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1.
Adv Exp Med Biol ; 1232: 375-381, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31893434

RESUMO

The value of optical redox imaging (ORI) of cells/tissues based on the intrinsic fluorescences of NADH (nicotinamide adenine dinucleotide) and oxidized flavoproteins (containing flavin adenine dinucleotide, i.e., FAD) has been demonstrated for potential biomedical applications including diagnosis, prognosis, and determining treatment response. However, the Chance redox scanner (a 3D cryogenic tissue imager) is limited by spatial resolution (~50 µm), and tissue ORI using fluorescence microscopy (single or multi-photon) is limited by the light penetration depth. Furthermore, viable or snap-frozen tissues are usually required. In this project, we aimed to study whether ORI may be achieved for unstained fixed tissue using a state-of-the-art modern Serial Two-Photon (STP) Tomography scanner that can rapidly acquire multi-plane images at micron resolution. Tissue specimens of mouse muscle, liver, and tumor xenografts were harvested and fixed in 4% paraformaldehyde (PFA) for 24 h. Tissue blocks were scanned by STP Tomography under room temperature to acquire the autofluorescence signals (NADH channel: excitation 750 nm, blue emission filter; FAD channel: excitation 860 nm, green emission filter). We observed remarkable signals with significant intra-tissue heterogeneity in images of NADH, FAD and redox ratio (FAD/(NADH+FAD)), which are worthy of further investigation for extracting biological information.


Assuntos
Tecnologia Biomédica , NAD , Imagem Óptica , Animais , Tecnologia Biomédica/instrumentação , Tecnologia Biomédica/métodos , Estudos de Viabilidade , Flavina-Adenina Dinucleotídeo , Xenoenxertos/diagnóstico por imagem , Camundongos , Oxirredução , Fótons
2.
J Agric Food Chem ; 68(2): 584-590, 2020 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-31623437

RESUMO

Flavor stability is a significant concern to brewers as the staling compounds impart unpleasant flavor to beer. Thus, yeasts with antistaling ability have been engineered to produce beer with improved flavor stability. Here, we proposed that increasing the NADH availability of yeast could improve the flavor stability of beer. By engineering endogenous pathways, we obtained an array of yeast strains with a higher reducing activity. Then, we carried out beer fermentation with these strains and found that the antistaling capacities of the beer samples were improved. For a better understanding of the underlying mechanism, we compared the flavor profiles of these strains. The production of staling components was significantly decreased, whereas the content of antistaling components, such as SO2, was increased, in line with the increased antistaling ability. The other aroma components were marginally changed, indicating that this concept was useful for improving the antistaling stability without changing the flavor of beer.


Assuntos
Cerveja/análise , Aromatizantes/metabolismo , NAD/metabolismo , Saccharomyces/metabolismo , Fermentação , Engenharia Genética , Odorantes/análise , Saccharomyces/genética
3.
Food Chem ; 305: 125439, 2020 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-31499287

RESUMO

Compared to the control longans, hydrogen peroxide (H2O2)-treated longans exhibited higher index of pulp breakdown, higher fruit respiration rate, higher activities of pulp phosphohexose isomerase (PGI), succinate dehydrogenase (SDH), cytochrome C oxidase (CCO), ascorbic acid oxidase (AAO) and polyphenol oxidase (PPO), but lower activity of pulp nicotinamide adenine dinucleotide kinase (NADK). H2O2-treated longans also exhibited lower total activities of pulp glucose-6-phosphate dehydrogenase (G-6-PDH) and 6-phosphogluconate dehydrogenase (6-PGDH), lower levels of pulp NADP(H), but higher levels of pulp NAD(H). These data indicated that H2O2-stimulated longan pulp breakdown was owing to a decreased proportion of pentose phosphate pathway (PPP), the increased proportions of Embden-Meyerhof-Parnas pathway (EMP), tricarboxylic acid (TCA) cycle and cytochrome pathway (CCP) in total respiratory pathways. These findings further revealed that H2O2 could enhance respiration rate, and thus accelerate pulp breakdown occurrence and shorten the shelf life of longan fruit.


