fatigue in Football

PubMed Central

Zurutuza, Unai; Castellano, Julen; Echeazarra, Ibon; Casamichana, David

2017-01-01

The aim of the study was to assess the relationship of external and internal training load (TL) indicators with the objective and subjective fatigue experienced by 15 semi-professional football players, over eight complete weeks of the competition period in the 2015–2016 season, which covered microcycles from 34th to 41st. The maximum heart rate (HRmax) and maximum speed (Vmax) of all the players were previously measured in specific tests. The TL was monitored via questionnaires on rating of perceived exertion (RPE), pulsometers and GPS devices, registering the variables: total distance (TD), player load 2D (PL2D), TD at >80% of the Vmax (TD80), TD in deceleration at < -2 m⋅sec-2 (TDD <-2), TD in acceleration >2 m⋅sec-2 (TDA >2), Edwards (ED), time spent at between 50 and 80% (50–80% HRmax), 80–90% (80–90% HRmax), and >90% of the HRmax (>90% HRmax), and RPE both respiratory/thoracic (RPEres) and leg/muscular (RPEmus). All the variables were analyzed taking into account both the absolute values accumulated over the week and the normalized values in relation to individual mean competition values. Neuromuscular fatigue was measured objectively using the countermovement jump test and subjectively via the Total Quality Recovery (TQR) scale questionnaire. Analytical correlation techniques were later applied within the general linear model. There is a correlation between the fatigue experienced by the player, assessed objectively and subjectively, and the load accumulated over the week, this being assessed in absolute and relative terms. Specifically, the load relative to competition correlated with the physical variables TD (-0.279), PL2D (-0.272), TDD < -2 (-0.294), TDA >2 (-0.309), and sRPEmus (-0.287). The variables related to heart rate produced a higher correlation with TQR. There is a correlation between objectively and subjectively assessed fatigue and the accumulated TL of a player over the week, with a higher sensitivity being shown when compared to the values related to the demands of competition. Monitoring load and assessing fatigue, we are closer to knowing what the prescription of an adequate dose of training should be in order for a player to be as fresh as possible and in top condition for a match. Normalizing training demands with respect to competition could be an appropriate strategy for individualizing player TL. PMID:28634456

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.

Molecular cloning and characterization of Siamese crocodile (Crocodylus siamensis) copper, zinc superoxide dismutase (CSI-Cu,Zn-SOD) gene.

PubMed

Sujiwattanarat, Penporn; Pongsanarakul, Parinya; Temsiripong, Yosapong; Temsiripong, Theeranan; Thawornkuno, Charin; Uno, Yoshinobu; Unajak, Sasimanas; Matsuda, Yoichi; Choowongkomon, Kiattawee; Srikulnath, Kornsorn

2016-01-01

Superoxide dismutase (SOD, EC 1.15.1.1) is an antioxidant enzyme found in all living cells. It regulates oxidative stress by breaking down superoxide radicals to oxygen and hydrogen peroxide. A gene coding for Cu,Zn-SOD was cloned and characterized from Siamese crocodile (Crocodylus siamensis; CSI). The full-length expressed sequence tag (EST) of this Cu,Zn-SOD gene (designated as CSI-Cu,Zn-SOD) contained 462bp encoding a protein of 154 amino acids without signal peptides, indicated as intracellular CSI-Cu,Zn-SOD. This agreed with the results from the phylogenetic tree, which indicated that CSI-Cu,Zn-SOD belonged to the intracellular Cu,Zn-SOD. Chromosomal location determined that the CSI-Cu,Zn-SOD was localized to the proximal region of the Siamese crocodile chromosome 1p. Several highly conserved motifs, two conserved signature sequences (GFHVHEFGDNT and GNAGGRLACGVI), and conserved amino acid residues for binding copper and zinc (His(47), His(49), His(64), His(72), His(81), Asp(84), and His(120)) were also identified in CSI-Cu,Zn-SOD. Real-time PCR analysis showed that CSI-Cu,Zn-SOD mRNA was expressed in all the tissues examined (liver, pancreas, lung, kidney, heart, and whole blood), which suggests a constitutively expressed gene in these tissues. Expression of the gene in Escherichia coli cells followed by purification yielded a recombinant CSI-Cu,Zn-SOD, with Km and Vmax values of 6.075mM xanthine and 1.4×10(-3)mmolmin(-1)mg(-1), respectively. This Vmax value was 40 times lower than native Cu,Zn-SOD (56×10(-3)mmolmin(-1)mg(-1)), extracted from crocodile erythrocytes. This suggests that cofactors, protein folding properties, or post-translational modifications were lost during the protein purification process, leading to a reduction in the rate of enzyme activity in bacterial expression of CSI-Cu,Zn-SOD. Copyright © 2015 Elsevier Inc. All rights reserved.

Population Pharmacokinetic Modeling of the Unbound Levofloxacin Concentrations in Rat Plasma and Prostate Tissue Measured by Microdialysis

PubMed Central

Hurtado, Felipe K.; Weber, Benjamin; Derendorf, Hartmut; Hochhaus, Guenther

2014-01-01

Levofloxacin is a broad-spectrum fluoroquinolone used in the treatment of both acute and chronic bacterial prostatitis. Currently, the treatment of bacterial prostatitis is still difficult, especially due to the poor distribution of many antimicrobials into the prostate, thus preventing the drug to reach effective interstitial concentrations at the infection site. Newer fluoroquinolones show a greater penetration into the prostate. In the present study, we compared the unbound levofloxacin prostate concentrations measured by microdialysis to those in plasma after a 7-mg/kg intravenous bolus dose to Wistar rats. Plasma and dialysate samples were analyzed using a validated high-pressure liquid chromatography-fluorescence method. Both noncompartmental analysis (NCA) and population-based compartmental modeling (NONMEM 6) were performed. Unbound prostate tissue concentrations represented 78% of unbound plasma levels over a period of 12 h by comparing the extent of exposure (unbound AUC0–∞) of 6.4 and 4.8 h·μg/ml in plasma and tissue, respectively. A three-compartment model with simultaneous passive diffusion and saturable distribution kinetics from the prostate to the central compartment gave the best results in terms of curve fitting, precision of parameter estimates, and model stability. The following parameter values were estimated by the population model: V1 (0.38 liter; where V1 represents the volume of the central compartment), CL (0.22 liter/h), k12 (2.27 h−1), k21 (1.44 h−1), k13 (0.69 h−1), Vmax (7.19 μg/h), kM (0.35 μg/ml), V3/fuprostate (0.05 liter; where fuprostate represents the fraction unbound in the prostate), and k31 (3.67 h−1). The interindividual variability values for V1, CL, Vmax, and kM were 21, 37, 42, and 76%, respectively. Our results suggest that levofloxacin is likely to be substrate for efflux transporters in the prostate. PMID:24217697

[Effect of phosphatidic acid on the reaction of linoleic acid oxidation by 5-lipooxygenase from potatoes].

PubMed

Skaterna, T D; Kharchenko, O V

2008-01-01

Influence of anionogenic phospholipid of phosphatidic acid (PA) on oxidation of linoleic acid by 5-lipoxygenase (5-LO) from Solanum tuberosum was studied. The influence of PA was studied in micellar system which consisted of mixed micelles of linolenic acid (LK), Lubrol PX and different quantity of enzyme effector PA. The reaction was initiated by addition of 5-LO. It was established that 5-LO had two pHopt. in the presence of 50 microM phosphatidic acid: pH 5.0 and 6.9. In concentration of 50 microM PA was able to activate 5-LO 15 times at pH 5.0. The reaction maximum velocity (Vmax) coincided with Vmax of lipoxygenase reaction without the effector at pH 6.9 under such conditions. It was found that 30-50 microM phospholipid in the reaction mixture decreased the concentration of half saturation by the substrate by 43-67%. The enzyme demonstrated positive cooperation in respect of the substrate, the reaction is described by the Hill equation. Hill coefficient value (h) of the substrate was 3.34 +/- 0.22 (pH 6.9) and 5.61 +/- 0.88 (pH 5.0), that is with the change of pH to acidic region the number of substrate molecules increased and they could interact with the enzyme molecule. In case of substrate insufficiency the enzyme demonstrated positive cooperation of PA, it added from 4 to 3 effectors' molecules at pH 5.0, that is the phospholipid acted as the allosteric regulator of 5-LO. A comparative analysis of the influence of 4-hydroxy-TEMPO displayed, that the level of nonenzymatic processes in the case of physiological pH values was lower by 15-50% in the presence of PA in the range of 30-80 microM than without the effector.

Environmentally relevant organophosphate triesters in herring gulls: In vitro biotransformation and kinetics and diester metabolite formation using a hepatic microsomal assay.

PubMed

Greaves, Alana K; Su, Guanyong; Letcher, Robert J

2016-10-01

The in vitro biotransformation and kinetics of six organophosphate triester (OPE) flame retardants were investigated in herring gulls (Larus argentatus) from the Great Lakes using a hepatic microsomal metabolism assay. Administration of each individual OPE (tri-n-butyl phosphate (TNBP), tris(2-butoxyethyl) phosphate (TBOEP), triphenyl phosphate (TPHP), triethyl phosphate (TEP), tris(1,3-dichloro-2-propyl) phosphate (TDCIPP) and tris(2-chloroisopropyl) phosphate (TCIPP)) to the in vitro assay (concentration range 0.01 to 10μM) resulted in rapid depletion with the exception of TEP. Following the Michaelis-Menten enzyme kinetics model, a preliminary 2-minute incubation period was used to estimate the Vmax (±SE) values (i.e., the maximal rate of reaction for a saturated enzyme system), which ranged from 5.0±0.4 (TPHP) to 29±18pmol/min/mg protein (TBOEP), as well as the KM (±SE) values (i.e., the OPE concentration corresponding to one half of the Vmax), which ranged from 9.8±1 (TPHP) to 189±135nM (TBOEP). Biotransformation assays over a 100-minute incubation period revealed that TNBP was metabolized most rapidly (with a depletion rate of 73±4pmol/min/mg protein), followed by TBOEP (53±8pmol/min/mg), TCIPP (27±1pmol/min/mg), TPHP (22±2pmol/min/mg) and TDCIPP (8±1pmol/min/mg). In vitro biotransformation of OP triesters was clearly structure-dependent where non-halogenated alkyl OP triesters were metabolized more rapidly than halogenated alkyl triesters. Halogenated OP triesters were transformed to their respective diesters more efficiently relative to non-halogenated OP triesters. To our knowledge, this is the first study to investigate OP triester metabolism and OP diester formation in an avian or wildlife model system, which is important to understand the fate and biological activity of OPEs in an exposed organism. Copyright © 2016 Elsevier Inc. All rights reserved.

Monkey liver cytochrome P450 2C19 is involved in R- and S-warfarin 7-hydroxylation.

PubMed

Hosoi, Yoshio; Uno, Yasuhiro; Murayama, Norie; Fujino, Hideki; Shukuya, Mitsunori; Iwasaki, Kazuhide; Shimizu, Makiko; Utoh, Masahiro; Yamazaki, Hiroshi

2012-12-15

Cynomolgus monkeys are widely used as primate models in preclinical studies. However, some differences are occasionally seen between monkeys and humans in the activities of cytochrome P450 enzymes. R- and S-warfarin are model substrates for stereoselective oxidation in humans. In this current research, the activities of monkey liver microsomes and 14 recombinantly expressed monkey cytochrome P450 enzymes were analyzed with respect to R- and S-warfarin 6- and 7-hydroxylation. Monkey liver microsomes efficiently mediated both R- and S-warfarin 7-hydroxylation, in contrast to human liver microsomes, which preferentially catalyzed S-warfarin 7-hydroxylation. R-Warfarin 7-hydroxylation activities in monkey liver microsomes were not inhibited by α-naphthoflavone or ketoconazole, and were roughly correlated with P450 2C19 levels and flurbiprofen 4-hydroxylation activities in microsomes from 20 monkey livers. In contrast, S-warfarin 7-hydroxylation activities were not correlated with the four marker drug oxidation activities used. Among the 14 recombinantly expressed monkey P450 enzymes tested, P450 2C19 had the highest activities for R- and S-warfarin 7-hydroxylations. Monkey P450 3A4 and 3A5 slowly mediated R- and S-warfarin 6-hydroxylations. Kinetic analysis revealed that monkey P450 2C19 had high V(max) and low K(m) values for R-warfarin 7-hydroxylation, comparable to those for monkey liver microsomes. Monkey P450 2C19 also mediated S-warfarin 7-hydroxylation with V(max) and V(max)/K(m) values comparable to those for recombinant human P450 2C9. R-warfarin could dock favorably into monkey P450 2C19 modeled. These results collectively suggest high activities for monkey liver P450 2C19 toward R- and S-warfarin 6- and 7-hydroxylation in contrast to the saturation kinetics of human P450 2C9-mediated S-warfarin 7-hydroxylation. Copyright © 2012 Elsevier Inc. All rights reserved.

Comparison of two laccases from Trametes versicolor for application in the decolorization of dyes.

PubMed

Li, Qi; Ge, Lin; Cai, Junli; Pei, Jianjun; Xie, Jingcong; Zhao, Linguo

2014-04-01

It has been previously demonstrated that laccases exhibit great potential for use in several industrial and environmental applications. In this paper, two laccase isoenzyme genes, lccB and lccC, were cloned and expressed in Pichia pastoris GS115. The sequence analysis indicated that the lccB and lccC genes consisted of 1,563 and 1,584 bp, and their open reading frames encoded 520 and 527 amino acids, respectively. They had 72.7% degree of identity in nucleotides and 86.7% in amino acids. The expression levels of LccB and LccC were up to 32,479 and 34,231 U/l, respectively. The recombinant laccases were purified by ultrafiltration and (NH4)2SO4 precipitation, showing a single band on SDS-PAGE, which had a molecular mass of 58 kDa. The optimal pH and temperature for LccB were 2.0 and 55°C with 2,2'-azino-bis-[3-ethylbenzthiazolinesulfonic acid (ABTS) as a substrate, whereas LccC exhibited optimal pH and temperature at 3.0 and 60°C. The apparent kinetic parameters of LccB were 0.43 mM for ABTS with a Vmax value of 51.28 U/mg, and the Km and Vmax values for LccC were 0.29 mM and 62.89 U/mg. The recombinant laccases were able to decolorize five types of dyes. Acid Violet 43 (100 g/ml) was completely decolorized by LccB or LccC (2 U/ml), and the decolorization of Reactive Blue KN-R (100 g/ml) was 91.6% by LccC (2 U/ml). Thus, the study characterizes useful laccase isoenzymes from T. versicolor that have the capability of being incorporated into the treatment of similar azo and anthraquinone dyes from dyeing industries.

Active Recovery After High-Intensity Interval-Training Does Not Attenuate Training Adaptation.

PubMed

Wiewelhove, Thimo; Schneider, Christoph; Schmidt, Alina; Döweling, Alexander; Meyer, Tim; Kellmann, Michael; Pfeiffer, Mark; Ferrauti, Alexander

2018-01-01

Objective: High-intensity interval training (HIIT) can be extremely demanding and can consequently produce high blood lactate levels. Previous studies have shown that lactate is a potent metabolic stimulus, which is important for adaptation. Active recovery (ACT) after intensive exercise, however, enhances blood lactate removal in comparison with passive recovery (PAS) and, consequently, may attenuate endurance performance improvements. Therefore, the aim of this study was to examine the influence of regular ACT on training adaptations during a HIIT mesocycle. Methods: Twenty-six well-trained male intermittent sport athletes (age: 23.5 ± 2.5 years; O 2 max: 55.36 ± 3.69 ml min kg -1 ) participated in a randomized controlled trial consisting of 4 weeks of a running-based HIIT mesocycle with a total of 12 HIIT sessions. After each training session, participants completed 15 min of either moderate jogging (ACT) or PAS. Subjects were matched to the ACT or PAS groups according to age and performance. Before the HIIT program and 1 week after the last training session, the athletes performed a progressive incremental exercise test on a motor-driven treadmill to determine O 2 max, maximum running velocity (vmax), the running velocity at which O 2 max occurs (vO 2 max), and anaerobic lactate threshold (AT). Furthermore, repeated sprint ability (RSA) were determined. Results: In the whole group the HIIT mesocycle induced significant or small to moderate changes in vmax ( p < 0.001, effect size [ES] = 0.65,), vO 2 max ( p < 0.001, ES = 0.62), and AT ( p < 0.001, ES = 0.56) compared with the values before the intervention. O 2 max and RSA remained unchanged throughout the study. In addition, no significant differences in the changes were noted in any of the parameters between ACT and PAS except for AT ( p < 0.05, ES = 0.57). Conclusion: Regular use of individualized ACT did not attenuate training adaptations during a HIIT mesocycle compared to PAS. Interestingly, we found that the ACT group obtained a significantly higher AT following the training program compared to the PAS group. This could be because ACT allows a continuation of the training at a low intensity and may activate specific adaptive mechanisms that are not triggered during PAS.

Active Recovery After High-Intensity Interval-Training Does Not Attenuate Training Adaptation

PubMed Central

Wiewelhove, Thimo; Schneider, Christoph; Schmidt, Alina; Döweling, Alexander; Meyer, Tim; Kellmann, Michael; Pfeiffer, Mark; Ferrauti, Alexander

2018-01-01

Objective: High-intensity interval training (HIIT) can be extremely demanding and can consequently produce high blood lactate levels. Previous studies have shown that lactate is a potent metabolic stimulus, which is important for adaptation. Active recovery (ACT) after intensive exercise, however, enhances blood lactate removal in comparison with passive recovery (PAS) and, consequently, may attenuate endurance performance improvements. Therefore, the aim of this study was to examine the influence of regular ACT on training adaptations during a HIIT mesocycle. Methods: Twenty-six well-trained male intermittent sport athletes (age: 23.5 ± 2.5 years; O2max: 55.36 ± 3.69 ml min kg-1) participated in a randomized controlled trial consisting of 4 weeks of a running-based HIIT mesocycle with a total of 12 HIIT sessions. After each training session, participants completed 15 min of either moderate jogging (ACT) or PAS. Subjects were matched to the ACT or PAS groups according to age and performance. Before the HIIT program and 1 week after the last training session, the athletes performed a progressive incremental exercise test on a motor-driven treadmill to determine O2max, maximum running velocity (vmax), the running velocity at which O2max occurs (vO2max), and anaerobic lactate threshold (AT). Furthermore, repeated sprint ability (RSA) were determined. Results: In the whole group the HIIT mesocycle induced significant or small to moderate changes in vmax (p < 0.001, effect size [ES] = 0.65,), vO2max (p < 0.001, ES = 0.62), and AT (p < 0.001, ES = 0.56) compared with the values before the intervention. O2max and RSA remained unchanged throughout the study. In addition, no significant differences in the changes were noted in any of the parameters between ACT and PAS except for AT (p < 0.05, ES = 0.57). Conclusion: Regular use of individualized ACT did not attenuate training adaptations during a HIIT mesocycle compared to PAS. Interestingly, we found that the ACT group obtained a significantly higher AT following the training program compared to the PAS group. This could be because ACT allows a continuation of the training at a low intensity and may activate specific adaptive mechanisms that are not triggered during PAS. PMID:29720949

Invertase immobilization onto radiation-induced graft copolymerized polyethylene pellets

NASA Astrophysics Data System (ADS)

de Queiroz, Alvaro Antonio Alencar; Vitolo, Michele; de Oliveira, Rômulo Cesar; Higa, Olga Zazuco

1996-06-01

The graft copolymer poly(ethylene-g-acrylic acid) (LDPE-g-AA) was prepared by radiation-induced graft copolymerization of acrylic acid onto low density polyethylene (LDPE) pellets, and characterized by infrared photoacoustic spectroscopy and scanning electron microscopy (SEM). The presence of the grafted poly(acrylic acid) (PAA) was established. Invertase was immobilized onto the graft polymer and the thermodynamic parameters of the soluble and immobilized enzyme were determined. The Michaelis constant, Km, and the maximum reaction velocity, Vmax, were determined for the free and the immobilized invertase. The Michaelis constant, Km was larger for the immobilized invertase than for the free enzyme, whereas Vmax was smaller for the immobilized invertase. The thermal stability of the immobilized invertase was higher than that of the free enzyme.

Effect of quantifying peptide release on ruminal protein degradation determined using the inhibitor in vitro system.

PubMed

Colombini, S; Broderick, G A; Clayton, M K

2011-04-01

The aim of this work was to compare use of an o-phthaldialdehyde (OPA) colorimetric assay (OPA-C), which responds to both free AA and peptides, with an OPA fluorimetric assay (OPA-F), which is insensitive to peptides, to quantify rates of ruminal protein degradation in the inhibitor in vitro system using Michaelis-Menten saturation kinetics. Four protein concentrates (expeller-extracted soybean meal, ESBM; 2 solvent-extracted soybean meals, SSBM1 and SSBM2; and casein) were incubated in a ruminal in vitro system treated with hydrazine and chloramphenicol to inhibit microbial uptake of protein degradation products. Proteins were weighed to give a range of N concentrations (from 0.15 to 3 mg of N/mL of inoculum) and incubated with 10 mL of ruminal inoculum and 5 mL of buffer; fermentations were stopped after 2 h by adding trichloroacetic acid (TCA). Proteins were analyzed for buffer-soluble N and buffer extracts were treated with TCA to determine N degraded at t=0 (FD0). The TCA supernatants were analyzed for ammonia (phenol-hypochlorite assay), total AA (TAA; OPA-F), and TAA plus oligopeptides (OPA-C) by flow injection analysis. Velocity of protein degradation was computed from extent of release of 1) ammonia plus free TAA or 2) ammonia plus free TAA and peptides. Rate of degradation (kd) was quantified using nonlinear regression of the integrated Michaelis-Menten equation. The parameters Km (Michaelis constant) and kd (Vmax/Km), where Vmax=maximum velocity, were estimated directly; kd values were adjusted (Akd) for the fraction FD0 using the equation Akd=kd-FD0/2. The OPA-C assay yielded faster degradation rates due to the contribution of peptides to the fraction degraded (overall mean=0.280/h by OPA-C and 0.219/h by OPA-F). Degradation rates for SSBM samples (0.231/h and 0.181/h) and ESBM (0.086/h) obtained by the OPA-C assay were more rapid than rates reported by the National Research Council (NRC). Both assays indicated that the 2 SSBM differed in rumen-undegradable protein (RUP) content; the more slowly degraded SSBM had RUP content (35% by OPA-C) similar to that reported by the NRC. The RUP content of ESBM (42% by OPA-C) was lower than the NRC value. Preliminary studies with 4 additional protein concentrates confirmed that accounting for peptide formation increased degradation rate; however, a trend for an interaction between assay and protein source suggested that peptide release made a smaller contribution to rate for more slowly degraded proteins. The OPA-C assay is a simple and reliable method to quantify formation of small peptides. Copyright © 2011 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

Echinococcus granulosus: specificity of amino acid transport systems in protoscoleces.

PubMed

Jeffs, S A; Arme, C

1987-08-01

Protoscoleces of Echinococcus granulosus absorb the L-amino acids proline, methionine, leucine, alanine, serine, phenylalanine, lysine and glutamic acid by a combination of mediated transport and diffusion. All eight amino acids were accumulated against a concentration gradient. Comparison of Kt and Vmax values suggests that a low affinity for a particular compound is compensated for by a relatively larger number of transport sites for that compound. Four systems serve for the transport of the eight substrates studied: 2 for neutral (EgN1, EgN2) and 1 each for acidic (EgA) and basic (EgB) amino acids. All eight amino acids are incorporated into protein to varying degrees and substantial portions of absorbed L-alanine and L-methionine are metabolized into other compounds.

A Novel Carbonyl Reductase with Anti-Prelog Stereospecificity from Acetobacter sp. CCTCC M209061: Purification and Characterization

PubMed Central

Wang, Xiao-Ting; Zong, Min-Hua; Lou, Wen-Yong

2014-01-01

A novel carbonyl reductase (AcCR) catalyzing the asymmetric reduction of ketones to enantiopure alcohols with anti-Prelog stereoselectivity was found in Acetobacter sp. CCTCC M209061 and enriched 27.5-fold with an overall yield of 0.4% by purification. The enzyme showed a homotetrameric structure with an apparent molecular mass of 104 kDa and each subunit of 27 kDa. The gene sequence of AcCR was cloned and sequenced, and a 762 bp gene fragment was obtained. Either NAD(H) or NADP(H) can be used as coenzyme. For the reduction of 4′-chloroacetophenone, the Km value for NADH was around 25-fold greater than that for NADPH (0.66 mM vs 0.026 mM), showing that AcCR preferred NADPH over NADH. However, when NADH was used as cofactor, the response of AcCR activity to increasing concentration of 4′-chloroacetophenone was clearly sigmoidal with a Hill coefficient of 3.1, suggesting that the enzyme might possess four substrate-binding sites cooperating with each other The Vmax value for NADH-linked reduction was higher than that for NADPH-linked reduction (0.21 mM/min vs 0.17 mM/min). For the oxidation of isopropanol, the similar enzymological properties of AcCR were found using NAD+ or NADP+ as cofactor. Furthermore, a broad range of ketones such as aryl ketones, α-ketoesters and aliphatic ketones could be enantioselectively reduced into the corresponding chiral alcohols by this enzyme with high activity. PMID:24740089

Biochemical properties of Glu-SH3 as a family 13 glycoside hydrolase with remarkable substrate specificity for trehalose: Implications to sequence-based classification of CAZymes.

PubMed

Ghadikolaei, Kamran Khalili; Shojaei, Maral; Ghaderi, Armin; Hojjati, Farzaneh; Noghabi, Kambiz Akbari; Zahiri, Hossein Shahbani

2016-08-01

A novel glycoside hydrolase from Exiguobacterium sp. SH3 was characterized. The enzyme, designated as Glu-SH3, was predicted by in silico analysis to have structural similarity with members of oligo-1,6-glucosidase and trehalose-6-phosphate hydrolase subfamilies in the GH-13 family of glycoside hydrolases. The gene was expressed in Escherichia coli and the recombinant enzyme was purified as a His-tagged protein of about 60 kDa. The enzyme was shown to have remarkable substrate specificity for trehalose. The characteristic ability of Glu-SH3 to hydrolyze trehalose was ascertained by zymography, thin layer chromatography, and NMR spectroscopy. The maximum activity of Glu-SH3 was obtained at 35 °C and pH 7, but it was able to exhibit more than 90% of the activity within the pH range of 5-8. The Vmax and Km values were estimated to be 170 U and 4.5 mg ml(-1), respectively. By comparison with trehalases, Glu-SH3 with Kcat and Kcat/Km values of 1552 s(-1) and 119.4 mM(-1) s(-1) can be recognized as a very efficient trehalose-hydrolyzing glycosidase. Given the phylogeny and the substrate specificity of Glu-SH3, it may be assumed that the enzyme shares a common ancestor with oligo-1,6-glucosidases but have evolved distinctly to serve a physiological function in trehalose metabolism. Copyright © 2016 Elsevier Inc. All rights reserved.

AhIRT1 and AhNRAMP1 metal transporter expression correlates with Cd uptake in peanuts under iron deficiency

PubMed Central

Xia, Shenglan; Deng, Rubo; Liu, Caifeng; Shi, Gangrong

2017-01-01

Fe deficiency may increase Cd accumulation in peanuts. However, the mechanisms are not yet fully understood. In the present study, two contrasting peanut cultivars, Luhua 8 (low seed-Cd cultivar) and Zhenghong 3 (high seed-Cd cultivar) were used to investigate the effect of Fe deficiency on the uptake and accumulation of cadmium (Cd) by hydroponic experiments. Under Fe-sufficient conditions, compared with Luhua 8, Zhenghong 3 had higher specific root length (SRL) and proportion of fine roots with a lower Km for Cd and showed slightly higher expression of AhIRT1 and AhNRAMP1 in the roots. These traits may be responsible for high capacity for Cd accumulation in Zhenghong 3. Under Fe deficiency, the increase of Cd accumulation was much larger in Zhenghong 3 than in Luhua 8. Kinetics studies revealed that the Vmax for Cd influx was 1.56-fold higher in Fe-deficient plants than in Fe-sufficient plants for Zhenghong 3, versus 0.48-fold higher for Luhua 8. Moreover, the increased expression levels of AhIRT1 and AhNRAMP1 induced by Fe deficiency was higher in Zhenghong 3 than in Luhua 8. Yeast complementation assays suggested that the AhIRT1 and AhNRAMP1 may function as transporters involved in Cd uptake. In conclusion, the different Cd accumulation between the two cultivars under Fe deficiency may be correlated with Vmax value for Cd uptake and the expression levels of AhIRT1 and AhNRAMP1 in the roots. PMID:28981520

A GUI-based Tool for Bridging the Gap between Models and Process-Oriented Studies

NASA Astrophysics Data System (ADS)

Kornfeld, A.; Van der Tol, C.; Berry, J. A.

2014-12-01

Models used for simulation of photosynthesis and transpiration by canopies of terrestrial plants typically have subroutines such as STOMATA.F90, PHOSIB.F90 or BIOCHEM.m that solve for photosynthesis and associated processes. Key parameters such as the Vmax for Rubisco and temperature response parameters are required by these subroutines. These are often taken from the literature or determined by separate analysis of gas exchange experiments. It is useful to note however that subroutines can be extracted and run as standalone models to simulate leaf responses collected in gas exchange experiments. Furthermore, there are excellent non-linear fitting tools that can be used to optimize the parameter values in these models to fit the observations. Ideally the Vmax fit in this way should be the same as that determined by a separate analysis, but it may not because of interactions with other kinetic constants and the temperature dependence of these in the full subroutine. We submit that it is more useful to fit the complete model to the calibration experiments rather as disaggregated constants. We designed a graphical user interface (GUI) based tool that uses gas exchange photosynthesis data to directly estimate model parameters in the SCOPE (Soil Canopy Observation, Photochemistry and Energy fluxes) model and, at the same time, allow researchers to change parameters interactively to visualize how variation in model parameters affect predicted outcomes such as photosynthetic rates, electron transport, and chlorophyll fluorescence. We have also ported some of this functionality to an Excel spreadsheet, which could be used as a teaching tool to help integrate process-oriented and model-oriented studies.

Phase I metabolism of 3-methylindole, an environmental pollutant, by hepatic microsomes from carp (Cyprinus carpio) and rainbow trout (Oncorhynchus mykiss).

PubMed

Zlabek, Vladimir; Burkina, Viktoriia; Borrisser-Pairó, Francesc; Sakalli, Sidika; Zamaratskaia, Galia

2016-05-01

We studied the in vitro metabolism of 3-methylindole (3MI) in hepatic microsomes from fish. Hepatic microsomes from juvenile and adult carp (Cyprinus carpio) and rainbow trout (Oncorhynchus mykiss) were included in the study. Incubation of 3MI with hepatic microsomes revealed the time-dependent formation of two major metabolites, 3-methyloxindole (3MOI) and indole-3-carbinol (I3C). The rate of 3MOI production was similar in both species at both ages. No differences in kinetic parameters were observed (p = 0.799 for Vmax, and p = 0.809 for Km). Production of I3C was detected only in the microsomes from rainbow trout. Km values were similar in juvenile and adult fish (p = 0.957); Vmax was higher in juvenile rainbow trout compared with adults (p = 0.044). In rainbow trout and carp, ellipticine reduced formation of 3MOI up to 53.2% and 81.9% and ketoconazole up to 65.8% and 91.3%, respectively. The formation of I3C was reduced by 53.7% and 51.5% in the presence of the inhibitors ellipticine and ketoconazole, respectively. These findings suggest that the CYP450 isoforms CYP1A and CYP3A are at least partly responsible for 3MI metabolism. In summary, 3MI is metabolised in fish liver to 3MOI and I3C by CYP450, and formation of these metabolites might be species-dependent. Copyright © 2016 Elsevier Ltd. All rights reserved.

Silver nanoparticle (AgNPs) doped gum acacia-gelatin-silica nanohybrid: an effective support for diastase immobilization.

PubMed

Singh, Vandana; Ahmed, Shakeel

2012-03-01

An effective carrier matrix for diastase alpha amylase immobilization has been fabricated by gum acacia-gelatin dual templated polymerization of tetramethoxysilane. Silver nanoparticle (AgNp) doping to this hybrid could significantly enhance the shelf life of the impregnated enzyme while retaining its full bio-catalytic activity. The doped nanohybrid has been characterized as a thermally stable porous material which also showed multipeak photoluminescence under UV excitation. The immobilized diastase alpha amylase has been used to optimize the conditions for soluble starch hydrolysis in comparison to the free enzyme. The optimum pH for both immobilized and free enzyme hydrolysis was found to be same (pH=5), indicating that the immobilization made no major change in enzyme conformation. The immobilized enzyme showed good performance in wide temperature range (from 303 to 323 K), 323 K being the optimum value. The kinetic parameters for the immobilized, (K(m)=10.30 mg/mL, V(max)=4.36 μmol mL(-1)min(-1)) and free enzyme (K(m)=8.85 mg/mL, V(max)=2.81 μmol mL(-1)min(-1)) indicated that the immobilization improved the overall stability and catalytic property of the enzyme. The immobilized enzyme remained usable for repeated cycles and did not lose its activity even after 30 days storage at 40°C, while identically synthesized and stored silver undoped hybrid lost its ~31% activity in 48 h. Present study revealed the hybrids to be potentially useful for biomedical and optical applications. Copyright © 2011 Elsevier B.V. All rights reserved.

Cloning, Sequencing, and Expression of the Gene Encoding Cyclic 2,3-Diphosphoglycerate Synthetase, the Key Enzyme of Cyclic 2,3-Diphosphoglycerate Metabolism in Methanothermus fervidus

PubMed Central

Matussek, Karl; Moritz, Patrick; Brunner, Nina; Eckerskorn, Christoph; Hensel, Reinhard

1998-01-01

Cyclic 2,3-diphosphoglycerate synthetase (cDPGS) catalyzes the synthesis of cyclic 2,3-diphosphoglycerate (cDPG) by formation of an intramolecular phosphoanhydride bond in 2,3-diphosphoglycerate. cDPG is known to be accumulated to high intracellular concentrations (>300 mM) as a putative thermoadapter in some hyperthermophilic methanogens. For the first time, we have purified active cDPGS from a methanogen, the hyperthermophilic archaeon Methanothermus fervidus, sequenced the coding gene, and expressed it in Escherichia coli. cDPGS purification resulted in enzyme preparations containing two isoforms differing in their electrophoretic mobility under denaturing conditions. Since both polypeptides showed the same N-terminal amino acid sequence and Southern analyses indicate the presence of only one gene coding for cDPGS in M. fervidus, the two polypeptides originate from the same gene but differ by a not yet identified modification. The native cDPGS represents a dimer with an apparent molecular mass of 112 kDa and catalyzes the reversible formation of the intramolecular phosphoanhydride bond at the expense of ATP. The enzyme shows a clear preference for the synthetic reaction: the substrate affinity and the Vmax of the synthetic reaction are a factor of 8 to 10 higher than the corresponding values for the reverse reaction. Comparison with the kinetic properties of the electrophoretically homogeneous, apparently unmodified recombinant enzyme from E. coli revealed a twofold-higher Vmax of the enzyme from M. fervidus in the synthesizing direction. PMID:9811660

Purification of 6-phosphogluconate dehydrogenase from parsley (Petroselinum hortense) leaves and investigation of some kinetic properties.

PubMed

Demir, Hülya; Ciftçi, Mehmet; Küfrevioğlu, O Irfan

2003-02-01

In this study, 6-phosphogluconate dehydrogenase (E.C.1.1.44; 6PGD) was purified from parsley (Petroselinum hortense) leaves, and analysis of the kinetic behavior and some properties of the enzyme were investigated. The purification consisted of three steps that are preparation of homogenate ammonium sulfate fractionation and on DEAE-Sephadex A50 ion exchange. The enzyme was obtained with a yield of 49% and had a specific activity of 18.3 U (mg proteins)(-1) (Lehninger, A.L.; Nelson, D.L.; Cox, M.M. Principles of Biochemistry, 2nd Ed.; Worth Publishers Inc.: N.Y., 2000, 558-560). The overall purification was about 339-fold. A temperature of +4 degrees C was maintained during the purification process. Enzyme activity was spectrophotometrically measured according to the Beutler method at 340 mn. In order to control the purification of the enzyme, SDS-polyacrylamide gel electrophoresis was carried out in 4% and 10% acrylamide for stacking and running gel, respectively. SDS-polyacrylamide gel electrophoresis showed a single band for enzyme. The molecular weight was found to be 97.5 kDa by Sephadex G-150 gel filtration chromatography. A protein band corresponding to a subunit molecular weight of 24.1 kDa was obtained on SDS-polyacrylamide gel electrophoresis. For the enzymes, the stable pH, optimum pH, and optimum temperature were found as 8.0, 8.0, and 50 degrees C, respectively. In addition, KM and Vmax values for NADP+ and G6-P at optimum pH and 25 degrees C were determined by means of Lineweaver-Burk plots.

A multipurpose immobilized biocatalyst with pectinase, xylanase and cellulase activities

PubMed Central

Dalal, Sohel; Sharma, Aparna; Gupta, Munishwar Nath

2007-01-01

Background The use of immobilized enzymes for catalyzing various biotransformations is now a widely used approach. In recent years, cross-linked enzyme aggregates (CLEAs) have emerged as a novel and versatile biocatalyst design. The present work deals with the preparation of a CLEA from a commercial preparation, Pectinex™ Ultra SP-L, which contains pectinase, xylanase and cellulase activities. The CLEA obtained could be used for any of the enzyme activities. The CLEA was characterized in terms of kinetic parameters, thermal stability and reusability in the context of all the three enzyme activities. Results Complete precipitation of the three enzyme activities was obtained with n-propanol. When resulting precipitates were subjected to cross-linking with 5 mM glutaraldehyde, the three activities initially present (pectinase, xylanase and cellulase) were completely retained after cross-linking. The Vmax/Km values were increased from 11, 75 and 16 to 14, 80 and 19 in case of pectinase, xylanase and cellulase activities respectively. The thermal stability was studied at 50°C, 60°C and 70°C for pectinase, xylanase and cellulase respectively. Half-lives were improved from 17, 22 and 32 minutes to 180, 82 and 91 minutes for pectinase, xylanase and cellulase respectively. All three of the enzymes in CLEA could be reused three times without any loss of activity. Conclusion A single multipurpose biocatalyst has been designed which can be used for carrying out three different and independent reactions; 1) hydrolysis of pectin, 2) hydrolysis of xylan and 3) hydrolysis of cellulose. The preparation is more stable at higher temperatures as compared to the free enzymes. PMID:17880745

Enzymic aromatization of 6-alkyl-substituted androgens, potent competitive and mechanism-based inhibitors of aromatase.

PubMed Central

Numazawa, M; Yoshimura, A; Oshibe, M

1998-01-01

To gain insight into the relationships between the aromatase inhibitory activity of 6-alkyl-substituted androgens, potent competitive inhibitors, and their ability to serve as a substrate of aromatase, we studied the aromatization of a series of 6alpha- and 6beta-alkyl (methyl, ethyl, n-propyl, n-pentyl and n-heptyl)-substituted androst-4-ene-3,17-diones (ADs) and their androsta-1,4-diene-3,17-dione (ADD) derivatives with human placental aromatase, by gas chromatography-mass spectrometry. Among the inhibitors examined, ADD and its 6alpha-alkyl derivatives with alkyl functions less than three carbons long, together with 6beta-methyl ADD, are suicide substrates of aromatase. All of the steroids, except for 6beta-n-pentyl ADD and its n-heptyl analogue as well as 6beta-n-heptyl AD, were found to be converted into the corresponding 6-alkyl oestrogens. The 6-methyl steroids were aromatized most efficiently in each series, and the aromatization rate essentially decreased in proportion to the length of the 6-alkyl chains in each series, where the 6alpha-alkyl androgens were more efficient substrates than the corresponding 6beta isomers. The Vmax of 6alpha-methyl ADD was approx. 2.5-fold that of the natural substrate AD and approx. 3-fold that of the parent ADD. On the basis of this, along with the facts that the rates of a mechanism-based inactivation of aromatase by ADD and its 6alpha-methyl derivative are similar, it is implied that alignment of 6alpha-methyl ADD in the active site could favour the pathway leading to oestrogen over the inactivation pathway, compared with that of ADD. The relative apparent Km values for the androgens obtained in this study are different from the relative Ki values obtained previously, indicating that there is a difference between the ability to serve as an inhibitor and the ability to serve as a substrate in the 6-alkyl androgen series. PMID:9405288

Glutamine, glutamate, and other possible regulators of alpha-ketoglutarate and malate uptake by synaptic terminals.

PubMed

Shank, R P; Campbell, G L

1984-04-01

The uptake of alpha-ketoglutarate and malate by rat brain synaptosomal preparations was found to be affected by a variety of substances at physiologically relevant concentrations. Glutamine altered the uptake of alpha-ketoglutarate by causing an apparent reduction in the substrate-carrier affinity and an increase in Vmax. In contrast, glutamine did not appear to affect the Vmax of malate uptake, but it did increase markedly the uptake velocity at low concentrations of malate. L-Glutamate and L-aspartate were comparatively strong inhibitors of alpha-ketoglutarate and malate uptake. N-Acetylaspartate was a weak inhibitor of alpha-ketoglutarate uptake, a finding that contrasts with our previous observation that this compound potently inhibited alpha-ketoglutarate uptake by synaptosomes obtained from the cerebellum of 8- to 14-day-old mice. Ca2+ exhibited a variable effect but usually enhanced the uptake of alpha-ketoglutarate. The addition of small amounts of postmicrosomal supernatant to the incubation medium enhanced the uptake of alpha-ketoglutarate by low-density synaptosomes. By comparison, the uptake of glutamate, glutamine, gamma-aminobutyric acid, and several other amino acids was not affected. The enhancement of alpha-ketoglutarate uptake by the supernatant was due to a heat labile substance that was retained by dialysis tubing (MW cutoff = 8,000) and Amicon filter cones (CF 25), and was precipitated by ammonium sulfate at 60% saturation. In experiments in which the metabolic conversion of [U-14C] alpha-ketoglutarate to glutamate, aspartate, glutamine, and gamma-aminobutyric acid was determined, the presence of glutamine and glutamate in the incubation medium did not affect the pattern of labelling appreciably.

Echocardiographic estimation of systemic systolic blood pressure in dogs with mild mitral regurgitation.

PubMed

Tou, Sandra P; Adin, Darcy B; Estrada, Amara H

2006-01-01

Systemic hypertension is likely underdiagnosed in veterinary medicine because systemic blood pressure is rarely measured. Systemic blood pressure can theoretically be estimated by echocardiography. According to the modified Bernoulli equation (PG = 4v(2)), mitral regurgitation (MR) velocity should approximate systolic left ventricular pressure (sLVP), and therefore systolic systemic blood pressure (sSBP) in the presence of a normal left atrial pressure (LAP) and the absence of aortic stenosis. The aim of this study was to evaluate the use of echocardiography to estimate sSBP by means of the Bernoulli equation. Systemic blood pressure can be estimated by echocardiography. Seventeen dogs with mild MR. No dogs had aortic or subaortic stenosis, and all had MR with a clear continuous-wave Doppler signal and a left atrial to aorta ratio of < or = 1.6. Five simultaneous, blinded continuous-wave measurements of maximum MR velocity (Vmax) and indirect sSBP measurements (by Park's Doppler) were obtained for each dog. Pressure gradient was calculated from Vmax by means of the Bernoulli equation, averaged, and added to an assumed LAP of 8 mm Hg to calculate sLVP. Calculated sLVP was significantly correlated with indirectly measured sSBP within a range of 121 to 218 mm Hg (P = .0002, r = .78). Mean +/- SD bias was 0.1 +/- 15.3 mm Hg with limits of agreement of -29.9 to 30.1 mm Hg. Despite the significant correlation, the wide limits of agreement between the methods hinder the clinical utility of echocardiographic estimation of blood pressure.

VizieR Online Data Catalog: Surface photometry of GHASP galaxies (Barbosa+, 2015)

NASA Astrophysics Data System (ADS)

Barbosa, C. E.; Mendes de Oliveira, C.; Amram, P.; Ferrari, F.; Russeil, D.; Epinat, B.; Perret, V.; Adami, C.; Marcelin, M.

2016-04-01

Our data set is constructed using new Rc-band observations taken at the Observatoire de Haute-Provence, supplemented with Sloan Digital Sky Survey archival data, obtained with the purpose of deriving homogeneous photometric profiles and parameters. Our results include Rc-band surface brightness profiles for 170 galaxies and ugriz profiles for 108 of these objects. We catalogue several parameters of general interest for further reference, such as total magnitude, effective radius and isophotal parameters (magnitude, position angle, ellipticity and inclination). We also perform a structural decomposition of the surface brightness profiles using a multi-component method to separate discs from bulges and bars, and to observe the main scaling relations involving luminosities, sizes and maximum velocities. We determine the Rc-band Tully-Fisher relation using maximum velocities derived solely from Hα rotation curves for a sample of 80 galaxies, resulting in a slope of -8.1+/-0.5, zero-point of -3.0+/-1.0 and an estimated intrinsic scatter of 0.28+/-0.07. We note that, unlike the Tully-Fisher relation in the near-infrared derived for the same sample, no change in the slope of the relation is seen at the low-mass end (for galaxies with Vmax<125km/s). We suggest that this different behaviour of the Tully-Fisher relation (with the optical relation being described by a single power law while the near-infrared has two), may be caused by differences in the stellar mass-to-light ratio for galaxies with Vmax<125km/s. (4 data files).

[Biomechanics and bio-energetics of smooth muscle contraction. Relation to bronchial hyperreactivity].

PubMed

Coirault, C; Blanc, F X; Chemla, D; Salmeron, S; Lecarpentier, Y

2000-06-01

Mechanical studies of isolated muscle and analysis of molecular actomyosin interactions have improved our understanding of the pathophysiology of airway smooth muscle. Mechanical properties of airway smooth muscle are similar to those of other smooth muscles. Airway smooth muscle exhibits spontaneous intrinsic tone and its maximum shortening velocity (Vmax) is 10-30 fold lower than in striated muscle. Smooth muscle myosin generates step size and elementary force per crossbridge interaction approximately similar to those of skeletal muscle myosin. Special slow cycling crossbridges, termed latch-bridges, have been attributed to myosin light chain dephosphorylation. From a mechanical point of view, it has been shown that airway hyperresponsiveness is characterized by an increased Vmax and an increased shortening capacity, with no significant change in the force-generating capacity.

Purification and properties of trimethylamine oxide reductase from Salmonella typhimurium.

PubMed Central

Kwan, H S; Barrett, E L

1983-01-01

The major inducible trimethylamine oxide reductase was purified from Salmonella typhimurium LT2. The molecular weights of the native enzyme were estimated to be 332,000 by gel filtration and 170,000 by nondenaturing disc gel electrophoresis. In sodium dodecyl sulfate-gel electrophoresis, the enzyme formed a single band of molecular weight 84,000. The isoelectric point was 4.28. Maximum activity was at pH 5.65 and 45 degrees C. Reduced flavin mononucleotide, but not reduced flavin adenine dinucleotide, served as an electron donor. The Km for trimethylamine oxide was 0.89 mM and Vmax was 1,450 U/mg of protein. The enzyme reduced chlorate with a Km of 2.2 mM and a Vmax of 350 U/mg of protein. Images PMID:6350272

Basal glycogenolysis in mouse skeletal muscle: in vitro model predicts in vivo fluxes

NASA Technical Reports Server (NTRS)

Lambeth, Melissa J.; Kushmerick, Martin J.; Marcinek, David J.; Conley, Kevin E.

2002-01-01

A previously published mammalian kinetic model of skeletal muscle glycogenolysis, consisting of literature in vitro parameters, was modified by substituting mouse specific Vmax values. The model demonstrates that glycogen breakdown to lactate is under ATPase control. Our criteria to test whether in vitro parameters could reproduce in vivo dynamics was the ability of the model to fit phosphocreatine (PCr) and inorganic phosphate (Pi) dynamic NMR data from ischemic basal mouse hindlimbs and predict biochemically-assayed lactate concentrations. Fitting was accomplished by optimizing four parameters--the ATPase rate coefficient, fraction of activated glycogen phosphorylase, and the equilibrium constants of creatine kinase and adenylate kinase (due to the absence of pH in the model). The optimized parameter values were physiologically reasonable, the resultant model fit the [PCr] and [Pi] timecourses well, and the model predicted the final measured lactate concentration. This result demonstrates that additional features of in vivo enzyme binding are not necessary for quantitative description of glycogenolytic dynamics.

Biochemical properties of Na+/K(+)-ATPase in axonal growth cone particles isolated from fetal rat brain.

PubMed

Mercado, R; Hernández, J

1994-08-01

Axonal growth cones (AGC) isolated from fetal rat brain have an important specific activity of N+/K(+)-ATPase. Kinetic assays of the enzyme in AGC showed that Km values for ATP or K+ are similar to those reported for the adult brain enzyme. For Na+ the affinity (Km) was lower. Vmax for the three substrates was several times lower in AGC as compared to the adult value. We also observed two apparent inhibition constants of Na+/K(+)-ATPase by ouabain, one of low affinity, possibly corresponding to the alpha 1 isoform and another of high affinity which is different to that described for the alpha 2 isoform of the enzyme. These results support an important role for the sodium pump in the maintainance of volume and cationic balance in neuronal differentiating structures. The functional differences observed also suggest that the enzymatic complex of Na+/K(+)-ATPase in AGC is in a transitional state towards the adult configuration.

Studies on the metabolism of benoxinate by human pseudocholinesterase.

PubMed

Dubbels, R; Schloot, W

1983-01-01

The local anesthetic drug benoxinate (oxybuprocaine, Novesine) is hydrolyzed to 3-butoxy-4-aminobenzoic acid. A rapid and simple spectrophotometric method for benoxinate hydrolysis by human plasma was developed. Benoxinate is hydrolyzed enzymatically by an esterase present in the serum. Heat stability characteristics and apparent affinity values of the benoxinate metabolizing enzyme were in the same range compared to benzoylcholine chloride hydrolysis. Apparent Vmax-values differ by a mean factor of about 18 between the hydrolysis of both substrates. Considerable interindividual variability of benoxinate hydrolysis and inhibition of the enzymatic reaction by dibucaine and sodium fluoride has been observed. Furthermore, enzyme activity with benoxinate as substrate is positively correlated (P less than 0.001) with benzoylcholine chloride hydrolysis. Therefore, we assume that benoxinate is metabolized by human pseudocholinesterase (PCHE, E.C. 3.1.1.8) and that ocular side effects after benoxinate application may be caused by altered metabolism of this drug, depending on genetically determined variants of pseudocholinesterase.

Carrier-mediated uptake of nobiletin, a citrus polymethoxyflavonoid, in human intestinal Caco-2 cells.

PubMed

Kimura, Osamu; Ohta, Chiho; Koga, Nobuyuki; Haraguchi, Koichi; Kato, Yoshihisa; Endo, Tetsuya

2014-07-01

The mechanism of intestinal absorption of nobiletin (NBL) was investigated using Caco-2 cells. The uptake of NBL from the apical membranes of Caco-2 cells was rapid and temperature-dependent and the presence of metabolic inhibitors, NaN3 and carbonylcyanide p-trifluoromethoxyphenylhydrazone, did not cause a decrease in NBL uptake. The relationship between the initial uptake of NBL and its concentration was saturable, suggesting the involvement of a carrier-mediated process. The Km and uptake clearance (Vmax/Km) values for NBL were 50.6 and 168.1μl/mg protein/min, respectively. This clearance value was about 9-fold greater than that of the non-saturable uptake clearance (Kd: 18.5μl/mg protein/min). The presence of structurally similar compounds, such as quercetin and luteolin, competitively inhibited NBL uptake. These results suggest that uptake of NBL from the apical membranes of Caco-2 cells is mainly mediated by an energy-independent facilitated diffusion process. Copyright © 2013 Elsevier Ltd. All rights reserved.

Cellular assessment of muscle in COPD: case studies of two males.

