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#28642093   2017/06/23 To Up

Functional characterization of 3-Ketosteroid 9α-Hydroxylases in Rhodococcus ruber strain Chol-4.

The 3-Ketosteroid-9α-Hydroxylase, also known as KshAB [androsta-1,4-diene-3,17-dione, NADH:oxygen oxidoreductase (9α-hydroxylating); EC 1.14.13.142)], is a key enzyme in the general scheme of the bacterial steroid catabolism in combination with a 3-ketosteroid-Δ(1)-dehydrogenase activity (KstD), being both responsible of the steroid nucleus (rings A/B) breakage. KshAB initiates the opening of the steroid ring by the 9α-hydroxylation of the C9 carbon of 4-ene-3-oxosteroids (e.g. AD) or 1,4-diene-3-oxosteroids (e.g. ADD), transforming them into 9α-hydroxy-4-androsten-3,17-dione (9OHAD) or 9α-hydroxy-1,4-androstadiene-3,17-dione (9OHADD), respectively. The redundancy of these enzymes in the actinobacterial genomes results in a serious difficulty for metabolic engineering this catabolic pathway to obtain intermediates of industrial interest. In this work, we have identified three homologous kshA genes and one kshB gen in different genomic regions of R. ruber strain Chol-4. We present a set of data that helps to understand their specific roles in this strain, including: i) description of the KshAB enzymes ii) construction and characterization of ΔkshB and single, double and triple ΔkshA mutants in R. ruber iii) growth studies of the above strains on different substrates and iv) genetic complementation and biotransformation assays with those strains. Our results show that KshA2 isoform is needed for the degradation of steroid substrates with short side chain, while KshA3 works on those molecules with longer side chains. KshA1 is a more versatile enzyme related to the cholic acid catabolism, although it also collaborates with KshA2 or KshA3 activities in the catabolism of steroids. Accordingly to what it is described for other Rhodococcus strains, our results also suggest that the side chain degradation is KshAB-independent.

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(3β)-3-(Acetyloxy)-chol- MOUSE ANTI BOVINE ROTAVIR Active Rat tPA Functional Active Mouse uPA Function Active Human uPA Function Total Human tPA Functiona Active human PAI-1 functi EpiSonic Multi Functional Active Rabbit PAI-1 Funct Active Porcine PAI-1 Func ELISA Human , IGFBP-3 (fu Active Mouse tPA Function

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#28643389   2017/06/23 To Up

Combined Molecular Modelling and 3D-QSAR Study for Understanding the Inhibition of NQO1 by Heterocyclic Quinone Derivatives.

A combination of three-dimensional quantitative structure-activity relationship (3D-QSAR), and molecular modelling methods were used to understand the potent inhibitory NAD(P)H:quinone oxidoreductase 1 (NQO1) activity of a set of 52 heterocyclic quinones. Molecular docking results indicated that some favourable interactions of key amino acid residues at the binding site of NQO1 with these quinones, would be responsible for an improvement of the NQO1 activity of these compounds. The main interactions involved are hydrogen bond of the amino group of residue Tyr128, π-stacking interactions with Phe106 and Phe178, and electrostatic interactions with flavin adenine dinucleotide (FADH) cofactor. Three models were prepared by 3D-QSAR analysis. The models derived from Model I and III, shown leave-one-out cross-validation correlation coefficients (q(2)loo ) of 0.75 and 0.73 as well as conventional correlation coefficients (R(2) ) of 0.93 and 0.95, respectively. In addition, the external predictive abilities of these models were evaluated using a test set, producing the predicted correlation coefficients (r(2)pred ) of 0.76 and 0.74, respectively. The good concordance between the docking results and 3D-QSAR contour maps provides helpful information about rational modification for new molecules based in quinone-scaffold, in order to design more potent NQO1 inhibitors, which would exhibit highly potent antitumor activity. This article is protected by copyright. All rights reserved.

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Carbohydrates or its deri Carbohydrates or its deri Mouse Anti-Human NQO1 Molecular & Cellular Biol Mouse Anti-Human NQO1, HR Goat Anti-Human, Mouse, D NQO1 antibody Source Rabb Directed In Vivo Angiogen  EpiQuik MBD2 Binding Ac EpiQuik MBD2 Binding Acti Agarose, Molecular Grade EpiQuik Histone Methyltra

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#28642839   2017/06/23 To Up

The Mitochondrial Complex(I)ty of Cancer.