Assuntos
Peróxido de Hidrogênio/farmacologia , Sapindaceae/efeitos dos fármacos , Aldeído Oxidase/metabolismo , Ciclo do Ácido Cítrico/efeitos dos fármacos , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Armazenamento de Alimentos , Frutas/metabolismo , Glucosefosfato Desidrogenase/metabolismo , Glicólise/efeitos dos fármacos , NAD/metabolismo , Via de Pentose Fosfato/efeitos dos fármacos , Sapindaceae/metabolismo
4.
J Agric Food Chem ; 67(51): 14121-14128, 2019 Dec 26.
Artigo em Inglês | MEDLINE | ID: mdl-31775508

RESUMO

Heliotropin, a compound with important roles in the spice and fragrance industries and broad application prospects, is mainly produced through chemical methods. Here, we established a novel process for the synthesis of heliotropin by Escherichia coli whole cells through biotransformation of isosafrole. Directed evolution and high-throughput screening based on 2,4-dinitrophenylhydrazine were used to improve the activity of trans-anethole oxygenase toward isosafrole, and a mutant (TAO3G2) was obtained that had a high ability to oxidize isosafrole. Formate dehydrogenase (FDH) and TAO3G2 were coexpressed in E. coli, significantly increasing the catalytic efficiency by regenerating more NADH to promote isosafrole oxidation. Furthermore, after optimizing the molar ratio of isosafrole to the auxiliary substrate, the final concentration of heliotropin was increased from 9.15 to 19.45 g/L, and the maximum yield and space-time yield reached 96.02% and 3.89 g/L/h, respectively. These results suggest that the biosynthesis of heliotropin should have excellent industrial application value.


Assuntos
Benzaldeídos/metabolismo , Benzodioxóis/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Formiato Desidrogenases/genética , Proteínas Fúngicas/genética , Oxigenases/genética , Candida/enzimologia , Formiato Desidrogenases/metabolismo , Proteínas Fúngicas/metabolismo , Engenharia Metabólica , NAD/metabolismo , Oxigenases/metabolismo , Safrol/metabolismo
5.
Biochemistry (Mosc) ; 84(11): 1403-1410, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31760926

RESUMO

Genomes of photoautotrophic organisms containing type I photosynthetic reaction center were searched for the rnf genes encoding Na+-translocating ferredoxin:NAD+ oxidoreductase (RNF). These genes were absent in heliobacteria, cyanobacteria, algae, and plants; however, genomes of many green sulfur bacteria (especially marine ones) were found to contain the full rnf operon. Analysis of RNA isolated from the marine green sulfur bacterium Chlorobium phaeovibrioides revealed a high level of rnf expression. It was found that the activity of Na+-dependent flavodoxin:NAD+ oxidoreductase detected in the membrane fraction of Chl. phaeovibrioides was absent in the membrane fraction of the freshwater green sulfur bacterium Chlorobaculum limnaeum, which is closely related to Chl. phaeovibrioides but whose genome lacks the rnf genes. Illumination of the membrane fraction of Chl. phaeovibrioides but not of Cba. limnaeum resulted in the light-induced NAD+ reduction. Based on the obtained data, we concluded that in some green sulfur bacteria, RNF may be involved in the NADH formation that should increase the efficiency of light energy conservation in these microorganisms and can serve as the first example of the use of Na+ energetics in photosynthetic electron transport chains.


Assuntos
Proteínas de Bactérias/metabolismo , Chlorobi/metabolismo , Oxirredutases/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Transporte de Elétrons , Luz , NAD/química , NAD/metabolismo , Oxirredutases/química , Oxirredutases/genética , Fotossíntese , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação
6.
Chem Soc Rev ; 48(23): 5596-5615, 2019 Nov 25.
Artigo em Inglês | MEDLINE | ID: mdl-31675020

RESUMO

A number of self-sufficient hydride transfer processes have been reported in biocatalysis, with a common feature being the dependence on nicotinamide as a cofactor. This cofactor is provided in catalytic amounts and serves as a hydride shuttle to connect two or more enzymatic redox events, usually ensuring overall redox neutrality. Creative systems were designed to produce synthetic sequences characterized by high hydride economy, typically going in hand with excellent atom economy. Several redox enzymes have been successfully combined in one-pot one-step to allow functionalization of a large variety of molecules while preventing by-product formation. This review analyzes and classifies the various strategies, with a strong focus on efficiency, which is evaluated here in terms of the hydride economy and measured by the turnover number of the nicotinamide cofactor(s). The review ends with a critical evaluation of the reported systems and highlights areas where further improvements might be desirable.