PubMed

Green, Howard J; Bombardier, Eric; Burnett, Margaret E; D'Arsigny, Christine L; Iqbal, Sobia; Webb, Katherine A; Ouyang, Jing; O'Donnell, Denis E

2009-12-29

The objective of this paper is to provide an overview of the recent developments in muscle physiology and biochemistry in general, and with respect to chronic obstructive pulmonary disease (COPD) specifically. As a way of illustration, we have presented data on the remodeling that occurs in vastus lateralis in two patients with COPD (COPD #1, forced expiratory volume in one second/forced vital capacity [FEV(1)/FVC] = 63%; COPD #2, FEV(1)/FVC = 41%) exhibiting differences in muscle wasting as compared to healthy controls (CON; FEV(1)/FVC = 111 +/- 2.2%, n = 4). Type I fibers percentages were lower in both COPD #1 (16.7) and COPD #2 (24.9) compared to CON (57.3 +/- 5.2). Cross sectional area of the type I fibers of the patients ranged between 65%-68% of CON and for the type II subtypes (IIA, IIAX, IIX) between 74% and 89% (COPD #1) and 17%-32% (COPD #2). A lower number of capillary contacts were observed for all fiber types in COPD #1 but not COPD #2. Lower concentrations of adenosine triphosphate (ATP) (24%-26%) and phosphocreatine (18%-20%), but not lactate occurred in COPD. In contrast to COPD #1, who displayed normal glucose transporter content, GLUT1 and GLUT4 were only 71% and 54%, respectively of CON in COPD #2. Lower monocarboxylate contents were found for MCT1 in both COPD #1 (63%) and COPD #2 (41%) and for MCT4 (78%) in COPD #1. Maximal oxidative enzyme activities (V(max)) for COPD #2 ranged between 37% (succinic dehydrogenase) and 70% (cytochrome C oxidase) of CON. For the cytosolic enzymes, V(max) ranged between 89% (hexokinase) to 31% (pyruvate kinase) of CON. Depressions were also observed in V(max) of the Na(+)-K(+)-ATPase for COPD #1 (66% of CON) but not COPD #2 (92% of CON) while V(max) of the Ca(2+)-ATPase was near normal in COPD #1 (84% CON). It is concluded that disturbances can occur in muscle to a wide range of excitation, contraction and metabolic processes in COPD.

β-Glucoside Activators of Mung Bean UDP-Glucose: β-Glucan Synthase 1

PubMed Central

Callaghan, Theresa; Ross, Peter; Weinberger-Ohana, Patricia; Benziman, Moshe

1988-01-01

n-Alkyl (C6-C12) β-d-monoglucopyranosides have been found to be highly potent activators of mung bean β-glucan synthase in vitro, increasing the Vmax of the enzyme as much as 60-fold and with Ka values as low as 10 micromolar. Activation is highly specific for the β-linked terminal glucose residue; other alkyl glycosides such as, octyl-α-glucoside, dodecyl β-maltoside, 6-lauryl sucrose, 6-lauryl glucose, which lack this structure, are ineffective as activators. Based on the similarities in their structure and effects on β-glucan synthesis under a variety of conditions, it is proposed that the alkyl β-glucosides are structural analogs of the native glucolipid activator of β-glucan synthase isolated from mung bean extracts. PMID:16666039

L-asparaginase activity in Aeromonas sp. isolated from freshwater mussel.

PubMed

Pattnaik, S; Kabi, R; Janaki Ram, K; Bhanot, K K

2000-11-01

Aeromonas sp. from Lamellidens marginalis produced L-asparaginase when grown at 37 degrees C. The optimum enzyme activity was at pH 9 when temperature was 45 degrees C. Half-life of partially purified enzyme at 50 degrees C and 55 degrees C was 35 and 20 min, respectively. Activation and deactivation energies of partially purified enzyme were 17.48 and 24.86 kcal mol-1 respectively. The enzyme exhibited a Km (L-asparagine) value of 4.9 x 10(-6) mol l-1 and a Vmax of 9.803 IU ml-1. Three metal ions inhibited the enzyme activity at 10-20 mumol l-1 concentrations. Catalytic activity was also inhibited by EDTA, iodoacetic acid, parachloromercuribenzoic acid and phenylmethylsulphonyl fluoride at 0.1 mumol l-1.

Substrate specificity and kinetic properties of alpha-galactosidases from Vicia faba.

PubMed

Dey, P M; Pridham, J B

1969-10-01

1. The hydrolysis of a variety of galactosides and other glycosides by alpha-galactosidases I and II of Vicia faba was studied. 2. The effect of temperature on kinetic parameters was also examined. 3. Both enzymes are inhibited by excess of substrate (p-nitrophenyl alpha-d-galactoside); with enzyme I this is competitive and is caused by the galactosyl moiety. 4. Enzyme I is inhibited by oligosaccharides possessing terminal non-reducing galactose residues and to a smaller extent by l-arabinose and d-fucose. 5. The effect of pH on K(m) and V(max.) values suggests that carboxyl and imidazole groups are involved in the catalytic activity of enzyme I. 6. Photo-oxidation experiments with enzyme I also suggest that an imidazole group is present at the active site.

Pre and post cloning characterization of a beta-1,4-endoglucanase from Bacillus sp.

PubMed

Afzal, Sumra; Saleem, Mahjabeen; Yasmin, Riffat; Naz, Mamoona; Imran, Muhammad

2010-04-01

Consistent with its precloning characterization from the cellulolytic Bacillus sp., beta-1,4-endoglucanase purified from the recombinant E. coli exhibited maximum activity at 60 degrees C and pH 7.0. It was highly specific for CMC hydrolysis, with stability up to 70 degrees C and over a pH range of 6.0-8.0. The K(m) and V(max) values for CMCase activity of the enzyme were 4.1 mg/ml and 25 micromole/ml min(-1), respectively. The purified enzyme was a monomer of 65 kDa, as determined by SDS-PAGE. The presence of sucrose and IPTG in fermentation media increased the endoglucanase activity of the recombinant enzyme to 5.2-folds as compared with that of the actual one.

Effect of a novel steroid (PM-9) on the inhibition of 5alpha-reductase present in Penicillium crustosum broths.

PubMed

Flores, Eugenio; Cabeza, Marisa; Quiroz, Alexandra; Bratoeff, Eugene; García, Genoveva; Ramírez, Elena

2003-03-01

The conversion of testosterone (T) to 5alpha-dihydrotestosterone (DHT) has been demonstrated in Penicillium crustosum broth obtained from fermented pistachios, lemons and corn tortillas. Furthermore, the presence of 5alpha-reductase enzyme, which is responsible for this conversion, has been established by electrophoretical techniques in these cultures.5alpha-Reductase enzyme is also present in animal and human androgen-dependent tissues as well as in prostate and seminal vesicles. The increase of the conversion of T to DHT in prostate gland, has been related to some illnesses such as benign prostate hyperplasia and prostate cancer. Furthermore, treatment with 5alpha-reductase inhibitors such as finasteride reduces the prostate growth. These data have stimulated research for the synthesis of new molecules with antiandrogenic activity, whose biological effect needs to be demonstrated. The purpose of this study is to determine the inhibition pattern of 5alpha-reductase in P. crustosum by finasteride and the new steroidal compound PM-9. K(m) and V(max) values for T, were determined in the broths by Lineweaver-Burk plots using different testosterone concentrations. The inhibition pattern of finasteride and PM-9 was also determined by Lineweaver-Burk using different concentrations of T and inhibitors. Results show that finasteride and PM-9 inhibit 5alpha-reductase present in the broth in a competitive manner.

Fluorescent Phosphatidylinositol 4,5-Bisphosphate Derivatives with Modified 6-Hydroxy Group as Novel Substrates for Phospholipase C

PubMed Central

Wang, Xiaoyang; Barrett, Matthew; Sondek, John; Harden, T. Kendall; Zhang, Qisheng

2013-01-01

The capacity to monitor spatiotemporal activity of phospholipase C (PLC) isozymes with a PLC-selective sensor would dramatically enhance understanding of the physiological function and disease relevance of these signaling proteins. Previous structural and biochemical studies defined critical roles for several of the functional groups of the endogenous substrate of PLC isozymes, phosphatidylinositol 4,5-bisphosphate (PIP2), indicating that these sites cannot be readily modified without compromising interactions with the lipase active site. However, the role of the 6-hydroxy group of PIP2 for interaction and hydrolysis by PLC has not been explored, possibly due to challenges in synthesizing 6-hydroxy derivatives. Here, we describe an efficient route for the synthesis of novel, fluorescent PIP2 derivatives modified at the 6-hydroxy group. Two of these derivatives were used in assays of PLC activity in which the fluorescent PIP2 substrates were separated from their diacylglycerol products and reaction rates quantified by fluorescence. Both PIP2 analogues effectively function as substrates of PLC-δ1, and the KM and Vmax values obtained with one of these are similar to those observed with native PIP2 substrate. These results indicate that the 6-hydroxy group can be modified to develop functional substrates for PLC isozymes, thereby serving as the foundation for further development of PLC-selective sensors. PMID:22703043

Production and characterization of alpha-amylase from mango kernel by Fusarium solani NAIMCC-F-02956 using submerged fermentation.

PubMed

Kumar, Devendra; Yadav, Kaushlesh K; Muthukumar, M; Garg, Neelima

2013-11-01

Microbial production of enzymes using low valued agro industrial wastes is gaining importance globally. Mango is one of the major fruit processed into a variety of products. During processing 40-50% of solid waste is generated in form of peel and stones. After decortications of mango stone, kernel is obtained which is a rich source of starch (upto 60%). It was utilized as a substrate for alpha-amylase production using Fusarium soloni. Maximum alpha-amylase production (0.889 U g(-1)) was recorded using a substrate concentration of 5% (w/v), pH-4 and temperature 30 degrees C on 9th day of incubation. Supplementation of production medium with micronutrients viz., Ca2+, Fe2+ or Mg2+ improved the enzyme production while, Zn2+, B3+ or Mn2+ ions exhibited inhibitory effect. The extracellular protein was precipitated by ammonium sulphate up to 70% saturation, dialyzed and purified (27.84 fold) by gel-exclusion (Sephadex G-75) chromatography. Protein profiling on 12% SDS-PAGE revealed three bands corresponding to 26, 27 and 30 kDa molecular sizes. The optimum amylase activity was achieved at pH 5.0 at 40 degrees C. The Michaelis constant (KM), Vmax and activation energy (-Ea) were found to be 3.7 mg ml(-1), 0.24 U mg(-1) and 42.39 kJ mole(-1), respectively.

Kinetic study of hydroxytyrosol oxidation and its related compounds by Red Globe grape polyphenol oxidase.

PubMed

García-García, María Inmaculada; Hernández-García, Samanta; Sánchez-Ferrer, Álvaro; García-Carmona, Francisco

2013-06-26

Red Globe grape polyphenol oxidase, partially purified using phase partitioning with Triton-X114, was used to study the oxidation of hydroxytytosol (HT) and its related compounds tyrosol (TS), tyrosol acetate (TSA), and hydroxytyrosol acetate (HTA). The enzyme showed activity toward both monophenols (monophenolase activity) and o-diphenols (diphenolase activity) with a pH optimum (pH 6.5) that was independent of the phenol used. However, the optimal temperature for diphenolase activity was substrate-dependent, with a broad optimum of 25-65 °C for HT, compared with the maximum obtained for HTA (40 °C). Monophenolase activity showed the typical lag period, which was modulated by pH, substrate and enzyme concentrations, and the presence of catalytic amounts of o-diphenols. When the catalytic power (Vmax/K(M)) was determined for both activities, higher values were observed for o-diphenols than for monophenols: 9-fold higher for the HT/TS pair and 4-fold higher for HTA/TSA pair. Surprisingly, this ratio was equally higher for TSA (2.2-fold) compared with that of TS, whereas no such effect was observed for o-diphenols. This higher efficiency of TSA could be related to its greater hydrophobicity. Acetyl modification of these phenols not only changes the kinetic parameters of the enzyme but also affects their antioxidant activity (ORAC-FL assays), which is lower in HTA than in HT.

Comparison of Free and Immobilized L-asparaginase Synthesized by Gamma-Irradiated Penicillium cyclopium.

PubMed

El-Refai, Heba A; Shafei, Mona S; Mostafa, Hanan; El-Refai, Abdel-Monem H; Araby, Eman M; El-Beih, Fawkia M; Easa, Saadia M; Gomaa, Sanaa K

2016-01-01

Gamma irradiation is used on Penicillium cyclopium in order to obtain mutant cells of high L-asparaginase productivity. Using gamma irradiation dose of 4 KGy, P. cyclopium cells yielded L-asparaginase with extracellular enzyme activity of 210.8 ± 3 U/ml, and specific activity of 752.5 ± 1.5 U/mg protein, which are 1.75 and 1.53 times, respectively, the activity of the wild strain. The enzyme was partially purified by 40-60% acetone precipitation. L-asparaginase was immobilized onto Amberlite IR-120 by ionic binding. Both free and immobilized enzymes exhibited maximum activity at pH 8 and 40 degrees C. The immobilization process improved the enzyme thermal stability significantly. The immobilized enzyme remained 100% active at temperatures up to 60 degrees C, while the free asparaginase was less tolerant to high temperatures. The immobilized enzyme was more stable at pH 9.0 for 50 min, retaining 70% of its relative activity. The maximum reaction rate (V(max)) and Michaelis-Menten constant (K(m)) of the free form were significantly changed after immobilization. The K(m) value for immobilized L-asparaginase was about 1.3 times higher than that of free enzyme. The ions K+, Ba2+ and Na+ showed stimulatory effect on enzyme activity with percentages of 110%, 109% and 106% respectively.

Purification and characterization of polyphenol oxidase from rape flower.

PubMed

Sun, Han-Ju; Wang, Jing; Tao, Xue-Ming; Shi, Juan; Huang, Mei-Ying; Chen, Zhe

2012-01-25

The purification and partial enzymology characteristics of polyphenol oxidase (PPO) from rape flower were studied. After preliminary treatments, the crude enzyme solution was in turn purified with ammonium sulfate, dialysis, and Sephadex G-75 gel chromatography. The optimal conditions and stability of PPO were examined at different pH values and temperatures. Subsequently, PPO was also characterized by substrate (catechol) concentrations, inhibitors, kinetic parameters, and molecular weight. Results showed that the optimal pH for PPO activity was 5.5 in the presence of catechol and that PPO was relatively stable at pH 3.5-5.5. PPO was moderately stable at temperatures from 60 to 70 °C, whereas it was easily denatured at 80-90 °C. Ethylenediaminetetraacetic acid, sodium chloride, and calcium chloride had little inhibitive effects on PPO, whereas citric acid, sodium sulfite, and ascorbic acid had strongly inhibitive effects. The Michaelis-Menten constant (K(m)) and maximal reaction velocity (V(max)) of PPO were 0.767 mol/L and 0.519 Ab/min/mL of the crude PPO solution, respectively. PPO was finally purified to homogeneity with a purification factor of 4.41-fold and a recovery of 12.41%. Its molecular weight was 60.4 kDa, indicating that the PPO is a dimer. The data obtained in this research may help to prevent the enzymatic browning of rape flower during its storage and processing.

Purification, characterization and kinetic properties of extracellular L-asparaginase produced by Cladosporium sp.

PubMed

Mohan Kumar, N S; Manonmani, H K

2013-04-01

L-asparaginase from Cladosporium sp. grown on wheat bran by SSF was purified. Enzyme appeared to be a trimer with homodimer of 37 kDa and another 47 kDa amounting to total mass of 121 kDa as estimated by SDS-PAGE and 120 kDa on gel filtration column. The optimum temperature and pH of the enzyme were 30 °C and 6.3, respectively with Vmax of 4.44 μmol/mL/min and Km of 0.1 M. Substrate specificity studies indicated that, L-asparaginase has greater affinity towards L-asparagine with substrate hydrolysis efficiency (Vmax/Km ratio) eightfold higher than that of L-glutamine. L-asparaginase activity in presence of thiols studied showed decrease in Vmax and increase in Km, indicating nonessential mode of inactivation. Among the thiols tested, β-mercaptomethanol, exerted inhibitory effect, suggesting a critical role of disulphide linkages in maintaining a suitable conformation of the enzyme. Metal ions such as Ca(2+), Co(2+), Cu(2+), Mg(2+), Na(+), K(+) and Zn(2+) significantly affected enzyme activity whereas presence of Fe(3+), Pb(2+) and KI stimulated the activity. Detergents studied also enhanced L-asparaginase activity. In-vitro half-life of purified L-asparaginase in mammalian blood serum was 93.69 h. The enzyme inhibited acrylamide formation in potato chips by 96 % making it a potential candidate for food industry to reduce acrylamide content in starchy fried food commodities.

Purification of an Exopolygalacturonase from Penicillium viridicatum RFC3 Produced in Submerged Fermentation.

PubMed

Gomes, Eleni; Leite, Rodrigo Simões Ribeiro; da Silva, Roberto; Silva, Dênis

2009-01-01

An exo-PG obtained from Penicillium viridicatum in submerged fermentation was purified to homogeneity. The apparent molecular weight of the enzyme was 92 kDa, optimum pH and temperature for activity were pH 5 and 50-55 degrees C. The exo-PG showed a profile of an exo-polygalacturonase, releasing galacturonic acid by hydrolysis of pectin with a high degree of esterification (D.E.). Ions Ca(2+) enhanced the stability of enzyme and its activity by 30%. The K(m) was 1.30 in absence of Ca(2+) and 1.16 mg mL(-1) in presence of this ion. In relation to the V(max) the presence of this ion increased from 1.76 to 2.07 mumol min(-1)mg(-1).

Tidal stripping and the structure of dwarf galaxies in the Local Group

NASA Astrophysics Data System (ADS)

Fattahi, Azadeh; Navarro, Julio F.; Frenk, Carlos S.; Oman, Kyle A.; Sawala, Till; Schaller, Matthieu

2018-05-01

The shallow faint-end slope of the galaxy mass function is usually reproduced in Λ cold dark matter (ΛCDM) galaxy formation models by assuming that the fraction of baryons that turn into stars drops steeply with decreasing halo mass and essentially vanishes in haloes with maximum circular velocities Vmax < 20-30 km s-1. Dark-matter-dominated dwarfs should therefore have characteristic velocities of about that value, unless they are small enough to probe only the rising part of the halo circular velocity curve (i.e. half-mass radii, r1/2 ≪ 1 kpc). Many dwarfs have properties in disagreement with this prediction: they are large enough to probe their halo Vmax but their characteristic velocities are well below 20 km s-1. These `cold faint giants' (an extreme example is the recently discovered Crater 2 Milky Way satellite) can only be reconciled with our ΛCDM models if they are the remnants of once massive objects heavily affected by tidal stripping. We examine this possibility using the APOSTLE cosmological hydrodynamical simulations of the Local Group. Assuming that low-velocity-dispersion satellites have been affected by stripping, we infer their progenitor masses, radii, and velocity dispersions, and find them in remarkable agreement with those of isolated dwarfs. Tidal stripping also explains the large scatter in the mass discrepancy-acceleration relation in the dwarf galaxy regime: tides remove preferentially dark matter from satellite galaxies, lowering their accelerations below the amin ˜ 10-11 m s-2 minimum expected for isolated dwarfs. In many cases, the resulting velocity dispersions are inconsistent with the predictions from Modified Newtonian Dynamics, a result that poses a possibly insurmountable challenge to that scenario.

A mathematical model of the influence of salivary urea on the pH of fasted dental plaque and on the changes occurring during a cariogenic challenge.

PubMed

Dibdin, G H; Dawes, C

1998-01-01

Urea diffusing from saliva into dental plaque is converted to ammonia and carbon dioxide by bacterial ureases. The influence of normal salivary urea levels on the pH of fasted plaque and on the depth and duration of a Stephan curve is uncertain. A numerical model which simulates a cariogenic challenge (a 10% sucrose rinse alone or one followed by use of chewing-gum with or without sugar) was modified to include salivary urea levels from 0 to 30 mmol/l. It incorporated: site-dependent exchange between bulk saliva and plaque surfaces via a salivary film; sugar and urea diffusion into plaque; pH-dependent rates of acid formation and urea breakdown; diffusion and dissociation of end-products and other buffers (acetate, lactate, phosphate, ammonia and carbonate); diffusion of protons and other ions; equilibration with fixed and mobile buffers; and charge-coupling between ionic flows. The Km (2.12 mmol/l) and Vmax (0.11 micromol urea/min/mg dry weight) values for urease activity and the pH dependence of Vmax were taken from the literature. From the results, it is predicted that urea concentrations normally present in saliva (3-5 mmol/l) will increase the pH at the base of a 0.5-mm-thick fasted plaque by up to 1 pH unit, and raise the pH minimum after a sucrose rinse or sugar-containing chewing-gum by at least half a pH unit. The results suggest that plaque cariogenicity may be inversely related to salivary urea concentrations, not only when the latter are elevated because of disease, but even when they are in the normal range.

Administration of progesterone after trauma and hemorrhagic shock prevents hepatocellular injury.

PubMed

Kuebler, Joachim F; Yokoyama, Yukihiro; Jarrar, Doraid; Toth, Balazs; Rue, Loring W; Bland, Kirby I; Wang, Ping; Chaudry, Irshad H

2003-07-01

Administration of a single dose of progesterone following trauma and hemorrhage in progesterone-deficient rats would ameliorate the inflammatory response and hepatocellular damage. A university laboratory. Ovariectomized female Sprague-Dawley rats (250-350 g; Charles River Laboratories, Wilmington, Mass) underwent a 5-cm midline laparotomy (ie, induction of soft tissue trauma), were bled to a mean arterial blood pressure of 35 mm Hg for about 90 minutes, and then were resuscitated using Ringer lactate solution. Progesterone (25 mg/kg of body weight) or vehicle was administered subcutaneously at the end of resuscitation. In additional animals, Kupffer cells were isolated following trauma, hemorrhage, and resuscitation and treated in vitro with progesterone, lipopolysaccharide, or both. Six hours following resuscitation, plasma tumor necrosis factor alpha (TNF-alpha) and interleukin 6 (IL-6) levels and liver myeloperoxidase activity were determined. Hepatocellular function (maximum velocity of indocyanine green clearance [Vmax] and the efficiency of the active transport or Michaelis-Menten constant [Km]) and plasma levels of transaminases were measured 20 hours after resuscitation. Kupffer cell IL-6 and TNF-alpha production were assessed. Plasma levels of TNF-alpha, IL-6, aspartate aminotransferase, and alanine aminotransferase, as well as hepatic myeloperoxidase activity were increased, whereas indocyanine green clearance was depressed in vehicle-treated rats following trauma-hemorrhage. Animals treated with progesterone showed significantly reduced levels of the TNF-alpha, IL-6, and transaminases as well as reduced myeloperoxidase activity in the liver. Progesterone-treated animals showed increased Vmax and Kmax values for indocyanine green. In vitro treatment of Kupffer cells with progesterone decreased TNF-alpha production but did not affect the production of IL-6. Progesterone administration following trauma-hemorrhage ameliorates the proinflammatory response and, subsequently, the hepatocellular injury via direct action on immunocompetent cells.

Identification and characterization of a Na+-dependent neutral amino acid transporter, ASCT1, in rabbit corneal epithelial cell culture and rabbit cornea.

PubMed

Katragadda, Suresh; Talluri, Ravi Sankar; Pal, Dhananjay; Mitra, Ashim K

2005-11-01

The aim of this study was to investigate the presence of a Na+-dependent neutral amino acid transporter, ASCT1, in rabbit primary corneal epithelial cell culture and rabbit cornea. Uptake studies were carried out on rabbit primary corneal epithelial culture (rPCEC) cells using 12-well plates. Transport studies were conducted with isolated rabbit corneas at 34 degrees C. Uptake and transport of L-alanine was determined at various concentrations. Inhibition studies were conducted in presence of various L- and D-amino acids, metabolic inhibitors like ouabain and sodium azide, and in the absence of sodium to delineate the functional characteristics of L-alanine uptake and transport. Reverse transcription-polymerase chain reaction (RT-PCR) was performed on total RNA harvested from rabbit cornea and rPCEC cells for identification of ASCT1. Uptake of L-Ala was found to be saturable with a Km of 0.71 mM and a Vmax value of 0.84 micromoles min(-1) mg(-1) protein. Uptake was independent of pH and energy but depends on sodium. It was inhibited by serine, threonine, cysteine, and glutamine but did not respond to BCH (2-aminobicyclo [2,2,1] heptane-2-carboxylic acid) and MeAIB (alpha -methylaminoisobutyric acid). Transport of L-Ala across rabbit cornea was also saturable (Km 6.52 mM and Vmax 1.09 x 10(-2) micromoles min(-1) cm(-2)), energy independent, and subject to similar competitive inhibition. Presence of ASCT1 on rPCEC and on rabbit cornea was identified by RT-PCR. L-Alanine, the chosen model substrate, was actively transported by Na+-dependent, neutral amino acid exchanger ASCT1, which was identified and functionally characterized on rPCEC cells and rabbit cornea.

Dissociable roles of dopamine and serotonin transporter function in a rat model of negative urgency.

PubMed

Yates, Justin R; Darna, Mahesh; Gipson, Cassandra D; Dwoskin, Linda P; Bardo, Michael T

2015-09-15

Negative urgency is a facet of impulsivity that reflects mood-based rash action and is associated with various maladaptive behaviors in humans. However, the underlying neural mechanisms of negative urgency are not fully understood. Several brain regions within the mesocorticolimbic pathway, as well as the neurotransmitters dopamine (DA) and serotonin (5-HT), have been implicated in impulsivity. Extracellular DA and 5-HT concentrations are regulated by DA transporters (DAT) and 5-HT transporters (SERT); thus, these transporters may be important molecular mechanisms underlying individual differences in negative urgency. The current study employed a reward omission task to model negative urgency in rats. During reward trials, a cue light signaled the non-contingent delivery of one sucrose pellet; immediately following the non-contingent reward, rats responded on a lever to earn sucrose pellets (operant phase). Omission trials were similar to reward trials, except that non-contingent sucrose was omitted following the cue light prior to the operant phase. As expected, contingent responding was higher following omission of expected reward than following delivery of expected reward, thus reflecting negative urgency. Upon completion of behavioral training, Vmax and Km were obtained from kinetic analysis of [(3)H]DA and [(3)H]5-HT uptake using synaptosomes prepared from nucleus accumbens (NAc), dorsal striatum (Str), medial prefrontal cortex (mPFC), and orbitofrontal cortex (OFC) isolated from individual rats. Vmax for DAT in NAc and for SERT in OFC were positively correlated with negative urgency scores. The current findings suggest that mood-based impulsivity (negative urgency) is associated with enhanced DAT function in NAc and SERT function in OFC. Copyright © 2015 Elsevier B.V. All rights reserved.

The Oncogene PDRG1 Is an Interaction Target of Methionine Adenosyltransferases

PubMed Central

Garrido, Francisco; Reytor, Edel; Portillo, Francisco; Pajares, María A.

2016-01-01

Methionine adenosyltransferases MAT I and MAT III (encoded by Mat1a) catalyze S-adenosylmethionine synthesis in normal liver. Major hepatic diseases concur with reduced levels of this essential methyl donor, which are primarily due to an expression switch from Mat1a towards Mat2a. Additional changes in the association state and even in subcellular localization of these isoenzymes are also detected. All these alterations result in a reduced content of the moderate (MAT I) and high Vmax (MAT III) isoenzymes, whereas the low Vmax (MAT II) isoenzyme increases and nuclear accumulation of MAT I is observed. These changes derive in a reduced availability of cytoplasmic S-adenosylmethionine, together with an effort to meet its needs in the nucleus of damaged cells, rendering enhanced levels of certain epigenetic modifications. In this context, the putative role of protein-protein interactions in the control of S-adenosylmethionine synthesis has been scarcely studied. Using yeast two hybrid and a rat liver library we identified PDRG1 as an interaction target for MATα1 (catalytic subunit of MAT I and MAT III), further confirmation being obtained by immunoprecipitation and pull-down assays. Nuclear MATα interacts physically and functionally with the PDRG1 oncogene, resulting in reduced DNA methylation levels. Increased Pdrg1 expression is detected in acute liver injury and hepatoma cells, together with decreased Mat1a expression and nuclear accumulation of MATα1. Silencing of Pdrg1 expression in hepatoma cells alters their steady-state expression profile on microarrays, downregulating genes associated with tumor progression according to GO pathway analysis. Altogether, the results unveil the role of PDRG1 in the control of the nuclear methylation status through methionine adenosyltransferase binding and its putative collaboration in the progression of hepatic diseases. PMID:27548429

[Origin of motion in the human ureter: mechanics, energetics and kinetics of the myosin molecular motors].

PubMed

Vargiu, Romina; Perinu, Anna; De Lisa, Antonello; Tintrup, Frank; Manca, Francesco; Mancinelli, Rino

2012-01-01

Ureteral peristalsis is the result of coordinated mechanical motor performance of longitudinal and circular smooth muscle layer of the ureter wall. The main aim of this study was to characterize in smooth muscle of proximal segments of human ureter, the mechanical properties at level of muscle tissue and at level of myosin molecular motors. Ureteral samples were collected from 15 patients, who underwent nephrectomy for renal cancer. Smooth muscle strips longitudinally and circularly oriented from proximal segments of human ureter were used for the in vitro experiments. Mechanical indices including the maximum unloaded shortening velocity (Vmax), and the maximum isometric tension (P0) normalized per cross-sectional area, were determined in vitro determined in electrically evoked contractions of longitudinal and circular smooth muscle strips. Myosin cross-bridge (CB) number per mm2 (Ψ) the elementary force per single CB (Ψ) and kinetic parameters were calculated in muscle strips, using Huxley's equations adapted to nonsarcomeric muscles. Longitudinal smooth muscle strips exhibited a significantly (p<0.05) faster Vmax (63%) and a higher P0 (40%), if compared to circular strips. Moreover, longitudinal muscle strips showed a significantly higher unitary force (Ψ) per CB. However, no significant differences were observed in CB number, the attachment (f1) and the detachment (g2) rate constants between longitudinal and circular muscle strips. The main result obtained in the present work documents that the mechanical, energetic and unitary forces per CB of longitudinal layer of proximal ureter are better compared to the circular one; these preliminary findings suggested, unlike intestinal smooth muscle, a major role of longitudinal smooth muscle layer in the ureter peristalsis.

[Establishment of an in vitro screening model for steroid 5 alpha-reductase inhibitors with the microplate reader].

PubMed

Wu, Jian-Hui; Sun, Zu-Yue

2013-06-01

To establish an in vitro screening model for steroid 5 alpha-reductase inhibitors using the microplate reader. Steroid 5 alpha-reductase was obtained from the liver of female rats, an in vitro screening model for steroid 5 alpha-reductase inhibitors established using the 96-well plate and microplate reader after determination of the enzymatic activity, and the reliability of the model verified with the known 5 alpha-reductase inhibitors epristeride and finasteride. Added to the 96-well plate were the final concentrations of testosterone (0-40 micromol/L), NADPH (22 micromol/L), epristeride (0-60 nmol/L) or finasteride (0-60 nmol/ L) and steroid 5 alpha-reductase (20 microl), the total volume of each well adjusted to 200 microl with Tris-Hcl buffer. The 96-well plate was placed in the microplate reader, mixed and incubated at 37 degrees C, followed by detection of the A340nm value at 0 and 10 min and analysis of the data. The Km value of steroid 5 alpha-reductase was 3.794 micromol/L, with a Vmax of 0.271 micromol/(L. min). The Ki of epristeride was 148.2 nmol/L, with an IC50 of 31.5 nmol/L, and the enzymatic reaction kinetic curve suggested that epristeride was an uncompetitive enzyme inhibitor. The Ki of finasteride was 158. 8 nmol/L, with an IC50 of 13.6 nmol/L. The enzymatic reaction kinetic curve showed that both epristeride and finasteride were competitive enzyme inhibitors, similar to those reported in the published literature. A screening model was successfully established, which could rapidly and effectively screen steroid 5 alpha-reductase inhibitors in vitro.

Age-related changes in the activity of the pyruvate carrier and in the lipid composition in rat-heart mitochondria.

PubMed

Paradies, G; Ruggiero, F M

1990-04-05

The effect of aging on the activity of the pyruvate translocator and on the lipid composition in rat-heart mitochondria has been investigated. It has been found that the rate of pyruvate transport in mitochondria from aged rats (28 months old) is markedly reduced (38%) as compared with that obtained with mitochondria from young adults rats (4 months old). Kinetic analysis of the pyruvate transport shows that only the Vmax of this process is decreased, while there is no change in the Km values. The age-related decrement in the activity of the pyruvate carrier is not due to a decrease in the transmembrane delta pH value, neither does it depend on a decrease in the total number of the pyruvate carrier molecules, titrated with radioactive alpha-cyanocinnamate. The lower activity of the pyruvate translocator in mitochondria from aged rats is associated to a parallel decrement of the rate of pyruvate-dependent oxygen uptake. There is, however no appreciable difference in either the respiratory control ratios or in the ADP/O ratios between these two types of mitochondrion. The Arrhenius plot characteristics differ for pyruvate transport activity in mitochondria from aged rats as compared with young rats in that the break point of the biphasic plot is shifted to a higher temperature. The heart mitochondrial lipid composition is significantly altered in aged rats. The total cholesterol increases (43%), the phospholipids decrease (15%) and the cholesterol/phospholipid molar ratio increases (68%). Among phospholipids, cardiolipin shows the greatest alteration (28% decrease in aged rats). The lower activity of the pyruvate carrier in mitochondria from aged rats may be ascribed to changes in the lipid domain surrounding the carrier molecule in the membrane.

Electrospun regenerated cellulose nanofibrous membranes surface-grafted with polymer chains/brushes via the atom transfer radical polymerization method for catalase immobilization.

PubMed

Feng, Quan; Hou, Dayin; Zhao, Yong; Xu, Tao; Menkhaus, Todd J; Fong, Hao

2014-12-10

In this study, an electrospun regenerated cellulose (RC) nanofibrous membrane with fiber diameters of ∼200-400 nm was prepared first; subsequently, 2-hydroxyethyl methacrylate (HEMA), 2-dimethylaminoethyl methacrylate (DMAEMA), and acrylic acid (AA) were selected as the monomers for surface grafting of polymer chains/brushes via the atom transfer radical polymerization (ATRP) method. Thereafter, four nanofibrous membranes (i.e., RC, RC-poly(HEMA), RC-poly(DMAEMA), and RC-poly(AA)) were explored as innovative supports for immobilization of an enzyme of bovine liver catalase (CAT). The amount/capacity, activity, stability, and reusability of immobilized catalase were evaluated, and the kinetic parameters (Vmax and Km) for immobilized and free catalase were determined. The results indicated that the respective amounts/capacities of immobilized catalase on RC-poly(HEMA) and RC-poly(DMAEMA) nanofibrous membranes reached 78 ± 3.5 and 67 ± 2.7 mg g(-1), which were considerably higher than the previously reported values. Meanwhile, compared to that of free CAT (i.e., 18 days), the half-life periods of RC-CAT, RC-poly(HEMA)-CAT, RC-poly(DMAEMA)-CAT, and RC-poly(AA)-CAT were 49, 58, 56, and 60 days, respectively, indicating that the storage stability of immobilized catalase was also significantly improved. Furthermore, the immobilized catalase exhibited substantially higher resistance to temperature variation (tested from 5 to 70 °C) and lower degree of sensitivity to pH value (tested from 4.0 and 10.0) than the free catalase. In particular, according to the kinetic parameters of Vmax and Km, the nanofibrous membranes of RC-poly(HEMA) (i.e., 5102 μmol mg(-1) min(-1) and 44.89 mM) and RC-poly(DMAEMA) (i.e., 4651 μmol mg(-1) min(-1) and 46.98 mM) had the most satisfactory biocompatibility with immobilized catalase. It was therefore concluded that the electrospun RC nanofibrous membranes surface-grafted with 3-dimensional nanolayers of polymer chains/brushes would be suitable/ideal as efficient supports for high-density and reusable enzyme immobilization.

In vivo maximal fascicle-shortening velocity during plantar flexion in humans.

PubMed

Hauraix, Hugo; Nordez, Antoine; Guilhem, Gaël; Rabita, Giuseppe; Dorel, Sylvain

2015-12-01

Interindividual variability in performance of fast movements is commonly explained by a difference in maximal muscle-shortening velocity due to differences in the proportion of fast-twitch fibers. To provide a better understanding of the capacity to generate fast motion, this study aimed to 1) measure for the first time in vivo the maximal fascicle-shortening velocity of human muscle; 2) evaluate the relationship between angular velocity and fascicle-shortening velocity from low to maximal angular velocities; and 3) investigate the influence of musculo-articular features (moment arm, tendinous tissues stiffness, and muscle architecture) on maximal angular velocity. Ultrafast ultrasound images of the gastrocnemius medialis were obtained from 31 participants during maximal isokinetic and light-loaded plantar flexions. A strong linear relationship between fascicle-shortening velocity and angular velocity was reported for all subjects (mean R(2) = 0.97). The maximal shortening velocity (V(Fmax)) obtained during the no-load condition (NLc) ranged between 18.8 and 43.3 cm/s. V(Fmax) values were very close to those of the maximal shortening velocity (V(max)), which was extrapolated from the F-V curve (the Hill model). Angular velocity reached during the NLc was significantly correlated with this V(Fmax) (r = 0.57; P < 0.001). This finding was in agreement with assumptions about the role of muscle fiber type, whereas interindividual comparisons clearly support the fact that other parameters may also contribute to performance during fast movements. Nevertheless, none of the biomechanical features considered in the present study were found to be directly related to the highest angular velocity, highlighting the complexity of the upstream mechanics that lead to maximal-velocity muscle contraction. Copyright © 2015 the American Physiological Society.

Purification and investigation of some kinetic properties of glucose-6-phosphate dehydrogenase from parsley (Petroselinum hortense) leaves.

PubMed

Coban, T Abdül Kadir; Ciftçi, Mehmet; Küfrevioğlu, O Irfan

2002-05-01

In this study, glucose-6-phosphate dehydrogenase (D-glucose-6-phosphate: NADP+ oxidoreductase, EC 1.1.1.49; G6PD) was purified from parsley (Petroselinum hortense) leaves, and analysis of the kinetic behavior and some properties of the enzyme were investigated. The purification consisted of three steps: preparation of homogenate, ammonium sulfate fractionation, and DEAE-Sephadex A50 ion exchange chromatography. The enzyme was obtained with a yield of 8.79% and had a specific activity of 2.146 U (mg protein)(-1). The overall purification was about 58-fold. Temperature of +4 degrees C was maintained during the purification process. Enzyme activity was spectrophotometrically measured according to the Beutler method, at 340 nm. In order to control the purification of enzyme, SDS-polyacrylamide gel electrophoresis was carried out in 4% and 10% acrylamide for stacking and running gel, respectively. SDS-polyacrylamide gel electrophoresis showed a single band for enzyme. The molecular weight was found to be 77.6 kDa by Sephadex G-150 gel filtration chromatography. A protein band corresponding to a molecular weight of 79.3 kDa was obtained on SDS-polyacrylamide gel electrophoresis. For the enzymes, the stable pH, optimum pH, and optimum temperature were found to be 6.0, 8.0, and 60 degrees C, respectively. Moreover, KM and Vmax values for NADP+ and G6-P at optimum pH and 25 degrees C were determined by means of Lineweaver-Burk graphs. Additionally, effects of streptomycin sulfate and tetracycline antibiotics were investigated for the enzyme activity of glucose-6-phosphate dehydrogenase in vitro.

Scaling Factor Variability and Toxicokinetic Outcomes in Children

EPA Science Inventory

Abstract title: Scaling Factor Variability and Toxicokinetic Outcomes in ChildrenBackgroundBiotransformation rates (Vmax) extrapolated from in vitro data are used increasingly in human physiologically based pharmacokinetic (PBPK) models. PBPK models are widely used in human hea...

Hidden Risks of Erectile Dysfunction "Treatments" Sold Online

MedlinePlus

... screen and stop these shipments from entering U.S. commerce," says Huascar Batista, team leader of OOC's Import- ... Free Rhino V Max V.Max True Man Energy Max HS Joy of Love NaturalUp Blue Steel ...

Partial purification and characterization of polyphenoloxidase from culinary-medicinal Royal Sun mushroom (the Himematsutake), Agaricus brasiliensis S. Wasser et al. (Agaricomycetideae).

PubMed

Matsumoto-Akanuma, Akiko; Akanuma, Satoshi; Motoi, Masuro; Yamagishi, Akihiko; Ohno, Naohito

2011-01-01

The Royal Sun mushroom, the Himematsutake culinary-medicinal mushroom, Agaricus brasiliensis has several polyphenoloxidase activities in a broad sense. Here we report the partial purification of tyrosinase-type polyphenoloxidase (PPO). PPO is purified from A. brasiliensis without browning using a two-phase partitioning with Triton X-114 and ammonium sulfate fractionation. Partially denaturing SDS-PAGE (sodium dodecyl sulfate-polyacrylamide electrophoresis) staining with L-3,4-dihydroxyphenylalanine was performed and the indicated molecular sizes were approximately 70 kDa and 45 kDa. The purified enzyme is in its latent state and can be activated maximally in the presence of 1.6 mM sodium dodecyl sulfate (SDS). This enzyme catalyzes two distinct reactions, monophenolase and diphenolase activity, and the monophenolase activity showed a lag time typical of polyphenoloxidase. The K(m) value for 4-tert-butylcatechol was quite similar in the presence and absence of SDS, but the apparent V(max) value was increased 2.0-fold by SDS. Mimosine was a typical competitive inhibitor with K(i) values of 138.2 microM and 281.0 microM n the presence and absence of SDS, respectively.

Simple predictions of maximum transport rate in unsaturated soil and rock

USGS Publications Warehouse

Nimmo, John R.

2007-01-01

In contrast with the extreme variability expected for water and contaminant fluxes in the unsaturated zone, evidence from 64 field tests of preferential flow indicates that the maximum transport speed Vmax, adjusted for episodicity of infiltration, deviates little from a geometric mean of 13 m/d. A model based on constant‐speed travel during infiltration pulses of actual or estimated duration can predict Vmax with approximate order‐of‐magnitude accuracy, irrespective of medium or travel distance, thereby facilitating such problems as the prediction of worst‐case contaminant traveltimes. The lesser variability suggests that preferential flow is subject to rate‐limiting mechanisms analogous to those that impose a terminal velocity on objects in free fall and to rate‐compensating mechanisms analogous to Le Chatlier's principle. A critical feature allowing such mechanisms to dominate may be the presence of interfacial boundaries confined by neither solid material nor capillary forces.

Isolation, Fractionation and Characterization of Catalase from Neurospora crassa (InaCC F226)

NASA Astrophysics Data System (ADS)

Suryani; Ambarsari, L.; Lindawati, E.

2017-03-01

Catalase from Indigenous isolate Neurospora crassa InaCC F226 has been isolated, fractionated and characterized. Production of catalase by Neurospora crassa was done by using PDA medium (Potato Dextrosa Agar) and fractionated with ammonium sulphate with 20-80% saturation. Fraction 60% was optimum saturation of ammonium sulphate and had highest specific activity 3339.82 U/mg with purity 6.09 times, total protein 0.920 mg and yield 88.57%. The optimum pH and temperature for catalase activity were at 40°C and pH 7.0, respectively. The metal ions that stimulated catalase activity acted were Ca2+, Mn2+ and Zn2+, and inhibitors were EDTA, Mg2+ and Cu2+. Based on Km and Vmax values were 0.2384 mM and 13.3156 s/mM.

Parameters for the Operation of Bacterial Thiosalt Oxidation Ponds

PubMed Central

Silver, M.

1985-01-01

Shake flask and pH-controlled reactor tests were used to determine the mathematical parameters for a mixed-culture bacterial thiosalt treatment pond. Values determined were as follows: Km and Vmax (thiosulfate), 9.83 g/liter and 243.9 mg/liter per h, respectively; Ki (lead), 3.17 mg/liter; Ki (copper), 1.27 mg/liter; Q10 between 10 and 30°C, 1.95. From these parameters, the required bioxidation pond volume and residence time could be calculated. Soluble zinc (0.2 g/liter) and particulate mill products and by-products (0.25 g/liter) were not inhibitory. Correlation with an operating thiosalt biooxidation pond showed the parameters used to be valid for thiosalt concentrations up to at least 2 g/liter, lead concentrations of at least 10 mg/liter, and temperatures of >2°C. PMID:16346885

Cloning of Arabidopsis serotonin N-acetyltransferase and its role with caffeic acid O-methyltransferase in the biosynthesis of melatonin in vitro despite their different subcellular localizations.

PubMed

Lee, Hyoung Yool; Byeon, Yeong; Lee, Kyungjin; Lee, Hye-Jung; Back, Kyoungwhan

2014-11-01

Serotonin N-acetyltransferase (SNAT) is the penultimate enzyme in melatonin biosynthesis. We cloned SNAT from Arabidopsis thaliana (AtSNAT) and functionally characterized this enzyme for the first time from dicotyledonous plants. Similar to rice SNAT, AtSNAT was found to localize to chloroplasts with peak enzyme activity at 45 °C (Km , 309 μm; Vmax , 1400 pmol/min/mg protein). AtSNAT also catalyzed 5-methoxytryptamine (5-MT) into melatonin with high catalytic activity (Km , 51 μm; Vmax , 5300 pmol/min/mg protein). In contrast, Arabidopsis caffeic acid O-methyltransferase (AtCOMT) localized to the cytoplasm. Interestingly, AtCOMT can methylate serotonin into 5-MT with low catalytic activity (Km , 3.396 mm; Vmax , 528 pmol/min/mg protein). These data suggest that serotonin can be converted into either N-acetylserotonin by SNAT or into 5-MT by COMT, after which it is metabolized into melatonin by COMT or SNAT, respectively. To support this hypothesis, serotonin was incubated in the presence of both AtSNAT and AtCOMT enzymes. In addition to melatonin production, the production of major intermediates depended on incubation temperatures; N-acetylserotonin was predominantly produced at high temperatures (45 °C), while low temperatures (37 °C) favored the production of 5-MT. Our results provide biochemical evidence for the presence of a serotonin O-methylation pathway in plant melatonin biosynthesis. © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Electrophysiological mechanisms of sophocarpine as a potential antiarrhythmic agent.

PubMed

Yang, Zhi-fang; Li, Ci-zhen; Wang, Wei; Chen, Ying-min; Zhang, Ying; Liu, Yuan-mou; Wang, Hong-wei

2011-03-01

To examine the electrophysiological effects of sophocarpine on action potentials (AP) and ionic currents of cardiac myocytes and to compare some of these effects with those of amiodarone. Langendorff perfusion set-up was used in isolated guinea pig heart, and responses to sophocarpine were monitored using electrocardiograph. Conventional microelectrode, voltage clamp technique and perforated patch were employed to record fast response AP (fAP), slow response AP (sAP) and ionic currents in guinea pig papillary muscle or rabbit sinus node cells. Tachyarrhythmia produced by isoprenaline (15 μmol/L) could be reversed by sophocarpine (300 μmol/L). Sophocarpine (10 μmol/L) decreased the amplitude by 4.0%, maximal depolarization velocity (V(max)) of the fAP by 24.4%, and Na(+) current (I(Na)) by 18.0%, while it prolonged the effective refractory period (ERP) by 21.1%. The same concentration of sophocarpine could also decrease the amplitude and V(max) of the sAP, by 26.8% and 25.7%, respectively, and attenuated the Ca(2+) current (I(CaL)) and the K(+) tail current substantially. Comparison of sophocarpine with amiodarone demonstrated that both prolonged the duration and the ERP of fAP and sAP, both decreased the amplitude and V(max) of the fAP and sAP, and both slowed the automatic heart rate. Sophocarpine could reverse isoprenaline-induced arrhythmia and inhibit I(Na), I(CaL), and I(Kr) currents. The electrophysiological effects of sophocarpine are similar to those of amiodarone, which might be regarded as a prospective antiarrhythmic agent.

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.

Differences in energy capacities between tennis players and runners.

PubMed

Novak, Dario; Vucetić, Vlatko; Zugaj, Sanja

2013-05-01

The primary purpose of this study was to determine differences between elite athletes and tennis players in order to provide a clearer picture regarding the energy demands in modern tennis. Forty-eight (48) athletes and 24 tennis players from Croatian national leagues were compared in morphological and physiological parameters of an all-out incremental treadmill test with gas exchange measurements. Tennis players' HRmax (192.96+/-7.75 bpm) shows values that are most different to 400-meters sprinters (200.13+/-6.95 bpm). Maximum running speed of tennis players on the treadmill (vmax) is no different with the speed achieved by sprinters, while there are significant differences among other athletes. Values in running speed at anaerobic threshold (vAnT) show no statistically significant difference with the values for athlete sprinters and 400-m sprinters. Values of RvO2max for tennis players indicate significant similarities with athlete sprinters and 400-m sprinters while the values of RvO2AnT are nearly identical with the values for sprinters and show no statistically significant differences (p<0.05). The results indicate that values achieved by tennis players approximate most different those of the middle and long distance runners. This singles out the possible importance of the anaerobic capacity and the high level of sprint endurance in tennis players. Knowing these characteristics is the basis for planning and implementing training systems that will enable the increase and optimal usage of energy capacities of tennis players in possibly improving sports results.

Characterization of deltamethrin metabolism by rat plasma and liver microsomes

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

Anand, Sathanandam S.; Bruckner, James V.; Haines, Wendy T.

2006-04-15

Deltamethrin, a widely used type II pyrethroid insecticide, is a relatively potent neurotoxicant. While the toxicity has been extensively examined, toxicokinetic studies of deltamethrin and most other pyrethroids are very limited. The aims of this study were to identify, characterize, and assess the relative contributions of esterases and cytochrome P450s (CYP450s) responsible for deltamethrin metabolism by measuring deltamethrin disappearance following incubation of various concentrations (2 to 400 {mu}M) in plasma (esterases) and liver microsomes (esterases and CYP450s) prepared from adult male rats. While the carboxylesterase metabolism in plasma and liver was characterized using an inhibitor, tetra isopropyl pyrophosphoramide (isoOMPA), CYP450more » metabolism was characterized using the cofactor, NADPH. Michaelis-Menten rate constants were calculated using linear and nonlinear regression as applicable. The metabolic efficiency of these pathways was estimated by calculating intrinsic clearance (Vmax/Km). In plasma, isoOMPA completely inhibited deltamethrin biotransformation at concentrations (2 and 20 {mu}M of deltamethrin) that are 2- to 10-fold higher than previously reported peak blood levels in deltamethrin-poisoned rats. For carboxylesterase-mediated deltamethrin metabolism in plasma, Vmax = 325.3 {+-} 53.4 nmol/h/ml and Km = 165.4 {+-} 41.9 {mu}M. Calcium chelation by EGTA did not inhibit deltamethrin metabolism in plasma or liver microsomes, indicating that A-esterases do not metabolize deltamethrin. In liver microsomes, esterase-mediated deltamethrin metabolism was completely inhibited by isoOMPA, confirming the role of carboxylesterases. The rate constants for liver carboxylesterases were Vmax = 1981.8 {+-} 132.3 nmol/h/g liver and Km = 172.5 {+-} 22.5 {mu}M. Liver microsomal CYP450-mediated biotransformation of deltamethrin was a higher capacity (Vmax = 2611.3 {+-} 134.1 nmol/h/g liver) and higher affinity (Km = 74.9 {+-} 5.9 {mu}M) process than carboxylesterase (plasma or liver) detoxification. Genetically engineered individual rat CYP450s (Supersomes) were used to identify specific CYP450 isozyme(s) involved in the deltamethrin metabolism. CYP1A2, CYP1A1, and CYP2C11 in decreasing order of importance quantitatively, metabolized deltamethrin. Intrinsic clearance by liver CYP450s (35.5) was more efficient than that by liver (12.0) or plasma carboxylesterases (2.4)« less

Purification and biochemical characterization of glucose 6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase and glutathione reductase from rat lung and inhibition effects of some antibiotics.

PubMed

Adem, Sevki; Ciftci, Mehmet

2016-12-01

G6PD, 6PGD and GR have been purified separately in the single step from rat lung using 2', 5'-ADP Sepharose 4B affinity chromatography. The purified enzymes showed a single band on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The molecular weights of the enzymes were estimated to be 134 kDa for G6PD, 107 kDa for 6PGD and 121 kDa for GR by Sephadex G-150 gel filtration chromatography, and the subunit molecular weights was respectively found to be 66, 52 and 63 kDa by SDS-PAGE. Optimum pH, stable pH, optimum ionic strength, optimum temperature, KM and Vmax values for substrates were determined. Product inhibition studies were also performed. The enzymes were inhibited by levofloxacin, furosemide, ceftazidime, cefuroxime and gentamicin as in vitro with IC50 values in the range of 0.07-30.13 mM. In vivo studies demonstrated that lung GR was inhibited by furosemide and lung 6PGD was inhibited by levofloxacin.