Recent evidence highlights that the cancer cell energy requirements vary greatly from normal cells and that cancer cells exhibit different metabolic phenotypes with variable participation of both glycolysis and oxidative phosphorylation. NADH-ubiquinone oxidoreductase (Complex I) is the largest complex of the mitochondrial electron transport chain and contributes about 40% of the proton motive force required for mitochondrial ATP synthesis. In addition, Complex I plays an essential role in biosynthesis and redox control during proliferation, resistance to cell death, and metastasis of cancer cells. Although knowledge about the structure and assembly of Complex I is increasing, information about the role of Complex I subunits in tumorigenesis is scarce and contradictory. Several small molecule inhibitors of Complex I have been described as selective anticancer agents; however, pharmacologic and genetic interventions on Complex I have also shown pro-tumorigenic actions, involving different cellular signaling. Here, we discuss the role of Complex I in tumorigenesis, focusing on the specific participation of Complex I subunits in proliferation and metastasis of cancer cells.

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ATP synthase H+ transport MOUSE ANTI APAAP COMPLEX, Rat AntiORC6 (origin reco Rat AntiORC3 (origin reco Rat AntiORC1 (origin reco Rat AntiORC2 (origin reco Mitochondrial (MA) Ab Mitochondrial DNA Isolati Cell Navigator™ Mitocho Mouse Anti-Human Inner Mi Cell Navigator™ Mitocho Cell Navigator™ Mitocho

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#28642848   2017/06/23 To Up

Mycobacterium tuberculosis Glyceraldehyde-3-Phosphate Dehydrogenase (GAPDH) Functions as a Receptor for Human Lactoferrin.

Iron is crucial for the survival of living cells, particularly the human pathogen Mycobacterium tuberculosis (M.tb) which uses multiple strategies to acquire and store iron. M.tb synthesizes high affinity iron chelators (siderophores), these extract iron from host iron carrier proteins such as transferrin (Tf) and lactoferrin (Lf). Recent studies have revealed that M.tb may also relocate several housekeeping proteins to the cell surface for capture and internalization of host iron carrier protein transferrin. One of the identified receptors is the glycolytic enzyme Glyceraldehyde-3-phosphate dehydrogenase (GAPDH). This conserved multifunctional protein has been identified as a virulence factor in several other bacterial species. Considering the close structural and functional homology between the two major human iron carrier proteins (Tf and Lf) and the fact that Lf is abundantly present in lung fluid (unlike Tf which is present in plasma), we evaluated whether GAPDH also functions as a dual receptor for Lf. The current study demonstrates that human Lf is sequestered at the bacterial surface by GAPDH. The affinity of Lf-GAPDH (31.7 ± 1.68 nM) is higher as compared to Tf-GAPDH (160 ± 24 nM). Two GAPDH mutants were analyzed for their enzymatic activity and interaction with Lf. Lastly, the present computational studies offer the first significant insights for the 3D structure of monomers and assembled tetramer with the associated co-factor NAD(+). Sequence analysis and structural modeling identified the surface exposed, evolutionarily conserved and functional residues and predicted the effect of mutagenesis on GAPDH.

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Mycobacterium Tuberculosi Mycobacterium tuberculosi Rabbit Polyclonal to Myco Glucose 6 Phosphate Dehyd rabbit IgG against Leucon Glucose-6-Phosphate Dehyd Goat Anti-Glycerol-3-Phos anti-Lactoferrin (1B8), M anti-Lactoferrin , Rabbit MAb to Glyceraldehyde 3 P D (+) Glyceraldehyde CAS Human Lactoferrin ELISA K

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#28642875   2017/06/23 To Up

Rapid Elimination of Blood Alcohol Using Erythrocytes: Mathematical Modeling and In Vitro Study.