Characterization of polyphenol oxidase from Cape gooseberry (Physalis peruviana L.) fruit.

PubMed

Bravo, Karent; Osorio, Edison

2016-04-15

Cape gooseberry (Physalis peruviana) is an exotic fruit highly valued, however it is a very rich source of polyphenol oxidase (PPO). In this study, Cape gooseberry PPO was isolated and biochemically characterized. The enzyme was extracted and purified using acetone and aqueous two-phase systems. The data indicated that PPO had the highest substrate affinity for chlorogenic acid, 4-methylcatechol and catechol. Chlorogenic acid was the most suitable substrate (Km=0.56±0.07 mM and Vmax=53.15±2.03 UPPO mL(-1) min(-1)). The optimal pH values were 5.5 for catechol and 4-methylcatechol and 5.0 for chlorogenic acid. Optimal temperatures were 40°C for catechol, 25°C for 4-methylcatechol and 20°C for chlorogenic acid. In inhibition tests, the most potent inhibitor was found to be ascorbic acid followed by L-cysteine and quercetin. This study shows possible treatments that can be implemented during the processing of Cape gooseberry fruits to prevent browning. Copyright © 2015 Elsevier Ltd. All rights reserved.

Taurine transport across hepatocyte plasma membranes: analysis in isolated rat liver sinusoidal plasma membrane vesicles.

PubMed

Inoue, M; Arias, I M

1988-07-01

To elucidate the mechanism of taurine transport across the hepatic plasma membranes, rat liver sinusoidal plasma membrane vesicles were isolated and the transport process was analyzed. In the presence of a sodium gradient across the membranes (vesicle inside less than vesicle outside), an overshooting uptake of taurine occurred. In the presence of other ion gradients (K+, Li+, and choline+), taurine uptake was very small and no such overshoot was observed. Sodium-dependent uptake of taurine occurred into an osmotically active intravesicular space. Taurine uptake was stimulated by preloading vesicles with unlabeled taurine (transstimulation) in the presence of NaCl, but not in the presence of KCl. Sodium-dependent transport followed saturation kinetics with respect to taurine concentration; double-reciprocal plots of uptake versus taurine concentration gave a straight line from which an apparent Km value of 0.38 mM and Vmax of 0.27 nmol/20 s x mg of protein were obtained. Valinomycin-induced K+-diffusion potential failed to enhance the rate of taurine uptake, suggesting that taurine transport does not depend on membrane potential. Taurine transport was inhibited by structurally related omega-amino acids, such as beta-alanine and gamma-aminobutyric acid, but not by glycine, epsilon-aminocaproic acid, or other alpha-amino acids, such as L-alanine. These results suggest that Na+-dependent uptake of taurine might occur across the hepatic sinusoidal plasma membranes via a transport system that is specific for omega-amino acids having 2-3 carbon chain length.

Laccase production by the aquatic ascomycete Phoma sp. UHH 5-1-03 and the white rot basidiomycete Pleurotus ostreatus DSM 1833 during submerged cultivation on banana peels and enzyme applicability for the removal of endocrine-disrupting chemicals.

PubMed

Libardi, Nelson; Gern, Regina Maria Miranda; Furlan, Sandra Aparecida; Schlosser, Dietmar

2012-07-01

This work aimed to study the production of laccase from Pleurotus ostreatus DSM 1833 and Phoma sp. UHH 5-1-03 using banana peels as alternative carbon source, the subsequent partial purification and characterization of the enzyme, as well the applicability to degrade endocrine disruptors. The laccase stability with pH and temperature, the optimum pH, the K (m) and V(max) parameters, and the molar mass were determined. Tests were conducted for assessing the ability of degradation of the endocrine disruptors t-nonylphenol, bisphenol A, and 17α-ethinylestradiol. Laccase production of 752 and 1,117 U L⁻¹ was obtained for Phoma sp. and P. ostreatus, respectively. Phoma sp. laccase showed higher stability with temperature and pH. The laccase from both species showed higher affinity by syringaldazine. The culture broth with banana peels induced the production of two isoforms of P. ostreatus (58.7 and 21 kDa) and one of Phoma sp. laccase (72 kDa). In the first day of incubation, the concentrations of bisphenol A and 17α-ethinylestradiol were reduced to values close to zero and after 3 days the concentration of t-nonylphenol was reduced in 90% by the P. ostreatus laccase, but there was no reduction in its concentration by the Phoma sp. laccase.

Functional and Biochemical Analysis of Chlamydia trachomatis MurC, an Enzyme Displaying UDP-N-Acetylmuramate:Amino Acid Ligase Activity

PubMed Central

Hesse, Lars; Bostock, Julieanne; Dementin, Sebastien; Blanot, Didier; Mengin-Lecreulx, Dominique; Chopra, Ian

2003-01-01

Chlamydiae are unusual obligate intracellular bacteria that cause serious infections in humans. Chlamydiae contain genes that appear to encode products with peptidoglycan biosynthetic activity. The organisms are also susceptible to antibiotics that inhibit peptidoglycan synthesis. However, chlamydiae do not synthesize detectable peptidoglycan. The paradox created by these observations is known as the chlamydial anomaly. The MurC enzyme of chlamydiae, which is synthesized as a bifunctional MurC-Ddl product, is expected to possess UDP-N-acetylmuramate (UDP-MurNAc):l-alanine ligase activity. In this paper we demonstrate that the MurC domain of the Chlamydia trachomatis bifunctional protein is functionally expressed in Escherichia coli, since it complements a conditional lethal E. coli mutant possessing a temperature-sensitive lesion in MurC. The recombinant MurC domain was overexpressed in and purified from E. coli. It displayed in vitro ATP-dependent UDP-MurNAc:l-alanine ligase activity, with a pH optimum of 8.0 and dependence upon magnesium ions (optimum concentration, 20 mM). Its substrate specificity was studied with three amino acids (l-alanine, l-serine, and glycine); comparable Vmax/Km values were obtained. Our results are consistent with the synthesis of a muramic acid-containing polymer in chlamydiae with UDP-MurNAc-pentapeptide as a precursor molecule. However, due to the lack of specificity of MurC activity in vitro, it is not obvious which amino acid is present in the first position of the pentapeptide. PMID:14594822

Functional and biochemical analysis of Chlamydia trachomatis MurC, an enzyme displaying UDP-N-acetylmuramate:amino acid ligase activity.

PubMed

Hesse, Lars; Bostock, Julieanne; Dementin, Sebastien; Blanot, Didier; Mengin-Lecreulx, Dominique; Chopra, Ian

2003-11-01

Chlamydiae are unusual obligate intracellular bacteria that cause serious infections in humans. Chlamydiae contain genes that appear to encode products with peptidoglycan biosynthetic activity. The organisms are also susceptible to antibiotics that inhibit peptidoglycan synthesis. However, chlamydiae do not synthesize detectable peptidoglycan. The paradox created by these observations is known as the chlamydial anomaly. The MurC enzyme of chlamydiae, which is synthesized as a bifunctional MurC-Ddl product, is expected to possess UDP-N-acetylmuramate (UDP-MurNAc):L-alanine ligase activity. In this paper we demonstrate that the MurC domain of the Chlamydia trachomatis bifunctional protein is functionally expressed in Escherichia coli, since it complements a conditional lethal E. coli mutant possessing a temperature-sensitive lesion in MurC. The recombinant MurC domain was overexpressed in and purified from E. coli. It displayed in vitro ATP-dependent UDP-MurNAc:L-alanine ligase activity, with a pH optimum of 8.0 and dependence upon magnesium ions (optimum concentration, 20 mM). Its substrate specificity was studied with three amino acids (L-alanine, L-serine, and glycine); comparable Vmax/Km values were obtained. Our results are consistent with the synthesis of a muramic acid-containing polymer in chlamydiae with UDP-MurNAc-pentapeptide as a precursor molecule. However, due to the lack of specificity of MurC activity in vitro, it is not obvious which amino acid is present in the first position of the pentapeptide.

Purification and characterization of an extracellular trypsin-like protease of Fusarium oxysporum var. lini.

PubMed

Barata, Ricardo Andrade; Andrade, Milton Hercules Guerra; Rodrigues, Roberta Dias; Castro, Ieso Miranda

2002-01-01

An alkaline serineprotease, capable of hydrolyzing Nalpha-benzoyl- dl arginine p-nitroanilide, was secreted by Fusarium oxysporum var. lini grown in the presence of gelatin as the sole nitrogen and carbon source. The protease was purified 65-fold to electrophoretic homogenity from the culture supernatant in a three-step procedure comprising QSepharose chromatography, affinity chromatography, and FPLC on a MonoQ column. SDS-PAGE analysis of the purified protein indicated an estimated molecular mass of 41 kDa. The protease had optimum activity at a reaction temperature of 45 degrees C and showed a rapid decrease of activity at 48 degrees C. The optimum pH was around 8.0. Characterization of the protease showed that Ca2+ and Mg2+ cations increased the activity, which was not inhibited by EDTA or 1,10-phenanthroline. The enzyme activity on Nalpha-benzoyl-DL arginine p-nitroanilide was inhibited by 4-(2-aminoethyl)-benzenesulfonyl fluoride hydrochloride, p-aminobenzamidine dihydrochloride, aprotinin, 3-4 dichloroisocoumarin, and N-tosyl-L-lysine chloromethyl ketone. The enzyme is also inhibited by substrate concentrations higher than 2.5 x 10(-4)M. The protease had a Michaelis-Menten constant of 0.16 mM and a V(max) of 0.60 mumol released product.min(-1).mg(-1) enzyme when assayed in a non-inhibiting substrate concentration. The activity on Nalpha-benzoyl- dl arginine p-nitroanilide was competitively inhibited by p-aminobenzamidine dihydrochoride. A K(i) value of 0.04 mM was obtained.

Biochemical and Functional Studies on the Burkholderia cepacia Complex bceN Gene, Encoding a GDP-D-Mannose 4,6-Dehydratase

PubMed Central

Pinheiro, Pedro F.; Leitão, Jorge H.

2013-01-01

This work reports the biochemical and functional analysis of the Burkholderia cenocepacia J2315 bceN gene, encoding a protein with GDP-D-mannose 4,6-dehydratase enzyme activity (E.C.4.2.1.47). Data presented indicate that the protein is active when in the tetrameric form, catalyzing the conversion of GDP-D-mannose into GDP-4-keto-6-deoxy-D-mannose. This sugar nucleotide is the intermediary necessary for the biosynthesis of GDP-D-rhamnose, one of the sugar residues of cepacian, the major exopolysaccharide produced by environmental and human, animal and plant pathogenic isolates of the Burkholderia cepacia complex species. Vmax and Km values of 1.5±0.2 µmol.min−1.mg−1 and 1024±123 µM, respectively, were obtained from the kinetic characterization of the B. cenocepacia J2315 BceN protein by NMR spectroscopy, at 25°C and in the presence of 1 mol MgCl2 per mol of protein. The enzyme activity was strongly inhibited by the substrate, with an estimated Ki of 2913±350 µM. The lack of a functional bceN gene in a mutant derived from B. cepacia IST408 slightly reduced cepacian production. However, in the B. multivorans ATCC17616 with bceN as the single gene in its genome with predicted GMD activity, a bceN mutant did not produce cepacian, indicating that this gene product is required for cepacian biosynthesis. PMID:23460819

Single-step purification and characterization of recombinant aspartase of Aeromonas media NFB-5.

PubMed

Singh, Ram Sarup; Yadav, Mukesh

2012-07-01

Aspartase (L-aspartate ammonia-lyase; EC 4.3.1.1) catalyzes the reversible amination of fumaric acid to produce L-aspartic acid. Aspartase coding gene (aspA) of Aeromonas media NFB-5 was cloned, sequenced, and expressed with His tag using pET-21b⁺ expression vector in Escherichia coli BL21. Higher expression was obtained with IPTG (1.5 mM) induction for 5 h at 37 °C in LB medium supplemented with 0.3% K₂HPO₄ and 0.3% KH₂PO₄. Recombinant His tagged aspartase was purified using Ni-NTA affinity chromatography and characterized for various biochemical and kinetic parameters. The purified aspartase showed optimal activity at pH 8.5 and 8.0 in the presence and absence of magnesium ions, respectively. The optimum temperature was determined to be 35 °C. The enzyme showed apparent K(m) and V(max) values for L-aspartate as 2.01 mM and 114 U/mg, respectively. The enzyme was stable in pH range of 6.5-9.5 and temperature up to 45 °C. Divalent metal ion requirement of enzyme was efficiently fulfilled by Mg²⁺, Mn²⁺, and Ca²⁺ ions. The cloned gene (aspA) product showed molecular weight of approximately 51 kDa by SDS-PAGE, which is in agreement with the molecular weight calculated from putative amino acid sequence. This is the first report on expression and characterization of recombinant aspartase from A. media.

Overexpression and characterization of laccase from Trametes versicolor in Pichia pastoris.

PubMed

Li, Q; Pei, J; Zhao, L; Xie, J; Cao, F; Wang, G

2014-01-01

A laccase-encoding gene of Trametes versicolor, lccA, was cloned and expressed in Pichia pastoris X33. The lccA gene consists ofa 1560 bp open reading frame encoding 519 amino acids, which was classified into family copper blue oxidase. To improve the expression level of recombinant laccase in P. pastoris, conditions of the fermentation were optimized by the single factor experiments. The optimal fermentation conditions for the laccase production in shake flask cultivation using BMGY medium were obtained: the optimal initial pH 7.0, the presence of 0.5 mM Cu2+, 0.6% methanol added into the culture every 24 h. The laccase activity was up to 11.972 U/L under optimal conditions after 16 days of induction in a medium with 4% peptone. After 100 h of large scale production in 5 L fermenter the enzyme activity reached 18.123 U/L. The recombinant laccase was purified by ultrafiltration and (NH4)2SO4 precipitation showing a single band on SDS-PAGE, which had a molecular mass of 58 kDa. The optimum pH and temperature for the laccase were pH 2.0 and 50 degrees C with 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) as a substrate. The recombinant laccase was stable over a pH range of 2.0-7.0. The K(m) and the V(max) value of LccA were 0.43 mM and 82.3 U/mg for ABTS, respectively.

Galaxy kinematics in the XMMU J2235-2557 cluster field at z 1.4

NASA Astrophysics Data System (ADS)

Pérez-Martínez, J. M.; Ziegler, B.; Verdugo, M.; Böhm, A.; Tanaka, M.

2017-09-01

Aims: The relationship between baryonic and dark components in galaxies varies with the environment and cosmic time. Galaxy scaling relations describe strong trends between important physical properties. A very important quantitative tool in case of spiral galaxies is the Tully-Fisher relation (TFR), which combines the luminosity of the stellar population with the characteristic rotational velocity (Vmax) taken as proxy for the total mass. In order to constrain galaxy evolution in clusters, we need measurements of the kinematic status of cluster galaxies at the starting point of the hierarchical assembly of clusters and the epoch when cosmic star formation peaks. Methods: We took spatially resolved slit FORS2 spectra of 19 cluster galaxies at z 1.4, and 8 additional field galaxies at 1 < z < 1.2 using the ESO Very Large Telescope. The targets were selected from previous spectroscopic and photometric campaigns as [OII] and Hα emitters. Our spectroscopy was complemented with HST/ACS imaging in the F775W and F850LP filters, which is mandatory to derive the galaxy structural parameters accurately. We analyzed the ionized gas kinematics by extracting rotation curves from the two-dimensional spectra. Taking into account all geometrical, observational, and instrumental effects, we used these rotation curves to derive the intrinsic maximum rotation velocity. Results: Vmax was robustly determined for six cluster galaxies and three field galaxies. Galaxies with sky contamination or insufficient spatial rotation curve extent were not included in our analysis. We compared our sample to the local B-band TFR and the local velocity-size relation (VSR), finding that cluster galaxies are on average 1.6 mag brighter and a factor 2-3 smaller. We tentatively divided our cluster galaxies by total mass (I.e., Vmax) to investigate a possible mass dependency in the environmental evolution of galaxies. The averaged deviation from the local TFR is ⟨ ΔMB ⟩ = -0.7 for the high-mass subsample (Vmax > 200 km s-1). This mild evolution may be driven by younger stellar populations (SP) of distant galaxies with respect to their local counterparts, and thus, an increasing luminosity is expected toward higher redshifts. However, the low-mass subsample (Vmax < 200 km s-1) is made of highly overluminous galaxies that show ⟨ ΔMB ⟩ = -2.4 mag. When we repeated a similar analysis with the stellar mass TFR, we did not find significant offsets in our subsamples with respect to recent results at similar redshift. While the B-band TFR is sensitive to recent episodes of star formation, the stellar mass TFR tracks the overall evolution of the underlying stellar population. In order to understand the discrepancies between these two incarnations of the TFR, the reported B-band offsets can no longer be explained only by the gradual evolution of stellar populations with lookback time. We suspect that we instead see compact galaxies whose star formation was enhanced during their infall toward the dense regions of the cluster through interactions with the intracluster medium. Based on observations with the European Southern Observatory Very Large Telescope (ESO-VLT), observing run ID 091.B-0778(B).

Purification and characterization of a novel cytosolic NADP(H)-dependent retinol oxidoreductase from rabbit liver.

PubMed

Huang, D Y; Ichikawa, Y

1997-03-07

Rabbit liver cytosol exhibits very high retinol dehydrogenase activity. At least two retinol dehydrogenases were demonstrated to exist in rabbit liver cytosol, and the major one, a cytosolic NADP(H)-dependent retinol dehydrogenase (systematic name: retinol oxidoreductase) was purified about 1795-fold to electrophoretic and column chromatographic homogeneity by a procedure involving column chromatography on AF-Red Toyopearl twice and then hydroxyapatite. Its molecular mass was estimated to be 34 kDa by SDS-PAGE, and 144 kDa by HPLC gel filtration, suggesting that it is a homo-tetramer. The enzyme uses free retinol and retinal, and their complexes with CRBP as substrates in vitro. The optimum pH values for retinol oxidation of free retinol and CRBP-retinol were 8.8-9.2 and 8.0-9.0, respectively, and those for retinal reduction of free retinal and retinal-CRBP were the same, 7.0-7.6. Km for free retinol and Vmax for retinal formation were 2.8 microM and 2893 nmol/min per mg protein at 37 degrees C (pH 9.0) and the corresponding values with retinol-CRBP as a substrate were 2.5 microM and 2428 nmol/min per mg protein at 37 degrees C (pH 8.6); Km for free retinal and Vmax for retinol formation were 6.5 microM and 4108 nmol/min per mg protein, and the corresponding values with retinal-CRBP as a substrate were 5.1 microM and 3067 nmol/min per mg protein at 37 degrees C, pH 7.4. NAD(H) was not effective as a cofactor. 4-Methylpyrazole was a weak inhibitor (IC50 = 28 mM) of the enzyme, and ethanol was neither a substrate nor an inhibitor of the enzyme. This enzyme exhibits relatively broad aldehyde reductase activity and some ketone reductase activity, the activity for aromatic substitutive aldehydes being especially high and effective. Whereas, except in the case of retinol, oxidative activity toward the corresponding alcohols was not detected. This novel cytosolic enzyme may play an important role in vivo in maintaining the homeostasis of retinal, the substrate of retinoic acid synthesis, at least in rabbit liver, since a high concentration of retinol in liver and the lower Km of the enzyme for retinol force the oxidative reaction, while higher activity of retinal reductase at physiological pH forces the reductive reaction.

Comparative study of invertases of Streptococcus mutans.

PubMed

Tanzer, J M; Brown, A T; McInerney, M F; Woodiel, F N

1977-04-01

Sucrase activity was studied in 13 strains of Streptococcus mutans representing the five Bratthall serotypes. Sucrose-adapted cells have sucrase activity in the 37,000 x g-soluble fraction of all strains. The enzyme was identified as invertase (beta-d-fructofuranoside fructohydrolase; EC 3.2.1.26) because it hydrolyzed the beta-fructofuranoside trisaccharide raffinose, giving fructose and melibiose as its products, and because it hydrolyzed the beta-fructofuranoside dissacharide sucrose, giving equimolar glucose and fructose as its products. Invertases of c and e strains exhibit two activity peaks by molecular exclusion chromatography with molecular weights of 45,000 to 50,000 and about 180,000; those of serotypes a, b, and d strains exhibit only a single component of 45,000 to 50,000 molecular weight. The electrophoretic mobility of invertases is different between the serotypes and the same within them. Inorganic orthophosphate (P(i)) has a weak positive effect on the V(max) of invertases of serotypes c and e cells but a strong positive effect on the invertases of serotype b cells; P(i) has a strong positive effect on the apparent K(m) of the invertases of serotype d cells, but has no effect on the V(max); P(i) has a strong positive effect on both the apparent K(m) and V(max) of the invertases of serotype a cells. Thus, the invertases were different between all of the serotypes but similar within the serotypes. These findings support the taxonomic schemes of Coykendall and of Bratthall. It was additionally noted that 37,000 x g-soluble fractions of only serotypes b and c but not serotypes a, d, and e cells have melibiase activity, and it could be deduced that serotype d cells lack an intact raffinose permease system.

Effect of chronic pre-treatment with angiotensin converting enzyme inhibition on skeletal muscle mitochondrial recovery after ischemia/reperfusion.

PubMed

Thaveau, Fabien; Zoll, Joffrey; Bouitbir, Jamal; N'guessan, Benoît; Plobner, Philippe; Chakfe, Nabil; Kretz, Jean-Georges; Richard, Ruddy; Piquard, François; Geny, Bernard

2010-06-01

Impaired skeletal muscle energetic participates in peripheral arterial disease (PAD) patient's morbidity and mortality. Angiotensin converting enzyme inhibition (ACEi), cornerstone for pharmacologic risk factor management in PAD patients, might also be interesting by protecting skeletal muscle energetic. We therefore determined whether chronic ACEi might reduce ischemia-induced mitochondrial respiratory chain dysfunction in the frequent setting of hindlimb ischemia-reperfusion. Ischemic legs of rats submitted to 5 h ischemia induced by a rubber band tourniquet applied on the root of the hindlimb followed by reperfusion without (IR, n = 11) or after ACEi (n = 14; captopril 40 mg/kg per day during 28 days before surgery) were studied and compared to that of sham-operated animals (n = 11). The effect of ACEi on the non-ischemic contralateral leg was also determined in the ACEi group. Maximal oxidative capacities (V(max)) and complexes I, II and IV activities of the mitochondrial respiratory chain of the gastrocnemius muscle were determined using glutamate-malate, succinate and TMPD-ascorbate substrates. Arterial blood pressure was significantly decreased after ACEi (124 +/- 2.8 vs. 108 +/- 4.19 mmHg; P = 0.01). Ischemia-reperfusion reduced V(max) (4.4 +/- 0.4 vs. 8.7 +/- 0.5 micromol O2/min/g dry weight, -49%, P < 0.001), affecting mitochondrial complexes I, II and IV activities. ACEi failed to modulate ischemia-induced dysfunction (V(max) 5.1 +/- 0.7 micromol O2/min/g dry weight) or the non-ischemic contralateral muscle respiratory rate. Ischemia-reperfusion significantly impaired the mitochondrial respiratory chain I, II and IV complexes of skeletal muscle. Pharmacologic pre-treatment with ACEi did not prevent or increase such alterations. Further studies might be useful to improve the pharmacologic conditioning of PAD patients needing arterial revascularization.

Effect of Calcium on the Oxidative Phosphorylation Cascade in Skeletal Muscle Mitochondria

PubMed Central

Glancy, Brian; Willis, Wayne T; Chess, David J; Balaban, Robert S

2014-01-01

Calcium is believed to regulate mitochondrial oxidative phosphorylation, thereby contributing to the maintenance of cellular energy homeostasis. Skeletal muscle, with an energy conversion dynamic range of up to 100-fold, is an extreme case for evaluating the cellular balance of ATP production and consumption. This study examined the role of Ca2+ on the entire oxidative phosphorylation reaction network in isolated skeletal muscle mitochondria and attempted to extrapolate these results back to the muscle, in vivo. Kinetic analysis was conducted to evaluate the dose response effect of Ca2+ on the maximum velocity of oxidative phosphorylation (VmaxO) and the ADP affinity. Force-flow analysis evaluated the interplay between energetic driving forces and flux to determine the conductance, or effective activity, of individual steps within oxidative phosphorylation. Measured driving forces (extramitochondrial phosphorylation potential (ΔGATP), membrane potential, and redox states of NADH and cytochromes bH, bL, c1, c, and a,a3) were compared with flux (oxygen consumption) at 37°C. 840 nM Ca2+ generated a ∼2 fold increase in VmaxO with no change in ADP affinity (∼43 μM). Force-flow analysis revealed that Ca2+ activation of VmaxO was distributed throughout the oxidative phosphorylation reaction sequence. Specifically, Ca2+ increased the conductance of Complex IV (2.3-fold), Complexes I+III (2.2-fold), ATP production/transport (2.4-fold), and fuel transport/dehydrogenases (1.7-fold). These data support the notion that Ca2+ activates the entire muscle oxidative phosphorylation cascade, while extrapolation of these data to the exercising muscle predicts a significant role of Ca2+ in maintaining cellular energy homeostasis. PMID:23547908

Organization of metabolic pathways in vastus lateralis of patients with chronic obstructive pulmonary disease.

PubMed

Green, Howard J; Bombardier, Eric; Burnett, Margaret; Iqbal, Sobia; D'Arsigny, Christine L; O'Donnell, Dennis E; Ouyang, Jing; Webb, Katherine A

2008-09-01

The objective of this study was to determine whether patients with chronic obstructive lung disease (COPD) display differences in organization of the metabolic pathways and segments involved in energy supply compared with healthy control subjects. Metabolic pathway potential, based on the measurement of the maximal activity (V(max)) of representative enzymes, was assessed in tissue extracted from the vastus lateralis in seven patients with COPD (age 67 +/- 4 yr; FEV(1)/FVC = 44 +/- 3%, where FEV(1) is forced expiratory volume in 1 s and FVC is forced vital capacity; means +/- SE) and nine healthy age-matched controls (age 68 +/- 2 yr; FEV(1)/FVC = 75 +/- 2%). Compared with control, the COPD patients displayed lower (P < 0.05) V(max) (mol.kg protein(-1).h(-1)) for cytochrome c oxidase (COX; 21.2 +/- 2.0 vs. 28.7 +/- 2.2) and 3-hydroxyacyl-CoA dehydrogenase (HADH; 2.54 +/- 0.14 vs. 3.74 +/- 0.12) but not citrate synthase (CS; 2.20 +/- 0.16 vs. 3.19 +/- 0.5). While no differences between groups were observed in V(max) for creatine phosphokinase, phosphorylase (PHOSPH), phosphofructokinase (PFK), pyruvate kinase, and lactate dehydrogenase, hexokinase (HEX) was elevated in COPD (P < 0.05). Enzyme activity ratios were higher (P < 0.05) for HEX/CS, HEX/COX, PHOSPH/HADH and PFK/HADH in COPD compared with control. It is concluded that COPD patients exhibit a reduced potential for both the electron transport system and fat oxidation and an increased potential for glucose phosphorylation while the potential for glycogenolysis and glycolysis remains normal. A comparison of enzyme ratios indicated greater potentials for glucose phosphorylation relative to the citric acid cycle and the electron transport chain and glycogenolysis and glycolysis relative to beta-oxidation.

Passive and active floating torque during swimming.

PubMed

Kjendlie, Per-Ludvik; Stallman, Robert Keig; Stray-Gundersen, James

2004-10-01

The purpose of this study was to examine the effect of passive underwater torque on active body angle with the horizontal during front crawl swimming and to assess the effect of body size on passive torque and active body angle. Additionally, the effects of passive torque, body angle and hydrostatic lift on maximal sprinting performance were addressed. Ten boys [aged 11.7 (0.8) years] and 12 male adult [aged 21.4 (3.7) years] swimmers volunteered to participate. Their body angle with the horizontal was measured at maximal velocity, and at two submaximal velocities using an underwater video camera system. Passive torque and hydrostatic lift were measured during an underwater weighing procedure, and the center of mass and center of volume were determined. The results showed that passive torque correlated significantly with the body angle at a velocity 63% of v(max) ( alpha(63) r=-0.57), and that size-normalized passive torque correlated significantly with the alpha(63) and alpha(77) (77% of v(max)) with r=-0.59 and r=-0.54 respectively. Hydrostatic lift correlated with alpha(63) with r=-0.45. The negative correlation coefficients are suggested to be due to the adults having learned to overcome passive torque when swimming at submaximal velocities by correcting their body angle. It is concluded that at higher velocities the passive torque and hydrostatic lift do not influence body angle during swimming. At a velocity of 63% of v(max), hydrostatic lift and passive torque influences body angle. Passive torque and size-normalized passive torque increases with body size. When corrected for body size, hydrostatic lift and passive torque did not influence the maximal sprinting velocity.

Kinetics of phosphate uptake, growth, and accumulation of cyclic diphosphoglycerate in a phosphate-limited continuous culture of Methanobacterium thermoautotrophicum.

PubMed Central

Krueger, R D; Harper, S H; Campbell, J W; Fahrney, D E

1986-01-01

The archaebacterium Methanobacterium thermoautotrophicum was grown in continuous culture at 65 degrees C in a phosphate-limited medium at specific growth rates from 0.06 to 0.28 h-1 (maximum growth rate [mu max] = 0.36 h-1). Cyclic-2,3-diphosphoglycerate (cyclic DPG) levels ranged from 2 to 20 mM in Pi-limited cells, compared with about 30 mM in batch-grown cells. The Monod constant for Pi-limited growth was 5 nM. Pi uptake rates were determined by following the disappearance of 32Pi from the medium. Interrupting the H2 supply stopped the uptake of Pi and the release of organic phosphates. Little or no efflux of Pi occurred in the presence or absence of H2. Pi uptake of cells adapted to nanomolar Pi concentrations could be accounted for by the operation of one uptake system with an apparent Km of about 25 nM and a Vmax of 58 nmol of Pi per min per g (dry weight). Uptake curves at 30 microM Pi or above were biphasic due to a sevenfold decrease in Vmax after an initial phase of rapid movement of Pi into the cell. Under these conditions the growth rate slowed to zero and the cyclic DPG pool expanded before growth resumed. Thus, three properties of M. thermoautotrophicum make it well adapted to live in a low-P environment: the presence of a low-Km, high-Vmax uptake system for Pi; the ability to accumulate cyclic DPG rapidly; and a growth strategy in which accumulation of Pi and cyclic DPG takes precedence over a shift-up in growth rate when excess Pi becomes available. PMID:3722128

Kinetics of phosphate uptake, growth, and accumulation of cyclic diphosphoglycerate in a phosphate-limited continuous culture of Methanobacterium thermoautotrophicum.

PubMed

Krueger, R D; Harper, S H; Campbell, J W; Fahrney, D E

1986-07-01

The archaebacterium Methanobacterium thermoautotrophicum was grown in continuous culture at 65 degrees C in a phosphate-limited medium at specific growth rates from 0.06 to 0.28 h-1 (maximum growth rate [mu max] = 0.36 h-1). Cyclic-2,3-diphosphoglycerate (cyclic DPG) levels ranged from 2 to 20 mM in Pi-limited cells, compared with about 30 mM in batch-grown cells. The Monod constant for Pi-limited growth was 5 nM. Pi uptake rates were determined by following the disappearance of 32Pi from the medium. Interrupting the H2 supply stopped the uptake of Pi and the release of organic phosphates. Little or no efflux of Pi occurred in the presence or absence of H2. Pi uptake of cells adapted to nanomolar Pi concentrations could be accounted for by the operation of one uptake system with an apparent Km of about 25 nM and a Vmax of 58 nmol of Pi per min per g (dry weight). Uptake curves at 30 microM Pi or above were biphasic due to a sevenfold decrease in Vmax after an initial phase of rapid movement of Pi into the cell. Under these conditions the growth rate slowed to zero and the cyclic DPG pool expanded before growth resumed. Thus, three properties of M. thermoautotrophicum make it well adapted to live in a low-P environment: the presence of a low-Km, high-Vmax uptake system for Pi; the ability to accumulate cyclic DPG rapidly; and a growth strategy in which accumulation of Pi and cyclic DPG takes precedence over a shift-up in growth rate when excess Pi becomes available.

A spectrophotometric assay for fatty acid amide hydrolase suitable for high-throughput screening.

PubMed

De Bank, Paul A; Kendall, David A; Alexander, Stephen P H

2005-04-15

Signalling via the endocannabinoids anandamide and 2-arachidonylglycerol appears to be terminated largely through the action of the enzyme fatty acid amide hydrolase (FAAH). In this report, we describe a simple spectrophotometric assay to detect FAAH activity in vitro using the ability of the enzyme to hydrolyze oleamide and measuring the resultant production of ammonia with a NADH/NAD+-coupled enzyme reaction. This dual-enzyme assay was used to determine Km and Vmax values of 104 microM and 5.7 nmol/min/mgprotein, respectively, for rat liver FAAH-catalyzed oleamide hydrolysis. Inhibitor potency was determined with the resultant rank order of methyl arachidonyl fluorophosphonate>phenylmethylsulphonyl fluoride>anandamide. This assay system was also adapted for use in microtiter plates and its ability to detect a known inhibitor of FAAH demonstrated, highlighting its potential for use in high-throughput screening.

Effects of turpentine-induced inflammation on the hypoxic stimulation of intestinal Fe3+ absorption in mice.

PubMed Central

Raja, K. B.; Duane, P.; Peters, T. J.

1990-01-01

Chronic subcutaneous turpentine administration (weekly for 6 weeks) induced a mild normocytic anaemia in mice. In-vitro and in-vivo intestinal Fe3+ absorption parameters were, however, not significantly altered from values in saline-treated or untreated mice. Normal mice, when exposed to 3 days hypoxia demonstrated a 2-3-fold increase in iron absorption in vivo, mainly due to changes in the amount of iron transferred from the mucosa to the plasma and thence to the carcass. A 2-3-fold increase in Vmax was also observed in in-vitro uptake experiments using isolated duodenal fragments. In contrast, turpentine-treated animals, though demonstrating an enhanced in-vitro maximal uptake capacity, failed to elicit an adaptive response in vivo following hypoxic exposure. These findings suggest that a circulating (humoral) factor may be responsible for the inhibition in absorption in vivo in this turpentine-induced inflammatory model. PMID:2278822

Simplified numerical approach for estimation of effective segregation coefficient at the melt/crystal interface

NASA Astrophysics Data System (ADS)

Prostomolotov, A. I.; Verezub, N. A.; Voloshin, A. E.

2014-09-01

A thermo-gravitational convection and impurity transfer in the melt were investigated using a simplified numerical model for Bridgman GaSb(Te) crystal growth in microgravity conditions. Simplifications were as follows: flat melt/crystal interface, fixed melt sizes and only lateral ampoule heating. Calculations were carried out by Ansys®Fluent® code employing a two-dimensional Navier-Stokes-Boussinesq and heat and mass transfer equations in a coordinate system moving with the melt/crystal interface. The parametric dependence of the effective segregation coefficient Keff at the melt/crystal interface was studied for various ampoule sizes and for microgravity conditions. For the uprising one-vortex flow, the resulting dependences were presented as Keff vs. Vmax-the maximum velocity value. These dependences were compared with the formulas by Burton-Prim-Slichter's, Ostrogorsky-Muller's, as well as with the semi-analytical solutions.

Guinea-pig liver testosterone 17 beta-dehydrogenase (NADP+) and aldehyde reductase exhibit benzene dihydrodiol dehydrogenase activity.

PubMed Central

Hara, A; Hayashibara, M; Nakayama, T; Hasebe, K; Usui, S; Sawada, H

1985-01-01

We have kinetically and immunologically demonstrated that testosterone 17 beta-dehydrogenase (NADP+) isoenzymes (EC 1.1.1.64) and aldehyde reductase (EC 1.1.1.2) from guinea-pig liver catalyse the oxidation of benzene dihydrodiol (trans-1,2-dihydroxycyclohexa-3,5-diene) to catechol. One isoenzyme of testosterone 17 beta-dehydrogenase, which has specificity for 5 beta-androstanes, oxidized benzene dihydrodiol at a 3-fold higher rate than 5 beta-dihydrotestosterone, and showed a more than 4-fold higher affinity for benzene dihydrodiol and Vmax. value than did another isoenzyme, which exhibits specificity for 5 alpha-androstanes, and aldehyde reductase. Immunoprecipitation of guinea-pig liver cytosol with antisera against the testosterone 17 beta-dehydrogenase isoenzymes and aldehyde reductase indicated that most of the benzene dihydrodiol dehydrogenase activity in the tissue is due to testosterone 17 beta-dehydrogenase. PMID:2983661

An acidic pectin lyase from Aspergillus niger with favourable efficiency in fruit juice clarification.

PubMed

Xu, S X; Qin, X; Liu, B; Zhang, D Q; Zhang, W; Wu, K; Zhang, Y H

2015-02-01

The pectin lyase gene pnl-zj5a from Aspergillus niger ZJ5 was identified and expressed in Pichia pastoris. PNL-ZJ5A was purified by ultrafiltration, anion exchange and gel chromatography. The Km and Vmax values determined using citrus pectin were 0.66 mg ml(-1) and 32.6 μmol min(-1) mg(-1) , respectively. PNL-ZJ5A exhibited optimal activity at 43°C and retained activity over 25-50°C. PNL-ZJ5A was optimally active at pH 5 and effective in apple juice clarification. Compared with controls, PNL-ZJ5A increased the fruit juice yield significantly. Furthermore, PNL-ZJ5A reduced the viscosity of apple juice by 38.8% and increased its transmittance by 86.3%. PNL-ZJ5A combined with a commercial pectin esterase resulted in higher juice volume. © 2014 The Society for Applied Microbiology.

Characterization, gene cloning, and sequencing of a fungal phytase, PhyA, from Penicillium oxalicum PJ3.

PubMed

Lee, Seung Ho; Cho, Jaiesoon; Bok, Jinduck; Kang, Seungha; Choi, Yunjaie; Lee, Peter C W

2015-01-01

A phytase from Penicillium oxalicum PJ3, PhyA, was purified near to homogeneity with 427-fold increase in specific phytase activity by ammonium sulfate precipitation, gel filtration, and ion-exchange chromatographies. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and zymogram analysis of the purified enzyme indicated an estimated molecular mass of 65 kD. The optimal pH and temperature of the purified enzyme were pH 4.5 and 55°C, respectively. The enzyme activity was strongly inhibited by Ca(2+), Cu(2+), Zn(2+), and phenylmethylsulfonyl fluoride (PMSF). The Km value for sodium phytate was 0.545 mM with a Vmax of 600 U/mg of protein. The phyA gene was cloned, and it contains an open reading frame of 1,383 with a single intron (118 bp), and encodes a protein of 461 amino acids.

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

NASA Astrophysics Data System (ADS)

Steen, A. D.

2015-12-01

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

Influence of lifestyle habits, nutritional status and insulin resistance in NAFLD.

PubMed

Malavolti, Marcella; Battistini, Nino Carlo; Miglioli, Lucia; Bagni, Ilaria; Borelli, Luca; Marino, Mariano; Scaglioni, Federica; Bellentani, Stefano

2012-01-01

Non alcoholic fatty liver disease (NAFLD) is associated with obesity, diabetes and insulin resistance (IR). The aim of our study was to assess the relationship between IR, anthropometry, lifestyle habits, resting energy expenditure (REE) and degree of fatty liver at ultrasound in 48 overweight patients with NAFLD as compared to 24 controls without fatty liver, matched for age. Nutritional status, alcohol intake and physical activity were assessed by skinfold thickness measurements, a 7-day diary, and SenseWear armband (SWA). REE was assessed by both SWA (REE-SWA) and a Vmax metabolic cart (REE-Vmax). Fatty liver was measured by US and the Doppler Power Index was calculated. IR was assessed using the HOMA index. There was significant correlation between waist circumference, HOMA, Doppler power index and fatty liver grade at US. Multivariate analysis showed that alteration of waist circumference, Doppler power index, and HOMA were the major significant predictors of fatty liver. Our data demonstrated a significant association between NAFLD and central adiposity and IR.

Insights into ligand binding to a Glutathione S-transferase from mango: structure, thermodynamics and kinetics

PubMed Central

Valenzuela-Chavira, Ignacio; Contreras-Vergara, Carmen A.; Arvizu-Flores, Aldo A.; Serrano-Posada, Hugo; Lopez-Zavala, Alonso A.; García-Orozco, Karina D.; Hernandez-Paredes, Javier; Rudiño-Piñera, Enrique; Stojanoff, Vivian; Sotelo-Mundo, Rogerio R.; Islas-Osuna, Maria A.

2017-01-01

We studied a mango glutathione S-transferase (GST) (Mangifera indica) bound to glutathione (GSH) and S-hexyl glutathione (GSX). This GST Tau class (MiGSTU) had a molecular mass of 25.5 kDa. MiGSTU Michaelis-Menten kinetic constants were determined for their substrates obtaining a Km, Vmax and kcat for CDNB of 0.792 mM, 80.58 mM·min−1 and 68.49 s−1 respectively and 0.693 mM, 105.32 mM·min−1 and 89.57 s−1, for reduced GSH respectively. MiGSTU had a micromolar affinity towards GSH (5.2 μM) or GSX (7.8 μM). The crystal structure of the MiGSTU in apo or bound to GSH or GSX generated a model that explains the thermodynamic signatures of binding and showed the importance of enthalpic-entropic compensation in ligand binding to Tau-class GST enzymes. PMID:28104507

Enzyme Activity Experiments Using a Simple Spectrophotometer

ERIC Educational Resources Information Center

Hurlbut, Jeffrey A.; And Others

1977-01-01

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

[Evaluation of the arterial blood flow parameters in the eye of myopic patients].

PubMed

Mrugacz, Małgorzata; Bryl, Anna

2013-04-01

Myopia is a common refractive defect. Has a good vision from near and deterioration of vision with increasing distance. The main reason for its occurrence is too long axis of the eyeball. The consequence of elongation of the eyeball is the development of degenerative changes in the retina. Despite much research has failed to clearly identify the causes of degenerative changes in those short-sighted. The aim of the study was to evaluate the maximum and minimum speed in arterial blood vessels of the eye in people with myopia. The study included 70 patients (138 eyes), 53 women and 17 men, aged from 18 to 79 years, with myopia of -0.25 to -18.0 Dsph and length of the eyeball from 22.61 to 33.36 mm. Depending on the kind and the degree of the progress of degenerative changes, patients were divided in 4 groups: I - without degenerative changes on the fundus (n=32; K-23, M-9); II- with the short-sighted sickle (n=20; K-14, M-6); Ill - with the structure thinned down of the retina, accompanying the short-sighted sickle (n = 8; K-6, M-2); IV - with extensive choroidal-retina disappearances in the fundus (n = 10; K-7, M-3). In all individuals enrolled underwent Color Doppler ultrasound with apparatus SSA 770A Toshiba Aplio with linear probe frequency 12 MHz, judging maximum (Vmax) and minimum (Vmin) speed in the arteries of the eye: ophthalmic artery (OA), central retinal artery (CRA) and short posterior ciliary arteries (SPCA) located on the nasal and temporal side of the optic disc. The results were statistically analyzed. No statistically significant relationship between the nature of degenerative changes of the eye, and blood velocity in the OA. There was a increase in Vmax and Vmin blood in OA in Group IV, but these changes were not statistically significant. Statistically significant correlation was observed while in the CRA. With a decrease in Vmax and Vmin of blood flowing through a vessel exacerbation of retinal degeneration. Vmax and Vmin changes in the blood did not correlate significantly SPCA with retinal degeneration, although the results were much worse in the temporal vessels. With the deterioration of blood flow parameters of the central retinal artery and short posterior ciliary arteries comes to the severity of the retinal degeneration in myopic patients. More severe impairment of blood circulation in the temporal ciliary explain higher incidence of degenerative changes in the temporal side of the optic disc.

Differential in vitro inhibition of M3G and M6G formation from morphine by (R)- and (S)-methadone and structurally related opioids

PubMed Central

Morrish, Glynn A; Foster, David J R; Somogyi, Andrew A

2006-01-01

Aims To determine the in vitro kinetics of morphine-3-glucuronide (M3G) and morphine-6-glucuronide (M6G) formation and the inhibition potential by methadone enantiomers and structurally related opioids. Methods M3G and M6G formation kinetics from morphine were determined using microsomes from five human livers. Inhibition of glucuronide formation was investigated with eight inhibitors (100 µm) and the mechanism of inhibition determined for (R)- and (S)-methadone (70–500 µm) using three microsomal samples. Results Glucuronide formation displayed single enzyme kinetics. The M3G Vmax (mean ± SD) was 4.8-fold greater than M6G Vmax (555 ± 110 vs. 115 ± 19 nmol mg−1 protein h−1; P = 0.006, mean of difference 439; 95% confidence interval 313, 565 nmol mg−1 protein h−1). Km values for M3G and M6G formation were not significantly different (1.12 ± 0.37 vs. 1.11 ± 0.31 mm; P = 0.89, 0.02; −0.29, 0.32 mm). M3G and M6G formation was inhibited (P < 0.01) with a significant increase in the M3G/M6G ratio (P < 0.01) for all compounds tested. Detailed analysis with (R)- and (S)-methadone revealed noncompetitive inhibition with (R)-methadone Ki of 320 ± 42 µm and 192 ± 12 µm for M3G and M6G, respectively, and (S)-methadone Ki of 226 ± 30 µm and 152 ± 20 µm for M3G and M6G, respectively. Ki values for M3G inhibition were significantly greater than for M6G for (R)-methadone (P = 0.017, 128; 55, 202 µm) and (S)-methadone (P = 0.026, 75; 22, 128 µm). Conclusions Both methadone enantiomers noncompetitively inhibited the formation of morphine's primary metabolites, with greater inhibition of M6G formation compared with M3G. These findings indicate a mechanism for reduced morphine clearance in methadone-maintained patients and reduced relative formation of the opioid active M6G compared with M3G. PMID:16487227

Calcium transport in gill cells of Ucides cordatus, a mangrove crab living in variable salinity environments.

PubMed

Leite, V P; Zanotto, F P

2013-10-01

Crustaceans show discontinuous growth and have been used as a model system for studying cellular mechanisms of calcium transport, which is the main mineral found in their exoskeleton. Ucides cordatus, a mangrove crab, is naturally exposed to fluctuations in calcium and salinity. To study calcium transport in this species during isosmotic conditions, dissociated gill cells were marked with fluo-3 and intracellular Ca(2+) change was followed by adding extracellular Ca(2+) as CaCl2 (0, 0.1, 0.25, 0.50, 1.0 and 5mM), together with different inhibitors. For control gill cells, Ca(2+) transport followed Michaelis-Menten kinetics with Vmax=0.137±0.001 ∆Ca(2+)i (μM×22.10(4)cells(-1)×180s(-1); N=4; r(2)=0.99); Km=0.989±0.027mM. The use of different inhibitors for gill cells showed that amiloride (Na(+)/Ca(2+) exchange inhibitor) inhibited 80% of Ca(2+) transport in gill cells (Vmax). KB-R, an inhibitor of Ca influx in vertebrates, similarly caused a decrease in Ca(2+) transport and verapamil (Ca(2+) channel inhibitor) had no effect on Ca(2+) transport, while nifedipine (another Ca(2+) channel inhibitor) caused a 20% decrease in Ca(2+) affinity compared to control values. Ouabain, on the other hand, caused no change in Ca(2+) transport, while vanadate increased the concentration of intracellular calcium through inhibition of Ca(2+) efflux probably through the plasma membrane Ca(2+)-ATPase. Results show that transport kinetics for Ca(2+) in these crabs under isosmotic conditions is lower compared to a hyper-regulator freshwater crab Dilocarcinus pagei studied earlier using fluorescent Ca(2+) probes. These kinds of studies will help understanding the comparative mechanisms underlying the evolution of Ca transport in crabs living in different environments. © 2013.

Toxicological and biochemical characterizations of AChE in phosalone-susceptible and resistant populations of the common pistachio psyllid, Agonoscena pistaciae

PubMed Central

Alizadeh, Ali; Talebi-Jahromi, Khalil; Hosseininaveh, Vahid; Ghadamyari, Mohammad

2014-01-01

Abstract The toxicological and biochemical characteristics of acetylcholinesterases (AChE) in nine populations of the common pistachio psyllid, Agonoscena pistaciae Burckhardt and Lauterer (Hemiptera: Psyllidae), were investigated in Kerman Province, Iran. Nine A. pistaciae populations were collected from pistachio orchards, Pistacia vera L. (Sapindales: Anacardiaceae), located in Rafsanjan, Anar, Bam, Kerman, Shahrbabak, Herat, Sirjan, Pariz, and Paghaleh regions of Kerman province. The previous bioassay results showed these populations were susceptible or resistant to phosalone, and the Rafsanjan population was most resistant, with a resistance ratio of 11.3. The specific activity of AChE in the Rafsanjan population was significantly higher than in the susceptible population (Bam). The affinity ( KM ) and hydrolyzing efficiency ( Vmax ) of AChE on acetylthiocholine iodide, butyrylthiocholine iodide, and propionylthiocholine odide as artificial substrates were clearly lower in the Bam population than that in the Rafsanjan population. These results indicated that the AChE of the Rafsanjan population had lower affinity to these substrates than that of the susceptible population. The higher Vmax value in the Rafsanjan population compared to the susceptible population suggests a possible over expression of AChE in the Rafsanjan population. The in vitro inhibitory effect of several organophosphates and carbamates on AChE of the Rafsanjan and Bam populations was determined. Based on I50, the results showed that the ratios of AChE insensitivity of the resistant to susceptible populations were 23 and 21.7-fold to monocrotophos and phosphamidon, respectively. Whereas, the insensitivity ratios for Rafsanjan population were 0.86, 0.8, 0.78, 0.46, and 0.43 for carbaryl, eserine, propoxur, m-tolyl methyl carbamate, and carbofuran, respectively, suggesting negatively correlated sensitivity to organophosphate-insensitive AChE. Therefore, AChE from the Rafsanjan population showed negatively correlated sensitivity, being insensitive to phosphamidon and monocrotophos and sensitive to N -methyl carbamates. PMID:25373165

Renal functional reserve and renal hemodynamics in hypertensive patients.

PubMed

Gaipov, Abduzhappar; Solak, Yalcin; Zhampeissov, Nurlan; Dzholdasbekova, Aliya; Popova, Nadezhda; Molnar, Miklos Z; Tuganbekova, Saltanat; Iskandirova, Elmira

2016-10-01

The renal functional reserve (RFR) is the ability of the kidneys to increase renal plasma flow and glomerular filtration rate (GFR) in response to protein intake. It is a measure of functional and anatomic integrity of nephrons. It is not known what relation between RFR and kidney Doppler parameters. We aimed to study the relation between the RFR and renal hemodynamic parameters in hypertensive patients with and without nephropathy who had normal kidney function. Twenty-four hypertensive subjects with nephropathy (HTN-n, n = 10) and hypertension without nephropathy (HTN, n = 14) were included in the study. Control group included 11 healthy subjects. Baseline GFR (GFR1) and GFR after intake of egg protein 1 mg/kg of body weight were determined (GFR2). RFR was calculated by the following formula: (GFR2-GFR1)/GFR1 × 100%. Doppler ultrasonography was performed. Arterial blood pressure (BP), body mass index (BMI), and estimated GFR were also recorded. HTN and HTN-n groups had impaired levels of RFR compared with controls (p < 0.05), significantly decreased value of flow velocity parameters (Vmax, Vmin), and increased RRI compared with controls. There was significant negative correlation of RFR with blood pressure levels (sBP, r = -0.435, p = 0.009; dBP, r = -0.504, p = 0.002), RRI (r = -0.456, p = 0.008), micro albuminuria (MAU, r = -0.366, p = 0.031) and positive correlation with Vmax and Vmin (r = 0.556, p = 0.001 and r = 0.643, respectively, p < 0.001). Linear regression showed that RRI and MAU were independent predictors of decreased RFR. RFR is lower in hypertensive patients despite near-normal level of kidney function and is related to particular level of BP. RRI and MAU were independent predictors of decreased RFR.

Modeling Reduction of Uranium U(VI) under Variable Sulfate Concentrations by Sulfate-Reducing Bacteria

PubMed Central

Spear, John R.; Figueroa, Linda A.; Honeyman, Bruce D.

2000-01-01

The kinetics for the reduction of sulfate alone and for concurrent uranium [U(VI)] and sulfate reduction, by mixed and pure cultures of sulfate-reducing bacteria (SRB) at 21 ± 3°C were studied. The mixed culture contained the SRB Desulfovibrio vulgaris along with a Clostridium sp. determined via 16S ribosomal DNA analysis. The pure culture was Desulfovibrio desulfuricans (ATCC 7757). A zero-order model best fit the data for the reduction of sulfate from 0.1 to 10 mM. A lag time occurred below cell concentrations of 0.1 mg (dry weight) of cells/ml. For the mixed culture, average values for the maximum specific reaction rate, Vmax, ranged from 2.4 ± 0.2 μmol of sulfate/mg (dry weight) of SRB · h−1) at 0.25 mM sulfate to 5.0 ± 1.1 μmol of sulfate/mg (dry weight) of SRB · h−1 at 10 mM sulfate (average cell concentration, 0.52 mg [dry weight]/ml). For the pure culture, Vmax was 1.6 ± 0.2 μmol of sulfate/mg (dry weight) of SRB · h−1 at 1 mM sulfate (0.29 mg [dry weight] of cells/ml). When both electron acceptors were present, sulfate reduction remained zero order for both cultures, while uranium reduction was first order, with rate constants of 0.071 ± 0.003 mg (dry weight) of cells/ml · min−1 for the mixed culture and 0.137 ± 0.016 mg (dry weight) of cells/ml · min−1 (U0 = 1 mM) for the D. desulfuricans culture. Both cultures exhibited a faster rate of uranium reduction in the presence of sulfate and no lag time until the onset of U reduction in contrast to U alone. This kinetics information can be used to design an SRB-dominated biotreatment scheme for the removal of U(VI) from an aqueous source. PMID:10966381

Acute Effects of Lateral Thigh Foam Rolling on Arterial Tissue Perfusion Determined by Spectral Doppler and Power Doppler Ultrasound.

PubMed

Hotfiel, Thilo; Swoboda, Bernd; Krinner, Sebastian; Grim, Casper; Engelhardt, Martin; Uder, Michael; Heiss, Rafael U

2017-04-01

Hotfiel, T, Swoboda, B, Krinner, S, Grim, C, Engelhardt, M, Uder, M, and Heiss, R. Acute effects of lateral thigh foam rolling on arterial tissue perfusion determined by spectral Doppler and power Doppler ultrasound. J Strength Cond Res 31(4): 893-900, 2017-Foam rolling has been developed as a popular intervention in training and rehabilitation. However, evidence on its effects on the cellular and physiological level is lacking. The aim of this study was to assess the effect of foam rolling on arterial blood flow of the lateral thigh. Twenty-one healthy participants (age, 25 ± 2 years; height, 177 ± 9 cm; body weight, 74 ± 9 kg) were recruited from the medical and sports faculty. Arterial tissue perfusion was determined by spectral Doppler and power Doppler ultrasound, represented as peak flow (Vmax), time average velocity maximum (TAMx), time average velocity mean (TAMn), and resistive index (RI), and with semiquantitative grading that was assessed by 4 blindfolded investigators. Measurement values were assessed under resting conditions and twice after foam rolling exercises of the lateral thigh (0 and 30 minutes after intervention). The trochanteric region, mid portion, and distal tibial insertion of the lateral thigh were representative for data analysis. Arterial blood flow of the lateral thigh increased significantly after foam rolling exercises compared with baseline (p ≤ 0.05). We detected a relative increase in Vmax of 73.6% (0 minutes) and 52.7% (30 minutes) (p < 0.001), in TAMx of 53.2% (p < 0.001) and 38.3% (p = 0.002), and in TAMn of 84.4% (p < 0.001) and 68.2% (p < 0.001). Semiquantitative power Doppler scores at all portions revealed increased average grading of 1.96 after intervention and 2.04 after 30 minutes compared with 0.75 at baseline. Our results may contribute to the understanding of local physiological reactions to self-myofascial release.

Purification, substrate specificity, and classification of tripeptidyl peptidase II.

PubMed

Bålöw, R M; Tomkinson, B; Ragnarsson, U; Zetterqvist, O

1986-02-15

An extralysosomal tripeptide-releasing aminopeptidase was recently discovered in rat liver (Bålöw, R.-M., Ragnarsson, U., and Zetterqvist, O. (1983) J. Biol. Chem. 258, 11622-11628). In the present work this tripeptidyl peptidase is shown to occur in several rat tissues and in human erythrocytes. The erythrocyte enzyme was purified about 80,000-fold from a hemolysate while the rat liver enzyme was purified about 4,000-fold from a homogenate. Upon polyacrylamide gel electrophoresis in sodium dodecyl sulfate under reducing conditions more than 90% of the protein was represented by a polypeptide of Mr 135,000 in both cases. In addition, the two enzymes eluted at similar positions in the various chromatographic steps, showed similar specific activity, and had a pH optimum around 7.5. A tryptic pentadecapeptide from the alpha-chain of human hemoglobin, Val-Gly-Ala-His-Ala-Gly-Glu-Tyr-Gly-Ala-Glu-Ala-Leu-Glu-Arg, i.e. residues 17-31, was found to be sequentially cleaved by the erythrocyte enzyme into five tripeptides, beginning from the NH2 terminus. Chromogenic tripeptidylamides showed various rates of hydrolysis at pH 7.5. With Ala-Ala-Phe-4-methyl-7-coumarylamide, Km was 16 microM and Vmax 13 mumol min-1 . mg-1, comparable to the standard substrate Arg-Arg-Ala-Ser(32P)-Val-Ala values (Km 13 microM and Vmax 24 mumol . min-1 . mg-1). The tripeptidyl peptidase of human erythrocytes was classified as a serine peptidase from its irreversible inhibition by phenylmethanesulfonyl fluoride and diisopropyl fluorophosphate. The rate of inhibition was decreased by the presence of an efficient competitive inhibitor, Val-Leu-Arg-Arg-Ala-Ser-Val-Ala (Ki 1.5 microM). [3H]Diisopropylphosphate was incorporated to the extent of 0.7-0.9 mol/mol of Mr 135,000 subunit, which confirms the high purity of the enzyme.

Characterization of the co-purified invertase and β-glucosidase of a multifunctional extract from Aspergillus terreus.

PubMed

Giraldo, Marielle Aleixo; Gonçalves, Heloísa Bressan; Furriel, Rosa Dos Prazeres Melo; Jorge, João Atílio; Guimarães, Luis Henrique Souza

2014-05-01

The filamentous fungus Aspergillus terreus secretes both invertase and β-glucosidase when grown under submerged fermentation containing rye flour as the carbon source. The aim of this study was to characterize the co-purified fraction, especially the invertase activity. An invertase and a β-glucosidase were co-purified by two chromatographic steps, and the isolated enzymatic fraction was 139-fold enriched in invertase activity. SDS-PAGE analysis of the co-purified enzymes suggests that the protein fraction with invertase activity was heterodimeric, with subunits of 47 and 27 kDa. Maximal invertase activity, which was determined by response surface methodology, occurred in pH and temperature ranges of 4.0-6.0 and 55-65 °C, respectively. The invertase in co-purified enzymes was stable for 1 h at pH 3.0-10.0 and maintained full activity for up to 1 h at 55 °C when diluted in water. Invertase activity was stimulated by 1 mM concentrations of Mn²⁺ (161 %), Co²⁺ (68 %) and Mg²⁺ (61 %) and was inhibited by Al³⁺, Ag⁺, Fe²⁺ and Fe³⁺. In addition to sucrose, the co-purified enzymes hydrolyzed cellobiose, inulin and raffinose, and the apparent affinities for sucrose and cellobiose were quite similar (K(M) = 22 mM). However, in the presence of Mn²⁺, the apparent affinity and V(max) for sucrose hydrolysis increased approximately 2- and 2.9-fold, respectively, while for cellobiose, a 2.6-fold increase in V(max) was observed, but the apparent affinity decreased 5.5-fold. Thus, it is possible to propose an application of this multifunctional extract containing both invertase and β-glucosidase to degrade plant biomass, thus increasing the concentration of monosaccharides obtained from sucrose and cellobiose.

Detoxification of Acetylcholinesterase Inhibitors.

DTIC Science & Technology

1987-02-19

IIB:HB-o Mvse three machanisms can be distinguished by appropriate labelling experiments in oxygen-18 H.O. If mechanism C is operating, hydrolysis...enzyue reveals that there is a single break at pa 6.2 relative to both Vmax and Vfax/Km. This would appear to represent the titration of the basic residue

Satellite retrievals of leaf chlorophyll and photosynthetic capacity for improved modeling of GPP

USDA-ARS?s Scientific Manuscript database

This study investigates the utility of in-situ and satellite-based leaf chlorophyll (Chl) estimates for quantifying leaf photosynthetic capacity and for constraining model simulations of Gross Primary Productivity (GPP) over a corn field in Maryland, U.S.A. The maximum rate of carboxylation (Vmax) r...

Taurine: A Potential Ergogenic Aid for Preventing Muscle Damage and Protein Catabolism and Decreasing Oxidative Stress Produced by Endurance Exercise

PubMed Central

De Carvalho, Flávia G.; Galan, Bryan S. M.; Santos, Priscila C.; Pritchett, Kelly; Pfrimer, Karina; Ferriolli, Eduardo; Papoti, Marcelo; Marchini, Júlio S.; de Freitas, Ellen C.

2017-01-01

The aim of this study was to evaluate the effects of taurine and chocolate milk supplementation on oxidative stress and protein metabolism markers, and aerobic parameters in triathletes. Methods: A double-blind, crossover study was conducted with 10 male triathletes, aged 30.9 ± 1.3 year, height 1.79 ± 0.01 m and body weight 77.45 ± 2.4 kg. Three grams of taurine and 400 ml of chocolate milk (TAUchoc), or a placebo (chocolate milk) (CHOC) was ingested post exercise for 8 weeks. Oxidative stress marker levels, and 24 h urinary nitrogen, creatinine, and urea excretion were measured before and after 8 weeks of training and supplementation with TAUchoc or CHOC. A maximal incremental running test on a treadmill was performed in order to evaluate aerobic parameters: Vmax, heart rate (HR) and rate of perceived exertion (RPE). Results: TAUchoc treatment during the 8 weeks resulted in increased taurine plasma levels (PRE 201.32 ± 29.03 μmol/L and POST 234.36 ± 35.51 μmol/L, p = 0.01), decreased malondialdehyde levels (19.4%, p = 0.03) and urinary nitrogen excretion (−33%, p = 0.03), and promoted positive nitrogen balance (p = 0.01). There were no changes in reduced glutathione (TAUchoc PRE 0.72 ± 0.08 mmol/L and POST 0.83 ± 0.08 mmol/L; CHOC PRE 0.69 ± 0.08 mmol/L and POST 0.81 ± 0.06 mmol/L), vitamin E plasma levels (TAUchoc PRE 33.99 ± 2.52 μmol/L and 35.95 ± 2.80 μmol/L and CHOC PRE 31.48 ± 2.12 μmol/L and POST 33.77 ± 3.64 μmol/L), or aerobic parameters, which were obtained in the last phase of the maximal incremental running test (Vmax TAUchoc PRE 13 ± 1.4 km/h and POST 13.22 ± 1.34 km/h; CHOC PRE 13.11 ± 2.34 km/h and POST 13.11 ± 2.72 km/h), the heart rate values were TAUchoc PRE 181.89 ± 24.18 bpm and POST 168.89 ± 46.56 bpm; CHOC PRE 181.56 ± 2.14 bpm and POST 179.78 ± 3.4 bpm, and the RPE were TAUchoc PRE 8.33 ± 2.4 AU and POST 9.1 ± 2.1 AU; CHOC PRE 8.11 ± 4.94 AU and POST 8.78 ± 2.78 AU). Conclusion: Taurine supplementation did not improve aerobic parameters, but was effective in increasing taurine plasma levels and decreasing oxidative stress markers, which suggests that taurine may prevent oxidative stress in triathletes. PMID:28979213

Characterizing Isozymes of Chlorite Dismutase for Water Treatment

PubMed Central

Mobilia, Kellen C.; Hutchison, Justin M.; Zilles, Julie L.

2017-01-01

This work investigated the potential for biocatalytic degradation of micropollutants, focusing on chlorine oxyanions as model contaminants, by mining biology to identify promising biocatalysts. Existing isozymes of chlorite dismutase (Cld) were characterized with respect to parameters relevant to this high volume, low-value product application: kinetic parameters, resistance to catalytic inactivation, and stability. Maximum reaction velocities (Vmax) were typically on the order of 104 μmol min-1 (μmol heme)-1. Substrate affinity (Km) values were on the order of 100 μM, except for the Cld from Candidatus Nitrospira defluvii (NdCld), which showed a significantly lower affinity for chlorite. NdCld also had the highest susceptibility to catalytic inactivation. In contrast, the Cld from Ideonella dechloratans was least susceptible to catalytic inactivation, with a maximum turnover number of approximately 150,000, more than sevenfold higher than other tested isozymes. Under non-reactive conditions, Cld was quite stable, retaining over 50% of activity after 30 days, and most samples retained activity even after 90–100 days. Overall, Cld from I. dechloratans was the most promising candidate for environmental applications, having high affinity and activity, a relatively low propensity for catalytic inactivation, and excellent stability. PMID:29312158

WOR5, a Novel Tungsten-Containing Aldehyde Oxidoreductase from Pyrococcus furiosus with a Broad Substrate Specificity

PubMed Central

Bevers, Loes E.; Bol, Emile; Hagedoorn, Peter-Leon; Hagen, Wilfred R.

2005-01-01

WOR5 is the fifth and last member of the family of tungsten-containing oxidoreductases purified from the hyperthermophilic archaeon Pyrococcus furiosus. It is a homodimeric protein (subunit, 65 kDa) that contains one [4Fe-4S] cluster and one tungstobispterin cofactor per subunit. It has a broad substrate specificity with a high affinity for several substituted and nonsubstituted aliphatic and aromatic aldehydes with various chain lengths. The highest catalytic efficiency of WOR5 is found for the oxidation of hexanal (Vmax = 15.6 U/mg, Km = 0.18 mM at 60°C). Hexanal-incubated enzyme exhibits S = 1/2 electron paramagnetic resonance signals from [4Fe-4S]1+ (g values of 2.08, 1.93, and 1.87) and W5+ (g values of 1.977, 1.906, and 1.855). Cyclic voltammetry of ferredoxin and WOR5 on an activated glassy carbon electrode shows a catalytic wave upon addition of hexanal, suggesting that ferredoxin can be a physiological redox partner. The combination of WOR5, formaldehyde oxidoreductase, and aldehyde oxidoreductase forms an efficient catalyst for the oxidation of a broad range of aldehydes in P. furiosus. PMID:16199576

Evaluation of the nature of camel retinal acetylcholinesterase: inhibition by hexamethonium.

PubMed

Alhomida, A S; Kamal, M A; al-Jafari, A A

1997-12-01

Acetylcholinesterase (AChE, EC 3.1.1.7) has been demonstrated in retinas of several species, however, the nature of the interaction of AChE with specific inhibitors are very limited in the literature and the mode of inhibition of camel retinal AChE by hexamethonium has been studied. Hexamethonium reversibly inhibited AChE in a concentration dependent manner, the IC50 value being c. 2.52 mM. The Km for the hydrolysis of acetylthiocholine iodide was found to be 0.087 mM and the Vmax was 0.63 mumol/min/mg protein. Dixon, as well as Lineweaver-Burk, plots and their secondary replots indicated that the nature of the inhibition is of the hyperbolic (partial) mixed type, which is considered to be a partial competitive and non-competitive mixture. The values of Ki(slope) and KI(intercept) from a Lineweaver-Burk plot were estimated as 0.30 mM and 0.17 mM, respectively, while Ki from a Dixon plot was estimated as 0.725 mM. The Ki was greater than KI indicating that hexamethonium has a greater affinity of binding for the active site than the peripheral site of the camel retina AChE.

Purified reconstituted lac carrier protein from Escherichia coli is fully functional.

PubMed

Viitanen, P; Garcia, M L; Kaback, H R

1984-03-01

Proteoliposomes reconstituted with lac carrier protein purified from the plasma membrane of Escherichia coli catalyze each of the translocation reactions typical of the beta-galactoside transport system (i.e., active transport, counterflow, facilitated influx and efflux) with turnover numbers and apparent Km values comparable to those observed in right-side-out membrane vesicles. Furthermore, detailed kinetic studies show that the reconstituted system exhibits properties analogous to those observed in membrane vesicles. Imposition of a membrane potential (delta psi, interior negative) causes a marked decrease in apparent Km (by a factor of 7 to 10) with a smaller increase in Vmax (approximately equal to 3-fold). At submaximal values of delta psi, the reconstituted carrier exhibits biphasic kinetics, with one component manifesting the kinetic parameters of active transport and the other exhibiting the characteristics of facilitated diffusion. Finally, at low lactose concentrations, the initial velocity of influx varies linearly with the square of the proton electro-chemical gradient. The results provide quantitative support for the contention that a single polypeptide species, the product of the lac y gene, is responsible for each of the transport reactions typical of the beta-galactoside transport system.

Purification and Biochemical and Kinetic Properties of an Endo-Polygalacturonase from the Industrial Fungus Aspergillus sojae.

PubMed

Fratebianchi, Dante; Cavello, Ivana Alejandra; Cavalitto, Sebastián Fernando

2017-01-01

An endo-polygalacturonase secreted by Aspergillus sojae was characterized after being purified to homogeneity from submerged cultures with orange peel as the sole carbon source by gel filtration and ion-exchange chromatographies. According to SDS-PAGE and analytical isoelectric focusing analyses, the enzyme presents a molecular weight of 47 kDa and pI value of 4.2. This enzyme exhibits considerable stability under highly acidic to neutral conditions (pH 1.5-6.5) and presents a half-life of 2 h at 50°C. Besides its activity towards pectin and polygalacturonic acid, the enzyme displays pectin-releasing activity, acting best in a pH range of 3.3-5.0. Thin-layer chromatographic analysis revealed that tri-galacturonate is the main enzymatic end product of polygalacturonic acid hydrolysis, indicating that it is an endo-polygalacturonase. The enzyme exhibits Michaelis-Menten kinetics, with KM and VMAX values of 0.134 mg/mL and 9.6 µmol/mg/min, respectively, and remained stable and active in the presence of SO2, ethanol, and various cations assayed except Hg2+. © 2017 S. Karger AG, Basel.

High-level expression of a novel thermostable and mannose-tolerant β-mannosidase from Thermotoga thermarum DSM 5069 in Escherichia coli

PubMed Central

2013-01-01

Background Mannan is one of the primary polysaccharides in hemicellulose and is widely distributed in plants. β-Mannosidase is an important constituent of the mannan-degrading enzyme system and it plays an important role in many industrial applications, such as food, feed and pulp/paper industries as well as the production of second generation bio-fuel. Therefore, the mannose-tolerant β-mannosidase with high catalytic efficiency for bioconversion of mannan has a great potential in the fields as above. Results A β-mannosidase gene (Tth man5) of 1,827 bp was cloned from the extremely thermophilic bacterium Thermotoga thermarum DSM 5069 that encodes a protein containing 608 amino acid residues, and was over-expressed in Escherichia coli BL21 (DE3). The results of phylogenetic analysis, amino acid alignment and biochemical properties indicate that the Tth Man5 is a novel β-mannosidase of glycoside hydrolase family 5. The optimal activity of the Tth Man5 β-mannosidase was obtained at pH 5.5 and 85°C and was stable over a pH range of 5.0 to 8.5 and exhibited 2 h half-life at 90°C. The kinetic parameters Km and Vmax values for p-nitrophenyl-β-D-mannopyranoside and 1,4-β-D-mannan were 4.36±0.5 mM and 227.27±1.59 μmol min-1 mg-1, 58.34±1.75 mg mL-1 and 285.71±10.86 μmol min-1 mg-1, respectively. The kcat/Km values for p-nitrophenyl-β-D-mannopyranoside and 1,4-β-D-mannan were 441.35±0.04 mM-1 s-1 and 41.47±1.58 s-1 mg-1 mL, respectively. It displayed high tolerance to mannose, with a Ki value of approximately 900 mM. Conclusions This work provides a novel and useful β-mannosidase with high mannose tolerance, thermostability and catalytic efficiency, and these characteristics constitute a powerful tool for improving the enzymatic conversion of mannan through synergetic action with other mannan-degrading enzymes. PMID:24099409

Fatty acid ethyl esters (FAEE); comparative accumulation in human and guinea pig hair as a biomarker for prenatal alcohol exposure.

PubMed

Kulaga, Vivian; Caprara, Daniela; Iqbal, Umar; Kapur, Bhushan; Klein, Julia; Reynolds, James; Brien, James; Koren, Gideon

2006-01-01

To compare the incorporation rate (ICR) of fatty acid ethyl esters (FAEE) in hair between guinea pigs and humans, and to assess the relationship between ethanol exposure and FAEE concentrations in hair. Published data from pregnant guinea pigs, including maximum blood ethanol concentration (BEC), dosage regimen, and total hair FAEE concentration, were compared with published data from alcoholic patients, where dose of ethanol consumed and total hair FAEE concentration were reported. Mean values of ethanol Vmax for pregnant guinea pigs and humans were obtained from published data (26.2 and 24 mg/dl/h, respectively). Total and individual FAEE ICRs, defined as the ratio of hair FAEE to the area under the BEC-time curve (total systemic ethanol exposure), were found to be on average an order of magnitude lower in the guinea pig than in the human. The profiles of ester incorporation also differed slightly between species, with ethyl stearate being highly incorporated in guinea pig hair and less so in human hair. The results may reflect in the human greater FAEE production, greater FAEE deposition in hair, slower FAEE catabolism, differential sebum production and composition, or a combination thereof. Also, ethyl oleate was found to correlate with total systemic ethanol exposure for both guinea pigs and humans, correlation coefficients equalling 0.67 (P < 0.05) and 0.49 (P < 0.05), respectively. No other ethyl esters, nor total FAEE, were found to correlate with systemic ethanol exposure. When extrapolating FAEE concentrations in hair from guinea pigs to humans, an order of magnitude difference should be considered, with humans incorporating more FAEE per unit of ethanol exposure. Also, the results suggest caution should be taken when interpreting values of single esters because of their differential incorporation among species. Lastly, our findings suggest ethyl oleate may be of keen interest in FAEE hair analysis, particularly across species.

Biotransformation of nitroso aromatic compounds and 2-oxo acids to N-hydroxy-N-arylacylamides by thiamine-dependent enzymes in rat liver.

PubMed

Yoshioka, T; Uematsu, T

1998-07-01

The formation of N-hydroxy-N-arylacylamides from nitroso aromatic compounds and 2-oxo acids was investigated using rat liver subcellular fractions. Activities were found in both mitochondria and cytosol, except for activities for phenylpyruvate and glyoxylate; the former did not produce N-hydroxy-N-phenylphenylacetamide and the latter nonenzymatically produced N-hydroxy-N-phenylformamide with nitrosobenzene (NOB). The cytosolic activity of N-hydroxy-N-phenylglycolamide formation was indicated to be due to transketolase, which utilized hydroxypyruvate as a glycolic aldehyde donor to NOB. With mitochondria, 2-oxo acids (including hydroxypyruvate) served as substrates for the biotransformation of NOB to the corresponding N-hydroxy-N-phenylacylamides. The substrate preference was 2-oxobutyrate > pyruvate > 2-oxoisovalerate > 2-oxoisocaproate > 2-oxovalerate > 2-oxo-3-methylvalerate, judging from Vmax/half-saturating concentration for mitochondria values. The half-saturating concentrations for NOB were nearly constant. The mitochondrial activity was due to pyruvate dehydrogenase complex and branched-chain 2-oxo acid dehydrogenase complex (BCDHC). By using partially purified BCDHC, pyruvate and 2-oxobutyrate were found to be common substrates for both of the enzymes, and 2-oxoisovalerate was shown to be the most effective substrate for BCDHC. Analysis by the Taft equation indicated that the polar effects, rather than the steric effects, of the alkyl groups of 2-oxo acids are important for BCDHC-catalyzed formation of N-hydroxy-N-phenylacylamides. A positive Hammett constant obtained for the formation of N-hydroxy-N-arylisobutyramides indicates that an electron-withdrawing substituent makes the nitroso compounds susceptible to BCDHC-catalyzed biotransformation.

Left Atrial Mechanical Functions in Professional Soccer Players: A Pilot Study

ERIC Educational Resources Information Center

Kartal, Alper; Güngör, Hasan; Kartal, Resat; Ergin, Esin

2017-01-01

Long-term regular exercise is associated with physiologic and morphologic alterations in the heart chambers. The aim of this study to evaluate left atrium (LA) phasic functions in professional football players and compare with control subjects. Left atrial volume was calculated at end-systole (Vmax), end-diastole and pre-atrial contraction by…

Human beta-glucuronidase. Measurement of its activity in gallbladder bile devoid of intrinsic interference.

PubMed

Ho, Y C; Ho, K J

1988-04-01

Our purpose is to develop a standard method for preparing the bile for beta-glucuronidase determination by removal of bile acids and conjugated bilirubin which interfere with its activity. The bile acids and conjugated bilirubin in their purified solutions and in the diluted gallbladder biles could be extracted completely with cholestyramine in powder form or tetrahexylammonium chloride (THAC) in chloroform or ethyl acetate. The enzyme was, however, partially precipitated with cholestyramine and denatured by chloroform but not by ethyl acetate. A standard procedure, therefore, includes extraction of the diluted gallbladder bile with THAC in ethyl acetate, followed by determination of the maximal velocity (Vmax) of the enzyme by a kinetic method employing phenolphthalein glucuronide as the substrate. The average Vmax of beta-glucuronidase in the 20 normal gallbladder biles was 165 +/- 86 nmol/min/ml (mean +/- SD), a 23.5-fold increase over the activity before extraction. The measured activity represented the true activity of the enzyme in the bile for recovery of activity of the enzyme added to the bile was practically complete.

Phosphate-dependent glutaminase in enterocyte mitochondria and its regulation by ammonium and other ions.

PubMed

Masola, B; Zvinavashe, E

2003-06-01

The effects of ammonium and other ions on phosphate dependent glutaminase (PDG) activity in intact rat enterocyte mitochondria were investigated. Sulphate and bicarbonate activated the enzyme in absence and presence of added phosphate. In presence of 10 mM phosphate, ammonium at concentrations <1 mM inhibited the enzyme. This inhibition was reversed by increased concentration of phosphate or sulphate. The inhibition of PDG by ammonium in presence of 10 mM phosphate was biphasic with respect to glutamine concentration, its effect being through a lowering of V(max) at glutamine concentration of

GAMA/H-ATLAS: The Local Dust Mass Function and Cosmic Density as a Function of Galaxy Type - A Benchmark for Models of Galaxy Evolution

NASA Astrophysics Data System (ADS)

Beeston, R. A.; Wright, A. H.; Maddox, S.; Gomez, H. L.; Dunne, L.; Driver, S. P.; Robotham, A.; Clark, C. J. R.; Vinsen, K.; Takeuchi, T. T.; Popping, G.; Bourne, N.; Bremer, M. N.; Phillipps, S.; Moffett, A. J.; Baes, M.; Bland-Hawthorn, J.; Brough, S.; De Vis, P.; Eales, S. A.; Holwerda, B. W.; Loveday, J.; Liske, J.; Smith, M. W. L.; Smith, D. J. B.; Valiante, E.; Vlahakis, C.; Wang, L.

2018-06-01

We present the dust mass function (DMF) of 15,750 galaxies with redshift z < 0.1, drawn from the overlapping area of the GAMA and H-ATLAS surveys. The DMF is derived using the density corrected Vmax method, where we estimate Vmax using: (i) the normal photometric selection limit (pVmax) and (ii) a bivariate brightness distribution (BBD) technique, which accounts for two selection effects. We fit the data with a Schechter function, and find M^{*}=(4.65 ± 0.18)× 107 h^2_{70} M_{⊙ }, α = ( - 1.22 ± 0.01), φ ^{*}=(6.26 ± 0.28)× 10^{-3} h^3_{70} Mpc^{-3} dex^{-1}. The resulting dust mass density parameter integrated down to 104 M⊙ is Ωd = (1.11 ± 0.02) × 10-6 which implies the mass fraction of baryons in dust is f_{m_b}=(2.40± 0.04)× 10^{-5}; cosmic variance adds an extra 7-17 per cent uncertainty to the quoted statistical errors. Our measurements have fewer galaxies with high dust mass than predicted by semi-analytic models. This is because the models include too much dust in high stellar mass galaxies. Conversely, our measurements find more galaxies with high dust mass than predicted by hydrodynamical cosmological simulations. This is likely to be from the long timescales for grain growth assumed in the models. We calculate DMFs split by galaxy type and find dust mass densities of Ωd = (0.88 ± 0.03) × 10-6 and Ωd = (0.060 ± 0.005) × 10-6 for late-types and early-types respectively. Comparing to the equivalent galaxy stellar mass functions (GSMF) we find that the DMF for late-types is well matched by the GMSF scaled by (8.07 ± 0.35) × 10-4.

Movement velocity as a measure of exercise intensity in three lower limb exercises.

PubMed

Conceição, Filipe; Fernandes, Juvenal; Lewis, Martin; Gonzaléz-Badillo, Juan José; Jimenéz-Reyes, Pedro

2016-01-01

The purpose of this study was to investigate the relationship between movement velocity and relative load in three lower limbs exercises commonly used to develop strength: leg press, full squat and half squat. The percentage of one repetition maximum (%1RM) has typically been used as the main parameter to control resistance training; however, more recent research has proposed movement velocity as an alternative. Fifteen participants performed a load progression with a range of loads until they reached their 1RM. Maximum instantaneous velocity (Vmax) and mean propulsive velocity (MPV) of the knee extension phase of each exercise were assessed. For all exercises, a strong relationship between Vmax and the %1RM was found: leg press (r(2)adj = 0.96; 95% CI for slope is [-0.0244, -0.0258], P < 0.0001), full squat (r(2)adj = 0.94; 95% CI for slope is [-0.0144, -0.0139], P < 0.0001) and half squat (r(2)adj = 0.97; 95% CI for slope is [-0.0135, -0.00143], P < 0.0001); for MPV, leg press (r(2)adj = 0.96; 95% CI for slope is [-0.0169, -0.0175], P < 0.0001, full squat (r(2)adj = 0.95; 95% CI for slope is [-0.0136, -0.0128], P < 0.0001) and half squat (r(2)adj = 0.96; 95% CI for slope is [-0.0116, 0.0124], P < 0.0001). The 1RM was attained with a MPV and Vmax of 0.21 ± 0.06 m s(-1) and 0.63 ± 0.15 m s(-1), 0.29 ± 0.05 m s(-1) and 0.89 ± 0.17 m s(-1), 0.33 ± 0.05 m s(-1) and 0.95 ± 0.13 m s(-1) for leg press, full squat and half squat, respectively. Results indicate that it is possible to determine an exercise-specific %1RM by measuring movement velocity for that exercise.

Process optimization for production and purification of a thermostable, organic solvent tolerant lipase from Acinetobacter sp. AU07.

PubMed

Gururaj, P; Ramalingam, Subramanian; Nandhini Devi, Ganesan; Gautam, Pennathur

2016-01-01

The purpose of this study was to isolate, purify and optimize the production conditions of an organic solvent tolerant and thermostable lipase from Acinetobacter sp. AU07 isolated from distillery waste. The lipase production was optimized by response surface methodology, and a maximum production of 14.5U/mL was observed at 30°C and pH 7, using a 0.5% (v/v) inoculum, 2% (v/v) castor oil (inducer), and agitation 150rpm. The optimized conditions from the shake flask experiments were validated in a 3L lab scale bioreactor, and the lipase production increased to 48U/mL. The enzyme was purified by ammonium sulfate precipitation and ion exchange chromatography and the overall yield was 36%. SDS-PAGE indicated a molecular weight of 45kDa for the purified protein, and Matrix assisted laser desorption/ionization time of flight analysis of the purified lipase showed sequence similarity with GDSL family of lipases. The optimum temperature and pH for activity of the enzyme was found to be 50°C and 8.0, respectively. The lipase was completely inhibited by phenylmethylsulfonyl fluoride but minimal inhibition was observed when incubated with ethylenediaminetetraacetic acid and dithiothreitol. The enzyme was stable in the presence of non-polar hydrophobic solvents. Detergents like SDS inhibited enzyme activity; however, there was minimal loss of enzyme activity when incubated with hydrogen peroxide, Tween 80 and Triton X-100. The kinetic constants (Km and Vmax) revealed that the hydrolytic activity of the lipase was specific to moderate chain fatty acid esters. The Vmax, Km and Vmax/Km ratio of the enzyme were 16.98U/mg, 0.51mM, and 33.29, respectively when 4-nitrophenyl palmitate was used as a substrate. Copyright © 2016 Sociedade Brasileira de Microbiologia. Published by Elsevier Editora Ltda. All rights reserved.

Metabolism of endosulfan-alpha by human liver microsomes and its utility as a simultaneous in vitro probe for CYP2B6 and CYP3A4.

PubMed

Casabar, Richard C T; Wallace, Andrew D; Hodgson, Ernest; Rose, Randy L

2006-10-01

Endosulfan-alpha is metabolized to a single metabolite, endosulfan sulfate, in pooled human liver microsomes (Km = 9.8 microM, Vmax = 178.5 pmol/mg/min). With the use of recombinant cytochrome P450 (P450) isoforms, we identified CYP2B6 (Km = 16.2 microM, Vmax = 11.4 nmol/nmol P450/min) and CYP3A4 (Km = 14.4 microM, Vmax = 1.3 nmol/nmol P450/min) as the primary enzymes catalyzing the metabolism of endosulfan-alpha, although CYP2B6 had an 8-fold higher intrinsic clearance rate (CL(int) = 0.70 microl/min/pmol P450) than CYP3A4 (CL(int) = 0.09 microl/min/pmol P450). Using 16 individual human liver microsomes (HLMs), a strong correlation was observed with endosulfan sulfate formation and S-mephenytoin N-demethylase activity of CYP2B6 (r(2) = 0.79), whereas a moderate correlation with testosterone 6 beta-hydroxylase activity of CYP3A4 (r(2) = 0.54) was observed. Ticlopidine (5 microM), a potent CYP2B6 inhibitor, and ketoconazole (10 microM), a selective CYP3A4 inhibitor, together inhibited approximately 90% of endosulfan-alpha metabolism in HLMs. Using six HLM samples, the percentage total normalized rate (% TNR) was calculated to estimate the contribution of each P450 in the total metabolism of endosulfan-alpha. In five of the six HLMs used, the percentage inhibition with ticlopidine and ketoconazole in the same incubation correlated with the combined % TNRs for CYP2B6 and CYP3A4. This study shows that endosulfan-alpha is metabolized by HLMs to a single metabolite, endosulfan sulfate, and that it has potential use, in combination with inhibitors, as an in vitro probe for CYP2B6 and 3A4 catalytic activities.

Purification and characterization of the extracellular laccase produced by Trametes polyzona WR710-1 under solid-state fermentation.

PubMed

Chairin, Thanunchanok; Nitheranont, Thitinard; Watanabe, Akira; Asada, Yasuhiko; Khanongnuch, Chartchai; Lumyong, Saisamorn

2014-01-01

Laccase from Trametes polyzona WR710-1 was produced under solid-state fermentation using the peel from the Tangerine orange (Citrus reticulata Blanco) as substrate, and purified to homogeneity. This laccase was found to be a monomeric protein with a molecular mass of about 71 kDa estimated by SDS-PAGE. The optimum pH was 2.0 for ABTS, 4.0 for L-DOPA, guaiacol, and catechol, and 5.0 for 2,6-DMP. The K(m) value of the enzyme for the substrate ABTS was 0.15 mM, its corresponding V(max) value was 1.84 mM min(-1), and the k(cat)/K(m) value was about 3960 s(-1)  mM(-1). The enzyme activity was stable between pH 6.0 and 8.0, at temperatures of up to 40 °C. The laccase was inhibited by more than 50% in the presence of 20 mM NaCl, by 95% at 5 mM of Fe(2+), and it was completely inhibited by 0.1 mM NaN(3). The N-terminal amino acid sequence of this laccase is AVTPVADLQISNAGISPDTF, which is highly similar to those of laccases from other white-rot basidiomycetes. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Effects of acute L-carnitine intake on metabolic and blood lactate levels of elite badminton players.

PubMed

Eroğlu, Hüseyin; Senel, Omer; Güzel, Nevin A

2008-04-01

Purpose of this study is to research the effects of acute L-Carnitine intake on badminton players' metabolic and blood lactate values. A total of 16 Turkish national badminton players (8 male, 8 female) were voluntarily participated into study. MaxVO2, MET, energy consumption, HR (heart rate), VE (minute ventilation), R (respiratory exchange ratio), AT (anaerobic threshold), oxygen pulse and blood lactate (LA) of subjects were measured by Sensormedics VmaxST and Accutrend Lactate Analyzer. The participants were subjected to the test protocol twice before and after 2g of L-Carnitine intake. The data were evaluated by the use of SPSS 13.0 for Windows. No significant differences were found between 1st. (without L-Carnitine intake) and 2nd. (with L-Carnitine intake) measurements of female participants as regards to all measured parameters. There was a significant difference in EMHR (exercise maximum heart rate) of males between two measurements (p<0.05). However the differences in other parameters were not significant. AT values of female subjects were not significant difference (p>0.05). Respiratory exchange ratio of males was significantly different at anaerobic threshold (p<0.05). Results of this study show that L-carnitine intake one hour prior to the exercise has no effect on the metabolic and blood lactate values of badminton players.

Testing the coherence between occupational exposure limits for inhalation and their biological limit values with a generalized PBPK-model: the case of 2-propanol and acetone.

PubMed

Huizer, Daan; Huijbregts, Mark A J; van Rooij, Joost G M; Ragas, Ad M J

2014-08-01

The coherence between occupational exposure limits (OELs) and their corresponding biological limit values (BLVs) was evaluated for 2-propanol and acetone. A generic human PBPK model was used to predict internal concentrations after inhalation exposure at the level of the OEL. The fraction of workers with predicted internal concentrations lower than the BLV, i.e. the 'false negatives', was taken as a measure for incoherence. The impact of variability and uncertainty in input parameters was separated by means of nested Monte Carlo simulation. Depending on the exposure scenario considered, the median fraction of the population for which the limit values were incoherent ranged from 2% to 45%. Parameter importance analysis showed that body weight was the main factor contributing to interindividual variability in blood and urine concentrations and that the metabolic parameters Vmax and Km were the most important sources of uncertainty. This study demonstrates that the OELs and BLVs for 2-propanol and acetone are not fully coherent, i.e. enforcement of BLVs may result in OELs being violated. In order to assess the acceptability of this "incoherence", a maximum population fraction at risk of exceeding the OEL should be specified as well as a minimum level of certainty in predicting this fraction. Copyright © 2014 Elsevier Inc. All rights reserved.

Inhibition of thiopurine S-methyltransferase activity by impurities in commercially available substrates: a factor for differing results of TPMT measurements.

PubMed

Kröplin, T; Fischer, C; Iven, H

1999-06-01

Thiopurine S-methyltransferase (TPMT) activity, when measured in red blood cells (RBC) with a recently published TPMT activity assay using 6-thioguanine (6-TG) as substrate, could not be reproduced in another laboratory. We investigated factors which could influence the results of the TPMT activity measurement. We tested twelve 6-TG and four 6-mercaptopurine (6-MP) compounds from different suppliers as substrates and determined the enzyme kinetic parameters Km and Vmax. Furthermore, we studied the influence of different 6-TG compounds on the affinity of the methyl donor S-adenosyl-L-methionine (SAM) to the TPMT enzyme. All 6-TG products were of equal purity (declared >98% by the supplier): this was ascertained by HPLC. However, the rate of methylation obtained following incubation with 6-TG from different suppliers ranged from 10% to 100% when incubated with the same RBC lysate. The lowest apparent Km value for a 6-TG was 22.3 micromol x l(-1), while the product with the highest methylation rate showed a Km of 156 micromol x l(-1). From these results we assume that there is a contaminant in some 6-TG products, which acts as a strong inhibitor of TPMT activity. Compounds possibly used for the synthesis of 6-TG (guanine, pyridine, 6-chloroguanine) did not affect the methylation rate. Thioxanthine, which is known to be a strong inhibitor of TPMT when added to the assay system to give a 2% contamination, reduced TPMT activity from 100% to 72%. Using 6-MP from different suppliers as substrate resulted in Km values ranging from 110 to 162 micromol x l(-1) and Vmox values ranging from 54 to 68 nmol 6-MMP x g(-1)Hb x h(-1). The Km value for the methyl donor SAM was similar to and independent from the thiopurine substrates tested (range 4.9-11 micromol-l(-1) SAM). In contrast to other investigators, we found non-enzymatic S-methylation, which was negligible under our assay conditions (3% with 128 micromol x l(-1) SAM), but could become relevant in experiments using higher SAM concentrations. TPMT enzyme activity determined with 6-TG as substrate may be strongly inhibited by a contaminant in some of the 6-TG lots distributed.

Enzymatic extract containing lignin peroxidase immobilized on carbon nanotubes: Potential biocatalyst in dye decolourization.

PubMed

Oliveira, Sabrina Feliciano; da Luz, José Maria Rodrigues; Kasuya, Maria Catarina Megumi; Ladeira, Luiz Orlando; Correa Junior, Ary

2018-05-01

The majority of the textile dyes are harmful to the environment and potentially carcinogenic. Among strategies for their exclusion, the treatment of dye contaminated wastewater with fungal extract, containing lignin peroxidase (LiP), may be useful. Two fungi isolates, Pleurotus ostreatus (PLO9) and Ganoderma lucidum (GRM117), produced the enzymatic extract by fermentation in the lignocellulosic residue, Jatropha curcas seed cake. The extracts from PLO9 and GRM117 were immobilized on carbon nanotubes and showed an increase of 18 and 27-fold of LiP specific activity compared to the free enzyme. Also, LiP from both fungi extracts showed higher Vmax and lower Km values. Only the immobilized extracts could be efficiently reused in the dye decolourization, contrary, the carbon nanotubes became saturated and they should be discarded over time. This device may offer a final biocatalyst with higher catalytic efficiency and capability to be reused in the dye decolourization process.

Passage of delta sleep-inducing peptide (DSIP) across the blood-cerebrospinal fluid barrier

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

Zlokovic, B.V.; Segal, M.B.; Davson, H.

1988-05-01

Unidirectional flux of /sup 125/I-labeled DSIP at the blood-tissue interface of the blood-cerebrospinal fluid (CSF) barrier was studied in the perfused in situ choroid plexuses of the lateral ventricles of the sheep. Arterio-venous loss of /sup 125/I-radioactivity suggested a low-to-moderate permeability of the choroid epithelium to the intact peptide from the blood side. A saturable mechanism with Michaelis-Menten type kinetics with high affinity and very low capacity (approximate values: Kt = 5.0 +/- 0.4 nM; Vmax = 272 +/- 10 fmol.min-1) was demonstrated at the blood-tissue interface of the choroid plexus. The clearance of DSIP from the ventricles during ventriculo-cisternalmore » perfusion in the rabbit indicated no significant flux of the intact peptide out of the CSF. The results suggest that DSIP crosses the blood-CSF barrier, while the system lacks the specific mechanisms for removal from the CSF found with most, if not all, amino acids and several peptides.« less

Polyphenol oxidase activity and antioxidant properties of Yomra apple (Malus communis L.) from Turkey.

PubMed

Can, Zehra; Dincer, Barbaros; Sahin, Huseyin; Baltas, Nimet; Yildiz, Oktay; Kolayli, Sevgi

2014-12-01

In this study, firstly, antioxidant and polyphenol oxidase (PPO) properties of Yomra apple were investigated. Seventeen phenolic constituents were measured by reverse phase-high-performance liquid chromatography (RP-HPLC). Total phenolic compounds (TPCs), ferric reducing antioxidant power (FRAP) and 2, 2-diphenyl-1-picrylhydrazyl radical (DPPH) scavenging activities were performed to measure antioxidant capacity. Some kinetic parameters (Km, Vmax), and inhibition behaviors against five different substrates were measured in the crude extract. Catechin and chlorogenic acid were found as the major components in the methanolic extract, while ferulic acid, caffeic acid, p-hydroxybenzoic acid, quercetin and p-coumaric acid were small quantities. Km values ranged from 0.70 to 10.10 mM in the substrates, and also 3-(4-hydroxyphenyl) propanoic acid (HPPA) and L-DOPA showed the highest affinity. The inhibition constant of Ki were ranged from 0.05 to 14.90 mM against sodium metabisulphite, ascorbic acid, sodium azide and benzoic acid, while ascorbic acid and sodium metabisulphite were the best inhibitors.

Characterization of procaine metabolism as probe for the butyrylcholinesterase enzyme investigation by simultaneous determination of procaine and its metabolite using capillary electrophoresis with electrochemiluminescence detection.

PubMed

Yuan, Jipei; Yin, Jianyuan; Wang, Erkang

2007-06-22

Capillary electrophoresis with electrochemiluminescene detection was used to characterize procaine hydrolysis as a probe for butyrylcholinesterase by in vitro procaine metabolism in plasma with butyrylcholinesterase acting as bioscavenger. Procaine and its metabolite N,N-diethylethanolamine were separated at 16 kV and then detected at 1.25 V in the presence of 5.0 mM Ru(bpy)(3)2+, with the detection limits of 2.4x10(-7) and 2.0x10(-8) mol/L (S/N=3), respectively. The Michaelis constant Km value was 1.73x10(-4) mol/L and the maximum velocity Vmax was 1.62x10(-6) mol/L/min. Acetylcholine bromide and choline chloride presented inhibition effects on the enzymatic cleavage of procaine, with the 50% inhibition concentration (IC50) of 6.24x10(-3) and 2.94x10(-4) mol/L.

Differentiated effect of ageing on the enzymes of Krebs' cycle, electron transfer complexes and glutamate metabolism of non-synaptic and intra-synaptic mitochondria from cerebral cortex.

PubMed

Villa, R F; Gorini, A; Hoyer, S

2006-11-01

The effect of ageing on the activity of enzymes linked to Krebs' cycle, electron transfer chain and glutamate metabolism was studied in three different types of mitochondria of cerebral cortex of 1-year old and 2-year old male Wistar rats. We assessed the maximum rate (V(max)) of the mitochondrial enzyme activities in non-synaptic perikaryal mitochondria, and in two populations of intra-synaptic mitochondria. The results indicated that: (i) in normal, steady-state cerebral cortex the values of the catalytic activities of the enzymes markedly differed in the various populations of mitochondria; (ii) in intra-synaptic mitochondria, ageing affected the catalytic properties of the enzymes linked to Krebs' cycle, electron transfer chain and glutamate metabolism; (iii) these changes were more evident in intra-synaptic "heavy" than "light" mitochondria. These results indicate a different age-related vulnerability of subpopulations of mitochondria in vivo located into synapses than non-synaptic ones.

[Effects of 2-(p-dimethylaminostyryl) pyridine methycholide (DSPM-Ci) on ECG, left atrium contractivity and on papillary muscle action potentials].

PubMed

Jiang, X Y; Zhou, C M; Li, D M; Zhang, K J

1996-01-01

The effects of DSPM-Cl on ECG in rats, on the dose-effect curve in guinea pig left atria and on the fast action potential (AP), high-K+ depolarized slow action potential (SAP) in guinea pigs papillary muscle were examined electrophysiologically. DSPM-Cl (2 mg.kg-1) showed significant nagative frequency, negative conductivity effect, and prolonged the PP and PR interval. DSPM-CI (30-50 mumol.L-1) was shown to inhibit left atria contractility and shift the concentration-response curve of Iso and CaCl2 to the right with PD2' values of 4.60 and 4.13, respectively. In addition, DSPM-Cl was found to prolong the duration of action potential 90 (APD90) and effective refractory period (ERP), and decrease the maximal upstroke velocity (Vmax) in K(+)-depolarized guinea pigs papillary muscles. The results suggest that, like verpamil, DSPM-Cl might be a calcium antagonist.

Effectiveness of Vegetated Drainage Ditches for Domestic Sewage Effluent Mitigation.

PubMed

Kumwimba, Mathieu Nsenga; Zhu, Bo

2017-05-01

Plant species have an important role in eco-ditches; however, the Michaelis-Menten kinetic parameters of nutrient uptake, growth rate and purification efficiency of ditch plants and their influences on domestic sewage treatment efficiency are still unclear. Growth rates of all nine species, but especially Lemna gibba, Cladophora and Myriophyllum verticillatum were best in undiluted domestic sewage as opposed to a mixture of domestic sewage. Performance of species to accumulate nutrients was not only species-specific, but was also affected by both sewage treatments. Removal efficiency of nutrients was dependent on both plant species and treatment. Uptake kinetic parameters were significantly affected by both nutrient form and plant species. The maximum uptake rate (Vmax) of NH 4 -N was higher than NO 3 -N. Similarly, Km values for NH 4 -N were greater than NO 3 -N. These results could be used to identify plants for sewage treatment efficiency and enhance water quality in eco-ditch treatment systems.

Purification and characterization of broccoli (Brassica oleracea var. italica) myrosinase (β-thioglucosidase glucohydrolase).

PubMed

Mahn, Andrea; Angulo, Alejandro; Cabañas, Fernanda

2014-12-03

Myrosinase (β-thioglucosidase glucohydrolase, EC 3.2.1.147) from broccoli (Brassica oleracea var. italica) was purified by ammonium sulfate precipitation followed by concanavalin A affinity chromatography, with an intermediate dialysis step, resulting in 88% recovery and 1318-fold purification. These are the highest values reported for the purification of any myrosinase. The subunits of broccoli myrosinase have a molecular mass of 50-55 kDa. The native molecular mass of myrosinase was 157 kDa, and accordingly, it is composed of three subunits. The maximum activity was observed at 40 °C and at pH below 5.0. Kinetic assays demonstrated that broccoli myrosinase is subjected to substrate (sinigrin) inhibition. The Michaelis-Menten model, considering substrate inhibition, gave Vmax equal to 0.246 μmol min(-1), Km equal to 0.086 mM, and K(I) equal to 0.368 mM. This is the first study about purification and characterization of broccoli myrosinase.

Substrate specificity and pH dependence of homogeneous wheat germ acid phosphatase.

PubMed

Van Etten, R L; Waymack, P P

1991-08-01

The broad substrate specificity of a homogeneous isoenzyme of wheat germ acid phosphatase (WGAP) was extensively investigated by chromatographic, electrophoretic, NMR, and kinetic procedures. WGAP exhibited no divalent metal ion requirement and was unaffected upon incubation with EDTA or o-phenanthroline. A comparison of two catalytically homogeneous isoenzymes revealed little difference in substrate specificity. The specificity of WGAP was established by determining the Michaelis constants for a wide variety of substrates. p-Nitrophenyl phosphate, pyrophosphate, tripolyphosphate, and ATP were preferred substrates while lesser activities were seen toward sugar phosphates, trimetaphosphate, phosphoproteins, and (much less) phosphodiesters. An extensive table of Km and Vmax values is given. The pathway for the hydrolysis of trimetaphosphate was examined by colorimetric and 31P NMR methods and it was found that linear tripolyphosphate is not a free intermediate in the enzymatic reaction. In contrast to literature reports, homogeneous wheat germ acid phosphatase exhibits no measurable carboxylesterase activity, nor does it hydrolyze phenyl phosphonothioate esters or phytic acid at significant rates.

Catalytic and immunochemical properties of arylsulphatase A from urine, modified by potassium ferrate.