Erythrocytes (RBCs) loaded with alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALD) can metabolize plasma ethanol and acetaldehyde but with low efficiency. We investigated the rate-limiting factors in ethanol oxidation by these enzymes loaded into RBCs. Mathematical modeling and in vitro experiments on human RBCs loaded simultaneously with ADH and ALD (by hypoosmotic dialysis) were performed. The simulation showed that the rate of nicotinamide-adenine dinucleotide (NAD(+)) generation in RBC glycolysis, but not the activities of the loaded enzymes, is the rate-limiting step in external ethanol oxidation. The rate of oxidation could be increased if RBCs are supplemented by NAD(+) and pyruvate. Our experimental data verified this theoretical conclusion. RBCs loaded with the complete system of ADH, ALD, NAD(+), and pyruvate metabolized ethanol 20-40 times faster than reported in previous studies. The one-step procedure of hypoosmotic dialysis is the optimal method to encapsulate ADH and ALD in RBCs after cell recovery, encapsulation yield, osmotic resistance, and RBC-indexes. Consequently, transfusion of the RBCs loaded with the complete metabolic system, including ADH, ALD, pyruvate, and NAD(+) in the patients with alcohol intoxication, may be a promising method for rapid detoxification of blood alcohol based on metabolism.

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4 Hydroxy 3 methoxybenzyl Catalase, Human Erythrocy Sheep Anti-SOD, Cu Zn (Er Rabbit Anti-Bovine Carbon In vitro HUVECs Angiogene In vitro Human Lymphangio NICKEL RAPID RUN COBALT RAPID RUN COBALT RAPID RUN NICKEL RAPID RUN In vitro Human Glomerular In vitro Human Retina Mic

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#28646682   2017/06/24 To Up

Sensing NADH conformation using phasor analysis on fluorescence spectra.

Phasor analysis on fluorescence signals is a sensitive approach for analyzing multicomponent systems. Initially developed for time-resolved measurements, a spectral version has been used for the rapid identification of regions during the spectral imaging of biological systems. Here we show that quantitative information regarding conformation can be obtained from phasor analysis of fluorescence spectrum shape. Methanol denaturation of NADH and NADH binding to various dehydrogenase proteins are used as model reactions. Thermodynamic constants are calculated and compared with previous studies based on more direct measures of conformation. Next, the quantitative monitoring of UV-excited autofluorescence spectrum shape during chemically-induced metabolic transitions is presented and discussed in terms of NADH-utilizing pathways. Results show how phasor analysis is useful in assessing two-state behavior, and in interpreting autofluorescence as emission from an ensemble of cellular NADH forms.

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Amplite™ Fluorimetric N Amplite™ Fluorimetric N Amplite™ Fluorimetric N EnzyChrom™ NAD NADH Ass Cell Meter™ Cell Viabil NAD NADH Quantitation Kit EnzyChrom™ NAD NADH Ass EnzyFluo™ NAD NADH Assa Amplite™ Colorimetric N NADH disodium salt trihyd NADH disodium salt trihyd Rabbit Anti-Nox4 NADH Pol

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#28644902   2017/06/23 To Up

Sirt6 alters adult hippocampal neurogenesis.

Sirtuins are pleiotropic NAD+ dependent histone deacetylases involved in metabolism, DNA damage repair, inflammation and stress resistance. SIRT6, a member of the sirtuin family, regulates the process of normal aging and increases the lifespan of male mice over-expressing Sirt6 by 15%. Neurogenesis, the formation of new neurons within the hippocampus of adult mammals, involves several complex stages including stem cell proliferation, differentiation, migration and network integration. During aging, the number of newly generated neurons continuously declines, and this is correlated with a decline in neuronal plasticity and cognitive behavior. In this study we investigated the involvement of SIRT6 in adult hippocampal neurogenesis. Mice over-expressing Sirt6 exhibit increased numbers of young neurons and decreased numbers of mature neurons, without affecting glial differentiation. This implies of an involvement of SIRT6 in neuronal differentiation and maturation within the hippocampus. This work adds to the expanding body of knowledge on the regulatory mechanisms underlying adult hippocampal neurogenesis, and describes novel roles for SIRT6 as a regulator of cell fate during adult hippocampal neurogenesis.