PubMed

Laidler, P M; Steczko, J

1986-01-01

Arylsulphatase A (EC 3.1.6.1.) from urine was inactivated with potassium ferrate, a strong oxidizing agent. The inhibition could be prevented by competitive inhibitors, tetraborate and orthophosphate. Tetraborate which was shown to be a powerful competitive inhibitor (determined Ki = 4 X 10(-5) M) gave more efficient protection. The partially inactivated enzyme exhibited a Km value similar to that of the unmodified arylsulphatase A, and its Vmax decreased in proportion to the loss of enzymatic activity. The partially modified enzyme did not lose its ability to catalyse hydrolysis of p-nitrocatechol sulphate according to the "anomalous kinetics" exhibited towards this substrate and characteristic for arylsulphatase A. The immunochemical properties of arylsulphatase A either fully or partially inactivated were similar to those of the native enzyme. The results allow to conclude that ferrate reacts with arylsulphatase A in its active site. Thus ferrate seems to be a very sensitive probe for amino acid residues essential for catalytic activity of arylsulphatase A.

Intelligent Hybrid Vehicle Power Control - Part 1: Machine Learning of Optimal Vehicle Power

DTIC Science & Technology

2012-06-30

time window ),[ tWt DT : vave, vmax, vmin, ac, vst and vend, where the first four parameters are, respectively, the average speed, maximum speed...minimum speed and average acceleration, during the time period ),[ tWt DT , vst is the vehicle speed at )( DTWt  , and vend is the vehicle

Enzymatic carotenoid cleavage in star fruit (Averrhoa carambola).

PubMed

Fleischmann, Peter; Watanabe, Naoharu; Winterhalter, Peter

2003-05-01

This paper presents the first description of an enzyme fraction exhibiting carotenoid cleavage activity isolated from fruit skin of Averrhoa carambola. Partial purification of the enzyme could be achieved by acetone precipitation, ultrafiltration (300 kDa, 50 kDa), isoelectric focusing (pH 3-10) and sodium dodecyl sulfate polyacrylamide gel electrophoresis (7.5%). In this way, an enzymatically active protein fraction was obtained, consisting of four proteins in the molecular weight range of between 12 and 90 kDa. Using beta-carotene as substrate, the enzyme activity was detected spectrophotometrically at 505 nm. The main reaction product, detected by GC analysis, was beta-ionone. This proves that the isolated enzymes are closely related to aroma metabolism and release of star fruit. The time constant of the reaction was 16.6 min, the Michaelis Constant K(m)=3.6 micromol 1(-1) and the maximum velocity V(max)=10.5 x 10(-3) micromol l(-1) s(-1) mg((Protein))(-1). The optimum temperature was 45 degrees C.

Biodegradation of o-nitrophenol by aerobic granules with glucose as co-substrate.

PubMed

Basheer, Farrukh; Isa, M H; Farooqi, I H

2012-01-01

Aerobic granules to treat wastewater containing o-nitrophenol were successfully developed in a sequencing batch reactor (SBR) using activated sludge as inoculum. Stable aerobic granules were obtained with a clearly defined shape and diameters ranging from 2 to 6 mm after 122 days of operation. The integrity coefficient (IC) and granules density was found to be 98% and 1,054 kg m(-3) respectively. After development of aerobic granules, o-nitrophenols were successfully degraded to an efficiency of 78% at a concentration of 70 mg L(-1). GC-MS study revealed that the biodegradation of o-nitrophenol occurred via catechol via nitrobenzene pathway. Specific o-nitrophenol biodegradation rates followed the Haldane model and the associated kinetic parameters were found as follows: V(max) = 3.96 g o-nitrophenol g(-1)VSS(-1)d(-1), K(s) = 198.12 mg L(-1), and K(i) = 31.16 mg L(-1). The aerobic granules proved to be a feasible and effective way to degrade o-nitrophenol containing wastewater.

Space Density Of Optically-Selected Type II Quasars From The SDSS

NASA Astrophysics Data System (ADS)

Reyes, Reinabelle; Zakamska, N. L.; Strauss, M. A.; Green, J.; Krolik, J. H.; Shen, Y.; Richards, G. T.

2007-12-01

Type II quasars are luminous Active Galactic Nuclei (AGN) whose central regions are obscured by large amounts of gas and dust. In this poster, we present a catalog of 887 type II quasars with redshifts z<0.83 from the Sloan Digital Sky Survey (SDSS), selected based on their emission lines, and derive the 1/Vmax [OIII] 5007 luminosity function from this sample. Since some objects may not be included in the sample because they lack strong emission lines, the derived luminosity function is only a lower limit. We also derive the [OIII] 5007 luminosity function for a sample of type I (broad-line) quasars in the same redshift range. Taking [OIII] 5007 luminosity as a tracer of intrinsic luminosity in both type I and type II quasars, we obtain lower limits to the type II quasar fraction as a function of [OIII] 5007 luminosity, from L[OIII] = 108.3 to 1010 Lsun, which roughly correspond to bolometric luminosities of 1044 to 1046 erg/s.

Unbiased estimates of galaxy scaling relations from photometric redshift surveys

NASA Astrophysics Data System (ADS)

Rossi, Graziano; Sheth, Ravi K.

2008-06-01

Many physical properties of galaxies correlate with one another, and these correlations are often used to constrain galaxy formation models. Such correlations include the colour-magnitude relation, the luminosity-size relation, the fundamental plane, etc. However, the transformation from observable (e.g. angular size, apparent brightness) to physical quantity (physical size, luminosity) is often distance dependent. Noise in the distance estimate will lead to biased estimates of these correlations, thus compromising the ability of photometric redshift surveys to constrain galaxy formation models. We describe two methods which can remove this bias. One is a generalization of the Vmax method, and the other is a maximum-likelihood approach. We illustrate their effectiveness by studying the size-luminosity relation in a mock catalogue, although both methods can be applied to other scaling relations as well. We show that if one simply uses photometric redshifts one obtains a biased relation; our methods correct for this bias and recover the true relation.

Probing the mechanism of proton coupled electron transfer to dioxygen: the oxidative half-reaction of bovine serum amine oxidase.

PubMed

Su, Q; Klinman, J P

1998-09-08

Bovine serum amine oxidase (BSAO) catalyzes the oxidative deamination of primary amines, concomitant with the reduction of molecular oxygen to hydrogen peroxide via a ping-pong mechanism. A protocol has been developed for an analysis of chemical and kinetic mechanisms in the conversion of dioxygen to hydrogen peroxide. Steady-state kinetics show that two groups need to be deprotonated to facilitate the oxidative half-reaction. The pH dependence of Vmax/Km(O2) reveals pKa's of 6.2 +/- 0.3 and 7.0 +/- 0.2, respectively. A pKa of 7.2 +/- 0.1 has been obtained from a titration of anaerobically reduced BSAO using UV-vis spectrophotometry. The near identity of the pKa obtained from the reduced enzyme titration with the second pKa from steady-state kinetics suggests that this second pKa arises from the reduced cofactor. The assignment of pKa is supported by the observed pH dependence for formation of the cofactor semiquinone signal, detected by EPR spectroscopy under anaerobic conditions. To address the nature of rate-limiting steps in the oxidative half-reaction, the solvent isotope effect, viscosity effect, and O-18 isotope effect on Vmax/Km(O2) have been determined. The solvent isotope effect is indistinguishable from unity, ruling out a proton transfer as a rate-determining step. Use of glucose as a solvent viscosogen shows no viscosity effect, indicating that binding of oxygen is not in the rate-determining step. The O-18 kinetic isotope effect is independent of pH with an average value of 18(V/K) = 1.0097 +/- 0. 0010. This has been compared to calculated equilibrium O-18 isotope effects for various dioxygen intermediate species [Tian and Klinman (1993) J. Am. Chem. Soc. 115, 8891], leading to the conclusion that either the first electron transfer to dioxygen or the desorption of product peroxide from a Cu(II)-OOH complex could be the rate-limiting step. The distribution of steady-state enzyme species was, therefore, analyzed through a combination of stopped-flow experiments and analysis of DV and D(V/K) for benzylamine oxidation. We conclude that the major species accumulating in the steady state are the oxidized cofactor-substrate Schiff base complex and the reduced, aminoquinol form of cofactor. These data rule out a slow release of product hydroperoxide from the aminoquinone form of enzyme, leading to the conclusion that the first electron transfer from substrate-reduced cofactor to dioxygen is the rate-determining step in the oxidative half-reaction. This step is also estimated to be 40% rate-limiting in kcat. An important mechanistic conclusion from this study is that dioxygen binding is a separate step from the rate-limiting electron-transfer step to form superoxide. On the basis of a recently determined X-ray structure for the active form of a yeast amine oxidase from Hansenula polymorpha [Li et al. (1998) Structure 6, 293], a hydrophobic space has been identified near the O-2 position of reduced cofactor as the putative dioxygen binding site. Movement of superoxide from this site onto the Cu(II) at the active site may occur prior to further electron transfer from cofactor to superoxide.

Inhibition of Baicalin on Metabolism of Phenacetin, a Probe of CYP1A2, in Human Liver Microsomes and in Rats

PubMed Central

Gao, Na; Qi, Bing; Liu, Fang-jun; Fang, Yan; Zhou, Jun; Jia, Lin-jing; Qiao, Hai-ling

2014-01-01

Baicalin has been used as mainly bioactive constituent of about 100 kinds of traditional Chinese medicines in Chinese pharmacopoeia. The effect of baicalin on cytochrome P450 should be paid more attention because baicalin was used widely. The aim of this study was to investigate whether baicalin could inhibit CYP1A2 in pooled human liver microsomes (HLMs) and in rats in vivo and the gene polymorphisms could affect inter-individual variation in IC50 in 28 human livers. Phenacetin was used as probe of CYP1A2. Kinetic parameter of CYP1A2 and IC50 of baicalin on CYP1A2 to each sample were measured and the common CYP1A2 polymorphisms (−3860G>A and −163C>A) were genotyped. The results showed that baicalin exhibited a mixed-type inhibition in pooled HLMs, with a Ki value of 25.4 µM. There was substantial variation in Km, Vmax, CLint of CYP1A2 and IC50 of baicalin on CYP1A2 (3∼10-fold). The range was from 26.6 to 114.8 µM for Km, from 333 to 1330 pmol·min−1·mg−1protein for Vmax and from 3.8 to 45.3 µL·min−1·mg−1 protein for CLint in HLMs (n = 28). The Mean (range) value of IC50 in 28 HLMs was 36.3 (18.9 to 56.1) µM. The genotypes of −3860G>A and −163C>A had no significant effect on the inhibition of baicalin on CYP1A2. The animal experiment results showed that baicalin (450 mg/kg, i.v.) significantly decreased the Cmax and CL of phenacetin, and increased C60 min, t1/2, Vd and AUC (P<0.05). There were significant correlations between percentage of control in C60 min, t1/2, CL, AUC of phenacetin and Cmax of baicalin in 11 rats (P<0.05). Protein binding experiments in vitro showed that baicalin (0–2000 mg/L) increased the unbound phenacetin from 14.5% to 28.3%. In conclusion, baicalin can inhibit the activity of CYP1A2 in HLMs and exhibit large inter-individual variation that has no relationship with gene polymorphism. Baicalin can change the pharmacokinetics of phenacetin in rats. PMID:24587011

Estimating Tropical Cyclone Surface Wind Field Parameters with the CYGNSS Constellation

NASA Astrophysics Data System (ADS)

Morris, M.; Ruf, C. S.

2016-12-01

A variety of parameters can be used to describe the wind field of a tropical cyclone (TC). Of particular interest to the TC forecasting and research community are the maximum sustained wind speed (VMAX), radius of maximum wind (RMW), 34-, 50-, and 64-kt wind radii, and integrated kinetic energy (IKE). The RMW is the distance separating the storm center and the VMAX position. IKE integrates the square of surface wind speed over the entire storm. These wind field parameters can be estimated from observations made by the Cyclone Global Navigation Satellite System (CYGNSS) constellation. The CYGNSS constellation consists of eight small satellites in a 35-degree inclination circular orbit. These satellites will be operating in standard science mode by the 2017 Atlantic TC season. CYGNSS will provide estimates of ocean surface wind speed under all precipitating conditions with high temporal and spatial sampling in the tropics. TC wind field data products can be derived from the level-2 CYGNSS wind speed product. CYGNSS-based TC wind field science data products are developed and tested in this paper. Performance of these products is validated using a mission simulator prelaunch.

Mechanisms and time course of impaired skeletal muscle glucose transport activity in streptozocin diabetic rats.

PubMed Central

Napoli, R; Hirshman, M F; Horton, E S

1995-01-01

Skeletal muscle glucose transport is altered in diabetes in humans, as well as in rats. To investigate the mechanisms of this abnormality, we measured glucose transport Vmax, the total transporter number, their average intrinsic activity, GLUT4 and GLUT1 contents in skeletal muscle plasma membrane vesicles from basal or insulin-stimulated streptozocin diabetic rats with different duration of diabetes, treated or not with phlorizin. The glucose transport Vmax progressively decreased with the duration of diabetes. In the basal state, this decrease was primarily associated with the reduction of transporter intrinsic activity, which appeared earlier than any change in transporter number or GLUT4 and GLUT1 content. In the insulin-stimulated state, the decrease of transport was mainly associated with severe defects in transporter translocation. Phlorizin treatment partially increased the insulin-stimulated glucose transport by improving the transporter translocation defects. In conclusion, in streptozocin diabetes (a) reduction of intrinsic activity plays a major and early role in the impairment of basal glucose transport; (b) a defect in transporter translocation is the mechanism responsible for the decrease in insulin-stimulated glucose transport; and (c) hyperglycemia per se affects the insulin-stimulated glucose transport by altering the transporter translocation. PMID:7615815

A mutation Ser213/Asn in the hexokinase 1 from Schizosaccharomyces pombe increases its affinity for glucose.

PubMed

Petit, T; Herrero, P; Gancedo, C

1998-10-29

Alignment of amino acids of the region implicated in glucose binding from a series of hexokinases showed that Schizosaccharomyces pombe hexokinase 1 had a Ser residue in a place where all other kinases had an Asn. We changed an AGT codon to AAT to place an Asn in the Ser213 position. This mutation decreased Km for glucose from 9.4 mM to 1.6 mM and the ratio Vmax (Fructose)/Vmax (Glucose) from 5 to 2.5. Also the Km for 2-deoxyglucose decreased from 2.7 mM to 0.8 mM. A mutation in the similar position of S. pombe hexokinase 2 (Asn196/Ser) increased the Km for glucose from 0.16 mM to 0.56 mM. Fermentation of glucose is not detectable in a S. pombe mutant with only hexokinase 1 activity but expression of the hxk1(S213/N) gene conferred ability to ferment the sugar. While the mutated hexokinase 1 partially mimicked S. cerevisiae hexokinase II in catabolite repression of invertase, the wild type one could not substitute for it. Copyright 1998 Academic Press.

Cloning and characterization of a Weissella confusa dextransucrase and its application in high fibre baking.

PubMed

Kajala, Ilkka; Shi, Qiao; Nyyssölä, Antti; Maina, Ndegwa Henry; Hou, Yaxi; Katina, Kati; Tenkanen, Maija; Juvonen, Riikka

2015-01-01

Wheat bran offers health benefits as a baking ingredient, but is detrimental to bread textural quality. Dextran production by microbial fermentation improves sourdough bread volume and freshness, but extensive acid production during fermentation may negate this effect. Enzymatic production of dextran in wheat bran was tested to determine if dextran-containing bran could be used in baking without disrupting bread texture. The Weissella confusa VTT E-90392 dextransucrase gene was sequenced and His-tagged dextransucrase Wc392-rDSR was produced in Lactococcus lactis. Purified enzyme was characterized using (14)C-sucrose radioisotope and reducing value-based assays, the former yielding K(m) and V(max) values of 14.7 mM and 8.2 μmol/(mg ∙ min), respectively, at the pH optimum of 5.4. The structure and size of in vitro dextran product was similar to dextran produced in vivo. Dextran (8.1% dry weight) was produced in wheat bran in 6 h using Wc392-rDSR. Bran with and without dextran was used in wheat baking at 20% supplementation level. Dextran presence improved bread softness and neutralized bran-induced volume loss, clearly demonstrating the potential of using dextransucrases in bran bioprocessing for use in baking.

Molecular characterization of Streptomyces coelicolor A(3) SCO6548 as a cellulose 1,4-β-cellobiosidase.

PubMed

Lim, Ju-Hyeon; Lee, Chang-Ro; Dhakshnamoorthy, Vijayalakshmi; Park, Jae Seon; Hong, Soon-Kwang

2016-02-01

Genomic sequencing analysis and previous studies have shown that there are eight genes in Streptomyces coelicolor A3(2) encoding putative cellulases. One of these genes, sco6548, was cloned into the Streptomyces/Escherichia coli shuttle vector pUWL201PW. The recombinant protein was successfully overexpressed in S. lividans TK24 under the control of the strong ermE promoter. Sco6548 was 1740 bp in length, and encoded a 579-amino acid-, 60.8-kDa protein with strong hydrolyzing activity toward Avicel and filter paper, yielding cellobiose as the final product. SCO6548 showed optimal activity at 50°C and pH 5. The Km values of SCO6548 toward Avicel and filter paper were 15.38 and 16.1 mg/mL, respectively. The Vmax values toward Avicel and filter paper were 0.432 and 0.084 μM/min, respectively. EDTA did not affect cellulase activity; however, several divalent cations, including Co(2+), Cu(2+), Ni(2+) and Mn(2+) (at 10 mM) had severe inhibitory effects on enzyme activity. Our analysis showed that SCO6548 is a cellulose 1,4-β-cellobiosidase that hydrolyzes cellulose into cellobiose. © FEMS 2015. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Nucleoside pyrophosphatase activity associated with pig kidney alkaline phosphatase

PubMed Central

Wass, Milica; Butterworth, P. J.

1971-01-01

1. A study was made of the hydrolysis, at pH9.0, of ATP and ADP catalysed by pig kidney alkaline phosphatase. Both of these nucleoside pyrophosphates are substrates for the enzyme; Km values are 4×10−5m for ATP and 6.3×10−5m for ADP. Vmax. for ADP is approximately double that of ATP. 2. Above 0.1mm approximately, both ATP and ADP are inhibitory, but the inhibition is reversible by the addition of Mg2+ ions to form MgATP2− or MgADP− complexes. The complexes, besides being non-inhibitory, are also substrates for the enzyme with Km values identical with those of the respective free nucleotides. 3. Mg2+ ions are inhibitory when present in excess of ATP or ADP. The degree of inhibition is greater with ATP as substrate, but with both ATP and ADP a mixed competitive–non-competitive type of inhibition is observed. 4. It is suggested that under normal conditions the enzyme is inhibited by cellular concentrations of ATP plus ADP but that an increase in the concentration of Mg2+ ions stimulates activity by relieving nucleoside pyrophosphate inhibition. The properties may be of importance in the regulation of the transport of bivalent cations. PMID:4331861

Cyanide-insensitive quinol oxidase (CIO) from Gluconobacter oxydans is a unique terminal oxidase subfamily of cytochrome bd.

PubMed

Miura, Hiroshi; Mogi, Tatsushi; Ano, Yoshitaka; Migita, Catharina T; Matsutani, Minenosuke; Yakushi, Toshiharu; Kita, Kiyoshi; Matsushita, Kazunobu

2013-06-01

Cyanide-insensitive terminal quinol oxidase (CIO) is a subfamily of cytochrome bd present in bacterial respiratory chain. We purified CIO from the Gluconobacter oxydans membranes and characterized its properties. The air-oxidized CIO showed some or weak peaks of reduced haemes b and of oxygenated and ferric haeme d, differing from cytochrome bd. CO- and NO-binding difference spectra suggested that haeme d serves as the ligand-binding site of CIO. Notably, the purified CIO showed an extraordinary high ubiquinol-1 oxidase activity with the pH optimum of pH 5-6. The apparent Vmax value of CIO was 17-fold higher than that of G. oxydans cytochrome bo3. In addition, compared with Escherichia coli cytochrome bd, the quinol oxidase activity of CIO was much more resistant to cyanide, but sensitive to azide. The Km value for O2 of CIO was 7- to 10-fold larger than that of G. oxydans cytochrome bo3 or E. coli cytochrome bd. Our results suggest that CIO has unique features attributable to the structure and properties of the O2-binding site, and thus forms a new sub-group distinct from cytochrome bd. Furthermore, CIO of acetic acid bacteria may play some specific role for rapid oxidation of substrates under acidic growth conditions.

Relationship between CHA2DS2-VASc score and atrial electromechanical function in patients with paroxysmal atrial fibrillation: A pilot study.

PubMed

Vatan, Mehmet Bülent; Yılmaz, Sabiye; Ağaç, Mustafa Tarık; Çakar, Mehmet Akif; Erkan, Hakan; Aksoy, Murat; Demirtas, Saadet; Varım, Ceyhun; Akdemir, Ramazan; Gündüz, Hüseyin

2015-11-01

CHA2DS2-VASc score is the most widely preferred method for prediction of stroke risk in patients with atrial fibrillation. We hypothesized that CHA2DS2-VASc score may represent atrial remodeling status, and therefore echocardiographic evaluation of left atrial electromechanical remodeling can be used to identify patients with high risk. A total of 65 patients who had documented diagnosis of paroxysmal atrial fibrillation (PAF) were divided into three risk groups according to the CHA2DS2-VASc score: patients with low risk (score=0, group 1), with moderate risk (score=1, group 2), and with high risk score (score ≥2, group 3). We compared groups according to atrial electromechanical intervals and left atrium mechanical functions. Atrial electromechanical intervals including inter-atrial and intra-atrial electromechanical delay were not different between groups. However, parameters reflecting atrial mechanical functions including LA phasic volumes (Vmax, Vmin and Vp) were significantly higher in groups 2 and 3 compared with group 1. Likewise, LA passive emptying volume (LATEV) in the groups 2 and 3 was significantly higher than low-risk group (14.12±8.13ml/m(2), 22.36±8.78ml/m(2), 22.89±7.23ml/m(2), p: 0.031). Univariate analysis demonstrated that Vmax, Vmin and Vp were significantly correlated with CHA2DS2-VASc score (r=0.428, r=0.456, r=0.451 and p<0.001). Also, LATEV (r=0.397, p=0.016) and LA active emptying volume (LAAEV) (r=0.281, p=0.023) were positively correlated with CHA2DS2-VASc score. In the ROC analysis, Vmin≥11ml/m(2) has the highest predictive value for CHA2DS2-VASc score ≥2 (88% sensitivity and 89% specificity; ROC area 0.88, p<0.001, CI [0.76-0.99]). Echocardiographic evaluation of left atrial electromechanical function might represent a useful method to identify patients with high risk. Copyright © 2015 Japanese College of Cardiology. Published by Elsevier Ltd. All rights reserved.

Evaluation of NT-proBNP concentrations during exercise in asymptomatic patients with severe high-gradient aortic stenosis.

PubMed

Dobrowolski, Piotr; Lech, Agnieszka; Klisiewicz, Anna; Hoffman, Piotr

2016-08-11

INTRODUCTION The effect of asymptomatic severe aortic stenosis (ASAS) on N-terminal pro-B-type natriuretic peptide (NT-proBNP) levels ar rest and during exercise, as well as their relevance for clinical practice remain controversial.  OBJECTIVES The aim of this study was to test the hypothesis of whether the evaluation of NT-proBNP concentrations during exercise provides additional information about the severity of aortic stenosis and left ventricular remodeling in patients with ASAS. PATIENTS AND METHODS A total of 50 patients with ASAS (mean age, 38.4 ±18.1 years) and 21 healthy subjects (mean age, 43.4 ±10.6 years) were enrolled. Rest and exercise echocardiography was performed to evaluate maximum velocity (Vmax), mean aortic gradient (AG), and aortic valve area (AVA). The left ventricular mass index (LVMI) was calculated. NT-proBNP concentrations at rest and during exercise were assessed, and the difference between the 2 values was calculated (ΔNT-proBNP). RESULTS NT-proBNP and ΔNT-proBNP levels at rest and during exercise were significantly higher in the ASAS group compared with the control group. In the ASAS group, NT-proBNP levels at rest significantly correlated with LVMI (r = 0.432; P <0.0001), AVA (r = -0.408; P <0.0001), Vmax (r = 0.375; P = 0.002), and mean AG (r = 0.257; P = 0.03). NT-proBNP levels during exercise significantly correlated with LVMI (r = 0.432; P <0.0001), mean AG (r = 0.401; P = 0.001), and AVA (r = -0.375; P = 0.001). In the multivariate logistic regression model, the factors independently associated with NT-proBNP both at rest and during exercise were age, AVA, and LVMI. CONCLUSIONS NT-proBNP levels at rest provide valuable information for identifying patients with more advanced left ventricular hypertrophy secondary to severe aortic stenosis. NT-proBNP levels during exercise do not provide new information on the severity of AS.

Comparison of biochemical properties of liver arginase from streptozocin-induced diabetic and control mice.

PubMed

Spolarics, Z; Bond, J S

1989-11-01

Arginase activity is elevated in livers of diabetic animals compared to controls and there is evidence that this is due in part to increased specific activity (activity/mg arginase protein). To investigate the molecular basis of this increased activity, the physicochemical and kinetic properties of hepatic arginase from diabetic and control mice were compared. Two types of arginase subunits with molecular weights of 35,000 and 38,000 were found in both the diabetic and control animals and the subunits in these animals had similar, multiple ionic forms. Kinetic parameters of purified preparations of arginase for arginine (apparent Km and Vmax values) and the thermal stability of these preparations from diabetics and controls were also similar. Furthermore, no difference was found in the distribution of arginase activity among different subcellular liver fractions. Separation of basic and acidic oligomeric forms of arginase by fast-protein liquid chromatography resulted in a slightly different distribution of activity among the forms in the normal and diabetic group. The apparent Km values for Mn2+ of the basic form of the enzyme were 25 and 33 microM for the enzyme from normal and diabetic animals, respectively; for acidic forms, for which two apparent Km values were measured, the values were 8 and 197 microM for arginase from controls and 35 and 537 microM from diabetics. These results indicate that in diabetes, while no marked changes in the physicochemical characteristics of arginase are obvious, some changes are found in the interaction of arginase with its cofactor Mn.

A Quantitative Description of Suicide Inhibition of Dichloroacetic Acid in Rats and Mice

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

Keys, Deborah A.; Schultz, Irv R.; Mahle, Deirdre A.

Dichloroacetic acid (DCA), a minor metabolite of trichloroethylene (TCE) and water disinfection byproduct, remains an important risk assessment issue because of its carcinogenic potency. DCA has been shown to inhibit its own metabolism by irreversibly inactivating glutathione transferase zeta (GSTzeta). To better predict internal dosimetry of DCA, a physiologically based pharmacokinetic (PBPK) model of DCA was developed. Suicide inhibition was described dynamically by varying the rate of maximal GSTzeta mediated metabolism of DCA (Vmax) over time. Resynthesis (zero-order) and degradation (first-order) of metabolic activity were described. Published iv pharmacokinetic studies in native rats were used to estimate an initial Vmaxmore » value, with Km set to an in vitro determined value. Degradation and resynthesis rates were set to estimated values from a published immunoreactive GSTzeta protein time course. The first-order inhibition rate, kd, was estimated to this same time course. A secondary, linear non-GSTzeta-mediated metabolic pathway is proposed to fit DCA time courses following treatment with DCA in drinking water. The PBPK model predictions were validated by comparing predicted DCA concentrations to measured concentrations in published studies of rats pretreated with DCA following iv exposure to 0.05 to 20 mg/kg DCA. The same model structure was parameterized to simulate DCA time courses following iv exposure in native and pretreated mice. Blood and liver concentrations during and postexposure to DCA in drinking water were predicted. Comparisons of PBPK model predicted to measured values were favorable, lending support for the further development of this model for application to DCA or TCE human health risk assessment.« less

Clarithromycin accumulation by phagocytes and its effect on killing of Aggregatibacter actinomycetemcomitans.

PubMed

Iskandar, Irma; Walters, John D

2011-03-01

Clarithromycin inhibits several periodontal pathogens and is concentrated inside gingival fibroblasts and epithelial cells by an active transporter. We hypothesized that polymorphonuclear leukocytes (PMNs) and less mature myeloid cells possess a similar transporter for clarithromycin. It is feasible that clarithromycin accumulation inside PMNs could enhance their ability to kill Aggregatibacter actinomycetemcomitans (previously Actinobacillus actinomycetemcomitans). To test the first hypothesis, purified PMNs and cultured HL-60 cells were incubated with [(3)H]-clarithromycin. Clarithromycin transport was assayed by measuring changes in cell-associated radioactivity over time. The second hypothesis was examined with PMNs loaded by incubation with clarithromycin (5 μg/ml). Opsonized bacteria were incubated at 37°C with control and clarithromycin-loaded PMNs. Mature human PMNs, HL-60 cells differentiated into granulocytes, and undifferentiated HL-60 cells all took up clarithromycin in a saturable manner. The kinetics of uptake by all yielded linear Lineweaver-Burk plots. HL-60 granulocytes transported clarithromycin with a K(m) of ≈250 μg/ml and a V(max) of 473 ng/min/10(6) cells, which were not significantly different from the values obtained with PMNs. At steady state, clarithromycin levels inside HL-60 granulocytes and PMNs were 28- to 71-fold higher than extracellular levels. Clarithromycin-loaded PMNs killed significantly more A. actinomycetemcomitans and achieved shorter half-times for killing than control PMNs when assayed at a bacteria-to-PMN ratio of 100:1 (P <0.04). At a ratio of 30:1, these differences were not consistently significant. PMNs and less mature myeloid cells possess a transporter that takes up and concentrates clarithromycin. This system could help PMNs cope with an overwhelming infection by A. actinomycetemcomitans.

Kinetic characterization, optimum conditions for catalysis and substrate preference of secretory phospholipase A2 from Glycine max in model membrane systems.

PubMed

Mariani, María Elisa; Madoery, Ricardo Román; Fidelio, Gerardo Daniel

2015-01-01

Two secretory phospholipase A2 (sPLA2s) from Glycine max, GmsPLA2-IXA-1 and GmsPLA2-XIB-2, have been purified as recombinant proteins and the activity was evaluated in order to obtain the optimum conditions for catalysis using mixed micelles and lipid monolayers as substrate. Both sPLA2s showed a maximum enzyme activity at pH 7 and a requirement of Ca(2+) in the micromolar range. These parameters were similar to those found for animal sPLA2s but a surprising optimum temperature for catalysis at 60 °C was observed. The effect of negative interfacial charges on the hydrolysis of organized substrates was evaluated through initial rate measurements using short chain phospholipids with different head groups. The enzymes showed subtle differences in the specificity for phospholipids with different head groups (DLPC, DLPG, DLPE, DLPA) in presence or absence of NaCl. Both recombinant enzymes showed lower activity toward anionic phospholipids and a preference for the zwitterionic ones. The values of the apparent kinetic parameters (Vmax and KM) demonstrated that these enzymes have more affinity for phosphatidylcholine compared with phosphatidylglycerol, in contrast with the results observed for pancreatic sPLA2. A hopping mode of catalysis was proposed for the action of these sPLA2 on mixed phospholipid/triton micelles. On the other hand, Langmuir-monolayers assays indicated an optimum lateral surface pressure for activity in between 13 and 16 mN/m for both recombinant enzymes. Copyright © 2014 Elsevier B.V. and Société française de biochimie et biologie Moléculaire (SFBBM). All rights reserved.

Removal of cyanide by woody plants.

PubMed

Larsen, Morten; Trapp, Stefan; Pirandello, Alessandro

2004-01-01

Hydrogen cyanide is a high volume production chemical that causes severe environmental problems. The toxicity of potassium cyanide (KCN) to basket willow trees (Salix viminalis) was tested. In aqueous solution, 2 mg CN l(-1) as KCN depressed the transpiration after 72 h about 50%. Trees exposed to 0.4 mg CN l(-1) in aqueous solution showed initially a depression of transpiration, but recovered. Doses of 8 and 20 mg CN l(-1) in aqueous solution were quickly mortal to the trees. At the end of the test, almost all cyanide had disappeared from the solutions. Levels of cyanide in plants were related to the toxicity, with no elevated levels of cyanide in plants exposed to 0.4 mg CN l(-1). Willows grown in sand survived 423.5 h irrigation with 20 mg CN l(-1). Willows grown in sand irrigated with 50 mg CN l(-1) died within a few days. The roots of the surviving willows were able to consume about 10 mg CN kg fresh weight(-1)h(-1). Vascular plants possess the enzymes beta-cyanoalanine synthase and beta-cyanoalanine hydrolase, which convert free cyanide to the amino acid asparagine. The in vivo capacity of woody plants (willow, poplar, elder, rose, birch) to remove cyanide was evaluated. Tests were performed with detached leaves and roots in KCN solutions of different concentrations. The highest removal capacity was obtained for basket willow hybrids (Salix viminalis x schwerinii). The Michaelis-Menten kinetics was determined. Realistic values of the half-saturation constant, K(M), were between 0.6 and 1.7 mg CN l(-1); the maximum metabolic capacity, v(max), was around 9.3 mg CN kg fresh weight(-1)h(-1). The removal of cyanide by plants might be useful in phytoremediation and treatment of wastewater from gold mining.

Toward Enhancing the Enzymatic Activity of a Novel Fungal Polygalacturonase for Food Industry: Optimization and Biochemical Analyses.

PubMed

Shetaia, Yousseria M H; El-Baz, Ashraf F; ElMekawy, Ahmed M

2017-08-11

The review of literature and patents shows that enhancing the PG production and activity are still required to fulfill the increasing demands. A dual optimization process, which involved Plackett-Burman design (PBD), with seven factors, and response surface methodology, was applied to optimize the production of extracellular polygalacturonase (PG) enzyme produced by a novel strain of Aspergillus flavus isolated from rotten orange fruit. The fungal PG was purified and biochemically characterized. Three variables (harvesting time, pH and orange pomace concentration), that were verified to be significant by the PBD analysis, were comprehensively optimized via Box-Behnken design. According to this optimization, the highest PG activity (4073 U/mL) was obtained under pH 7 after 48 h using 40 g/L orange pomace as a substrate, with enhancement in PG activity by 51% compared to the first PBD optimization step. The specific activity of the purified PG was 1608 U/mg with polygalacturonic acid and its molecular weight was 55 kDa. The optimum pH was 5 with relative thermal stability (80%) at 50˚C after 30 min. The PG activity improved in the presence of Cu2+ and Ca2+, while Ba2+, Fe2+ and Zn2+ greatly inhibited the enzyme activity. The obvious Km and Vmax values were 0.8 mg/mL and 2000 µmol/min, respectively. This study is a starting point for initial research in the field of optimization and characterization of A. flavus PG. The statistical optimization of A. flavus PG and its biochemical characterization clearly revealed that this fungal strain can be a potential producer of PG which has a wide range of industrial applications. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Monocarboxylate transporters, blood lactate removal after supramaximal exercise, and fatigue indexes in humans.

PubMed

Thomas, C; Perrey, S; Lambert, K; Hugon, G; Mornet, D; Mercier, J

2005-03-01

The present study investigated whether muscular monocarboxylate transporter (MCT) 1 and 4 contents are related to the blood lactate removal after supramaximal exercise, fatigue indexes measured during different supramaximal exercises, and muscle oxidative parameters in 15 humans with different training status. Lactate recovery curves were obtained after a 1-min all-out exercise. A biexponential time function was then used to determine the velocity constant of the slow phase (gamma(2)), which denoted the blood lactate removal ability. Fatigue indexes were calculated during 1-min all-out (FI(AO)) and repeated 10-s (FI(Sprint)) cycling sprints. Biopsies were taken from the vastus lateralis muscle. MCT1 and MCT4 contents were quantified by Western blots, and maximal muscle oxidative capacity (V(max)) was evaluated with pyruvate + malate and glutamate + malate as substrates. The results showed that the blood lactate removal ability (i.e., gamma(2)) after a 1-min all-out test was significantly related to MCT1 content (r = 0.70, P < 0.01) but not to MCT4 (r = 0.50, P > 0.05). However, greater MCT1 and MCT4 contents were negatively related with a reduction of blood lactate concentration at the end of 1-min all-out exercise (r = -0.56, and r = -0.61, P < 0.05, respectively). Among skeletal muscle oxidative indexes, we only found a relationship between MCT1 and glutamate + malate V(max) (r = 0.63, P < 0.05). Furthermore, MCT1 content, but not MCT4, was inversely related to FI(AO) (r = -0.54, P < 0.05) and FI(Sprint) (r = -0.58, P < 0.05). We concluded that skeletal muscle MCT1 expression was associated with the velocity constant of net blood lactate removal after a 1-min all-out test and with the fatigue indexes. It is proposed that MCT1 expression may be important for blood lactate removal after supramaximal exercise based on the existence of lactate shuttles and, in turn, in favor of a better tolerance to muscle fatigue.

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

Microbial respiration and kinetics of extracellular enzymes activities through rhizosphere and detritusphere at agricultural site

NASA Astrophysics Data System (ADS)

Löppmann, Sebastian; Blagodatskaya, Evgenia; Kuzyakov, Yakov

2014-05-01

Rhizosphere and detritusphere are soil microsites with very high resource availability for microorganisms affecting their biomass, composition and functions. In the rhizosphere low molecular compounds occur with root exudates and low available polymeric compounds, as belowground plant senescence. In detritusphere the substrate for decomposition is mainly a polymeric material of low availability. We hypothesized that microorganisms adapted to contrasting quality and availability of substrates in the rhizosphere and detritusphere are strongly different in affinity of hydrolytic enzymes responsible for decomposition of organic compounds. According to common ecological principles easily available substrates are quickly consumed by microorganisms with enzymes of low substrate affinity (i.e. r-strategists). The slow-growing K-strategists with enzymes of high substrate affinity are better adapted for growth on substrates of low availability. Estimation of affinity of enzyme systems to the substrate is based on Michaelis-Menten kinetics, reflecting the dependency of decomposition rates on substrate amount. As enzymes-mediated reactions are substrate-dependent, we further hypothesized that the largest differences in hydrolytic activity between the rhizosphere and detritusphere occur at substrate saturation and that these differences are smoothed with increasing limitation of substrate. Affected by substrate limitation, microbial species follow a certain adaptation strategy. To achieve different depth gradients of substrate availability 12 plots on an agricultural field were established in the north-west of Göttingen, Germany: 1) 4 plots planted with maize, reflecting lower substrate availability with depth; 2) 4 unplanted plots with maize litter input (0.8 kg m-2 dry maize residues), corresponding to detritusphere; 3) 4 bare fallow plots as control. Maize litter was grubbed homogenously into the soil at the first 5 cm to ensure comparable conditions for the herbivore and detritivore communities in the soil. The kinetics (Km and Vmax) of four extracellular hydrolytic enzymes responsible for C- and phosphorous-cycle (β-glucosidase, β-xylosidase, β-cellobiohydrolase and acid phosphatase), microbial biomass, basal respiration (BR) and substrate-induced respiration (SIR) were measured in rhizosphere, detritusphere and control from 0 - 10 and 10 - 20 cm. The metabolic quotient (qCO2) was calculated as specific indicator for efficiency of microbial substrate utilization. We observed clear differences in enzymes activities at low and high concentrations of substrate. At substrate saturation enzyme activity rates of were significantly higher in rooted plots compared to litter amended plots, whereas at lower concentration no treatment effect could be found. The BR, SIR and qCO2 values were significantly higher at 0 - 10 cm of the planted treatment compared to litter and control plots, revealing a significantly higher respiration at lower efficiency of microbial substrate utilization in the rhizosphere. The Michaelis-Menten constant (Km) decreased with depth, especially for β-glucosidase, acid phosphatase and β-xylosidase, indicating higher substrate affinity of microorganisms in deeper soil and therefore different enzyme systems functioning. The substrate affinity factor (Vmax/Km) increased 2-fold with depth for various enzymes, reflecting a switch of predominantly occurring microbial strategies. Vmax/Km ratio indicated relative domination of zymogenous microbial communities (r-strategists) in 0 - 10 cm depth as compared with 10 - 20 cm depth where the K-strategists dominated.

Inhibition of purified enolases from oral bacteria by fluoride.

PubMed

Guha-Chowdhury, N; Clark, A G; Sissons, C H

1997-04-01

Enolase activity in strains of oral streptococci previously has been found to be inhibited by 50% (Ki) by fluoride concentrations ranging from 50 to 300 microM or more in the presence of 0.5 to 1.0 mM inorganic phosphate ions. In this study, enolase was extracted and partly purified by a two-step process from five oral bacterial species and the effect of fluoride on the kinetics of enolase examined. The molecular weight of the putative enolase proteins was 46-48 kDa. The Vmax values ranged from 20 to 323 IU/mg and K(m) for glycerate-2-phosphate from 0.22 to 0.74 mM. Enolase activity was inhibited competitively by fluoride, with Ki values ranging from 16 to 54 microM in the presence of 5 mM inorganic phosphate ions. Ki values for phosphate ranged from 2 to 8 mM. The enolase from Streptococcus sanguis ATCC 10556 was more sensitive to fluoride (Ki = 16 +/- 2) than was enolase from Streptococcus salivarius ATCC 10575 (Ki = 19 +/- 2) or Streptococcus mutans NCTC 10449 (Ki = 40 +/- 4) and all three streptococcal strains were more sensitive to fluoride than either Actinomyces naeslundii WVU 627 (Ki = 46 +/- 6) or Lactobacillus rhamnosus ATCC 7469 (Ki = 54 +/- 6) enolases. The levels of fluoride found to inhibit the streptococcal enolases in this study are much lower than previously reported and are likely to be present in plaque, especially during acidogenesis, and could exert an anti-glycolytic effect.

Nitrification exhibits Haldane kinetics in an agricultural soil treated with ammonium sulfate or dairy-waste compost.

PubMed

Koper, Teresa E; Stark, John M; Habteselassie, Mussie Y; Norton, Jeanette M

2010-11-01

An agricultural soil was treated with dairy-waste compost, ammonium-sulfate fertilizer or no added nitrogen (control) and planted to silage corn for 6 years. The kinetics of nitrification were determined in laboratory-shaken slurry assays with a range of substrate concentrations (0-20 mM NH(4)(+)) over a 24-h period for soils from the three treatments. Determined concentrations of substrate and product were fit to Michaelis-Menten and Haldane models. For all the treatments, the Haldane model was a better fit, suggesting that significant nitrification inhibition may occur in soils under high ammonium conditions similar to those found immediately after fertilization or waste applications. The maximum rate of nitrification (V(max)) was significantly higher for the fertilized and compost-treated soils (1.74 and 1.50 mmol N kg(-1) soil day(-1)) vs. control soil (0.98 mmol kg(-1) soil day(-1)). The K(m) and K(i) values were not significantly different, with average values of 0.02 and 27 mM NH(4)(+), respectively. Our results suggest that both N sources increased nitrifier community size, but did not shift the nitrifier community structure in ways that influenced enzyme affinity or sensitivity to ammonium. The K(m) values are comparable to those determined directly in other soils, but are substantially lower than those from most pure cultures of ammonia-oxidizing bacteria. © 2010 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. No claim to original US government works.

Polymeric amylase nanoparticles as a new semi-synthetic enzyme system for hydrolysis of starch.

PubMed

Say, R; Şenay, R Hilal; Biçen, Özlem; Ersöz, Arzu; Şişman Yılmaz, Filiz; Akgöl, Sinan; Denizli, Adil

2013-05-01

α-Amylase (EC 3.2.1.1; α-D-1,4,glucan glucanohydrolase) catalyzes the hydrolysis of α-D-(1,4)-glucosidic linkages in starch, glycogen, and various malto-oligosaccharides, by releasing α-anomeric products. In this study, a novel method has been developed to prepare nanoprotein particles that carry α-amylase as a monomer by using a photosensitive microemulsion polymerization process. The nanostructured α-amylase with photosensitive features have been characterized by fluorescence spectroscopy, transmission electron microscopy (TEM) and Zeta Sizer. The fluorescence intensity of amylase nanoparticles was determined to be 658 a.u. at 610 nm and the average particle size of nanoamylase was found to be about 71.8 nm. Both free α-amylase and nanoparticles were used in the hydrolysis of starch under varying reaction conditions such as pH and temperature that affect enzyme activity and the results were compared to each other. Km values were 0.26 and 0.87 mM and Vmax values were 0.36 IU mg(-1) and 22.32 IU mg(-1) for nanoenzyme and free enzyme, respectively. Then, thermal stability, storage stability and reusability were investigated and according to the results, activity was preserved 60% at 60 °C; 20% at 70-80 °C temperature values and 80% after 105 days storage. Finally after 10 cycles, the activity was preserved 90% and this novel enzymatic polymeric amylase nanoparticle has showed considerable potential as reusable catalyst. Copyright © 2012 Elsevier B.V. All rights reserved.

Assessment of microbial methane oxidation above a petroleum-contaminated aquifer using a combination of in situ techniques

NASA Astrophysics Data System (ADS)

Urmann, Karina; Schroth, Martin H.; Noll, Matthias; Gonzalez-Gil, Graciela; Zeyer, Josef

2008-06-01

Emissions of the greenhouse gas CH4, which is often produced in contaminated aquifers, are reduced or eliminated by microbial CH4 oxidation in the overlying vadose zone. The aim of this field study was to estimate kinetic parameters and isotope fractionation factors for CH4 oxidation in situ in the vadose zone above a methanogenic aquifer in Studen, Switzerland, and to characterize the involved methanotrophic communities. To quantify kinetic parameters, several field tests, so-called gas push-pull tests (GPPTs), with CH4 injection concentrations ranging from 17 to 80 mL L-1 were performed. An apparent Vmax of 0.70 ± 0.15 mmol CH4 (L soil air)-1 h-1 and an apparent Km of 0.28 ± 0.09 mmol CH4 (L soil air)-1 was estimated for CH4 oxidation at 2.7 m depth, close to the groundwater table. At 1.1 m depth, Km (0.13 ± 0.02 mmol CH4 (L soil air)-1) was in a similar range, but Vmax (0.076 ± 0.006 mmol CH4 (L soil air)-1 h-1) was an order of magnitude lower. At 2.7 m, apparent first-order rate constants determined from a CH4 gas profile (1.9 h-1) and from a single GPPT (2.0 ± 0.03 h-1) were in good agreement. Above the groundwater table, a Vmax much higher than the in situ CH4 oxidation rate prior to GPPTs indicated a high buffer capacity for CH4. At both depths, known methanotrophic species affiliated with Methylosarcina and Methylocystis were detected by cloning and sequencing. Apparent stable carbon isotope fractionation factors α for CH4 oxidation determined during GPPTs ranged from 1.006 to 1.032. Variability was likely due to differences in methanotrophic activity and CH4 availability leading to different degrees of mass transfer limitation. This complicates the use of stable isotopes as an independent quantification method.

Thermodynamics of gas and steam-blast eruptions

USGS Publications Warehouse

Mastin, L.G.

1995-01-01

Eruptions of gas or steam and non-juvenile debris are common in volcanic and hydrothermal areas. From reports of non-juvenile eruptions or eruptive sequences world-wide, at least three types (or end-members) can be identified: (1) those involving rock and liquid water initially at boiling-point temperatures ('boiling-point eruptions'); (2) those powered by gas (primarily water vapor) at initial temperatures approaching magmatic ('gas eruptions'); and (3) those caused by rapid mixing of hot rock and ground- or surface water ('mixing eruptions'). For these eruption types, the mechanical energy released, final temperatures, liquid water contents and maximum theoretical velocities are compared by assuming that the erupting mixtures of rock and fluid thermally equilibrate, then decompress isentropically from initial, near-surface pressure (???10 MPa) to atmospheric pressure. Maximum mechanical energy release is by far greatest for gas eruptions (??????1.3 MJ/kg of fluid-rock mixture)-about one-half that of an equivalent mass of gunpowder and one-fourth that of TNT. It is somewhat less for mixing eruptions (??????0.4 MJ/kg), and least for boiling-point eruptions (??????0.25 MJ/kg). The final water contents of crupted boiling-point mixtures are usually high, producing wet, sloppy deposits. Final erupted mixtures from gas eruptions are nearly always dry, whereas those from mixing eruptions vary from wet to dry. If all the enthalpy released in the eruptions were converted to kinetic energy, the final velocity (vmax) of these mixtures could range up to 670 m/s for boiling-point eruptions and 1820 m/s for gas eruptions (highest for high initial pressure and mass fractions of rock (mr) near zero). For mixing eruptions, vmax ranges up to 1150 m/s. All observed eruption velocities are less than 400 m/s, largely because (1) most solid material is expelled when mr is high, hence vmax is low; (2) observations are made of large blocks the velocities of which may be less than the average for the mixture; (3) heat from solid particles is not efficiently transferred to the fluid during the eruptions; and (4) maximum velocities are reduced by choked flow or friction in the conduit. ?? 1995 Springer-Verlag.

N-Acetyl-2-Aminofluorene (AAF) Processing in Adult Rat Hepatocytes in Primary Culture Occurs by High-Affinity Low-Velocity and Low-Affinity High-Velocity AAF Metabolite-Forming Systems.

PubMed

Koch, Katherine S; Moran, Tom; Shier, W Thomas; Leffert, Hyam L

2018-05-01

N-acetyl-2-aminofluorene (AAF) is a procarcinogen used widely in physiological investigations of chemical hepatocarcinogenesis. Its metabolic pathways have been described extensively, yet little is known about its biochemical processing, growth cycle expression, and pharmacological properties inside living hepatocytes-the principal cellular targets of this hepatocarcinogen. In this report, primary monolayer adult rat hepatocyte cultures and high specific-activity [ring G-3 H]-N-acetyl-2-aminofluorene were used to extend previous observations of metabolic activation of AAF by highly differentiated, proliferation-competent hepatocytes in long-term cultures. AAF metabolism proceeded by zero-order kinetics. Hepatocytes processed significant amounts of procarcinogen (≈12 μg AAF/106 cells/day). Five ring-hydroxylated and one deacetylated species of AAF were secreted into the culture media. Extracellular metabolite levels varied during the growth cycle (days 0-13), but their rank quantitative order was time invariant: 5-OH-AAF > 7-OH-AAF > 3-OH-AAF > N-OH-AAF > aminofluorene (AF) > 1-OH-AAF. Lineweaver-Burk analyses revealed two principal classes of metabolism: System I (high-affinity and low-velocity), Km[APPARENT] = 1.64 × 10-7  M and VMAX[APPARENT] = 0.1 nmol/106 cells/day and System II (low-affinity and high-velocity), Km[APPARENT] = 3.25 × 10-5  M and VMAX[APPARENT] = 1000 nmol/106 cells/day. A third system of metabolism of AAF to AF, with Km[APPARENT] and VMAX[APPARENT] constants of 9.6 × 10-5  M and 4.7 nmol/106 cells/day, was also observed. Evidence provided in this report and its companion paper suggests selective roles and intracellular locations for System I- and System II-mediated AAF metabolite formation during hepatocarcinogenesis, although some of the molecules and mechanisms responsible for multi-system processing remain to be fully defined.

Transport of the placental estriol precursor 16α-hydroxy-dehydroepiandrosterone sulfate (16α-OH-DHEAS) by stably transfected OAT4-, SOAT-, and NTCP-HEK293 cells.

PubMed

Schweigmann, H; Sánchez-Guijo, A; Ugele, B; Hartmann, K; Hartmann, M F; Bergmann, M; Pfarrer, C; Döring, B; Wudy, S A; Petzinger, E; Geyer, J; Grosser, G

2014-09-01

16α-Hydroxy-dehydroepiandrosterone sulfate (16α-OH-DHEAS) mainly originates from the fetus and serves as precursor for placental estriol biosynthesis. For conversion of 16α-OH-DHEAS to estriol several intracellular enzymes are required. However, prior to enzymatic conversion, 16α-OH-DHEAS must enter the cells by carrier mediated transport. To identify these carriers, uptake of 16α-OH-DHEAS by the candidate carriers organic anion transporter OAT4, sodium-dependent organic anion transporter SOAT, Na(+)-taurocholate cotransporting polypeptide NTCP, and organic anion transporting polypeptide OATP2B1 was measured in stably transfected HEK293 cells by LC-MS-MS. Furthermore, the study aimed to localize SOAT in the human placenta. Stably transfected OAT4-HEK293 cells revealed a partly sodium-dependent transport for 16α-OH-DHEAS with an apparent Km of 23.1 ± 5.1 μM and Vmax of 485.0 ± 39.1 pmol/mg protein/min, while stably transfected SOAT- and NTCP-HEK293 cells showed uptake only under sodium conditions with Km of 319.0 ± 59.5 μM and Vmax of 1465.8 ± 118.8 pmol/mg protein/min for SOAT and Km of 51.4 ± 9.9 μM and Vmax of 1423.3 ± 109.6 pmol/mg protein/min for NTCP. In contrast, stably transfected OATP2B1-HEK293 cells did not transport 16α-OH-DHEAS at all. Immunohistochemical studies and in situ hybridization of formalin fixed and paraffin embedded sections of human late term placenta showed expression of SOAT in syncytiotrophoblasts, predominantly at the apical membrane as well as in the vessel endothelium. In conclusion, OAT4, SOAT, and NTCP were identified as carriers for the estriol precursor 16α-OH-DHEAS. At least SOAT and OAT4 seem to play a functional role for the placental estriol synthesis as both are expressed in the syncytiotrophoblast of human placenta. Copyright © 2014 Elsevier Ltd. All rights reserved.

Recessive constitutive mutant Chinese hamster ovary cells (CHO-K1) with an altered A system for amino acid transport and the mechanism of gene regulation of the A system.

PubMed Central

Moffett, J; Englesberg, E

1984-01-01

Chinese hamster ovary cells (CHO-K1) starved for 24 h for amino acids show a severalfold increase in velocity of proline transport through the A system (Vmax is five times that of unstarved cells). This increase is inhibited by cycloheximide, actinomycin D, N-methyl-alpha-amino isobutyric acid (MeAIB, a non-metabolizable specific A system amino acid analog), and by other amino acids that are generally transported by the A system. However, transport by the A system is not a prerequisite for this repression, and all compounds that have affinity for the A system do not necessarily act as "co-repressors." The addition of proline, MeAIB, or other amino acids, as described above, to derepressed cells results in a rapid decrease in A system activity. As shown with proline and MeAIB, this decrease in activity is in part due to a rapid trans-inhibition and a slow, irreversible inactivation of the A system. Neither process is inhibited by cycloheximide or actinomycin D. Alanine antagonizes the growth of CHO-K1 pro cells by preventing proline transport, and alanine-resistant mutants (alar) have been isolated (Moffett et al., Somatic Cell Genet. 9:189-213, 1983). alar2 and alar4 are partial and full constitutive mutants for the A system and have two and six times the Vmax for proline uptake by the A system, respectively. The A system in alar4 is also immune to the co-repressor-induced inactivation. Both alar2 and alar4 phenotypes are recessive. Alar3 shows an increase in Vmax and Km for proline transport through the A system, and this phenotype is codominant. All three mutants have a pleiotropic effect, producing increases in activity of the ASC and P systems of amino acid transport. This increase is not due to an increase in the Na+ gradient. The ASC and P phenotypes behave similarly to the A system in hybrids. A model has been proposed incorporating these results. PMID:6538929

Number of Players and Relative Pitch Area per Player: Comparing Their Influence on Heart Rate and Physical Demands in Under-12 and Under-13 Football Players.

PubMed

Castellano, Julen; Puente, Asier; Echeazarra, Ibon; Usabiaga, Oidui; Casamichana, David

2016-01-01

The aim of the present study is to analyse the influence of different large-sided games (LSGs) on the physical and physiological variables in under-12s (U12) and -13s (U13) soccer players. The effects of the combination of different number of players per team, 7, 9, and 11 (P7, P9, and P11, respectively) with three relative pitch areas, 100, 200, and 300 m(2) (A100, A200, and A300, respectively), were analysed in this study. The variables analysed were: 1) global indicator such as total distance (TD); work:rest ratio (W:R); player-load (PL) and maximal speed (Vmax); 2) heart rate (HR) mean and time spent in different intensity zones of HR (<75%, 75-84%, 84-90% and >90%), and; 3) five absolute (<8, 8-13, 13-16 and >16 Km h(-1)) and three relative speed categories (<40%, 40-60% and >60% Vmax). The results support the theory that a change in format (player number and pitch dimensions) affects no similarly in the two players categories. Although it can seem that U13 players are more demanded in this kind of LSG, when the work load is assessed from a relative point of view, great pitch dimensions and/or high number of player per team are involved in the training task to the U12 players. The results of this study could alert to the coaches to avoid some types of LSGs for the U12 players such as: P11 played in A100, A200 or A300, P9 played in A200 or A300 and P7 played in A300 due to that U13>U12 in several physical and physiological variables (W:R, time spent in 84-90%HRmax, distance in 8-13 and 13-16 Km h(-1) and time spent in 40-60%Vmax). These results may help youth soccer coaches to plan the progressive introduction of LSGs so that task demands are adapted to the physiological and physical development of participants.

Is bacteriostatic saline superior to normal saline as an echocardiographic contrast agent?

PubMed

Cardozo, Shaun; Gunasekaran, Prasad; Patel, Hena; McGorisk, Timothy; Toosi, Mehrdad; Faraz, Haroon; Zalawadiya, Sandip; Alesh, Issa; Kottam, Anupama; Afonso, Luis

2014-12-01

Objective data on the performance characteristics and physical properties of commercially available saline formulations [normal saline (NS) vs. bacteriostatic normal saline (bNS)] are sparse. This study sought to compare the in vitro physical properties and in vivo characteristics of two commonly employed echocardiographic saline contrast agents in an attempt to assess superiority. Nineteen patients undergoing transesophageal echocardiograms were each administered agitated regular NS and bNS injections in random order and in a blinded manner according to a standardized protocol. Video time-intensity (TI) curves were constructed from a representative region of interest, placed paraseptally within the right atrium, in the bicaval view. TI curves were analyzed for maximal plateau acoustic intensity (Vmax, dB) and dwell time (DT, s), defined as time duration between onset of Vmax and decay of video intensity below clinically useful levels, reflecting the duration of homogenous opacification of the right atrium. To further characterize the physical properties of the bubbles in vitro, fixed aliquots of similarly agitated saline were injected into a glass well slide-cover slip assembly and examined using an optical microscope to determine bubble diameter in microns (µm) and concentration [bubble count/high power field (hpf)]. A higher acoustic intensity (a less negative dB level), higher bubble concentration and longer DT were considered properties of a superior contrast agent. For statistical analysis, a paired t test was conducted to evaluate the differences in means of Vmax and DT. Compared to NS, bNS administration was associated with superior opacification (video intensity -8.69 ± 4.7 vs. -10.46 ± 4.1 dB, P = 0.002), longer DT (17.3 ± 6.1 vs. 10.2 ± 3.7 s) in vivo and smaller mean bubble size (43.4 vs. 58.6 μm) and higher bubble concentration (1,002 vs. 298 bubble/hpf) in vitro. bNS provides higher intensity and more sustained opacification of the right atrium compared to NS. Higher bubble concentration and stability appear to be additional desirable rheological characteristics favoring bNS as a contrast agent.

Purification and partial characterization of an exo-polygalacturonase from Paecilomyces variotii liquid cultures.

PubMed

de Lima Damásio, Andre Ricardo; da Silva, Tony Márcio; Maller, Alexandre; Jorge, João Atílio; Terenzi, Hector Francisco; Polizeli, Maria de Lourdes Teixeira de Moraes

2010-03-01

An extracellular polygalacturonase (PG) produced from Paecilomyces variotii was purified to homogeneity through two chromatography steps using DEAE-Fractogel and Sephadex G-100. The molecular weight of P. variotii PG was 77,300 Da by gel filtration and SDS-PAGE. PG had isoelectric point of 4.37 and optimum pH 4.0. PG was very stable from pH 3.0 to 6.0. The extent of hydrolysis of different pectins by the purified enzyme was decreased with an increase in the degree of esterification. PG had no activity toward non-pectic polysaccharides. The apparent K(m) and V(max) values for hydrolyzing sodium polypectate were 1.84 mg/mL and 432 micromol/min/mg, respectively. PG was found to have temperature optimum at 65 degrees Celsius and was totally stable at 45 degrees Celsius for 90 min. Half-life at 55 degrees Celsius was 50.6 min. Almost all the examined metal cations showed partial inhibitory effects under enzymatic activity, except for Na(+1), K(+1), and Co(+2) (1 mM) and Cu(+2) (1 and 10 mM).

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

PubMed

Gonnelli, Margherita; Vestri, Stefano; Santinelli, Chiara

2013-12-01

This study reports the first information on extracellular enzymatic activity (EEA) combined with a study of DOM dynamics at the Arno River mouth. DOM dynamics was investigated from both a quantitative (dissolved organic carbon, DOC) and a qualitative (absorption and fluorescence of chromophoric DOM, CDOM) perspective. The data here reported highlight that the Arno River was an important source of both DOC and CDOM for this coastal area. CDOM optical properties suggested that terrestrial DOM did not undergo simple dilution at the river mouth but, other physical-chemical and biological processes were probably at work to change its molecular characteristics. This observation was further supported by the "potential" enzymatic activity of β-glucosidase (BG) and leucine aminopeptidase (LAP). Their Vmax values were markedly higher in the river water than in the seawater and their ratio suggested that most of the DOM used by microbes in the Arno River was polysaccharide-like, while in the seawater it was mainly protein-like. © 2013. Published by Elsevier B.V. All rights reserved.

Different shades of cholesterol: Gold nanoparticles supported on MoS2 nanoribbons for enhanced colorimetric sensing of free cholesterol.

PubMed

Nirala, Narsingh R; Pandey, Shobhit; Bansal, Anushka; Singh, Vijay K; Mukherjee, Bratindranath; Saxena, Preeti S; Srivastava, Anchal

2015-12-15

In the present study, we manifest that traditionally used gold nanoparticles when supported on molybdenum disulfide nanoribbons matrix (MoS2 NRs-Au NPs) show synergistically enhanced intrinsic peroxidase like catalytic activity and can catalyze the oxidation of 3,3',5,5' tetramethyl benzidine by H2O2 to produce a highly sensitive blue shade product depending on level of free cholesterol, when tested on complex system of human serum. Further the system attests appreciable kinetics, owing to Km value as low as 0.015 mM and better loading capacity (Vmax=6.7×10(-6) M s(-1)). Additionally, the proposed system is stable for weeks with ability to perform appreciably in wide pH (3-6) and temperature range (25-60 °C). Utilizing this potential, the present work proposes a cholesterol detection color wheel which is used along with cost effective cholesterol detection strips fabricated out of proposed MoS2 NRs-Au NPs system for quick and reliable detection of free cholesterol using unaided eye. Copyright © 2015 Elsevier B.V. All rights reserved.

Inhibitory effect of chlorogenic acid on digestion of potato starch.

PubMed

Karim, Zida; Holmes, Melvin; Orfila, Caroline

2017-02-15

The effect of the chlorogenic acid isomer 5-O-caffeoylquinic acid (5-CQA) on digestion of potato starch by porcine pancreatic alpha amylase (PPAA) was investigated using isolated starch and cooked potato tuber as substrates. In vitro digestion was performed on five varieties of potato with varying phenolic content. Co- and pre-incubation of PPAA with 5-CQA significantly reduced PPAA activity in a dose dependent manner with an IC50 value of about 2mgmL(-1). Lineweaver-Burk plots indicated that 5-CQA exerts a mixed type inhibition as km increased and Vmax decreased. The total polyphenol content (TPC) of peeled tuber tissue ranged from 320.59 to 528.94mg 100g(-1)dry weight (DW) in raw tubers and 282.03-543.96mg 100g(-1)DW in cooked tubers. With the exception of Désirée, TPC and 5-CQA levels decreased after cooking. Principle component analysis indicated that digestibility is affected by multiple factors including phenolic, dry matter and starch content. Copyright © 2016 Elsevier Ltd. All rights reserved.

Classical Michaelis-Menten and system theory approach to modeling metabolite formation kinetics.

PubMed

Popović, Jovan

2004-01-01

When single doses of drug are administered and kinetics are linear, techniques, which are based on the compartment approach and the linear system theory approach, in modeling the formation of the metabolite from the parent drug are proposed. Unlike the purpose-specific compartment approach, the methodical, conceptual and computational uniformity in modeling various linear biomedical systems is the dominant characteristic of the linear system approach technology. Saturation of the metabolic reaction results in nonlinear kinetics according to the Michaelis-Menten equation. The two compartment open model with Michaelis-Menten elimination kinetics is theorethicaly basic when single doses of drug are administered. To simulate data or to fit real data using this model, one must resort to numerical integration. A biomathematical model for multiple dosage regimen calculations of nonlinear metabolic systems in steady-state and a working example with phenytoin are presented. High correlation between phenytoin steady-state serum levels calculated from individual Km and Vmax values in the 15 adult epileptic outpatients and the observed levels at the third adjustment of phenytoin daily dose (r=0.961, p<0.01) were found.

Applicability of recombinant β-xylosidase from the extremely thermophilic bacterium Geobacillus thermodenitrificans in synthesizing alkylxylosides.

PubMed

Jain, Ira; Kumar, Vikash; Satyanarayana, T

2014-10-01

The β-xylosidase encoding gene (XsidB) of the extremely thermophilic bacterium Geobacillus thermodenitrificans has been cloned and expressed in Escherichia coli. The homotrimeric recombinant XsidB is of 204.0kDa, which is optimally active at 60°C and pH 7.0 with T1/2 of 58min at 70°C. The β-xylosidase remains unaffected in the presence of most metal ions and organic solvents. The Km [p-nitrophenyl β-xyloside (pNPX)], Vmax and kcat values of the enzyme are 2×10(-3)M, 1250μmolesmg(-1)min(-1) and 13.20×10(5)min(-1), respectively. The enzyme catalyzes transxylosylation reactions in the presence of alcohols as acceptors. The pharmaceutically important β-methyl-d-xylosides could be produced using pNPX as the donor and methanol as acceptor. The products of transxylosylation were identified by TLC and HPLC, and the structure was confirmed by (1)H NMR analysis. The enzyme is also useful in synthesizing transxylosylation products from the wheat bran hydrolysate. Copyright © 2014 Elsevier Ltd. All rights reserved.

Characterization of dipeptidylcarboxypeptidase of Leishmania donovani: a molecular model for structure based design of antileishmanials

NASA Astrophysics Data System (ADS)

Baig, Mirza Saqib; Kumar, Ashutosh; Siddiqi, Mohammad Imran; Goyal, Neena

2010-01-01

Leishmania donovani dipeptidylcarboxypeptidsae (LdDCP), an angiotensin converting enzyme (ACE) related metallopeptidase has been identified and characterized as a putative drug target for antileishmanial chemotherapy. The kinetic parameters for LdDCP with substrate, Hip-His-Leu were determined as, Km, 4 mM and Vmax, 1.173 μmole/ml/min. Inhibition studies revealed that known ACE inhibitors (captopril and bradykinin potentiating peptide; BPP1) were weak inhibitors for LdDCP as compared to human testicular ACE (htACE) with Ki values of 35.8 nM and 3.9 μM, respectively. Three dimensional model of LdDCP was generated based on crystal structure of Escherichia coli DCP (EcDCP) by means of comparative modeling and assessed using PROSAII, PROCHECK and WHATIF. Captopril docking with htACE, LdDCP and EcDCP and analysis of molecular electrostatic potentials (MEP) suggested that the active site domain of three enzymes has several minor but potentially important structural differences. These differences could be exploited for designing selective inhibitor of LdDCP thereby antileishmanial compounds either by denovo drug design or virtual screening of small molecule databases.

Kinetics study of invertase covalently linked to a new functional nanogel.

PubMed

Raj, Lok; Chauhan, Ghanshyam S; Azmi, Wamik; Ahn, J-H; Manuel, James

2011-02-01

Nanogels are promising materials as supports for enzyme immobilization. A new hydrogel comprising of methacrylic acid (MAAc) and N-vinyl pyrrolidone (N-VP) and ethyleneglycol dimethacrylate (EGDMA) was synthesized and converted to nanogel by an emulsification method. Nanogel was further functionalized by Curtius azide reaction for use as support for the covalent immobilization of invertase (Saccharomyces cerevisiae). As-prepared or invertase-immobilized nanogel was characterized by FTIR, XRD, TEM and nitrogen analysis. The characterization of both free and the immobilized-invertase were performed using a spectrophotometric method at 540 nm. The values of V(max), maximum reaction rate, (0.123 unit/mg), k(m), Michaelis constant (7.429 mol/L) and E(a), energy of activation (3.511 kj/mol) for the immobilized-invertase are comparable with those of the free invertase at optimum conditions (time 70 min, pH 6.0 and temperature 45°C). The covalent immobilization enhanced the pH and thermal stability of invertase. The immobilized biocatalyst was efficiently reused up to eight cycles. Copyright © 2010 Elsevier Ltd. All rights reserved.

Isolation and Characterization of a Glycosyl Hydrolase Family 16 β-Agarase from a Mangrove Soil Metagenomic Library

PubMed Central

Mai, Zhimao; Su, Hongfei; Zhang, Si

2016-01-01

A mangrove soil metagenomic library was constructed and a β-agarase gene designated as AgaML was isolated by functional screening. The gene encoded for a 659-amino-acids polypeptide with an estimated molecular mass of 71.6 kDa. The deduced polypeptide sequences of AgaML showed the highest identity of 73% with the glycoside hydrolase family 16 β-agarase from Microbulbifer agarilyticus in the GenBank database. AgaML was cloned and highly expressed in Escherichia coli BL21(DE3). The purified recombinant protein, AgaML, showed optimal activity at 50 °C and pH 7.0. The kinetic parameters of Km and Vmax values toward agarose were 4.6 mg·mL−1 and 967.5 μM·min−1·mg−1, respectively. AgaML hydrolyzed the β-1,4-glycosidic linkages of agar to generate neoagarotetraose (NA4) and neoagarohexaose (NA6) as the main products. These characteristics suggest that AgaML has potential application in cosmetic, pharmaceuticals and food industries. PMID:27548158

An efficient ribitol-specific dehydrogenase from Enterobacter aerogenes.

PubMed

Singh, Ranjitha; Singh, Raushan; Kim, In-Won; Sigdel, Sujan; Kalia, Vipin C; Kang, Yun Chan; Lee, Jung-Kul

2015-05-01

An NAD(+)-dependent ribitol dehydrogenase from Enterobacter aerogenes KCTC 2190 (EaRDH) was cloned and successfully expressed in Escherichia coli. The complete 729-bp gene was amplified, cloned, expressed, and subsequently purified in an active soluble form using nickel affinity chromatography. The enzyme had an optimal pH and temperature of 11.0 and 45°C, respectively. Among various polyols, EaRDH exhibited activity only toward ribitol, with Km, Vmax, and kcat/Km values of 10.3mM, 185Umg(-1), and 30.9s(-1)mM(-1), respectively. The enzyme showed strong preference for NAD(+) and displayed no detectable activity with NADP(+). Homology modeling and sequence analysis of EaRDH, along with its biochemical properties, confirmed that EaRDH belongs to the family of NAD(+)-dependent ribitol dehydrogenases, a member of short-chain dehydrogenase/reductase (SCOR) family. EaRDH showed the highest activity and unique substrate specificity among all known RDHs. Homology modeling and docking analysis shed light on the molecular basis of its unusually high activity and substrate specificity. Copyright © 2015 Elsevier Inc. All rights reserved.

Inhibition of angiotensin I converting enzyme by subtilisin NAT (nattokinase) in natto, a Japanese traditional fermented food.

PubMed

Murakami, Keiko; Yamanaka, Naoki; Ohnishi, Katsunori; Fukayama, Minoru; Yoshino, Masataka

2012-06-01

Angiotensin I converting enzyme (ACE) was inhibited by the culture medium of Bacillus subtilis subsp. natto, which ferments boiled soy beans to natto, a Japanese traditional food. Subtilisin NAT (nattokinase) produced by B. subtilis also inhibited ACE, and the inhibition was markedly stimulated by heat treatment of subtilisin at 120 °C for 15 min. Inhibition of ACE by subtilisin was of a mixed type: the decrease in V(max) and the increase in K(m) value. SDS-polyacrylamide gel electrophoresis showed that heat treatment of subtilisin caused inactivation with fragmentation of the enzyme protein into small peptides. The inhibitory action of subtilisin was not due to an enzymatic action of protease, but may be ascribed to the potent ACE-inhibitory peptides such as LY and FY, amino acid sequences in subtilisin. HPLC-MS analysis of heat-inactivated subtilisin confirmed that LY and FY were liberated by fragmentation of the enzyme. Inhibition of ACE by subtilisin and its degradation peptides such as LY and FY may participate in the suppression of blood pressure by ingestion of natto.

Induction of calcite precipitation through heightened production of extracellular carbonic anhydrase by CO2 sequestering bacteria.

PubMed

Sundaram, Smita; Thakur, Indu Shekhar

2018-04-01

The thermo-alkalotolerant bacterium exhibiting heightened extracellular carbonic anhydrase (CA) activity, survived at 100 mM sodium bicarbonateand 5% gaseous CO 2 was identified as Bacillus sp. by 16S rRNA sequencing. Extracellular carbonic anhydrase was purified by ammonium sulfate precipitation, gel filtration chromatography and affinity chromatography with a yield of 46.61% and specific activity of 481.66 U/mg. The size of purified carbonic anhydrase was approximately 28 kDa in SDS-PAGE gel filtration and further their role in calcium carbonate production was correlated. The purified enzyme was stable with half-life of 25.36 min at 90 °C and pH 8. K M and Vmax values of the enzyme were 1.77 mg/mL and 385.69 U/mg respectively. The production of calcite was confirmed by Scanning Electron Microscopy (SEM) analysis, FTIR, and Energy-Dispersive X-ray (EDX) analysis. Carbonic anhydrase and calcite deposition coupled with CO 2 fixingbacteria is a significant approach for CO 2 sequestration. Copyright © 2018 Elsevier Ltd. All rights reserved.

Effect of common NAT2 variant alleles in the acetylation of the major clonazepam metabolite, 7-aminoclonazepam.

PubMed

Olivera, M; Martínez, C; Gervasini, G; Carrillo, J A; Ramos, S; Benítez, J; García-Martin, E; Agúndez, J A G

2007-01-01

We investigated the role of NAT2 on clonazepam acetylation, using transiently expressed human NAT2 alleles. The NAT25*B and the NAT2*6A variant alleles cause a 20 and 22-fold reduction in the Vmax, respectively. We conclude that NAT2 is responsible for 7-aminoclonazepam acetylation and that NAT2 gene polymorphisms impair such metabolic pathway.

Purification and characterization of soluble (cytosolic) and bound (cell wall) isoforms of invertases in barley (Hordeum vulgare) elongating stem tissue

NASA Technical Reports Server (NTRS)

Karuppiah, N.; Vadlamudi, B.; Kaufman, P. B.

1989-01-01

Three different isoforms of invertases have been detected in the developing internodes of barley (Hordeum vulgare). Based on substrate specificities, the isoforms have been identified to be invertases (beta-fructosidases EC 3.2.1.26). The soluble (cytosolic) invertase isoform can be purified to apparent homogeneity by diethylaminoethyl cellulose, Concanavalin-A Sepharose, organo-mercurial Sepharose, and Sephacryl S-300 chromatography. A bound (cell wall) invertase isoform can be released by 1 molar salt and purified further by the same procedures as above except omitting the organo-mercurial Sepharose affinity chromatography step. A third isoform of invertase, which is apparently tightly associated with the cell wall, cannot be isolated yet. The soluble and bound invertase isoforms were purified by factors of 60- and 7-fold, respectively. The native enzymes have an apparent molecular weight of 120 kilodaltons as estimated by gel filtration. They have been identified to be dimers under denaturing and nondenaturing conditions. The soluble enzyme has a pH optimum of 5.5, Km of 12 millimolar, and a Vmax of 80 micromole per minute per milligram of protein compared with cell wall isozyme which has a pH optimum of 4.5, Km of millimolar, and a Vmax of 9 micromole per minute per milligram of protein.

[Specific aspects of thrombocyte system of serotonin in patients with different manifestations of schizoaffective psychosis].

PubMed

Brusov, O S; Dikaia, V I; Zlobina, G P; Faktor, M I; Pavlova, O A; Bologov, P V; Korenev, A N

2000-01-01

45 women with different manifestations of schizoaffective psychosis (SAP) were examined. The diagnosis corresponded to ICD-10 (F25). According to the classification elaborated in Mental Health Research Centre of Russian Academy of Medical Sciences, groups of patients were identified with different variants of the psychoses course: a nuclear SAP type; a borderline SAP variation with phasic-recurrent course; SAP with progredient variation (schizoaffective variation of schizophrenia). The patients were examined both during the attack and remission. A rate of serotonine uptake (Vmax) in blood platelets, a specific imipramine binding (Bmax) and the level of serotonin in blood platelets were evaluated. It was found that dynamics of both Vmax and the level of serotonin in different SAP types were different, that was related to clinical and biological SAP heterogeneity. A tendency to decreasing of serotonin system functional activity was found in progredient SAP variations, especially during the remission, which was of low quality in these cases. On the contrary, in the borderline variations the indices of the decreased function of serotonin system corresponded well to those of acute psychosis. In nuclear type--a type with the most favourable course of psychosis--any significant changes weren't revealed as compared with the normal parameters.

Lipid changes in hepatic microsomes and its relationship to P-nitrophenol glucuronidation in an experimental model of portal hypertension.

PubMed

Ghanem, C; Ghisolfi, C; Marabotto, L; Ouviña, G; Rubio, M; Perazzo, J; Lemberg, A; Bengochea, L

1997-10-01

The liver is responsible for the most important metabolic pathway of non polar compounds. The aim of the present work was to study the p-nitrophenol glucuronidation and its relationship with lipidic composition of microsomal membrane in a model of hepatic portal hypertension and hepatocellular damage induced by monocrotaline. A global increment in liver microsomal phospholipids as well as changes in the phospholipid pattern (phosphatidylethanolamine and sphingomyelin increased up to 156 +/- 13 and 195 +/- 14% respectively) were detected in monocrotaline intoxicated rats when it were compared to control rats. The microsomal cholesterol content showed a decrease in monocrotaline intoxicated rats. (4.1 +/- 0.7 against 6.6 +/- 1.5 micrograms/mg of microsomal protein, in control rats). When p-nitrophenol activity was measured, Km from monocrotaline intoxicated rats was 0.137 mM, and Vmax was 2.9 nmol of p-nitrophenol/mg microsomal protein since in control group Km was 0.322 mM, and Vmax was 4.5 nmol of p-nitrophenol/mg microsomal protein. It is concluded that monocrotaline intoxicated rats showed a different behavior in the kinetics of p-nitrophenol UDP-glucuronyltransferase, as well as a different microsomal lipidic profile, when compared to control group.

Characterization and regulation of glycine transport in Fusarium oxysporum var. lini.

PubMed

Castro, I M; Lima, A A; Nascimento, A F; Ruas, M M; Nicoli, J R; Brandão, R L

1996-08-01

Glycine was transported in Fusarium oxysporum cells, grown on glycine as the sole source of carbon and nitrogen, by a facilitated diffusion transport system with a half-saturation constant (Ks) of 11 mM and a maximum velocity (Vmax) of 1.2 mM (g dry weight)-1 h-1 at pH 5.0 and 26 degrees C. Under conditions of nitrogen starvation, the same system was present together with a high-affinity one (Ks) of about 47 microM and Vmax of about 60 microM (g dry weight)-1 h-1). The low-affinity system was more specific than the high-affinity system. Cells grown on gelatine showed the same behavior. In cells grown on glucose-gelatine medium, the low-affinity system was poorly expressed even after carbon and nitrogen starvation. Moreover, addition of glucose to cells grown on glycine and resuspended in mineral medium caused an increase of the glycine transport probably due to a boost in protein synthesis. This stimulation did not affect the Ks of the low-affinity system. These results demonstrate that, as is the case for other eukaryotic systems, F. oxysporum glycine transport is under control of nitrogen sources but its regulation by carbon sources appears to be more complex.

Hydrolysis is the dominating in vivo metabolism pathway for arctigenin: identification of novel metabolites of arctigenin by LC/MS/MS after oral administration in rats.

PubMed

Gao, Qiong; Zhang, Yufeng; Wo, Siukwan; Zuo, Zhong

2013-04-01

The phenylpropanoid dibenzylbutyrolactone lignan arctigenin, a key component found in Arctium lappa, or burdock, has been reported with a variety of therapeutic effects including anticancer, anti-inflammation, and antivirus effects. Using LC/MS/MS, three novel metabolites of arctigenin, namely, arctigenic acid, arctigenin-4-O'-glucuronide, and 4-O-demethylarctigenin were identified after oral administration of arctigenin in rats for the first time. Another potential metabolite of arctigenin, arctigenin-4'-O-sulfate, was identified in vitro but not in vivo. Structure of arctigenic acid, the major metabolite of arctigenin, was confirmed by 13C-NMR and 1H-NMR. Rapid hydrolysis in plasma was identified as the major metabolic pathway of arctigenin after its oral administration, with Vmax, Km, and Clint in rat plasma determined to be 2.21 ± 0.12 nmol/min/mg, 89.12 ± 9.44 µM, and 24.74 µL/min/mg, respectively. Paraoxonase 1 was further confirmed to be the enzyme responsible for arctigenin hydrolysis, with Vmax, Km, and Clint determined to be 55.39 ± 1.49 nmol/min/mg, 300.3 ± 10.86 µM, and 184.45 µL/min/mg, respectively. Georg Thieme Verlag KG Stuttgart · New York.

Cloning and expression of the Aspergillus oryzae glucan 1,3-beta-glucosidase A (exgA) in Pichia pastoris.

PubMed

Boonvitthya, Nassapat; Tanapong, Phatrapan; Kanngan, Patcharaporn; Burapatana, Vorakan; Chulalaksananukul, Warawut

2012-10-01

The glucan 1,3-beta-glucosidase A gene (exgA) from Aspergillus oryzae and fused to the Saccharomyces cerevisiae signal peptide (α-factor) was expressed under the control of either a constitutive (GAP) or an inducible (AOX1) promoter in Pichia pastoris. A 1.4-fold higher extracellular enzyme activity (2 U/ml) was obtained using the AOX1 inducible expression system than with the GAP constitutive promoter (1.4 U/ml). The purified recombinant ExgA enzyme, with a yield of 10 mg protein/l culture supernatant, was about 40 kDa by SDS-PAGE analysis with a specific activity of 289 U/mg protein. The enzyme was optimally active at 35 °C and pH 5.0 and displayed a K(M) and V(max) of 0.56 mM and 10,042 μmol/(min mg protein), respectively, with p-nitrophenyl-β-D-glucopyranoside as the substrate. Moreover, it was tolerant to glucose inhibition with a K(i) of 365 mM.

Effects of Acute Lymphoblastic Leukemia on Ceruloplasmin Oxidase, Copper and Several Markers of Oxidative Damage, in Children.

PubMed

Mehdi, Wesen Adel; Yusof, Faridah; Mehde, Atheer Awad; Zainulabdeen, Jwan Abdulmohsin; Raus, Raha Ahmed; Abdulbari, Alaa Shawqi

2015-01-01

Acute leukaemia is characterized by fast growth of abnormal clones of haemopoietic precursor cells inside bone marrow leading to undue accumulation in the bone marrow. Acute lymphoblastic leukemia (ALL) is the most common form of childhood cancer. The study concerned 50 children diagnosed with ALL (mean age, 8.55±2.54) compared to 40 healthy controls (mean age, 8.00±1.85). The Hb, serum copper, ceruloplasmin oxidase, advanced oxidation protein products (AOPPs), total antioxidant activity (TAA) and protein were measured in all groups. One proteinous component was isolated by gel filtration chromatography from the precipitate produced by polyethylene glycol. Significantly higher levels of AOPP, copper and decrease in total antioxidant activity were noted in the cases. Statistical analysis also showed a significant increase (p<0.01) in the activity of serum ceruloplasmin oxidase in patients with ALL compared to normal subjects. The maximum velocity (Vmax) and Michaelis constant had values of 104.2 U/L and 11.7 mM, respectively. The ΔH* values for ceruloplasmin oxidase in ALL patients were positive, confirming the reaction to be endothermic. The results from this study showed a significant increase in AOPP, ceruloplasmine oxidase and decrease in total antioxidant activity .These parameters may play a role in development of DNA damage in childhood patients with acute lymphoblastic leukemia (ALL). The ΔS* and ΔG* values were negative, these refer that the reaction of ES formation is spontaneous, but needs energy in a so-called endergonic reaction. Also the negative ΔS* value of ceruloplasmin oxidase indicates that the complex [ES*] is further modulated through increasing structure arrangement.

[Effects of methomyl on acetylcholinesterase in erythrocyte membrane and various brain areas].

PubMed

Zhao, Fei; Li, Tao; Zhang, Changchun; Xu, Yiping; Xu, Hangong; Shi, Nian

2015-06-01

To study the toxicity of methomyl to acetylcholinesterase (AChE) in different regions. The optimal temperature and time for measurement of AChE activity were determined in vitro. The dose- and time-response relationships of methomyl with AChE activity in human erythrocyte membrane, rat erythrocyte membrane, cortical synapses, cerebellar synapses, hippocampal synapses, and striatal synapses were evaluated. The half maximal inhibitory concentration (IC50) and bimolecular rate constant (K) of methomyl for AChE activity in different regions were calculated, and the type of inhibition of AChE activity by methomyl was determined. AChE achieved the maximum activity at 370 °C, and the optimal time to determine initial reaction velocity was 0-17 min. There were dose- and time-response relationships between methomyl and AChE activity in the erythrocyte membrane and various brain areas. The IC50 value of methomyl for AChE activity in human erythrocyte membrane was higher than that in rat erythrocyte membrane, while the Ki value of methomyl for AChE activity in rat erythrocyte membrane was higher than that in human erythrocyte membrane. Among synapses in various brain areas, the striatum had the highest IC50 value, followed by the cerebellum, cerebral cortex, and hippocampus, while the cerebral cortex had the highest Ki value, followed by the hippocampus, striatum, and cerebellum. Lineweaver-Burk diagram demonstrated that with increasing concentration of methomyl, the maximum reaction velocity (Vmax) of AChE decreased, and the Michaelis constant (Km) remained the same. Methomyl is a reversible non-competitive inhibitor of AChE. AChE of rat erythrocyte membrane is more sensitive to methomyl than that of human erythrocyte membrane; the cerebral cortical synapses have the most sensitive AChE to methomyl among synapses in various brain areas.

Detergent-compatible, organic solvent-tolerant alkaline protease from Bacillus circulans MTCC 7942: Purification and characterization.

PubMed

Patil, Ulhas; Mokashe, Narendra; Chaudhari, Ambalal

2016-01-01

Proteases are now recognized as the most indispensable industrial biocatalyst owing to their diverse microbial sources and innovative applications. In the present investigation, a thermostable, organic solvent-tolerant, alkaline serine protease from Bacillus circulans MTCC 7942, was purified and characterized. The protease was purified to 37-fold by a three-step purification scheme with 39% recovery. The optimum pH and temperature for protease was 10 and 60 °C, respectively. The apparent molecular mass of the purified enzyme was 43 kD as revealed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The Km and Vmax values using casein-substrate were 3.1 mg/mL and 1.8 µmol/min, respectively. The protease remained stable in the presence of organic solvents with higher (>3.2) log P value (cyclohexane, n-octane, n-hexadecane, n-decane, and n-dodecane), as compared to organic solvents with lower (<3.2) log P value (acetone, butanol, benzene, chloroform, toluene). Remarkably, the protease showed profound stability even in the presence of organic solvents with less log P values (glycerol, dimethyl sulfate [DMSO], p-xylene), indicating the possibility of nonaqueous enzymatic applications. Also, protease activity was improved in the presence of metal ions (Ca(2+), Mg(2+), Mn(2+)); enhanced by biosurfactants; hardly affected by bleaching agents, oxidizing agents, and chemical surfactants; and stable in commercial detergents. In addition, a protease-detergent formulation effectively washed out egg and blood stains as compared to detergent alone. The protease was suitable for various commercial applications like processing of gelatinous film and as a compatible additive to detergent formulation with its operative utility in hard water.

Immobilization and kinetics of catalase on calcium carbonate nanoparticles attached epoxy support.

PubMed

Preety; Hooda, Vinita

2014-01-01

A novel hybrid epoxy/nano CaCO3 composite matrix for catalase immobilization was prepared by polymerizing epoxy resin in the presence of CaCO3 nanoparticles. The hybrid support was characterized using scanning electron microscopy and Fourier transform infrared spectroscopy. Catalase was successfully immobilized onto epoxy/nano CaCO3 support with a conjugation yield of 0.67 ± 0.01 mg/cm(2) and 92.63 ± 0.80 % retention of activity. Optimum pH and optimum temperature of free and immobilized catalases were found to be 7.0 and 35 °C. The value of Km for H2O2 was higher for immobilized enzyme (31.42 mM) than native enzyme (27.73 mM). A decrease in Vmax value from 1,500 to 421.10 μmol (min mg protein)(-1) was observed after immobilization. Thermal and storage stabilities of catalase improved immensely after immobilization. Immobilized enzyme retained three times than the activity of free enzyme when kept at 75 °C for 1 h and the half-life of enzyme increased five times when stored in phosphate buffer (0.01 M, pH 7.0) at 5 °C. The enzyme could be reused 30 times without any significant loss of its initial activity. Desorption of catalase from the hybrid support was minimum at pH 7.0.

The impact of variation in scaling factors on the estimation of ...

EPA Pesticide Factsheets

Many physiologically based pharmacokinetic (PBPK) models include values for metabolic rate parameters extrapolated from in vitro metabolism studies using scaling factors such as mg of microsomal protein per gram of liver (MPPGL) and liver mass (FVL). Variation in scaling factor values impacts metabolic rate parameter estimates (Vmax) and hence estimates of internal dose used in dose response analysis. The impacts of adult human variation in MPPGL and FVL on estimates of internal dose were assessed using a human PBPK model for BDCM for several internal dose metrics for two exposure scenarios (single 0.25 liter drink of water or 10 minute shower) under plausible (5 micrograms/L) and high level (20 micrograms/L) water concentrations. For both concentrations, all internal dose metrics were changed less than 5% for the showering scenario (combined inhalation and dermal exposure). In contrast, a 27-fold variation in area under the curve for BDCM in venous blood was observed at both oral exposure concentrations, whereas total amount of BDCM metabolized in liver was relatively unchanged. This analysis demonstrates that variability in the scaling factors used for in vitro to in vivo extrapolation (IVIVE) for metabolic rate parameters can have a significant route-dependent impact on estimates of internal dose under environmentally relevant exposure scenarios. This indicates the need to evaluate both uncertainty and variability for scaling factors used for IVIVE. Sca

A novel tannase from the xerophilic fungus Aspergillus niger GH1.

PubMed

Mata-Gomez, Marco; Rodriguez, Luis V; Ramos, Erika L; Renovato, Jacqueline; Cruz-Hernandez, Mario A; Rodriguez, Raul; Contreras, Juan; Aguilar, Cristobal N

2009-09-01

Aspergillus niger GH1 previously isolated and identified by our group as a wild tannase producer was grown under solid-state (SSC) and submerged culture (SmC) conditions to select the enzyme production system. For tannase purification, extracellular tannase was produced under SSC using polyurethane foam as the inert support. Tannase was purified to apparent homogeneity by ultrafiltration, anion-exchange chromatography, and gel filtration that led to a purified enzyme with a specific activity of 238.14 IU/mg protein with a final yield of 0.3% and a purification fold of 46. Three bands were found on the SDS-PAG with molecular masses of 50, 75, and 100 kDa. PI of 3.5 and 7.1% Nglycosylation were noted. Temperature and pH optima were 60 degrees and 6.0 [methyl 3,4,5-trihydroxybenzoate (MTB) as substrate], respectively. Tannase was found with a KM value of 0.41 x 10-4 M and the value of Vmax was 11.03 micromoL/min at 60 degrees for MTB. Effects of several metal salts, solvents, surfactants, and typical enzyme inhibitors on tannase activity were evaluated to establish the novelty of the enzyme. Finally, the tannase from A. niger GH1 was significantly inhibited by PMSF (phenylmethylsulfonyl fluoride), and therefore, it is possible to consider the presence of a serine or cysteine residue in the catalytic site.

Purification and characterization of extracellular lipase from a new strain: Pseudomonas aeruginosa SRT 9

PubMed Central

Borkar, Prita S.; Bodade, Ragini G.; Rao, Srinivasa R.; Khobragade, C.N.

2009-01-01

An extra cellular lipase was isolated and purified from the culture broth of Pseudomonas aeruginosa SRT 9 to apparent homogeneity using ammonium sulfate precipitation followed by chromatographic techniques on phenyl Sepharose CL- 4B and Mono Q HR 5/5 column, resulting in a purification factor of 98 fold with specific activity of 12307.8 U/mg. The molecular weight of the purified lipase was estimated by SDS-PAGE to be 29 kDa with isoelectric point of 4.5. Maximum lipase activity was observed in a wide range of temperature and pH values with optimum temperature of 55ºC and pH 6.9. The lipase preferably acted on triacylglycerols of long chain (C14-C16) fatty acids. The lipase was inhibited strongly by EDTA suggesting the enzyme might be metalloprotein. SDS and metal ions such as Hg2+, Zn2+, Cu2+, Ag2+ and Fe2+ decreased the lipase activity remarkedly. Its marked stability and activity in organic solvents suggest that this lipase is highly suitable as a biotechnological tool with a variety of applications including organo synthetic reactions and preparation of enantiomerically pure pharmaceuticals. The Km and Vmax value of the purified enzyme for triolein hydrolysis were calculated to be 1.11 mmol/L and 0.05 mmol/L/min respectively. PMID:24031373

Decreased activity and expression of intestinal oligopeptide transporter PEPT1 in rats with hyperthyroidism in vivo.

PubMed

Ashida, Kayoko; Katsura, Toshiya; Saito, Hideyuki; Inui, Ken-ichi

2004-06-01

To examine the effect of thyroid hormone status on PEPT1 in vivo, the activity and expression of PEPT1 in the small intestine were examined in euthyroid and hyperthyroid rats. Hyperthyroidism was induced by treating rats with L-thyroxine (12 mg/L) in the drinking water for 21 days. Transport activity was measured by everted small intestinal preparations and in situ intestinal loop technique. Expressions of PEPT1 mRNA and protein were evaluated by competitive polymerase chain reaction and Western blotting, respectively. The uptake of [14C]glycylsarcosine by everted small intestinal preparations was significantly decreased in hyperthyroid rats, whereas that of methyl-alpha-D-[14C(U)]-glucopyranoside was not altered. Kinetic analysis showed that the Vmax value for [14C]glycylsarcosine uptake was significantly decreased in hyperthyroid rats, whereas the Km value was not affected. The mean portal vein concentrations after intrajejunal administration of [14C]glycylsarcosine were also decreased in hyperthyroid rats. Moreover, hyperthyroidism caused a significant decrease in the expression of PEPT1 mRNA in the small intestine, whereas the expression of Na+/glucose cotransporter (SGLT1) mRNA was not changed. The level of PEPT1 protein was also decreased in the small intestine of hyperthyroid rats. These results indicate that in hyperthyroid rats, the activity and expression of PEPT1 were decreased in the small intestine.

Thermodynamics and kinetic properties of halostable endoglucanase from Aspergillus fumigatus ABK9.

PubMed

Das, Arpan; Jana, Arijit; Paul, Tanmay; Halder, Suman Kumar; Ghosh, Kuntal; Maity, Chiranjit; Mohapatra, Pradeep Kumar Das; Pati, Bikas Ranjan; Mondal, Keshab Chandra

2014-07-01

An endoglucanase from Aspergillus fumigatus ABK9 was purified from the culture extract of solid-state fermentation and its some characteristics were evaluated. The molecular weight of the purified enzyme (56.3 kDa) was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, zymogram analysis and confirmed by MALDI-TOF mass spectrometry. The enzyme was active optimally at 50 °C, pH 5.0 and stable over a broad range of pH (4.0-7.0) and NaCl concentration of 0-3.0 M. The pKa1 and pKa2 of the ionizable groups of the active sites were 2.94 and 6.53, respectively. The apparent Km , Vmax , and Kcat values for carboxymethyl cellulose were 6.7 mg ml(-1), 775.4 µmol min(-1) , and 42.84 × 10(4)  s(-1), respectively. Thermostability of the enzyme was evidenced by the high activation energy (91.45 kJ mol(-1)), large enthalpy for activation of denaturation (88.77 kJ mol(-1)), longer half-life (T1/2) (433 min at 50 °C), higher melting temperature (Tm ) (73.5 °C), and Q10 (1.3) values. All the characteristics favors its suitability as halotolerant and thermostable enzyme during bioprocessing of lignocellulosic materials. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Gaucher disease types 1, 2, and 3: differential mutations of the acid beta-glucosidase active site identified with conduritol B epoxide derivatives and sphingosine.

PubMed Central

Grabowski, G A; Dinur, T; Osiecki, K M; Kruse, J R; Legler, G; Gatt, S

1985-01-01

To elucidate the genetic heterogeneity in Gaucher disease, the residual beta-glucosidase in cultured fibroblasts from affected patients with each of the major phenotypes was investigated in vitro and/or in viable cells by inhibitor studies using the covalent catalytic site inhibitors, conduritol B epoxide or its bromo derivative, and the reversible cationic inhibitor, sphingosine. These studies delineated three distinct groups (designated A, B, and C) of residual activities with characteristic responses to these inhibitors. Group A residual enzymes had normal I50 values (i.e., the concentration of inhibitor that results in 50% inhibition) for the inhibitors and normal or nearly normal t1/2 values for conduritol B epoxide. All neuronopathic (types 2 and 3) and most non-Jewish nonneuronopathic (type 1) patients had group A residual activities and, thus, could not be distinguished by these inhibitor studies. Group B residual enzymes had about four- to fivefold increased I50 values for the inhibitors and similarly increased t1/2 values for conduritol B epoxide. All Ashkenazi Jewish type 1 and only two non-Jewish type 1 patients had group B residual activities. The differences in I50 values between groups A and B also were confirmed by determining the uninhibited enzyme activity after culturing the cells in the presence of bromo-conduritol B epoxide. Group C residual activity had intermediate I50 values for the inhibitors and represented a single Afrikaner type 1 patient: this patient was a genetic compound for the group A (type 2) and group B (type 1) mutations. These inhibition studies indicated that: Gaucher disease type 1 is biochemically heterogeneous, neuronopathic and non-Jewish nonneuronopathic phenotypes cannot be reliably distinguished by these inhibitor studies, and the Ashkenazi Jewish form of Gaucher disease type 1 results from a unique mutation in a specific active site domain of acid beta-glucosidase that leads to a defective enzyme with a decreased Vmax. PMID:4003396

Modification of uptake and antiproliferative effect of methylglyoxal bis(guanylhydrazone) by treatment with alpha-difluoromethylornithine in rodent cell lines with different sensitivities to methylglyoxal bis(guanylhydrazone).

PubMed

Alhonen-Hongisto, L; Levin, V A; Marton, L J

1985-02-01

Uptake characteristics and growth-inhibitory effects of methylglyoxal bis(guanylhydrazone) (MGBG), a competitive inhibitor of S-adenosylmethionine decarboxylase, were investigated in 9L rat brain tumor cells and in V79 hamster lung cells. Proliferation of 9L cells was only slightly inhibited by treatment with 40 microM MGBG alone, but when used in combination with 0.5 mM alpha-difluoromethylornithine (DFMO), an irreversible inhibitor of ornithine decarboxylase, proliferation was much more effectively inhibited. The intracellular concentration of MGBG was approximately 2-fold higher 4 days after cells were treated with both DFMO and MGBG, either simultaneously or when MGBG was added after a 48-hr DFMO pretreatment, than that in cells treated with MGBG alone. Polyamine levels in DFMO- and MGBG-treated cells correlated with the antiproliferative effects of the drugs. Used either alone or in combination with 1 mM DFMO, 0.5 microM MGBG inhibited the growth of and eventually killed V79 cells. Simultaneous or sequential treatment with DFMO and MGBG increased intracellular concentrations of MGBG at 4 days by 2- and 3-fold, respectively, compared to treatment with MGBG alone. Intracellular polyamine levels did not correlate with the antiproliferative effect of the two drugs in V79 cells. In both cell lines, polyamines and MGBG share a common transport system. The net transport of polyamines and MGBG was more temperature dependent and up to 10-fold more active in V79 cells than in 9L cells. The Km and Vmax values for spermidine and MGBG measured 10 sec after addition (initial permeation) were not affected by DFMO pretreatment in either cell line. However, 1 hr after administration, the Vmax values for spermidine and MGBG uptake were doubled in V79 cells pretreated for 48 hr with DFMO; no significant change occurred in 9L cells. Mitochondrial function, assessed by pyruvate oxidation, was substantially impaired by MGBG in V79 cells but not in 9L cells when the intracellular concentrations of MGBG were equal in each cell line. Pretreatment with DFMO did not increase MGBG-induced inhibition of pyruvate oxidation in V79 cells. These results show that, compared with V79 cells, the decreased sensitivity of 9L cells to MGBG may be related to decreased intracellular MGBG accumulation but not to cellular permeation such as carrier transport. Results of measurements of both polyamine levels and mitochondrial function indicate that V79 cells may be more susceptible to nonpolyamine-dependent effects of MGBG than are 9L cells.

Comparison of the UDP-N-Acetylmuramate:l-Alanine Ligase Enzymes from Mycobacterium tuberculosis and Mycobacterium leprae

PubMed Central

Mahapatra, Sebabrata; Crick, Dean C.; Brennan, Patrick J.

2000-01-01

In the peptidoglycan of Mycobacterium leprae, l-alanine of the side chain is replaced by glycine. When expressed in Escherichia coli, MurC (UDP-N-acetyl-muramate:l-alanine ligase) of M. leprae showed Km and Vmax for l-alanine and glycine similar to those of Mycobacterium tuberculosis MurC, suggesting that another explanation should be sought for the presence of glycine. PMID:11073931

VizieR Online Data Catalog: Luminosity and redshift of galaxies from WISE/SDSS (Toba+, 2014)

NASA Astrophysics Data System (ADS)

Toba, Y.; Oyabu, S.; Matsuhara, H.; Malkan, M. A.; Gandhi, P.; Nakagawa, T.; Isobe, N.; Shirahata, M.; Oi, N.; Ohyama, Y.; Takita, S.; Yamauchi, C.; Yano, K.

2017-07-01

We selected 12 and 22 um flux-limited galaxies based on the WISE (Cat. II/311) and SDSS (Cat. II/294) catalogs, and these galaxies were then classified into five types according to their optical spectroscopic information in the SDSS catalog. For spectroscopically classified galaxies, we constructed the luminosity functions using the 1/Vmax method, considering the detection limit of the WISE and SDSS catalogs. (1 data file).

Computer program for the reservoir model of metabolic crossroads.

PubMed

Ribeiro, J M; Juzgado, D; Crespo, E; Sillero, A

1990-01-01

A program containing 344 sentences, written in BASIC and adapted to run in personal computers (PC) has been developed to simulate the reservoir model of metabolic crossroads. The program draws the holes of the reservoir with shapes reflecting the Vmax, Km (S0.5) and cooperativity coefficients (n) of the enzymes and calculates both the actual velocities and the percentage of contribution of every enzyme to the overall removal of their common substrate.

14 CFR Appendix D to Part 23 - Wheel Spin-Up and Spring-Back Loads

Code of Federal Regulations, 2014 CFR

2014-01-01

... (0.80 may be used); F Vmax=maximum vertical force on wheel (pounds)=n j W e, where W e and n j are... 14 Aeronautics and Space 1 2014-01-01 2014-01-01 false Wheel Spin-Up and Spring-Back Loads D.... D Appendix D to Part 23—Wheel Spin-Up and Spring-Back Loads D23.1 Wheel spin-up loads. (a) The...

14 CFR Appendix D to Part 23 - Wheel Spin-Up and Spring-Back Loads

Code of Federal Regulations, 2013 CFR

2013-01-01

... (0.80 may be used); F Vmax=maximum vertical force on wheel (pounds)=n j W e, where W e and n j are... 14 Aeronautics and Space 1 2013-01-01 2013-01-01 false Wheel Spin-Up and Spring-Back Loads D.... D Appendix D to Part 23—Wheel Spin-Up and Spring-Back Loads D23.1 Wheel spin-up loads. (a) The...

14 CFR Appendix D to Part 23 - Wheel Spin-Up and Spring-Back Loads

Code of Federal Regulations, 2011 CFR

2011-01-01

... (0.80 may be used); F Vmax=maximum vertical force on wheel (pounds)=n j W e, where W e and n j are... 14 Aeronautics and Space 1 2011-01-01 2011-01-01 false Wheel Spin-Up and Spring-Back Loads D.... D Appendix D to Part 23—Wheel Spin-Up and Spring-Back Loads D23.1 Wheel spin-up loads. (a) The...