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#28645327   2017/06/24 To Up

Impact of growth matrix on pharmacodynamics of antimicrobial drugs for pig pneumonia pathogens.

The most widely used measure of potency of antimicrobial drugs is Minimum Inhibitory Concentration (MIC). MIC is usually determined under standardised conditions in broths formulated to optimise bacterial growth on a species-by-species basis. This ensures comparability of data between laboratories. However, differences in values of MIC may arise between broths of differing chemical composition and for some drug classes major differences occur between broths and biological fluids such as serum and inflammatory exudate. Such differences must be taken into account, when breakpoint PK/PD indices are derived and used to predict dosages for clinical use. There is therefore interest in comparing MIC values in several broths and, in particular, in comparing broth values with those generated in serum. For the pig pneumonia pathogens, Actinobacillus pleuropneumoniae and Pasteurella multocida, MICs were determined for three drugs, florfenicol, oxytetracycline and marbofloxacin, in five broths [Mueller Hinton Broth (MHB), cation-adjusted Mueller Hinton Broth (CAMHB), Columbia Broth supplemented with NAD (CB), Brain Heart Infusion Broth (BHI) and Tryptic Soy Broth (TSB)] and in pig serum.

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#28646144   2017/06/24 To Up

Using Fractional Intensities of Time-resolved Fluorescence to Sensitively Quantify NADH/NAD(+) with Genetically Encoded Fluorescent Biosensors.

In this paper, we propose a novel and sensitive ratiometric analysis method that uses the fractional intensities of time-resolved fluorescence of genetically encoded fluorescent NADH/NAD(+) biosensors, Peredox, SoNar, and Frex. When the conformations of the biosensors change upon NADH/NAD(+) binding, the fractional intensities (α i τ i ) have opposite changing trends. Their ratios could be exploited to quantify NADH/NAD(+) levels with a larger dynamic range and higher resolution versus commonly used fluorescence intensity and lifetime methods. Moreover, only one excitation and one emission wavelength are required for this ratiometric measurement. This eliminates problems of traditional excitation-ratiometric and emission-ratiometric methods. This method could be used to simplify the design and achieve highly sensitive analyte quantification of genetically encoded fluorescent biosensors. Wide potential applications could be developed for imaging live cell metabolism based on this new method.

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Amplite™ Fluorimetric N Amplite™ Fluorimetric N Amplite™ Fluorimetric N Rainbow Fluorescent Parti N,N,N Trimethyl 4 (6 phen EnzyChrom™ NAD NADH Ass MarkerGene™ FDG Bacteri MarkerGene™ Cellular Se Fluorescent 100 bp DNA La Cell Meter™ Cell Viabil Anti PDX1 Polyclonal Anti pCMVLuxA Mammalian LuxA E

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#28646149   2017/06/24 To Up

Crucial role of 4-deoxy-L-erythro-5-hexoseulose uronate reductase for alginate utilization revealed by adaptive evolution in engineered Saccharomyces cerevisiae.

In brown macroalgae, alginate and D-mannitol are promising carbohydrates for biorefinery. Saccharomyces cerevisiae is widely used as a microbial cell factory, but this budding yeast is unable to utilize either alginate or D-mannitol. Alginate can be depolymerized by both endo-type and exo-type alginate lyases, yielding a monouronate, 4-deoxy-L-erythro-5-hexoseulose uronate (DEH), a key intermediate in the metabolism of alginate. Here, we constructed engineered two S. cerevisiae strains that are able to utilize both DEH and D-mannitol on two different strain backgrounds, and we also improved their aerobic growth in a DEH liquid medium through adaptive evolution. In both evolved strains, one of the causal mutations was surprisingly identical, a c.50A > G mutation in the codon-optimized NAD(P)H-dependent DEH reductase gene, one of the 4 genes introduced to confer the capacity to utilize DEH. This mutation resulted in an E17G substitution at a loop structure near the coenzyme-binding site of this reductase, and enhanced the reductase activity and aerobic growth in both evolved strains. Thus, the crucial role for this reductase reaction in the metabolism of DEH in the engineered S. cerevisiae is demonstrated, and this finding provides significant information for synthetic construction of a S. cerevisiae strain as a platform for alginate utilization.

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