14 CFR Appendix D to Part 23 - Wheel Spin-Up and Spring-Back Loads

Code of Federal Regulations, 2010 CFR

2010-01-01

... (0.80 may be used); F Vmax=maximum vertical force on wheel (pounds)=n j W e, where W e and n j are... 14 Aeronautics and Space 1 2010-01-01 2010-01-01 false Wheel Spin-Up and Spring-Back Loads D.... D Appendix D to Part 23—Wheel Spin-Up and Spring-Back Loads D23.1 Wheel spin-up loads. (a) The...

14 CFR Appendix D to Part 23 - Wheel Spin-Up and Spring-Back Loads

Code of Federal Regulations, 2012 CFR

2012-01-01

... (0.80 may be used); F Vmax=maximum vertical force on wheel (pounds)=n j W e, where W e and n j are... 14 Aeronautics and Space 1 2012-01-01 2012-01-01 false Wheel Spin-Up and Spring-Back Loads D.... D Appendix D to Part 23—Wheel Spin-Up and Spring-Back Loads D23.1 Wheel spin-up loads. (a) The...

Organic Nitrogen Utilization by Phytoplankton: The Role of Cell-Surface Deaminases

DTIC Science & Technology

1989-06-01

Pleurochrysis carterae (Coccoll-N) is a coccolithless clone isolated by the authors from Coccoll. Emiliania huxleyi (12-1) was isolated from the Sargasso Sea...another coccolithophorid, Emiliania huxleyi from the Sargasso Sea (now 12-1, CCMP) for L-amino acid oxidase activity. No activity was found under log...acid oxidase regulation. Saturated oxidase rate constants (Vmax) are shown for Pleurochrysis isolates and one Emiliania huxleyi isolate (12-1). Nlim

Optimized extraction by cetyl trimethyl ammonium bromide reversed micelles of xylose reductase and xylitol dehydrogenase from Candida guilliermondii homogenate.

PubMed

Cortez, Ely Vieira; Pessoa, Adalberto; das Graças de Almeida Felipe, Maria; Roberto, Inês Conceição; Vitolo, Michele

2004-07-25

The intracellular enzymes xylose reductase (XR, EC 1.1.1.21) and xylitol dehydrogenase (XD, EC 1.1.1.9) from Candida guilliermondii, grown in sugar cane bagasse hydrolysate, were separated by reversed micelles of cetyl trimethyl ammonium bromide (CTAB) cationic surfactant. An experimental design was employed to optimize the extraction conditions of both enzymes. Under these conditions (temperature = 5 degree C, hexanol: isooctane proportion = 5% (v/v), 22 %, surfactant concentration = 0.15M, pH = 7.0 and electrical conductivity = 14 mScm(-1)) recovery values of about 100 and 80% were achieved for the enzymes XR and XD, respectively. The purity of XR and XD increased 5.6- and 1.8-fold, respectively. The extraction process caused some structural modifications in the enzymes molecules, as evidenced by the alteration of K(M) values determined before and after extraction, either in regard to the substrate (up 35% for XR and down 48% for XD) or cofactor (down 29% for XR and up 11% for XD). However, the average variation of V(max) values for both enzymes was not higher than 7%, indicating that the modified affinity of enzymes for their respective substrates and cofactors, as consequence of structural modifications suffered by them during the extraction, are compensated in some extension. This study demonstrated that liquid-liquid extraction by CTAB reversed micelles is an efficient process to separate the enzymes XR and XD present in the cell extract, and simultaneously increase the enzymatic activity and the purity of both enzymes produced by C. guilliermondii.

Number of Players and Relative Pitch Area per Player: Comparing Their Influence on Heart Rate and Physical Demands in Under-12 and Under-13 Football Players

PubMed Central

Castellano, Julen; Puente, Asier; Echeazarra, Ibon; Usabiaga, Oidui; Casamichana, David

2016-01-01

The aim of the present study is to analyse the influence of different large-sided games (LSGs) on the physical and physiological variables in under-12s (U12) and -13s (U13) soccer players. The effects of the combination of different number of players per team, 7, 9, and 11 (P7, P9, and P11, respectively) with three relative pitch areas, 100, 200, and 300 m2 (A100, A200, and A300, respectively), were analysed in this study. The variables analysed were: 1) global indicator such as total distance (TD); work:rest ratio (W:R); player-load (PL) and maximal speed (Vmax); 2) heart rate (HR) mean and time spent in different intensity zones of HR (<75%, 75–84%, 84–90% and >90%), and; 3) five absolute (<8, 8–13, 13–16 and >16 Km h-1) and three relative speed categories (<40%, 40–60% and >60% Vmax). The results support the theory that a change in format (player number and pitch dimensions) affects no similarly in the two players categories. Although it can seem that U13 players are more demanded in this kind of LSG, when the work load is assessed from a relative point of view, great pitch dimensions and/or high number of player per team are involved in the training task to the U12 players. The results of this study could alert to the coaches to avoid some types of LSGs for the U12 players such as: P11 played in A100, A200 or A300, P9 played in A200 or A300 and P7 played in A300 due to that U13>U12 in several physical and physiological variables (W:R, time spent in 84–90%HRmax, distance in 8–13 and 13–16 Km h-1 and time spent in 40–60%Vmax). These results may help youth soccer coaches to plan the progressive introduction of LSGs so that task demands are adapted to the physiological and physical development of participants. PMID:26752422

Energetic aspects of skeletal muscle contraction: implications of fiber types.

PubMed

Rall, J A

1985-01-01

In this chapter fundamental energetic properties of skeletal muscles as elucidated from isolated muscle preparations are described. Implications of these intrinsic properties for the energetic characterization of different fiber types and for the understanding of locomotion have been considered. Emphasis was placed on the myriad of physical and chemical techniques that can be employed to understand muscle energetics and on the interrelationship of results from different techniques. The anaerobic initial processes which liberate energy during contraction and relaxation are discussed in detail. The high-energy phosphate (approximately P) utilized during contraction and relaxation can be distributed between actomyosin ATPase or cross-bridge cycling (70%) and the Ca2+ ATPase of the sacroplasmic reticulum (30%). Muscle shortening increases the rate of approximately P hydrolysis, and stretching a muscle during contraction suppresses the rate of approximately P hydrolysis. The economy of an isometric contraction is defined as the ratio of isometric mechanical response to energetic cost and is shown to be a fundamental intrinsic parameter describing muscle energetics. Economy of contraction varies across the animal kingdom by over three orders of magnitude and is different in different mammalian fiber types. In mammalian skeletal muscles differences in economy of contraction can be attributed mainly to differences in the specific actomyosin and Ca2+ ATPase of muscles. Furthermore, there is an inverse relationship between economy of contraction and maximum velocity of muscle shortening (Vmax) and maximum power output. This is a fundamental relationship. Muscles cannot be economical at developing and maintaining force and also exhibit rapid shortening. Interestingly, there appears to be a subtle system of unknown nature that modulates the Vmax and economy of contraction. Efficiency of a work-producing contraction is defined and contrasted to the economy of contraction. Unlike economy, maximum efficiency of work production varies little across the animal kingdom. There are difficulties associated with the measurement of maximum efficiency of contraction, and it has yet to be determined unequivocally if the maximum efficiency of contraction varies in different fiber types. The intrinsic properties of force per cross-sectional area, economy, and Vmax determine the basic energetic properties of skeletal muscles. Nonetheless, the mechanics and energetics of skeletal muscles in the body are profoundly influenced by muscle architecture, attachment of muscles to the skeleton, and motor unit organization.(ABSTRACT TRUNCATED AT 400 WORDS)

In vitro metabolism and drug-drug interaction potential of UTL-5g, a novel chemo- and radioprotective agent.

PubMed

Wu, Jianmei; Shaw, Jiajiu; Dubaisi, Sarah; Valeriote, Frederick; Li, Jing

2014-12-01

N-(2,4-dichlorophenyl)-5-methyl-1,2-oxazole-3-carboxamide (UTL-5g), a potential chemo- and radioprotective agent, acts as a prodrug requiring bioactivation to the active metabolite 5-methylisoxazole-3-carboxylic acid (ISOX). UTL-5g hydrolysis to ISOX and 2,4-dichloroaniline (DCA) has been identified in porcine and rabbit liver esterases. The purpose of this study was to provide insights on the metabolism and drug interaction potential of UTL-5g in humans. The kinetics of UTL-5g hydrolysis was determined in human liver microsomes (HLM) and recombinant human carboxylesterases (hCE1b and hCE2). The potential of UTL-5g and its metabolites for competitive inhibition and time-dependent inhibition of microsomal cytochrome P450 (P450) was examined in HLM. UTL-5g hydrolysis to ISOX and DCA in HLM were NADPH-independent, with a maximum rate of reaction (Vmax) of 11.1 nmol/min per mg and substrate affinity (Km) of 41.6 µM. Both hCE1b and hCE2 effectively catalyzed UTL-5g hydrolysis, but hCE2 exhibited ∼30-fold higher catalytic efficiency (Vmax/Km) than hCE1b. UTL-5g and DCA competitively inhibited microsomal CYP1A2, CYP2B6, and CYP2C19 (IC50 values <50 µM), and exhibited time-dependent inhibition of microsomal CYP1A2 with the inactivation efficiency (kinact/KI) of 0.68 and 0.51 minute(-1)·mM(-1), respectively. ISOX did not inhibit or inactivate any tested microsomal P450. In conclusion, hCE1b and hCE2 play a key role in the bioactivation of UTL-5g. Factors influencing carboxylesterase activities may have a significant impact on the pharmacological and therapeutic effects of UTL-5g. UTL-5g has the potential to inhibit P450-mediated metabolism through competitive inhibition or time-dependent inhibition. Caution is particularly needed for potential drug interactions involving competitive inhibition or time-dependent inhibition of CYP1A2 in the future clinical development of UTL-5g. Copyright © 2014 by The American Society for Pharmacology and Experimental Therapeutics.

In Vitro Metabolism and Drug-Drug Interaction Potential of UTL-5g, a Novel Chemo- and Radioprotective Agent

PubMed Central

Wu, Jianmei; Shaw, Jiajiu; Dubaisi, Sarah; Valeriote, Frederick

2014-01-01

N-(2,4-dichlorophenyl)-5-methyl-1,2-oxazole-3-carboxamide (UTL-5g), a potential chemo- and radioprotective agent, acts as a prodrug requiring bioactivation to the active metabolite 5-methylisoxazole-3-carboxylic acid (ISOX). UTL-5g hydrolysis to ISOX and 2,4-dichloroaniline (DCA) has been identified in porcine and rabbit liver esterases. The purpose of this study was to provide insights on the metabolism and drug interaction potential of UTL-5g in humans. The kinetics of UTL-5g hydrolysis was determined in human liver microsomes (HLM) and recombinant human carboxylesterases (hCE1b and hCE2). The potential of UTL-5g and its metabolites for competitive inhibition and time-dependent inhibition of microsomal cytochrome P450 (P450) was examined in HLM. UTL-5g hydrolysis to ISOX and DCA in HLM were NADPH-independent, with a maximum rate of reaction (Vmax) of 11.1 nmol/min per mg and substrate affinity (Km) of 41.6 µM. Both hCE1b and hCE2 effectively catalyzed UTL-5g hydrolysis, but hCE2 exhibited ∼30-fold higher catalytic efficiency (Vmax/Km) than hCE1b. UTL-5g and DCA competitively inhibited microsomal CYP1A2, CYP2B6, and CYP2C19 (IC50 values <50 µM), and exhibited time-dependent inhibition of microsomal CYP1A2 with the inactivation efficiency (kinact/KI) of 0.68 and 0.51 minute−1·mM−1, respectively. ISOX did not inhibit or inactivate any tested microsomal P450. In conclusion, hCE1b and hCE2 play a key role in the bioactivation of UTL-5g. Factors influencing carboxylesterase activities may have a significant impact on the pharmacological and therapeutic effects of UTL-5g. UTL-5g has the potential to inhibit P450-mediated metabolism through competitive inhibition or time-dependent inhibition. Caution is particularly needed for potential drug interactions involving competitive inhibition or time-dependent inhibition of CYP1A2 in the future clinical development of UTL-5g. PMID:25249693

Effects of high-intensity interval training and moderate-intensity continuous training on glycaemic control and skeletal muscle mitochondrial function in db/db mice.

PubMed

Chavanelle, Vivien; Boisseau, Nathalie; Otero, Yolanda F; Combaret, Lydie; Dardevet, Dominique; Montaurier, Christophe; Delcros, Geoffrey; Peltier, Sébastien L; Sirvent, Pascal

2017-03-16

Physical activity is known as an effective strategy for prevention and treatment of Type 2 Diabetes. The aim of this work was to compare the effects of a traditional Moderate Intensity Continuous Training (MICT) with a High Intensity Interval Training (HIIT) on glucose metabolism and mitochondrial function in diabetic mice. Diabetic db/db male mice (N = 25) aged 6 weeks were subdivided into MICT, HIIT or control (CON) group. Animals in the training groups ran on a treadmill 5 days/week during 10 weeks. MICT group ran for 80 min (0° slope) at 50-60% of maximal speed (Vmax) reached during an incremental test. HIIT group ran thirteen times 4 minutes (20° slope) at 85-90% of Vmax separated by 2-min-rest periods. HIIT lowered fasting glycaemia and HbA1c compared with CON group (p < 0.05). In all mitochondrial function markers assessed, no differences were noted between the three groups except for total amount of electron transport chain proteins, slightly increased in the HIIT group vs CON. Western blot analysis revealed a significant increase of muscle Glut4 content (about 2 fold) and higher insulin-stimulated Akt phosphorylation ratios in HIIT group. HIIT seems to improve glucose metabolism more efficiently than MICT in diabetic mice by mechanisms independent of mitochondrial adaptations.

Neuraminidase activity of blue eye disease porcine rubulavirus: Specificity, affinity and inhibition studies.

PubMed

Santos-López, Gerardo; Borraz-Argüello, María T; Márquez-Domínguez, Luis; Flores-Alonso, Juan Carlos; Ramírez-Mendoza, Humberto; Priem, Bernard; Fort, Sébastien; Vallejo-Ruiz, Verónica; Reyes-Leyva, Julio; Herrera-Camacho, Irma

2017-10-01

Porcine rubulavirus (PorPV), also known as La Piedad Michoacan Virus (LPMV) causes encephalitis and reproductive failure in newborn and adult pigs, respectively. The hemagglutinin-neuraminidase (HN) glycoprotein is the most exposed and antigenic of the virus proteins. HN plays central roles in PorPV infection; i.e., it recognizes sialic acid-containing cell receptors that mediate virus attachment and penetration; in addition, its neuraminidase (sialic acid releasing) activity has been proposed as a virulence factor. This work describes the purification and characterization of PorPV HN protein (isolate PAC1). The specificity of neuraminidase is restricted to sialyl(α2,3)lactose (3SL). HN showed typical Michaelis-Menten kinetics with fetuin as substrate (km=0.029μM, Vmax=522.8nmolmin -1 mg -1 ). When 3SL was used as substrate, typical cooperative kinetics were found (S 50 =0.15μM, Vmax=154.3nmolmin -1 mg -1 ). The influenza inhibitor zanamivir inhibited the PorPV neuraminidase with IC 50 of 0.24μM. PorPV neuraminidase was activated by Ca 2+ and inhibited by nucleoside triphosphates with the level of inhibition depending on phosphorylation level. The present results open possibilities to study the role of neuraminidase in the pathogenicity of PorPV infection and its potential inhibitors. Copyright © 2017 Elsevier Ltd. All rights reserved.

Individual differences in impulsive action and dopamine transporter function in rat orbitofrontal cortex.

PubMed

Yates, J R; Darna, M; Beckmann, J S; Dwoskin, L P; Bardo, M T

2016-01-28

Impulsivity, which can be subdivided into impulsive action and impulsive choice, is implicated as a factor underlying drug abuse vulnerability. Although previous research has shown that dopamine (DA) systems in prefrontal cortex are involved in impulsivity and substance abuse, it is not known if inherent variation in DA transporter (DAT) function contributes to impulsivity. The current study determined if individual differences in either impulsive action or impulsive choice are related to DAT function in orbitofrontal (OFC) and/or medial prefrontal cortex (mPFC). Rats were first tested both for impulsive action in a cued go/no-go task and for impulsive choice in a delay-discounting task. Following behavioral evaluation, in vitro [(3)H]DA uptake assays were performed in OFC and mPFC isolated from individual rats. Vmax in OFC, but not mPFC, was correlated with performance in the cued go/no-go task, with decreased OFC DAT function being associated with high impulsive action. In contrast, Vmax in OFC and mPFC was not correlated with performance in the delay-discounting task. The current results demonstrate that impulsive behavior in cued go/no-go performance is associated with decreased DAT function in OFC, suggesting that hyperdopaminergic tone in this prefrontal subregion mediates, at least in part, increased impulsive action. Copyright © 2015 IBRO. Published by Elsevier Ltd. All rights reserved.

Kinetic studies of lipid oxidation induced by hemoglobin measured by consumption of dissolved oxygen in a liposome model system.

PubMed

Carvajal, Ana Karina; Rustad, Turid; Mozuraityte, Revilija; Storrø, Ivar

2009-09-09

The effect of hemoglobin (Hb) and lipid concentration, pH, temperature, and different antioxidants on heme-mediated lipid oxidation of liposomes from marine phospholipids was studied. The rate of lipid oxidation was measured by consumption of dissolved oxygen. Heme-mediated lipid oxidation at different Hb and lipid concentrations was modeled by Michaelis-Menten kinetics. The maximum rate (V(max)) for the reaction with cod and bovine Hb as a pro-oxidant was 66.2 +/- 3.4 and 56.6 +/- 3.4 microM/min, respectively. The Michaelis-Menten constant (K(m)) for the reaction with cod and bovine Hb was 0.67 +/- 0.09 and 1.2 +/- 0.2 microM, respectively. V(max) for the relationship between the oxygen uptake rate and lipid concentration was 43.2 +/- 1.5 microM/min, while the K(m) was 0.93 +/- 0.14 mg/mL. The effect of the temperature followed Arrhenius kinetics, and there was no significant difference in activation energy between cod and bovine Hb. The rate of lipid oxidation induced by bovine Hb was highest around pH 6. Ethylenediaminetetraacetic acid (EDTA) had no significant effect on heme-mediated lipid oxidation, but alpha-tocopherol and astaxanthin worked well as antioxidants. Kinetic differences were found between iron and Hb as pro-oxidants, and the efficacy of the antioxidants depended upon the pro-oxidant in the system.

Effects of metal ions on the catalytic degradation of dicofol by cellulase.

PubMed

Zhai, Zihan; Yang, Ting; Zhang, Boya; Zhang, Jianbo

2015-07-01

A new technique whereby cellulase immobilized on aminated silica was applied to catalyze the degradation of dicofol, an organochlorine pesticide. In order to evaluate the performance of free and immobilized cellulase, experiments were carried out to measure the degradation efficiency. The Michaelis constant, Km, of the reaction catalyzed by immobilized cellulase was 9.16 mg/L, and the maximum reaction rate, Vmax, was 0.40 mg/L/min, while that of free cellulase was Km=8.18 mg/L, and Vmax=0.79 mg/L/min, respectively. The kinetic constants of catalytic degradation were calculated to estimate substrate affinity. Considering that metal ions may affect enzyme activity, the effects of different metal ions on the catalytic degradation efficiency were explored. The results showed that the substrate affinity decreased after immobilization. Monovalent metal ions had no effect on the reaction, while divalent metal ions had either positive or inhibitory effects, including activation by Mn2+, reversible competition with Cd2+, and irreversible inhibition by Pb2+. Ca2+ promoted the catalytic degradation of dicofol at low concentrations, but inhibited it at high concentrations. Compared with free cellulase, immobilized cellulase was affected less by metal ions. This work provided a basis for further studies on the co-occurrence of endocrine-disrupting chemicals and heavy metal ions in the environment. Copyright © 2015. Published by Elsevier B.V.

Acute and chronic exposure of rat intestinal mucosa to dextran promotes SGLTI-mediated glucose transport.

PubMed

Debnam, E S; Denholm, E E; Grimble, G K

1998-08-01

The intestinal handling of dextran, an alpha-1,6-linked glucose polymer, is poor compared with starch, and some ingested dextran might therefore reach the lower small intestine. As luminal sugar up-regulates SGLT1 (sodium-dependent glucose transporter) locally, we report the effects of a dextran-enriched diet on jejunal and ileal brush border membrane (BBM) glucose uptake. Rats were maintained on a diet containing 65% maltodextrin or 32.5% maltodextrin + 32.5% dextran (10 kD or 40 kD) for 8-10 days, and the kinetics of phlorizin-sensitive [3H]-glucose uptake by purified BBM vesicles was determined. Ingestion of 40-kD but not 10-kD dextran increased Vmax for jejunal and ileal glucose uptake (+64.3% and +61.8% respectively, both P < 0.02). The transport response to 40-kD dextran was in keeping with lower levels of expired H2 at the end of the feeding period. High-performance liquid chromatography (HPLC) analysis of luminal contents indicated extensive hydrolysis of ingested dextran. Finally, 3-h jejunal exposure to 40-kD dextran in vivo increased the Vmax for glucose uptake by jejunal BBM. It is likely that increased SGLT1-mediated glucose uptake after short or longer term mucosal exposure to dextran results from luminal dextran per se or a hydrolysis product. The clinical implications of this up-regulation are discussed.

Methodological uncertainty in quantitative prediction of human hepatic clearance from in vitro experimental systems.

PubMed

Hallifax, D; Houston, J B

2009-03-01

Mechanistic prediction of unbound drug clearance from human hepatic microsomes and hepatocytes correlates with in vivo clearance but is both systematically low (10 - 20 % of in vivo clearance) and highly variable, based on detailed assessments of published studies. Metabolic capacity (Vmax) of commercially available human hepatic microsomes and cryopreserved hepatocytes is log-normally distributed within wide (30 - 150-fold) ranges; Km is also log-normally distributed and effectively independent of Vmax, implying considerable variability in intrinsic clearance. Despite wide overlap, average capacity is 2 - 20-fold (dependent on P450 enzyme) greater in microsomes than hepatocytes, when both are normalised (scaled to whole liver). The in vitro ranges contrast with relatively narrow ranges of clearance among clinical studies. The high in vitro variation probably reflects unresolved phenotypical variability among liver donors and practicalities in processing of human liver into in vitro systems. A significant contribution from the latter is supported by evidence of low reproducibility (several fold) of activity in cryopreserved hepatocytes and microsomes prepared from the same cells, between separate occasions of thawing of cells from the same liver. The large uncertainty which exists in human hepatic in vitro systems appears to dominate the overall uncertainty of in vitro-in vivo extrapolation, including uncertainties within scaling, modelling and drug dependent effects. As such, any notion of quantitative prediction of clearance appears severely challenged.

Effect of complete protein 4.1R deficiency on ion transportproperties of murine erythrocytes

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

Rivera, Alicia; De Franceschi, Lucia; Peters, Luanne L.

2006-06-02

Moderate hemolytic anemia, abnormal erythrocyte morphology(spherocytosis), and decreased membrane stability are observed in micewith complete deficiency of all erythroid protein 4.1 protein isoforms(4.1-/-; Shi TS et al., J. Clin. Invest. 103:331,1999). We have examinedthe effects of erythroid protein 4.1 (4.1R) deficiency on erythrocytecation transport and volume regulation. 4.1-/- mice exhibited erythrocytedehydration that was associated with reduced cellular K and increased Nacontent. Increased Na permeability was observed in these mice, mostlymediated by Na/H exchange with normal Na-K pump and Na-K-2Cl cotransportactivities. The Na/H exchange of 4.1-/- erythrocytes was markedlyactivated by exposure to hypertonic conditions (18.2+- 3.2 in 4.1 -/- vs.9.8 +-more » 1.3 mmol/1013 cell x h in control mice), with an abnormaldependence on osmolarity, (K0.5=417 +- 42 in 4.1 -/- vs. 460 +- 35 mOsmin control mice) suggestive of an up-regulated functional state. Whilethe affinity for internal protons was not altered (K0.5= 489.7 +- 0.7 vs.537.0+- 0.56 nM in control mice), the Vmax of the H-induced Na/H exchangeactivity was markedly elevated in 4.1-/- erythrocytes (Vmax 91.47Moderatehemolytic anemia, abnormal erythrocyte morphology (spherocytosis), anddecreased membrane stability are observed in mice with completedeficiency of all erythroid protein 4.1 protein isoforms (4.1-/-; Shi TSet al., J. Clin. Invest. 103:331,1999). We have examined the effects oferythroid protein 4.1 (4.1R) deficiency on erythrocyte cation transportand volume regulation. 4.1-/- mice exhibited erythrocyte dehydration thatwas associated with reduced cellular K and increased Na content.Increased Na permeability was observed in these mice, mostly mediated byNa/H exchange with normal Na-K pump and Na-K-2Cl cotransport activities.The Na/H exchange of 4.1-/- erythrocytes was markedly activated byexposure to hypertonic conditions (18.2 +- 3.2 in 4.1 -/- vs. 9.8 +- 1.3mmol/1013 cell x h in control mice), with an abnormal dependence onosmolarity, (K0.5=417 +- 42 in 4.1 -/- vs. 460 +- 35 mOsm in controlmice) suggestive of an up-regulated functional state. While the affinityfor internal protons was not altered (K0.5= 489.7 +- 0.7 vs. 537.0 +-0.56 nM in control mice), the Vmax of the H-induced Na/H exchangeactivity was markedly elevated in 4.1-/- erythrocytes (Vmax 91.47+-7.2compared to 46.52+-5.4 mmol/1013 cell x h in control mice). Na/H exchangeactivation by okadaic acid was absent in 4.1-/- erythrocytes. Altogether,these results suggest that erythroid protein 4.1 plays a major role involume regulation and physiologically down-regulates Na/H exchange inmouse erythrocytes. Up-regulation of the Na/H exchange is an importantcontributor to the elevated cell Na content of 4.1 -/- erythrocytes.-7.2compared to 46.52+-5.4 mmol/1013 cell x h in control mice). Na/H exchangeactivation by okadaic acid was absent in 4.1-/- erythrocytes. Altogether,these results suggest that erythroid protein 4.1 plays a major role involume regulation and physiologically down-regulates Na/H exchange inmouse erythrocytes. Up-regulation of the Na/H exchange is an importantcontributor to the elevated cell Na content of 4.1 -/-erythrocytes.« less

Expression of drug transporters and drug metabolizing enzymes in the bladder urothelium in man and affinity of the bladder spasmolytic trospium chloride to transporters likely involved in its pharmacokinetics.

PubMed

Bexten, Maria; Oswald, Stefan; Grube, Markus; Jia, Jia; Graf, Tanja; Zimmermann, Uwe; Rodewald, Kathrin; Zolk, Oliver; Schwantes, Ulrich; Siegmund, Werner; Keiser, Markus

2015-01-05

The cationic, water-soluble quaternary trospium chloride (TC) is incompletely absorbed from the gut and undergoes wide distribution but does not pass the blood-brain barrier. It is secreted by the kidneys, liver, and intestine. To evaluate potential transport mechanisms for TC, we measured affinity of the drug to the human uptake and efflux transporters known to be of pharmacokinetic relevance. Affinity of TC to the uptake transporters OATP1A2, -1B1, -1B3, -2B1, OCT1, -2, -3, OCTN2, NTCP, and ASBT and the efflux carriers P-gp, MRP2 and MRP3 transfected in HEK293 and MDCK2 cells was measured. To identify relevant pharmacokinetic mechanisms in the bladder urothelium, mRNA expression of multidrug transporters, drug metabolizing enzymes, and nuclear receptors, and the uptake of TC into primary human bladder urothelium (HBU) cells were measured. TC was shown to be a substrate of OATP1A2 (Km = 6.9 ± 1.3 μmol/L; Vmax = 41.6 ± 1.8 pmol/mg·min), OCT1 (Km = 106 ± 16 μmol/L; Vmax = 269 ± 18 pmol/mg·min), and P-gp (Km = 34.9 ± 7.5 μmol/L; Vmax = 105 ± 9.1 pmol/mg·min, lipovesicle assay). The genetic OATP1A2 variants *2 and *3 were loss-of-function transporters for TC. The mRNA expression analysis identified the following transporter proteins in the human urothelium: ABCB1 (P-gp), ABCC1-5 (MRP1-5), ABCG2 (BCRP), SLCO2B1 (OATP2B1), SLCO4A1 (OATP4A1), SLC22A1 (OCT1), SLC22A3 (OCT3), SLC22A4 (OCTN1), SLC22A5 (OCTN2), and SLC47A1 (MATE1). Immuno-reactive P-gp and OATP1A2 were localized to the apical cell layers. Drug metabolizing enzymes CYP3A5, -2B6, -2B7 -2E1, SULT1A1-4, UGT1A1-10, and UGT2B15, and nuclear receptors NR1H3 and NR1H4 were also expressed on mRNA level. TC was taken up into HBU cells (Km = 18.5 ± 4.8 μmol/L; Vmax = 106 ± 11.3 pmol/mg·min) by mechanisms that could be synergistically inhibited by naringin (IC50 = 10.8 (8.4; 13.8) μmol/L) and verapamil (IC50 = 4.6 (2.8; 7.5) μmol/L), inhibitors of OATP1A2 and OCT1, respectively. Affinity of TC to OCT1 and P-glycoprotein may be the reason for incomplete oral absorption, wide distribution into liver and kidneys, and substantial intestinal and renal secretions. Absence of brain distribution may result from affinity to P-gp and a low affinity to OATP1A2. The human urothelium expresses many drug transporters and drug metabolizing enzymes that may interact with TC and other drugs eliminated into the urine.

Significance of the enzymatic properties of yeast S39A enolase to the catalytic mechanism.

PubMed

Brewer, J M; Glover, C V; Holland, M J; Lebioda, L

1998-04-02

The S39A mutant of yeast enolase (isozyme 1), prepared by site-directed mutagenesis, has a relative Vmax of 0.01% and an activation constant for Mg2+ ca. 10-fold higher, compared with native enzyme. It is correctly folded. There is little effect of solvent viscosity on activity. We think that the loop Ser36-His43 fails to move to the 'closed' position upon catalytic Mg2+ binding, weakening several electrostatic interactions involved in the mechanism.

Kinetic characterization and radiation-target sizing of the glucose transporter in cardiac sarcolemmal vesicles.

PubMed

Dale, W E; Tsai, Y S; Jung, C Y; Hale, C C; Rovetto, M J; Kim, H D; Yung, C Y

1988-08-18

Stereospecific glucose transport was assayed and characterized in bovine cardiac sarcolemmal vesicles. Sarcolemmal vesicles were incubated with D-[3H]glucose or L-[3H]glucose at 25 degrees C. The reaction was terminated by rapid addition of 4 mM HgCl2 and vesicles were immediately collected on glass fiber filters for quantification of accumulated [3H]glucose. Non-specific diffusion of L-[3H]glucose was never more than 11% of total D-[3H]glucose transport into the vesicles. Stereospecific uptake of D-[3H]glucose reached a maximum level by 20 s. Cytochalasin B (50 microM) inhibited specific transport of D-[3H]glucose to the level of that for non-specific diffusion. The vesicles exhibited saturable transport (Km = 9.3 mM; Vmax = 2.6 nmol/mg per s) and the transporter turnover number was 197 glucose molecules per transporter per s. The molecular sizes of the cytochalasin B binding protein and the D-glucose transport protein in sarcolemmal vesicles were estimated by radiation inactivation. These values were 77 and 101 kDa, respectively, and by the Wilcoxen Rank Sum Test were not significantly different from each other.

Purification and characterization of peroxidase from cauliflower (Brassica oleracea L. var. botrytis) buds.

PubMed

Köksal, Ekrem; Gülçin, Ilhami

2008-01-01

Peroxidases (EC 1.11.1.7; donor: hydrogen peroxide oxidoreductase) are part of a large group of enzymes. In this study, peroxidase, a primer antioxidant enzyme, was purified with 19.3 fold and 0.2% efficiency from cauliflower (Brassica oleracea L.) by ammonium sulphate precipitation, dialysis, CM-Sephadex ion-exchange chromatography and Sephadex G-25 purification steps. The substrate specificity of peroxidase was investigated using 2,2'-azino-bis(3-ethylbenz-thiazoline-6-sulphonic acid) (ABTS), 2-methoxyphenol (guaiacol), 1,2-dihydroxybenzene (catechol), 1,2,3-trihyidroxybenzene (pyrogallol) and 4-methylcatechol. Also, optimum pH, optimum temperature, optimum ionic strength, stable pH, stable temperature, thermal inactivation conditions were determined for guaiacol/H(2)O(2), pyrogallol/H(2)O(2), ABTS/H(2)O(2), catechol/H(2)O(2) and 4-methyl catechol/H(2)O(2) substrate patterns. The molecular weight (M(w)) of this enzyme was found to be 44 kDa by gel filtration chromatography method. Native polyacrylamide gel electrophoresis (PAGE) was performed for isoenzyme determination and a single band was observed. K(m) and V(max) values were calculated from Lineweaver-Burk graph for each substrate patterns.

[Antibacterial activity of sulopenem, a new parenteral penem antibiotic].

PubMed

Inoue, E; Komoto, E; Taniyama, Y; Mitsuhashi, S

1996-04-01

Sulopenem, a new penem antibiotic, was compared with other antibiotics with regard to in vitro antibacterial and bactericidal activities, stabilization against beta-lactamases, and effect on the release of lipopolysaccharide from Gram-negative bacteria. The results are summarized as follows. 1. Sulopenem showed more potent activities than other antibiotics against both Gram-positive and Gram-negative bacteria except Pseudomonas aeruginosa. 2. Sulopenem showed potent bactericidal activities (MIC/MBC) against both Gram-positive and Gram-negative bacteria. Time kill studies against Staphylococcus aureus, Escherichia coli, Enterobacter cloacae and Citrobacter freundii showed potent bactericidal activities of sulopenem. 3. Sulopenem was found to possess a stronger activity than other antibiotics against beta-lactamase-producing strains except P. aeruginosa and Stenotrophomonas maltophilia. 4. In particular, sulopenem was found to be more stable to the hydrolysis by various beta-lactamases produced by Gram-negative bacteria than any other antibiotics tested. Vmax/Km values of sulopenem were smaller than those of cefotiam for all tested beta-lactamases, which reflected a broad antibacterial spectrum of sulopenem. 5. E. coli ML4707 exposed to sulopenem and imipenem released less endotoxin than did controls at all concentration ranges tested. In contrast, the strain exposed to ceftazidime at bacteriostatic concentrations released a large amount of endotoxin.

The key nickel enzyme of methanogenesis catalyses the anaerobic oxidation of methane.

PubMed

Scheller, Silvan; Goenrich, Meike; Boecher, Reinhard; Thauer, Rudolf K; Jaun, Bernhard

2010-06-03

Large amounts (estimates range from 70 Tg per year to 300 Tg per year) of the potent greenhouse gas methane are oxidized to carbon dioxide in marine sediments by communities of methanotrophic archaea and sulphate-reducing bacteria, and thus are prevented from escaping into the atmosphere. Indirect evidence indicates that the anaerobic oxidation of methane might proceed as the reverse of archaeal methanogenesis from carbon dioxide with the nickel-containing methyl-coenzyme M reductase (MCR) as the methane-activating enzyme. However, experiments showing that MCR can catalyse the endergonic back reaction have been lacking. Here we report that purified MCR from Methanothermobacter marburgensis converts methane into methyl-coenzyme M under equilibrium conditions with apparent V(max) (maximum rate) and K(m) (Michaelis constant) values consistent with the observed in vivo kinetics of the anaerobic oxidation of methane with sulphate. This result supports the hypothesis of 'reverse methanogenesis' and is paramount to understanding the still-unknown mechanism of the last step of methanogenesis. The ability of MCR to cleave the particularly strong C-H bond of methane without the involvement of highly reactive oxygen-derived intermediates is directly relevant to catalytic C-H activation, currently an area of great interest in chemistry.

Adsorption of peroxidase on Celite 545 directly from ammonium sulfate fractionated white radish (Raphanus sativus) proteins.

PubMed

Satar, Rukhsana; Husain, Qayyum

2009-03-01

This paper demonstrates the direct immobilization of peroxidase from ammonium sulfate fractionated white radish proteins on an inorganic support, Celite 545. The adsorbed peroxidase was crosslinked by using glutaraldehyde. The activity yield for white radish peroxidase was adsorbed on Celite 545 was 70% and this activity was decreased and remained 60% of the initial activity after crosslinking by glutaraldehyde. The pH and temperature-optima for both soluble and immobilized peroxidase was at pH 5.5 and 40 degrees C. Immobilized peroxidase retained higher stability against heat and water-miscible organic solvents. In the presence of 5.0 mM mercuric chloride, immobilized white radish peroxidase retained 41% of its initial activity while the free enzyme lost 93% activity. Soluble enzyme lost 61% of its initial activity while immobilized peroxidase retained 86% of the original activity when exposed to 0.02 mM sodium azide for 1 h. The K(m) values were 0.056 and 0.07 mM for free and immobilized enzyme, respectively. Immobilized white radish peroxidase exhibited lower V(max) as compared to the soluble enzyme. Immobilized peroxidase preparation showed better storage stability as compared to its soluble counterpart.

Comparative Characterization of Complete and Truncated Forms of Lactobacillus amylovorus α-Amylase and Role of the C-Terminal Direct Repeats in Raw-Starch Binding

PubMed Central

Rodriguez Sanoja, R.; Morlon-Guyot, J.; Jore, J.; Pintado, J.; Juge, N.; Guyot, J. P.

2000-01-01

Two constructs derived from the α-amylase gene (amyA) of Lactobacillus amylovorus were expressed in Lactobacillus plantarum, and their expression products were purified, characterized, and compared. These products correspond to the complete (AmyA) and truncated (AmyAΔ) forms of α-amylase; AmyAΔ lacks the 66-kDa carboxyl-terminal direct-repeating-unit region. AmyA and AmyAΔ exhibit similar amylase activities towards a range of soluble substrates (amylose, amylopectin and α-cyclodextrin, and soluble starch). The specific activities of the enzymes towards soluble starch are similar, but the KM and Vmax values of AmyAΔ were slightly higher than those of AmyA, whereas the thermal stability of AmyAΔ was lower than that of AmyA. In contrast to AmyA, AmyAΔ is unable to bind to β-cyclodextrin and is only weakly active towards glycogen. More striking is the fact that AmyAΔ cannot bind or hydrolyze raw starch, demonstrating that the carboxyl-terminal repeating-unit domain of AmyA is required for raw-starch binding activity. PMID:10919790

Novel thrombolytic protease from edible and medicinal plant Aster yomena (Kitam.) Honda with anticoagulant activity: purification and partial characterization.

PubMed

Choi, Jun-Hui; Kim, Dae-Won; Park, Se-Eun; Choi, Bong-Suk; Sapkota, Kumar; Kim, Seung; Kim, Sung-Jun

2014-10-01

A thrombolytic protease named kitamase possessing anticoagulant property was purified from edible and medicinal plant Aster yomena (Kitam.) Honda. Kitamase showed a molecular weight of 50 kDa by SDS-PAGE and displayed a strong fibrin zymogram lysis band corresponding to the similar molecular mass. The enzyme was active at high temperatures (50°C). The fibrinolytic activity of kitamase was strongly inhibited by EDTA, EGTA, TPCK and PMSF, inhibited by Zn(2+). The Km and Vmax values for substrate S-2251 were determined as 4.31 mM and 23.81 mM/mg respectively. It dissolved fibrin clot directly and specifically cleaved the α, Aα and γ-γ chains of fibrin and fibrinogen. In addition, kitamase delayed the coagulation time and increased activated partial thromboplastin time and prothrombin time. Kitamase exerted a significant protective effect against collagen and epinephrine induced pulmonary thromboembolism in mice. These results suggest that kitamase may have the property of metallo-protease like enzyme, novel fibrino(geno)lytic enzyme and a potential to be a therapeutic agent for thrombosis. Copyright © 2014 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

Metabolic efficiency and turnover of soil microbial communities in biodegradation tests.

PubMed Central

Shen, J; Bartha, R

1996-01-01

Biodegradability screening tests of soil commonly measure 14CO2 evolution from radiolabeled test compounds, and glucose has often served as a positive control. When constant amounts of radiolabel were added to soil in combination with increasing amounts of unlabeled substrates, glucose and some related hexoses behaved in an anomalous manner. In contrast to that of formate, benzoate, n-hexadecane, or bis(2-ethylhexyl) phthalate, dilution of glucose radiocarbon with unlabeled glucose increased rather than decreased the rate and extent of 14CO2 evolution. [14C]glucose incorporation into biomass and Vmax values were consistent with the interpretation that application of relatively high concentrations of glucose to soil shifts the balance of the soil microbial community from the autochthonous (humus-degrading) to the zymogeneous (opportunistic) segment. The higher growth and turnover rates that define zymogeneous microorganisms, combined with a lower level of carbon incorporation into their biomass, result in the evolution of disproportionate percentages of 14CO2. When used as positive controls, glucose and related hexoses may raise the expectations for percent 14CO2 evolution to levels that are not realistic for other biodegradable compounds. PMID:8779580

A novel xylan degrading β-D-xylosidase: purification and biochemical characterization.

PubMed

Michelin, Michele; Peixoto-Nogueira, Simone C; Silva, Tony M; Jorge, João A; Terenzi, Héctor F; Teixeira, José A; Polizeli, Maria de Lourdes T M

2012-11-01

Aspergillus ochraceus, a thermotolerant fungus isolated in Brazil from decomposing materials, produced an extracellular β-xylosidase that was purified using DEAE-cellulose ion exchange chromatography, Sephadex G-100 and Biogel P-60 gel filtration. β-xylosidase is a glycoprotein (39 % carbohydrate content) and has a molecular mass of 137 kDa by SDS-PAGE, with optimal temperature and pH at 70 °C and 3.0-5.5, respectively. β-xylosidase was stable in acidic pH (3.0-6.0) and 70 °C for 1 h. The enzyme was activated by 5 mM MnCl₂ (28 %) and MgCl₂ (20 %) salts. The β-xylosidase produced by A. ochraceus preferentially hydrolyzed p-nitrophenyl-β-D-xylopyranoside, exhibiting apparent K(m) and V(max) values of 0.66 mM and 39 U (mg protein)⁻¹ respectively, and to a lesser extent p-nitrophenyl-β-D-glucopyranoside. The enzyme was able to hydrolyze xylan from different sources, suggesting a novel β-D-xylosidase that degrades xylan. HPLC analysis revealed xylans of different compositions which allowed explaining the differences in specificity observed by β-xylosidase. TLC confirmed the capacity of the enzyme in hydrolyzing xylan and larger xylo-oligosaccharides, as xylopentaose.

Improvement of bread making quality by supplementation with a recombinant xylanase produced by Pichia pastoris.

PubMed

de Queiroz Brito Cunha, Carolina Cândida; Gama, Aline Rodrigues; Cintra, Lorena Cardoso; Bataus, Luiz Artur Mendes; Ulhoa, Cirano José

2018-01-01

Xylanases (EC 3.2.1.8) are hydrolytic enzymes, which randomly cleave the β-1,4-linked xylose residues from xylan. The synthetic gene xynBS27 from Streptomyces sp. S27 was successfully cloned and expressed in Pichia pastoris. The full-length gene consists of 729 bp and encodes 243 amino acids including 51 residues of a putative signal peptide. This enzyme was purified in two steps and was shown to have a molecular weight of 20 kDa. The purified r-XynBS27 was active against beechwood xylan and oat spelt xylan as expected for GH 11 family. The optimum pH and temperature values for the enzyme were 6.0 and 75 °C, respectively. The Km and Vmax were 12.38 mg/mL and 13.68 μmol min/mg, respectively. The r-XynBS27 showed high xylose tolerance and was inhibited by some metal ions and by SDS. r-XynBS27 was employed as an additive in the bread making process. A decrease in firmness, stiffness and consistency, and improvements in specific volume and reducing sugar content were recorded.

Characterization of a plasma membrane-associated prenylcysteine-directed alpha carboxyl methyltransferase in human neutrophils.

PubMed

Pillinger, M H; Volker, C; Stock, J B; Weissmann, G; Philips, M R

1994-01-14

Signal transduction in human neutrophils requires prenylcysteine-directed carboxyl methylation of ras-related low molecular weight GTP-binding proteins. We now report the subcellular localization and characterization of a neutrophil prenylcysteine alpha carboxyl methyltransferase. The highest carboxyl methyltransferase activity copurified with biotinylated neutrophil surface membranes, supporting a plasma membrane localization of the enzyme. Neutrophil nuclear fractions contained little or no methyltransferase activity. Methyltransferase activity was detergent-sensitive but could be reconstituted by removal of detergent in the presence of phosphatidyl choline and an anionic phospholipid. N-Acetyl-S-trans,trans-farnesyl-L-cysteine (AFC) and N-acetyl-S-all-trans-geranylgeranyl-L-cysteine (AGGC) were effective substrates for neutrophil prenylcysteine-directed methyltransferase; Vmax values for AFC and AGGC (16.4 and 22.1 pmol of methylated/mg protein/min, respectively) are among the highest yet reported. Although both GTP gamma S and the chemoattractant fMet-Leu-Phe stimulated methylation of ras-related proteins, neither affected methylation of AFC. These data suggest that neutrophil plasma membranes contain a phospholipid-dependent, prenylcysteine-directed carboxyl methyltransferase of relatively high specific activity that modifies ras-related protein substrates in the GTP-bound, activated state.

The dark side of galaxy colour

NASA Astrophysics Data System (ADS)

Hearin, Andrew P.; Watson, Douglas F.

2013-10-01

We present age distribution matching, a theoretical formalism for predicting how galaxies of luminosity L and colour C occupy dark matter haloes. Our model supposes that there are just two fundamental properties of a halo that determine the colour and brightness of the galaxy it hosts: the maximum circular velocity Vmax and the redshift zstarve that correlates with the epoch at which the star formation in the galaxy ceases. The halo property zstarve is intended to encompass physical characteristics of halo mass assembly that may deprive the galaxy of its cold gas supply and, ultimately, quench its star formation. The new, defining feature of the model is that, at fixed luminosity, galaxy colour is in monotonic correspondence with zstarve, with the larger values of zstarve being assigned redder colours. We populate an N-body simulation with a mock galaxy catalogue based on age distribution matching and show that the resulting mock galaxy distribution accurately describes a variety of galaxy statistics. Our model suggests that halo and galaxy assembly are indeed correlated. We make publicly available our low-redshift, Sloan Digital Sky Survey Mr < -19 mock galaxy catalogue, and main progenitor histories of all z = 0 haloes, at http://logrus.uchicago.edu/~aphearin

Branched-chain amino acid transport in Streptococcus mutans Ingbritt.

PubMed

Dashper, S G; Reynolds, E C

1993-06-01

Leucine transport in glucose-energized cells of Streptococcus mutans exhibited Michaelis-Menten-type kinetics at low extracellular concentrations, with a K1 of 15.3 microM and a Vmax of 6.1 nmol/mg dry weight/min. At high extracellular leucine concentrations, the transmembrane diffusion of leucine was not saturable, indicating that passive diffusion becomes a significant mechanism of leucine transmembrane movement under these conditions. The proton motive force (PMF) was measured in glucose-energized cells of S. mutans and was found to have a maximum value of 126 mV at an extracellular pH (pH0) of 5.0; this decreased to 45 mV at pH0 8.0. The intracellular accumulation of leucine was significantly correlated with the magnitude of the PMF. The addition of excess isoleucine or valine caused a marked decrease in the leucine transport rate. Maximal rates of leucine transport occurred at pH0 6.0, and the rate of leucine transport was independent of the growth medium. The results suggest that there is a PMF-driven, branched-chain amino acid carrier in S. mutans with a proton: substrate stoichiometry of 1.

Magnetized poly(STY-co-DVB) as a matrix for immobilizing microbial lipase to be used in biotransformation

NASA Astrophysics Data System (ADS)

Bento, H. B. S.; de Castro, H. F.; de Oliveira, P. C.; Freitas, L.

2017-03-01

Magnetized hydrophobic polymeric particles were prepared by suspension polymerization of styrene and divinylbenzene with the addition of magnetite (Fe3O4) functionalized with oleic acid (OA). The magnetic poly(STY-co-DVB) particles were characterized by Fourier transform infrared spectroscopy (FTIR), X-ray diffraction (XRD) and scanning electron microscopy (SEM). The results showed that the magnetic polymer particles fulfill the requirements for being used as matrix in the immobilization of microbial lipase from Candida rugosa by physical adsorption. The resulted immobilized derivative presented high catalytic activity in both aqueous and non-aqueous media. A comparative study between free and immobilized lipases showed a similar biochemical behavior, but with better hydrolytic activity at a pH range of 8.0-8.5. The patterns of heat stability indicated that the immobilization process also stabilizes the enzyme by a 50-fold improvement of thermal stability parameters (thermal deactivation and half-life time). Data on olive oil hydrolytic activities indicated that the Michaelis-Menten equation can be used to adjust data so as to calculate Km and Vmax, which attained values of 1766 mM and 5870 μM g-1 min-1, respectively. Such values indicated that the immobilized system was subjected to mass transfer limitations. High operational stability (t ½=1014 h) was achieved under repetitive batch runs in ester synthesis. The results indicated that the magnetized support particles can be very promising carriers for immobilizing enzymes in biotransformation reactions.

First report of the characterization of a snake venom apyrase (Ruviapyrase) from Indian Russell's viper (Daboia russelii) venom.

PubMed

Kalita, Bhargab; Patra, Aparup; Jahan, Shagufta; Mukherjee, Ashis K

2018-05-01

A novel apyrase from Russell's viper venom (RVV) was purified and characterized, and it was named Ruviapyrase (Russell's viper apyrase). It is a high molecular weight (79.4 kDa) monomeric glycoprotein that contains 2.4% neutral sugars and 58.4% N-linked oligosaccharides and strongly binds to Concanavalin A. The LC-MS/MS analysis did not identify any protein in NCBI protein database, nevertheless some de novo sequences of Ruviapyrase showed putative conserved domain of apyrase superfamily. Ruviapyrase hydrolysed adenosine triphosphate (ATP) to a significantly greater extent (p < .05) as compared to adenosine diphosphate (ADP); however, it was devoid of 5'-nucleotidase and phosphodiesterase activities. The Km and Vmax values for Ruviapyrase towards ATP were 2.54 μM and 615 μM of Pi released min -1 , respectively with a turnover number (Kcat) of 24,600 min -1 . Spectrofluorometric analysis demonstrated interaction of Ruviapyrase with ATP and ADP at Kd values of 0.92 nM and 1.25 nM, respectively. Ruviapyrase did not show cytotoxicity against breast cancer (MCF-7) cells and haemolytic activity, it exhibited marginal anticoagulant and strong antiplatelet activity, and dose-dependently reversed the ADP-induced platelet aggregation. The catalytic activity and platelet deaggregation property of Ruviapyrase was significantly inhibited by EDTA, DTT and IAA, and neutralized by commercial monovalent and polyvalent antivenom. Copyright © 2018 Elsevier B.V. All rights reserved.

Effect of antihypertensive agents - captopril and nifedipine - on the functional properties of rat heart mitochondria

PubMed Central

Kancirová, Ivana; Jašová, Magdaléna; Waczulíková, Iveta; Ravingerová, Táňa; Ziegelhöffer, Attila; Ferko, Miroslav

2016-01-01

Objective(s): Investigation of acute effect on cellular bioenergetics provides the opportunity to characterize the possible adverse effects of drugs more comprehensively. This study aimed to investigate the changes in biochemical and biophysical properties of heart mitochondria induced by captopril and nifedipine antihypertensive treatment. Materials and Methods: Male, 12-week-old Wistar rats in two experimental models (in vivo and in vitro) were used. In four groups, the effects of escalating doses of captopril, nifedipine and combination of captopril + nifedipine added to the incubation medium (in vitro) or administered per os to rat (in vivo) on mitochondrial ATP synthase activity and membrane fluidity were monitored. Results: In the in vitro model we observed a significant inhibitory effect of treatment on the ATP synthase activity (P<0.05) with nonsignificant differences in membrane fluidity. Decrease in the value of maximum reaction rate Vmax (P<0.05) without any change in the value of Michaelis-Menten constant Km, indicative of a noncompetitive inhibition, was presented. At the in vivo level, we did not demonstrate any significant changes in the ATP synthase activity and the membrane fluidity in rats receiving captopril, nifedipine, and combined therapy. Conclusion: In vitro kinetics study revealed that antihypertensive drugs (captopril and nifedipine) directly interact with mitochondrial ATP synthase. In vivo experiment did not prove any acute effect on myocardial bioenergetics and suggest that drugs do not enter cardiomyocyte and have no direct effect on mitochondria. PMID:27482342

Molecular cloning and functional characterization of a glucose transporter (CsGLUT) in Clonorchis sinensis.

PubMed

Ahn, Seong Kyu; Cho, Pyo Yun; Na, Byoung-Kuk; Hong, Sung-Jong; Nam, Ho-Woo; Sohn, Woon-Mok; Ardelli, Bernadette F; Park, Yun-Kyu; Kim, Tong-Soo; Cha, Seok Ho

2016-01-01

A complementary DNA (cDNA) encoding a glucose transporter of Clonorchis sinensis (CsGLUT) was isolated from the adult C. sinensis cDNA library. The open reading frame of CsGLUT cDNA consists of 1653 base pairs that encode a 550-amino acid residue protein. Hydropathy analysis suggested that CsGLUT possess 12 putative membrane-spanning domains. The Northern blot analysis result using poly(A)(+)RNA showed a strong band at ~2.1 kb for CsGLUT. When expressed in Xenopus oocytes, CsGLUT mediated the transport of radiolabeled deoxy-D-glucose in a time-dependent but sodium-independent manner. Concentration-dependency results showed saturable kinetics and followed the Michaelis-Menten equation. Nonlinear regression analyses yielded a Km value of 588.5 ± 53.0 μM and a Vmax value of 1500.0 ± 67.5 pmol/oocyte/30 min for [1,2-(3)H]2-deoxy-D-glucose. No trans-uptakes of bile acid (taurocholic acid), amino acids (tryptophan and arginine), or p-aminohippuric acid were observed. CsGLUT-mediated transport of deoxyglucose was significantly and concentration-dependently inhibited by radio-unlabeled deoxyglucose and D-glucose. 3-O-Methylglucose at 10 and 100 μM inhibited deoxyglucose uptake by ~50 % without concentration dependence. No inhibitory effects by galactose, mannose, and fructose were observed. This work may contribute to the molecular biological study of carbohydrate metabolism and new drug development of C. sinensis.

Biochemical characterization of recombinant mevalonate kinase from Bacopa monniera.

PubMed

Kumari, Uma; Vishwakarma, Rishi K; Sonawane, Prashant; Abbassi, Shakeel; Khan, Bashir M

2015-01-01

Mevalonate kinase (MK; ATP: mevalonate 5-phosphotransferase; EC 2.7.1.36) plays a key role in isoprenoid biosynthetic pathway in plants. MK catalyzes the phosphorylation of mevalonate to form mevalonate-5-phosphate. The recombinant BmMK was cloned and over-expressed in E. coli BL21 (DE3), and purified to homogeneity by affinity chromatography followed by gel filtration. Optimum pH and temperature for forward reaction was found to be 7.0 and 30 °C, respectively. The enzyme was most stable at pH 8 at 25 °C with deactivation rate constant (Kd*) 1.398 × 10(-4) and half life (t1/2) 49 h. pH activity profile of BmMK indicates the involvement of carboxylate ion, histidine, lysine, arginine or aspartic acid at the active site of enzyme. Activity of recombinant BmMK was confirmed by phosphorylation of RS-mevalonate in the presence of Mg(2+), having Km and Vmax 331.9 μM and 719.1 pKat μg(-1), respectively. The values of kcat and kcat/Km for RS-mevalonate were determined to be 143.82 s(-1) and 0.43332 M(-1) s(-1) and kcat and kcat/Km values for ATP were found 150.9 s(-1) and 1.023 M(-1) s(-1). The metal ion studies suggested that BmMK is a metal dependent enzyme and highly active in the presence of MgCl2. Copyright © 2014 Elsevier B.V. All rights reserved.

Bacopa monniera recombinant mevalonate diphosphate decarboxylase: Biochemical characterization.

PubMed

Abbassi, Shakeel J; Vishwakarma, Rishi K; Patel, Parth; Kumari, Uma; Khan, Bashir M

2015-08-01

Mevalonate diphosphate decarboxylase (MDD; EC 4.1.1.33) is an important enzyme in the mevalonic acid pathway catalyzing the Mg(2+)-ATP dependant decarboxylation of mevalonate 5-diphosphate (MVAPP) to isopentenyl diphosphate (IPP). Bacopa monniera recombinant MDD (BmMDD) protein was overexpressed in Escherichia coli BL21 (DE3) strain and purified to apparent homogeneity. Km and Vmax for MVAPP were 144 μM and 52 U mg(-1) respectively. The values of turnover (kcat) and kcat/Km for mevalonate 5-diphosphate were determined to be 40s(-1) and 2.77×10(5) M(-1) s(-1) and kcat and kcat/Km values for ATP were found to be 30 s(-1) and 2.20×10(4) M(-1) s(-1), respectively. pH activity profile indicated the involvement of carboxylate ion, lysine and arginine for the activity of enzyme. The apparent activation energy for the BmMDD catalyzed reaction was 12.7 kJ mol(-1). Optimum pH and temperature for the forward reaction was found to be 8.0 and 45 °C. The enzyme was most stable at pH 7 at 20 °C with the deactivation rate constant (Kd(*)) of 1.69×10(-4) and half life (t1/2) of 68 h. The cation studies suggested that BmMDD is a cation dependant enzyme and optimum activity was achieved in the presence of Mg(2+). Copyright © 2015 Elsevier B.V. All rights reserved.

Molecular modeling of the inhibition of enzyme PLA2 from snake venom by dipyrone and 1-phenyl-3-methyl-5-pyrazolone

NASA Astrophysics Data System (ADS)

Silva, S. L. Da; Comar, M., Jr.; Oliveira, K. M. T.; Chaar, J. S.; Bezerra, E. R. M.; Calgarotto, A. K.; Baldasso, P. A.; Veber, C. L.; Villar, J. A. F. P.; Oliveira, A. R. M.; Marangoni, S.

Phospholipases A2 (PLA2) are enzymes that trigger the degradation cascade of the arachidonic acid, leading to the formation of pro-inflammatory eicosanoids. The selective inhibition of PLA2s is crucial in the search for a more efficient anti-inflammatory drug with fewer side effects than the drugs currently used. Hence, we studied the influences caused by two pyrazolonic inhibitors: dipyrone (DIP) and 1-phenyl-3-methyl-5-pyrazolone (PMP) on the kinetic behavior of PLA2 from Crotalus adamanteus venom. Molecular modeling results, by DFT and MM approaches, showed that DIP is strongly associated to the active site of PLA2 through three hydrogen bonds, whereas PMP is associated to the enzyme just through hydrophobic interactions. In addition, only PMP presents an intramolecular hydrogen bond that make difficult the formation of more efficient interactions with PLA2. These results help in the understanding of the experimental observations. Experimentally, the results showed that PLA2 from C. adamanteus present a typical Michaelian behavior. In addition, the calculated kinetic parameters showed that, in the presence of DIP or PMP, the maximum enzymatic velocity (VMAX) value was kept constant, whereas the Michaelis constant (KM) values increased and the inhibition constant (KI) decreased, indicating competitive inhibition. These results show that the phenyl-pyrazolonic structures might help in the development and design of new drugs able to selectively inhibit PLA2.

Investigation of yeast invertase immobilization onto cupric ion-chelated, porous, and biocompatible poly(hydroxyethyl methacrylate-n-vinyl imidazole) microspheres.

PubMed

Sari, Müfrettin Murat

2011-04-01

Cupric ion-chelated poly(hydroxyethyl methacrylate-n-vinyl imidazole) (poly(HEMA-VIM)) microspheres prepared by suspension polymerization were investigated as a specific adsorbent for immobilization of yeast invertase in a batch system. They were characterized by scanning electron microscopy, surface area, and pore size measurements. They have spherical shape and porous structure. The specific surface area of the p(HEMA-VIM) spheres was found to be 81.2 m²/g with a size range of 70-120 μm in diameter, and the swelling ratio was 86.9%. Then, Cu(II) ion chelated on the microspheres (546 μmol Cu(II)/g), and they were used in the invertase adsorption. Maximum invertase adsorption was 51.2 mg/g at pH 4.5. Cu(II) chelation increases the tendency from Freundlich-type to Langmuir-type adsorption model. The optimum activity for both free and adsorbed invertase was observed at pH 4.5. The optimum temperature for the poly(HEMA-VIM)/Cu(II)-invertase system was found to be at 55 °C, 10 °C higher than that of the free enzyme at 45 °C. V(max) values were determined as 342 and 304 U/mg enzyme, for free and adsorbed invertase, respectively. K(m) values were found to be same for free and adsorbed invertase (20 mM). Thermal and pH stability and reusability of invertase increased with immobilization.

A novel bifunctional endo-/exo-type cellulase from an anaerobic ruminal bacterium.

PubMed

Ko, Kyong-Cheol; Han, Yunjon; Choi, Jong Hyun; Kim, Geun-Joong; Lee, Seung-Goo; Song, Jae Jun

2011-03-01

An anaerobic microorganism termed AN-C16-KBRB was isolated from the bovine rumen and demonstrated cellulolytic activity on a NB agar plate containing azo-carboxymethyl cellulose. The 16S rRNA gene of the strain was 98% similar to that of Clostridiaceae bacterium SK082 (AB298754) as the highest homology. A novel celEdx16 gene encoding a bifunctional endo-/exocellulase (CelEdx16) was cloned by the shotgun method from AN-C16-KBRB, and the enzyme was characterized. The celEdx16 gene had an open reading frame of 1,104-base pairs, which encoded 367 amino acids to yield a protein of molecular mass 40.4 kDa. The amino acid sequence was 53% identical to that of an endoglucanase from Clostridium thermocellum. CelEdx16 was overexpressed in Escherichia coli and purified using Ni-NTA affinity chromatography. The specific endocellulase and exocellulase activities of CelEdx16 were 15.9 and 3.6 x 10⁻² U mg⁻¹, respectively. The Michaelis-Menten constant (K (m) values) and the maximal reaction velocities (V(max) values) of CelEdx16 were 47.1 μM and 9.6 x 10⁻³ μmole min⁻¹ when endocellulase activity was measured and 106.3 μM and 2.1 x 10⁻⁵ μmol min⁻¹ when exocellulase activity was assessed. CelEdx16 was optimally active at pH 5.0 and 40 °C.

Design of epoxy-functionalized Fe3O4@MCM-41 core-shell nanoparticles for enzyme immobilization.

PubMed

Ulu, Ahmet; Ozcan, Imren; Koytepe, Suleyman; Ates, Burhan

2018-05-01

The scope of our research was to prepare the organosilane-modified Fe 3 O 4 @MCM-41 core-shell magnetic nanoparticles, used for L-ASNase immobilization and explored screening of immobilization conditions such as pH, temperature, thermal stability, kinetic parameters, reusability and storage stability. In this content, Fe 3 O 4 core-shell magnetic nanoparticles were prepared via co-precipitation method and coated with MCM-41. Then, Fe 3 O 4 @MCM-41 magnetic nanoparticles were functionalized by (3-glycidyloxypropyl) trimethoxysilane (GPTMS) as an organosilane compound. Subsequently, L-ASNase was covalently immobilized on epoxy-functionalized Fe 3 O 4 @MCM-41 magnetic nanoparticles. The immobilized L-ASNase had greater activity at high pH and temperature values. It also maintained >92% of the initial activity after incubation at 55 °C for 3 h. Regarding kinetic values, immobilized L-ASNase showed a higher Vmax and lower Km compared to native L-ASNase. In addition, it displayed excellent reusability for 12 successive cycles. After 30 days of storage at 4 °C and 25 °C, immobilized L-ASNase retained 54% and 26% of its initial activities while native L-ASNase lost about 68% and 84% of its initial activity, respectively. As a result, the immobilization of L-ASNase onto magnetic nanoparticles may provide an advantage in terms of removal of L-ASNase from reaction media. Copyright © 2018. Published by Elsevier B.V.

Monkey liver cytochrome P450 2C9 is involved in caffeine 7-N-demethylation to form theophylline.

PubMed

Utoh, Masahiro; Murayama, Norie; Uno, Yasuhiro; Onose, Yui; Hosaka, Shinya; Fujino, Hideki; Shimizu, Makiko; Iwasaki, Kazuhide; Yamazaki, Hiroshi

2013-12-01

Caffeine (1,3,7-trimethylxanthine) is a phenotyping substrate for human cytochrome P450 1A2. 3-N-Demethylation of caffeine is the main human metabolic pathway, whereas monkeys extensively mediate the 7-N-demethylation of caffeine to form pharmacological active theophylline. Roles of monkey P450 enzymes in theophylline formation from caffeine were investigated using individual monkey liver microsomes and 14 recombinantly expressed monkey P450 enzymes, and the results were compared with those for human P450 enzymes. Caffeine 7-N-demethylation activity in microsomes from 20 monkey livers was not strongly inhibited by α-naphthoflavone, quinidine or ketoconazole, and was roughly correlated with diclofenac 4'-hydroxylation activities. Monkey P450 2C9 had the highest activity for caffeine 7-N-demethylation. Kinetic analysis revealed that monkey P450 2C9 had a high Vmax/Km value for caffeine 7-N-demethylation, comparable to low Km value for monkey liver microsomes. Caffeine could dock favorably with monkey P450 2C9 modeled for 7-N-demethylation and with human P450 1A2 for 3-N-demethylation. The primary metabolite theophylline was oxidized to 8-hydroxytheophylline in similar ways by liver microsomes and by recombinant P450s in both humans and monkeys. These results collectively suggest a high activity for monkey liver P450 2C9 toward caffeine 7-N-demethylation, whereas, in humans, P450 1A2-mediated caffeine 3-N-demethylation is dominant.

CGP-37157 inhibits the sarcoplasmic reticulum Ca²+ ATPase and activates ryanodine receptor channels in striated muscle.

PubMed

Neumann, Jake T; Diaz-Sylvester, Paula L; Fleischer, Sidney; Copello, Julio A

2011-01-01

7-Chloro-5-(2-chlorophenyl)-1,5-dihydro-4,1-benzothiazepin-2(3H)-one [CGP-37157 (CGP)], a benzothiazepine derivative of clonazepam, is commonly used as a blocker of the mitochondrial Na+/Ca²+ exchanger. However, evidence suggests that CGP could also affect other targets, such as L-type Ca²+ channels and plasmalemma Na+/Ca²+ exchanger. Here, we tested the possibility of a direct modulation of ryanodine receptor channels (RyRs) and/or sarco/endoplasmic reticulum Ca²+-stimulated ATPase (SERCA) by CGP. In the presence of ruthenium red (inhibitor of RyRs), CGP decreased SERCA-mediated Ca²+ uptake of cardiac and skeletal sarcoplasmic reticulum (SR) microsomes (IC₅₀ values of 6.6 and 9.9 μM, respectively). The CGP effects on SERCA activity correlated with a decreased V(max) of ATPase activity of SERCA-enriched skeletal SR fractions. CGP (≥ 5 μM) also increased RyR-mediated Ca²+ leak from skeletal SR microsomes. Planar bilayer studies confirmed that both cardiac and skeletal RyRs are directly activated by CGP (EC(50) values of 9.4 and 12.0 μM, respectively). In summary, we found that CGP inhibits SERCA and activates RyR channels. Hence, the action of CGP on cellular Ca²+ homeostasis reported in the literature of cardiac, skeletal muscle, and other nonmuscle systems requires further analysis to take into account the contribution of all CGP-sensitive Ca²+ transporters.

CGP-37157 Inhibits the Sarcoplasmic Reticulum Ca2+ ATPase and Activates Ryanodine Receptor Channels in Striated Muscle

PubMed Central

Neumann, Jake T.; Diaz-Sylvester, Paula L.; Fleischer, Sidney

2011-01-01

7-Chloro-5-(2-chlorophenyl)-1,5-dihydro-4,1-benzothiazepin-2(3H)-one [CGP-37157 (CGP)], a benzothiazepine derivative of clonazepam, is commonly used as a blocker of the mitochondrial Na+/Ca2+ exchanger. However, evidence suggests that CGP could also affect other targets, such as L-type Ca2+ channels and plasmalemma Na+/Ca2+ exchanger. Here, we tested the possibility of a direct modulation of ryanodine receptor channels (RyRs) and/or sarco/endoplasmic reticulum Ca2+-stimulated ATPase (SERCA) by CGP. In the presence of ruthenium red (inhibitor of RyRs), CGP decreased SERCA-mediated Ca2+ uptake of cardiac and skeletal sarcoplasmic reticulum (SR) microsomes (IC50 values of 6.6 and 9.9 μM, respectively). The CGP effects on SERCA activity correlated with a decreased Vmax of ATPase activity of SERCA-enriched skeletal SR fractions. CGP (≥5 μM) also increased RyR-mediated Ca2+ leak from skeletal SR microsomes. Planar bilayer studies confirmed that both cardiac and skeletal RyRs are directly activated by CGP (EC50 values of 9.4 and 12.0 μM, respectively). In summary, we found that CGP inhibits SERCA and activates RyR channels. Hence, the action of CGP on cellular Ca2+ homeostasis reported in the literature of cardiac, skeletal muscle, and other nonmuscle systems requires further analysis to take into account the contribution of all CGP-sensitive Ca2+ transporters. PMID:20923851

Adsorption of Trametes versicolor laccase to soil iron and aluminum minerals: enzyme activity, kinetics and stability studies.

PubMed

Wu, Yue; Jiang, Ying; Jiao, Jiaguo; Liu, Manqiang; Hu, Feng; Griffiths, Bryan S; Li, Huixin

2014-02-01

Laccases play an important role in the degradation of soil phenol or phenol-like substance and can be potentially used in soil remediation through immobilization. Iron and aluminum minerals can adsorb extracellular enzymes in soil environment. In the present study, we investigated the adsorptive interaction of laccase, from the white-rot fungus Trametes versicolor, with soil iron and aluminum minerals and characterized the properties of the enzyme after adsorption to minerals. Results showed that both soil iron and aluminum minerals adsorbed great amount of laccase, independent of the mineral specific surface areas. Adsorbed laccases retained 26-64% of the activity of the free enzyme. Compared to the free laccase, all adsorbed laccases showed higher Km values and lower Vmax values, indicating a reduced enzyme-substrate affinity and a lower rate of substrate conversion in reactions catalyzed by the adsorbed laccase. Adsorbed laccases exhibited increased catalytic activities compared to the free laccase at low pH, implying the suitable application of iron and aluminum mineral-adsorbed T. versicolor laccase in soil bioremediation, especially in acid soils. In terms of the thermal profiles, adsorbed laccases showed decreased thermal stability and higher temperature sensitivity relative to the free laccase. Moreover, adsorption improved the resistance of laccase to proteolysis and extended the lifespan of laccase. Our results implied that adsorbed T. versicolor laccase on soil iron and aluminum minerals had promising potential in soil remediation. Crown Copyright © 2013. Published by Elsevier B.V. All rights reserved.

Changes in calcium uptake rate by rat cardiac mitochondria during postnatal development.

PubMed

Bassani, R A; Fagian, M M; Bassani, J W; Vercesi, A E

1998-10-01

Ca2+ uptake, transmembrane electrical potential (Deltapsim) and oxygen consumption were measured in isolated ventricular mitochondria of rats from 3 days to 5 months of age. Estimated values of ruthenium red-sensitive, succinate-supported maximal rate of Ca2+ uptake (Vmax, expressed as nmol Ca2+/min/mg protein) were higher in neonates and gradually fell during postnatal development (from 435+/-24 at 3-6 days, to 156+/-10 in adults,P<0.001), whereas K0.5 values (approximately 10 microM were not significantly affected by age. Under similar conditions, mitochondria from adults (5 months old) and neonates (4-6 days old) showed comparable state 4 (succinate and alpha-ketoglutarate as substrates) and state 3ADP (alpha-ketoglutarate-supported) respiration rates, as well as Deltapsim values (approximately-150 mV). Respiration-independent Deltapsim and Ca2+ uptake, supported by valinomycin-induced K+ efflux were also investigated at these ages. A transient Deltapsim (approximately -30 mV) was evoked by valinomycin in both neonatal and adult mitochondria. Respiration-independent Ca2+ uptake was also transient, but its initial rate was significantly higher in neonates than in adults (49. 4+/-10.0v 28.0+/-5.7 mmol Ca2+/min/mg protein,P<0.01). These results indicate that Ca2+ uptake capacity of rat cardiac mitochondria is remarkably high just after birth and declines over the first weeks of postnatal life, without change in apparent affinity of the transporter. Increased mitochondrial Ca2+ uptake rate in neonates appears to be related to the uniporter itself, rather than to modification of the driving force of the transport. Copyright 1998 Academic Press

Effect of hyperthyroidism on the transport of pyruvate in rat-heart mitochondria.

PubMed

Paradies, G; Ruggiero, F M

1988-08-17

A comparative study of the transport of pyruvate in heart mitochondria from normal and triiodothyronine-treated rats has been carried out. It has been found that the rate of carrier-mediated (alpha-cyanocinnamate-sensitive) pyruvate uptake is significantly enhanced in mitochondria from triiodothyronine-treated rats as compared with mitochondria from control rats. The kinetic parameters of the pyruvate uptake indicate that only the Vmax of this process is enhanced whilst there is no change in the Km value. The enhanced rate of pyruvate uptake is not dependent on the increase of the transmembrane delta pH value (both mitochondria from normal and triiodothyronine-treated rats exhibit the same delta pH value) neither does it depend on the increase of the pyruvate carrier molecules (titration of these last with alpha-cyanocinnamate gives the same total number of binding sites). the pyruvate-dependent oxygen uptake is stimulated by 35-40% in mitochondria from hyperthyroid rats when compared with mitochondria from control rats. There is, however, no difference in either the respiratory control ratios or in the ADP/O ratios between these two types of mitochondria. The heart mitochondrial phospholipid composition is altered significantly in hyperthyroid rats; in particular, negatively charged phospholipid such as cardiolipin and phosphatidylserine were found to increase by more than 50%. Minor alterations were found in the pattern of fatty acids with an increase of 20:4/18:2 ratio. It is suggested that the changes in the kinetic parameters of pyruvate transport in mitochondria from hyperthyroid rats involve hormone-mediated changes in the lipid composition of the mitochondrial membranes which in turn modulate the activity of the pyruvate carrier.

Light-Dependent Sulfide Oxidation in the Anoxic Zone of the Chesapeake Bay Can Be Explained by Small Populations of Phototrophic Bacteria

PubMed Central

Bennett, Alexa J.; Hanson, Thomas E.; Luther, George W.

2015-01-01

Microbial sulfide oxidation in aquatic environments is an important ecosystem process, as sulfide is potently toxic to aerobic organisms. Sulfide oxidation in anoxic waters can prevent the efflux of sulfide to aerobic water masses, thus mitigating toxicity. The contribution of phototrophic sulfide-oxidizing bacteria to anaerobic sulfide oxidation in the Chesapeake Bay and the redox chemistry of the stratified water column were investigated in the summers of 2011 to 2014. In 2011 and 2013, phototrophic sulfide-oxidizing bacteria closely related to Prosthecochloris species of the phylum Chlorobi were cultivated from waters sampled at and below the oxic-anoxic interface, where measured light penetration was sufficient to support populations of low-light-adapted photosynthetic bacteria. In 2012, 2013, and 2014, light-dependent sulfide loss was observed in freshly collected water column samples. In these samples, extremely low light levels caused 2- to 10-fold increases in the sulfide uptake rate over the sulfide uptake rate under dark conditions. An enrichment, CB11, dominated by Prosthecochloris species, oxidized sulfide with a Ks value of 11 μM and a Vmax value of 51 μM min−1 (mg protein−1). Using these kinetic values with in situ sulfide concentrations and light fluxes, we calculated that a small population of Chlorobi similar to those in enrichment CB11 can account for the observed anaerobic light-dependent sulfide consumption activity in natural water samples. We conclude that Chlorobi play a far larger role in the Chesapeake Bay than currently appreciated. This result has potential implications for coastal anoxic waters and expanding oxygen-minimum zones as they begin to impinge on the photic zone. PMID:26296727

Rabbit ventricular myocardium undergoing simulated ischemia and reperfusion in a double compartment tissue bath: a model to investigate both antiarrhythmic and arrhythmogenic likelihood.

PubMed

Alexandre, Joachim; Schiariti, Michele; Rouet, René; Puddu, Paolo Emilio

2013-01-01

An ischemia/reperfusion-simulating model in rabbit tissue should be right oriented and clinically relevant to provide a non expensive approach for manipulations of currents involved in the repolarization process. Standard right ventricular guinea-pig (N=18) and newly investigated rabbit (N=12) myocardial strips were placed in a special perfusion chamber allowing partition into two segments independently superfused with oxygenated Tyrode's solution or a modified Tyrode's solution mimicking ischemia by: 1) increased extracellular potassium concentration (12 mmol/L), 2) decreased HCO3 (-) concentration (9 mmol/L), leading to a decrease in pH (6.90 ± 0.05), 3) decreased pO2 by replacement of 95% O2 and 5% CO2 by 95% N2 and 5% CO2 gas mixture, and 4) complete withdrawal of glucose. There were significant differences in rabbit as compared to guinea-pig preparations in baseline (p<0.02) and post-ischemic-like (p<0.01) APA and RMP with lower values in the formers, and lower post-ischemic Vmax in rabbit preparations (25±15 versus 97±83 V/s, p<0.01) but neither baseline nor post-ischemic-like or absolute changes in APD50, APD90 were different. In ischemia- and reperfusion-like phases, there were high proportions of single spontaneous repetitive responses, both in guinea-pig (respectively 50 and 89%) and rabbit preparations (respectively 67 and 92%). Guinea-pig preparations showed higher incidence of severe spontaneous repetitive responses (61 versus 17%, p<0.02). This rabbit model is proposed to investigate both anti- and pro-arrhythmic effects of drugs acting at various levels electrophysiologically, which may be obtained with great power and relatively few (around 10 per group) preparations. This model should now be tested pharmacologically.

Interaction of GABA-mimetics with the taurine transporter (TauT, Slc6a6) in hyperosmotic treated Caco-2, LLC-PK1 and rat renal SKPT cells.

PubMed

Rasmussen, Rune Nørgaard; Lagunas, Candela; Plum, Jakob; Holm, René; Nielsen, Carsten Uhd

2016-01-20

The aim of the present study was to investigate if basic GABA-mimetics interact with the taurine transporter (TauT, Slc6a6), and to find a suitable cell based model that is robust towards extracellular changes in osmolality during uptake studies. Taurine uptake was measured in human Caco-2 cells, porcine LLC-PK1 cells, and rat SKPT cells using radiolabelled taurine. Hyperosmotic conditions were obtained by incubation with raffinose (final osmolality of 500mOsm) for 24h prior to the uptake experiments. Expression of the taurine transporter, TauT, was investigated at the mRNA level by real-time PCR. Uptake of the GABA-mimetics gaboxadol and vigabatrin was investigated in SKPT cells, and quantified by liquid scintillation or HPLC-MS/MS analysis, respectively. The uptake rate of [(3)H]-taurine was Na(+) and Cl(-) and concentration dependent with taurine with an apparent Vmax of 6.3±1.6pmolcm(-2)min(-1) and a Km of 24.9±15.0μM. β-alanine, nipecotic acid, gaboxadol, GABA, vigabatrin, δ-ALA and guvacine inhibited the taurine uptake rate in a concentration dependent manner. The order of affinity for TauT was β-alanine>GABA>nipecotic acid>guvacine>δ-ALA>vigabatrin>gaboxadol with IC50-values of 0.04, 1.07, 2.02, 4.19, 4.94, 31.4 and 39.9mM, respectively. In conclusion, GABA mimetics inhibited taurine uptake in hyperosmotic rat renal SKPT cells. SKPT cells, which seem to be a useful model for investigating taurine transport in the short-term presence of high concentrations of osmolytes. Furthermore, analogues of β-alanine appear to have higher affinities for TauT than GABA-analogues. Copyright © 2015 Elsevier B.V. All rights reserved.

[Characterization of a malic enzyme isoform V from Mucor circinelloides].

PubMed

Zhang, Yingtong; Chen, Haiqin; Song, Yuanda; Zhang, Hao; Chen, Yongquan; Chen, Wei

2016-02-04

We aimed at characterizing a malic enzyme isoform V from Mucor circinelloides. me1 gene encoding malic enzyme isoform V was amplified and cloned into expression vector pET28a. High-purity recombinant protein BLME1 was obtained by affinity chromatography using. Ni-NTA column and characterized subsequently. The optimum conditions were pH at 8.0 and temperature at 33 degrees C. Under optimum conditions, BLME1 activity achieved 92.8 U/mg. The K(m) for L-malate and NADP+ were 0.74960 ± 0.06120 mmol/L and 0.22070 ± 0.01810 mmol/L, the V(max) for L-malate and NADP+ were 72.820 ± 1.077 U/mg and 86.110 ± 1.665 U/mg, respectively. In addition, ions played important roles in BLME1 activity; several ions such as Mn2+, Mg2+, Co2+, Ni2+ could activate BLME1, whereas Ca2+, Cu2+ could be used as inhibitors. Additionally, the metabolic intermediates such as oxaloacetic acid and α-ketoglutaric acid inhibited the activity of BLME1, whereas succinic acid activated it. A malic enzyme isoform V from Mucor circinelloides was characterized, providing the references for further studies on this enzyme.

Rapid purification of the over-expressed membrane 3 β-hydroxysteroid dehydrogenase in the presence of detergents

NASA Astrophysics Data System (ADS)

Zhou, Ming; Breton, Rock; Azzi, Arezki; Lin, Sheng-Xiang

1996-10-01

Three-beta hydroxysteroid dehydrogenase / Δ 5-Δ 4 isomerase catalyses a key step in the transformation of all 5-prognen-3β-ol and 5-androsten-3β-ol steroids into the corresponding Δ 4-3-keto-steroids. Human type I 3β-HSD can be found in the subcellular fractions of mitochondria and microsome. A 1.5 kbp cDNA encoding human type I 3β-HSD was inserted into the transfer vector pBlueBac to form plasmid pBB / 3β-HSD. The recombinant baculovirus was obtained by co-transfection of wild type AcNPV genomic DNA and PBB / 3β-HSD in Sf9 cells, then used to infect Sf9 cells to over-express human 3β-HSD protein. The 3β-HSD sample was purified to homogeneity by a rapid procedure, consisting of an anion-exchange and an adsorbance chromatographies, based on FPLC and some detergents application. The whole process was successful with a purification rate of 90 fold and a high recovery (70%). The kinetic study showed a Vmax of 500 nmol/min · mg and a Km of 2.8 μM, being much more active than those reported.

[Purification, characterization and partial primary structure analysis of rutin-degrading enzyme in tartary buckwheat seeds].

PubMed

Zhang, Yuwei; Li, Jie; Yuan, Yong; Gu, Jijuan; Chen, Peng

2017-05-25

Rutin-degrading enzymes (RDE) can degrade rutin into poorly water soluble compound, quercetin, and cause the bitter taste in tartary buckwheat. In the present study RDE from Yu 6-21 tartary buckwheat seeds was purified by ammonium sulphate precipitation, followed by hydrophobic interaction chromatography on Phenyl Sepharose CL-4B, ion exchange chromatography on CM-Cellulose and gel filtration chromatography on Sephadex G-150. Purified RDE showed single band with molecular weight of 66 kDa on SDS-PAGE. The optimum pH and temperature of RDE were 5.0 and 50 ℃ respectively. The Km was 0.27 mmol/L, and the Vmax was 39.68 U/mg. The RDE activity could be inhibited by Cu²⁺, Zn²⁺, Mn²⁺ and EDTA, and showed tolerance to 50% methanol (V/V). The N terminal sequence (TVSRSSFPDGFLFGL) was obtained by Edman degradation method and 15 internal peptide sequences were determined by MALDI-TOF-MS (matrix-assisted laser desorption ionization time of flight mass spectrometry). These results established the foundations for identification of the candidate gene of RDE via transcriptome data and further studying RDE biological function.

Purification, immobilization and characterization of tannase from Penicillium variable.

PubMed

Sharma, Shashi; Agarwal, Lata; Saxena, Rajendra Kumar

2008-05-01

Tannase from Penicillium variable IARI 2031 was purified by a two-step purification strategy comprising of ultra-filtration using 100 kDa molecular weight cutoff and gel-filtration using Sephadex G-200. A purification fold of 135 with 91% yield of tannase was obtained. The enzyme has temperature and pH optima of 50 degrees C and 5 degrees C, respectively. However, the functional temperature range is from 25 to 80 degrees C and functional pH range is from 3.0 to 8.0. This tannase could successfully be immobilized on Amberlite IR where it retains about 85% of the initial catalytic activity even after ninth cycle of its use. Based on the Michaelis-Menten constant (Km) of tannase, tannic acid is the best substrate with Km of 32 mM and Vmax of 1.11 micromol ml(-1)min(-1). Tannase is inhibited by phenyl methyl sulphonyl fluoride (PMSF) and N-ethylmaleimide retaining only 28.1% and 19% residual activity indicating that this enzyme belongs to the class of serine hydrolases. Tannase in both crude and crude lyophilized forms is stable for one year retaining more than 60% residual activity.

Macromolecular crowding effect upon in vitro enzyme kinetics: mixed activation-diffusion control of the oxidation of NADH by pyruvate catalyzed by lactate dehydrogenase.

PubMed

Balcells, Cristina; Pastor, Isabel; Vilaseca, Eudald; Madurga, Sergio; Cascante, Marta; Mas, Francesc

2014-04-17

Enzyme kinetics studies have been usually designed as dilute solution experiments, which differ substantially from in vivo conditions. However, cell cytosol is crowded with a high concentration of molecules having different shapes and sizes. The consequences of such crowding in enzymatic reactions remain unclear. The aim of the present study is to understand the effect of macromolecular crowding produced by dextran of different sizes and at diverse concentrations in the well-known reaction of oxidation of NADH by pyruvate catalyzed by L-lactate dehydrogenase (LDH). Our results indicate that the reaction rate is determined by both the occupied volume and the relative size of dextran obstacles with respect to the enzyme present in the reaction. Moreover, we analyzed the influence of macromolecular crowding on the Michaelis-Menten constants, vmax and Km. The obtained results show that only high concentrations and large sizes of dextran reduce both constants suggesting a mixed activation-diffusion control of this enzymatic reaction due to the dextran crowding action. From our knowledge, this is the first experimental study that depicts mixed activation-diffusion control in an enzymatic reaction due to the effect of crowding.

Purification and Characterization of an Aminopeptidase from Lactococcus lactis subsp. cremoris AM2.

PubMed

Neviani, E; Boquien, C Y; Monnet, V; Thanh, L P; Gripon, J C

1989-09-01

An aminopeptidase was purified from cell extracts of Lactococcus lactis subsp. cremoris AM2 by ion-exchange chromatography. After electrophoresis of the purified enzyme in the presence or absence of sodium dodecyl sulfate, one protein band was detected. The enzyme was a 300-kilodalton hexamer composed of identical subunits not linked by disulfide bridges. Activity was optimal at 40 degrees C and pH 7 and was inhibited by classical thiol group inhibitors. The aminopeptidase hydrolyzed naphthylamide-substituted amino acids, as well as dipeptides and tripeptides. Longer protein chains such as the B chain of insulin were hydrolyzed, but at a much slower rate. The Michaelis constant (K(m)) and the maximal rate of hydrolysis (V(max)) were, respectively, 4.5 mM and 3,600 pkat/mg for the substrate l-histidyl-beta-naphthylamide. Amino acid analysis showed that the enzyme contained low levels of hydrophobic residues. The partial N-terminal sequence of the first 19 residues of the mature enzyme was determined. Polyclonal antibodies were obtained from the purified enzyme, and after immunoblotting, there was no cross-reaction between these antibodies and other proteins in the crude extract.

Does respiratory muscle training increase physical performance?

PubMed

Sperlich, Billy; Fricke, Hannes; de Marées, Markus; Linville, John W; Mester, Joachim

2009-09-01

Special force units and military personnel undergo demanding physical exercise and might benefit from high-intensity respiratory muscle training (RMT) by increasing their endurance performance. This study examined the effects of a 6-week high-intensity RMT on running performance and oxygen uptake (VO2max) in a group of German Special Force Squad members. 17 participants were randomly assigned to a training or control group. Baseline and post-testing included a ramp test, as well as an incremental test on a treadmill, performed to physical exhaustion. VO2, respiratory exchange ratio, and heart rate were measured breath by breath. Furthermore, maximum running speed (V(max)), 4 mmol x 1(-1) lactate threshold (V4) and perception of respiratory effort were determined. During pulmonary testing, sustained maximum inspiratory and expiratory pressure (PI(max) and PE(max)) were obtained. RMT was performed daily at approximately 90% PI(max) for 6 weeks with 2 x 30 breath cycles using an Ultrabreathe lung trainer. No statistical differences were detected between the groups for any parameter after RMT. High-intensity RMT did not show any benefits on VO2max and endurance performance and are unlikely to be of benefit to military or paramilitary training programs for an increase in endurance performance.

Expression and characterization of truncated human heme oxygenase (hHO-1) and a fusion protein of hHO-1 with human cytochrome P450 reductase.

PubMed

Wilks, A; Black, S M; Miller, W L; Ortiz de Montellano, P R

1995-04-04

A human heme oxygenase (hHO-1) gene without the sequence coding for the last 23 amino acids has been expressed in Escherichia coli behind the pho A promoter. The truncated enzyme is obtained in high yields as a soluble, catalytically-active protein, making it available for the first time for detailed mechanistic studies. The purified, truncated hHO-1/heme complex is spectroscopically indistinguishable from that of the rat enzyme and converts heme to biliverdin when reconstituted with rat liver cytochrome P450 reductase. A self-sufficient heme oxygenase system has been obtained by fusing the truncated hHO-1 gene to the gene for human cytochrome P450 reductase without the sequence coding for the 20 amino acid membrane binding domain. Expression of the fusion protein in pCWori+ yields a protein that only requires NADPH for catalytic turnover. The failure of exogenous cytochrome P450 reductase to stimulate turnover and the insensitivity of the catalytic rate toward changes in ionic strength establish that electrons are transferred intramolecularly between the reductase and heme oxygenase domains of the fusion protein. The Vmax for the fusion protein is 2.5 times higher than that for the reconstituted system. Therefore, either the covalent tether does not interfere with normal docking and electron transfer between the flavin and heme domains or alternative but equally efficient electron transfer pathways are available that do not require specific docking.

Temperature sensitivity of organic substrate decay varies with pH

NASA Astrophysics Data System (ADS)

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

2012-12-01

Cellulose is the most abundant biopolymer in soils and globally ubiquitous. It serves as a primary carbon source for myriad microbes able to release cellulases which cleave the cellulose into smaller molecules. For example, β-glucosidase, one type of cellulase, breaks down a terminal β-glycosidic bond of cellulose. The carbon of the liberated glucose becomes available for microbial uptake, after which it can then be mineralized and returned to the atmosphere via heterotrophic respiration. Thus, exoenzymes play an important role in the global cycling of carbon. Numerous studies suggest that global warming potentially increases the rate at which β-glucosidase breaks down cellulose, but it is not known how pH of the soil solution influences the effect of temperature on cellulose decomposition rates; this is important given the globally wide range of soil pH. Using fluorescence enzyme assay techniques, we studied the effect of temperature and pH on the reaction rate at which purified β-Glucosidase decays β-D-cellobioside (a compound often employed to simulate cellulose). We evaluated the temperature sensitivity of this reaction at five temperatures (5, 10, 15, 20, and 25°C) and six pH values (3.5, 4.5, 5.5, 6.5, 7.5, and 8.5)encompassing the naturally occurring range in soils, in a full-factorial design. First, we determined Vmax at 25°C and pH 6.5, standard conditions for measuring enzyme activities in many studies. The Vmax was 858.65 μmol h-1mg-1and was achieved at substrate concentration of 270 μM. At all pH values, the reaction rate slowed down at lower temperatures; at a pH of 3.5, no enzymatic activity was detected. The enzyme activity was significantly different between pH 4.5 and higher pHs. For example, enzyme reactivity at pH 4.5 was significantly lower than that at 7.5 at 20 and 25°C (Bonferroni-corrected P =0.0006, 0.0004, respectively), but not at lower temperatures. Similarly, enzyme reactivity at pH 4.5 was lower than that at pH 8.5 at 10, 15, and 25°C (P=0.0009, 0.0007, 0.0005, respectively), with a near-significant trend at 20°C (P=0.0023), and exhibited a nearly significant depression in response to temperature at 25°C compared to that at pH 6.5 (P=0.0015). Our results suggest that exoenzymatic cellulose decomposition with warming may be more enhanced in soil systems exhibiting higher pH. This work highlights the importance of soil solution pH as a driver of temperature sensitivity of substrate decay, and adds a level of complexity for developing accurate predictions of soil carbon cycling with climate change.

Does oxidative stress affect the activity of the sodium-proton exchanger?

PubMed

Bober, Joanna; Kedzierska, Karolina; Kwiatkowska, Ewa; Stachowska, Ewa; Gołembiewska, Edyta; Mazur, Olech; Staniewicz, Zdzisław; Ciechanowski, Kazimierz; Chlubek, Dariusz

2010-01-01

Accumulation of reactive oxygen species (ROS) takes place in patients with chronic renal failure (CRF). Oxidative stress causes disorders in the activity of the sodium-proton exchanger (NHE). Studies on NHE in CRF produced results that are discrepant and difficult to interpret. The aim of this study was to demonstrate that oxidative stress had an effect on the activity of NHE. We enrolled 87 subjects divided into 4 groups: patients with CRF treated conservatively; patients with CRF hemodialyzed without glucose--HD-g(-); patients with CRF hemodialyzed with glucose--HD-g(+); controls (C). The activity of NHE, the rate of proton efflux V(max), Michaelis constant (Km), and the concentration of thiobarbituric acid-reactive substances (TBARS, an indicator of oxidative stress) in plasma, as well as the concentration of reduced glutathione in blood were determined. The concentration of TBARS was significantly higher in hemodialyzed patients before and after dialysis and in patients with CRF on conservative treatment in comparison with group C. TBARS in plasma correlated negatively with VpH(i)6.4 in group C and with V(max) and VpH(i)6.4 after HD in group HD-g(-). We found that the concentration of creatinine correlated with TBARS (p < 0.0001; r = +0.51) in the conservatively treated group. We observed a marked oxidative stress and decreased NHE activity when dialysis was done without glucose, whereas patients dialyzed with glucose demonstrated a relatively low intensity of oxidative stress.

[Metabolic kinetics of MN9202 in Beagle dog liver microsomes].

PubMed

Yang, Zhi-fu; Zhou, Si-yuan; Mei, Qi-bing; Yang, Tie-hong; Liu, Zhen-guo

2005-11-01

To study the metabolic kinetics of MN9202 in Beagle dog liver microsome. Beagle dog liver microsomes were prepared by using ultracentrifuge method. After incubating 0.4 micromol x L(-1) MN9202 with 1 g x L(-1) microsomes for 30 min at 37 degrees C, the reaction was terminated by adding 0.5 mL alkalization. The RP-HPLC was used to determine the drug in the incubation mixture. The Michaelis-Menten parameters Km, and Vmax in Beagle dog liver microsomes were initially estimated by analyzing Lineweave-Brurk plot. Various selective CYP inhibitors were used to investigate their inhibitory effect on the metabolism of MN9202. The Km, Vmax and CLint of MN9202 were (22.6 +/- 8.0) micromol x L(-1), (0.54 +/- 0.17) micromol x g(-1) x min(-1) and (0.0242 +/- 0.0009) L x g(-1) x min(-1), respectively. The metabolism of MN9202 was significantly inhibited by ketoconazole (Ket) and troleandomycin (Tro) in Beagle dog liver microsomes. Tranylcypromine (Tra) could inhibit the metabolism of drug as well. While other inhibitors showed little inhibitory effect on the metabolism of MN9202. It was shown that CYP3A and CYP2C19 were involved in MN9202 metabolism. The inhibitors of human CYP3A and CYP2C19 may have potential interaction with MN9202, and this can reduce the metabolism rate and increase the toxicity of MN9202.

Characterization of arylalkylamine N-acetyltransferase (AANAT) activities and action spectrum for suppression in the band-legged cricket, Dianemobius nigrofasciatus (Orthoptera: Gryllidae).

PubMed

Izawa, Norimitsu; Suzuki, Takeshi; Watanabe, Masakatsu; Takeda, Makio

2009-04-01

Arylalkylamine N-acetyltransferase (AANAT), constituting a large family of enzymes, catalyzes the transacetylation from acetyl-CoA to monoamine substrates, although homology among species is not very high. AANAT in vertebrates is photosensitive and mediates circadian regulation. Here, we analyzed AANAT of the cricket, Dianemobius nigrofasciatus. The central nervous system contained AANAT activity. The optimum pHs were 6.0 (a minor peak) and 10.5 (a major peak) with crude enzyme solution. We analyzed the kinetics at pH 10.5 using the sample containing collective AANAT activities, which we term AANAT. Lineweaver-Burk plot and secondary plot yielded a K(m) for tryptamine as substrate of 0.42 microM, and a V(max) of 9.39 nmol/mg protein/min. The apparent K(m) for acetyl-CoA was 59.9 microM and the V(max) was 8.14 nmol/mg protein/min. AANAT of D. nigrofasciatus was light-sensitive. The activity was higher at night-time than at day-time as in vertebrates. To investigate most effective wavelengths on AANAT activity, a series of monochromatic lights was applied (350, 400, 450, 500, 550, 600 and 650 nm). AANAT showed the highest sensitivity to around 450 nm and 550 nm. 450 nm light was more effective than 550 nm light. Therefore, the most effective light affecting AANAT activity is blue light, which corresponds to the absorption spectrum of blue wave (BW)-opsin.

Molecular genetics of the platelet serotonin system in first-degree relatives of patients with autism

PubMed Central

Cross, Sarah; Kim, Soo-Jeong; Weiss, Lauren A.; Delahanty, Ryan J.; Sutcliffe, James S.; Leventhal, Bennett L.; Cook, Edwin H.; Veenstra-VanderWeele, Jeremy

2009-01-01

Elevated platelet serotonin (5-HT) is found in a subset of children with autism and in some of their first-degree relatives. Indices of the platelet serotonin system, including whole blood serotonin (5-HT), 5-HT binding affinity for the serotonin transporter (Km), 5-HT uptake (Vmax), and lysergic acid diethylamide (LSD) receptor binding, were previously studied in twenty-four first-degree relatives of probands with autism, half of whom were selected for elevated whole blood 5-HT levels. All subjects were then genotyped for selected polymorphisms at the SLC6A4, HTR7, HTR2A, ITGB3, and TPH1 loci. Previous studies allowed an a priori prediction of SLC6A4 haplotypes that separated the subjects into three groups that showed significantly different 5-HT binding affinity (Km, p = 0.005) and 5-HT uptake rate (Vmax, p = 0.046). Genotypes at four individual polymorphisms in SLC6A4 were not associated with platelet 5-HT indices. Haplotypes at SLC6A4 and individual genotypes of polymorphisms at SLC6A4, HTR7, HTR2A, ITGB3, and TPH1 showed no significant association with whole blood 5-HT. Haplotype analysis of two polymorphisms in TPH1 revealed a nominally significant association with whole blood 5-HT (p = 0.046). These initial studies of indices of the 5-HT system with several SNPs at loci in this system generate hypotheses for testing in other samples. PMID:17406648

Functional and Structural Correlates of Motor Speed in the Cerebellar Anterior Lobe

PubMed Central

Wenzel, Uwe; Taubert, Marco; Ragert, Patrick; Krug, Jürgen; Villringer, Arno

2014-01-01

In athletics, motor performance is determined by different abilities such as technique, endurance, strength and speed. Based on animal studies, motor speed is thought to be encoded in the basal ganglia, sensorimotor cortex and the cerebellum. The question arises whether there is a unique structural feature in the human brain, which allows “power athletes” to perform a simple foot movement significantly faster than “endurance athletes”. We acquired structural and functional brain imaging data from 32 track-and-field athletes. The study comprised of 16 “power athletes” requiring high speed foot movements (sprinters, jumpers, throwers) and 16 endurance athletes (distance runners) which in contrast do not require as high speed foot movements. Functional magnetic resonance imaging (fMRI) was used to identify speed specific regions of interest in the brain during fast and slow foot movements. Anatomical MRI scans were performed to assess structural grey matter volume differences between athletes groups (voxel based morphometry). We tested maximum movement velocity of plantarflexion (PF-Vmax) and acquired electromyographical activity of the lateral and medial gastrocnemius muscle. Behaviourally, a significant difference between the two groups of athletes was noted in PF-Vmax and fMRI indicates that fast plantarflexions are accompanied by increased activity in the cerebellar anterior lobe. The same region indicates increased grey matter volume for the power athletes compared to the endurance counterparts. Our results suggest that speed-specific neuro-functional and -structural differences exist between power and endurance athletes in the peripheral and central nervous system. PMID:24800742

Carnosine: effect on aging-induced increase in brain regional monoamine oxidase-A activity.

PubMed

Banerjee, Soumyabrata; Poddar, Mrinal K

2015-03-01

Aging is a natural biological process associated with several neurological disorders along with the biochemical changes in brain. Aim of the present investigation is to study the effect of carnosine (0.5-2.5μg/kg/day, i.t. for 21 consecutive days) on aging-induced changes in brain regional (cerebral cortex, hippocampus, hypothalamus and pons-medulla) mitochondrial monoamine oxidase-A (MAO-A) activity with its kinetic parameters. The results of the present study are: (1) The brain regional mitochondrial MAO-A activity and their kinetic parameters (except in Km of pons-medulla) were significantly increased with the increase of age (4-24 months), (2) Aging-induced increase of brain regional MAO-A activity including its Vmax were attenuated with higher dosages of carnosine (1.0-2.5μg/kg/day) and restored toward the activity that observed in young, though its lower dosage (0.5μg/kg/day) were ineffective in these brain regional MAO-A activity, (3) Carnosine at higher dosage in young rats, unlike aged rats significantly inhibited all the brain regional MAO-A activity by reducing their only Vmax excepting cerebral cortex, where Km was also significantly enhanced. These results suggest that carnosine attenuated the aging-induced increase of brain regional MAO-A activity by attenuating its kinetic parameters and restored toward the results of MAO-A activity that observed in corresponding brain regions of young rats. Copyright © 2014 Elsevier Ireland Ltd and the Japan Neuroscience Society. All rights reserved.

Kinetics of DCE and VC mineralization under methanogenic and Fe(III)- reducing conditions

USGS Publications Warehouse

Bradley, P.M.; Chapelle, F.H.

1997-01-01

The kinetics of anaerobic mineralization of DCE and VC under mathanogenic and Fe(III)-reducing conditions as a function of dissolved contaminant concentration were evaluated. Microorganisms indigenous to creek bed sediments, where groundwater contaminated with chlorinated ethenes continuously discharges, demonstrated significant mineralization of DCE and VC under methanogenic and Fe(III)- reducing conditions. Over 37 days, the recovery of [1,214C]VC radioactivity as 14CO2 ranged from 5% to 44% and from 8% to 100% under methanogenic and Fe(III)-reducing conditions, respectively. The recovery of [1,2-14C]DCE radioactivity as 14CO2 ranged from 4% to 14% and did not vary significantly between methanogenic and Fe(III)reducing conditions. VC mineralization was described by Michaelis- Menten kinetics. Under methanogenic conditions, V(max) was 0.19 ?? 0.01 ??mol L-1 d-1 and the half-saturation constant, k(m), was 7.6 ?? 1.7 ??M. Under Fe(III)-reducing conditions, V(max) was 0.76 ?? 0.07 ??mol L-1 d-1 and k(m) was 1.3 ?? 0.5 ??M. In contrast, DCE mineralization could be described by first-order kinetics. The first-order degradation rate constant for DCE mineralization was 0.6 ?? 0.2% d-1 under methanogenic and Fe(III)-reducing conditions. The results indicate that the kinetics of chlorinated ethane mineralization can vary significantly with the specific contaminant and the predominant redox conditions under which mineralization occurs.