Assuntos
Enzimas/metabolismo , NAD/metabolismo , Biocatálise , Enzimas/química , Isomerismo , NAD/química , Oxirredução , Especificidade por Substrato
7.
J Agric Food Chem ; 67(48): 13327-13332, 2019 Dec 04.
Artigo em Inglês | MEDLINE | ID: mdl-31715101

RESUMO

The biochemical basis of lower metmyoglobin reducing activity (MRA) in high-oxygen modified atmospheric packaged (HiOx-MAP) beef than those in vacuum and polyvinyl chloride (PVC) packaging is not clear. To explore this, the effects of lipid oxidation products with varying carbon chain length on lactate dehydrogenase (LDH) and NADH-dependent metmyoglobin reductase activity were evaluated. Surface color, MRA, and lipid oxidation of beef longissimus lumborum steaks (n = 10) were measured during 6-day display. Further, two enzymes, LDH and NADH-dependent metmyoglobin reductase (n = 5), critical for MRA were incubated with or without (control) lipid oxidation products of varying carbon chain length: malondialdehyde (3-carbon), hexenal (6-carbon), and 4-hydroxynonenal (9-carbon). Steaks in HiOx-MAP had greater (P < 0.05) redness than vacuum and PVC, but had lower (P < 0.05) MRA and greater (P < 0.05) lipid oxidation on day 6. LDH and NADH-dependent metmyoglobin reductase activities were differentially influenced by lipid oxidation products (P < 0.05). The results indicate that the difference in reactivity of various lipid oxidation products on LDH (HNE > MDA = hexenal) and NADH-dependent metmyoglobin reductase (HNE = MDA > hexenal) activity could be responsible for lower MRA in HiOx-MAP.


Assuntos
Carbono/química , L-Lactato Desidrogenase/química , Lipídeos/química , NADH NADPH Oxirredutases/química , Carne Vermelha/análise , Animais , Carbono/metabolismo , Bovinos , Embalagem de Alimentos , L-Lactato Desidrogenase/metabolismo , Metamioglobina/química , Metamioglobina/metabolismo , Músculo Esquelético/química , Músculo Esquelético/enzimologia , Músculo Esquelético/metabolismo , NAD/química , NAD/metabolismo , NADH NADPH Oxirredutases/metabolismo , Oxirredução
8.
J Phys Chem A ; 123(45): 9865-9873, 2019 Nov 14.
Artigo em Inglês | MEDLINE | ID: mdl-31638388

RESUMO

Phasor FLIM in cells undergoing oxidative stress and in mice liver sections have shown the presence of a third autofluorescent component indicative of lipid droplets along with free and enzyme-bound NADH with similar emissions. This third component affects the position and shape of the phasor distribution, pushing it away from the metabolic trajectory. Phasor rule of addition is still valid and was exploited here to create a multicomponent analysis where the phasor distribution can be reassigned to the metabolic trajectory and changes in metabolism can be detected independently of the intensity of this third component. Calculation of multiple components from FLIM imaging data of biological systems is a difficult process, especially if different fluorescent species are present at the same pixel. This paper describes the methodology that can be used to separate these multiple components when they are present in the phasor signature acquired in a single pixel of an image.


Assuntos
NAD/análise , Células HeLa , Humanos , Microscopia de Fluorescência , NAD/metabolismo , Imagem Óptica , Proteínas/metabolismo
9.
Phys Chem Chem Phys ; 21(44): 24572-24583, 2019 Nov 13.
Artigo em Inglês | MEDLINE | ID: mdl-31663551

RESUMO

In this work, poly(N,N'-dimethylaminoethylmethacrylate-co-N-isopropylacrylamide) copolymer films were polymerized on the surface of Au electrodes with a facile one-step method, and Au nanoclusters (AuNCs) and tetraphenylethene (TPE) were synchronously embedded in the films, designated as P(DMA-co-NIPA)/AuNCs/TPE. Ferrocene dicarboxylic acid (FDA), an electroactive probe in solution displayed inverse pH- and SO42--sensitive on-off cyclic voltammetric (CV) behaviors at the film electrodes. The electrocatalytic oxidation of nicotinamide adenine dinucleotide (NADH) mediated by FDA in solution could substantially amplify the CV response difference between the on and off states. Moreover, the two fluorescence emission (FL) signals from the TPE constituent at 450 nm and AuNCs component at 660 nm in the films also demonstrated SO42-- and pH-sensitive behaviors. Based on the aforementioned results, a 4-input/9-output biomolecular logic circuit was constructed with pH, Na2SO4, FDA and NADH as the inputs, and the CV signals and the FL responses at 450 and 660 nm at different levels as the outputs. Additionally, some functional non-Boolean devices were elaborately designed on an identical platform, including a 1-to-2 decoder, a 2-to-1 encoder, a 1-to-2 demultiplexer and different types of keypad locks. This work combines copolymer films, bioelectrocatalysis, and fluorescence together so that more complicated biocomputing systems could be established. This work may pave a new way to develop advanced and sophisticated biocomputing logic circuits and functional devices in the future.


Assuntos
Técnicas Eletroquímicas/métodos , Nanopartículas Metálicas/química , NAD/química , Polímeros/química , Estilbenos/química , Benzoatos/química , Eletrodos , Compostos Ferrosos/química , Ouro/química , Concentração de Íons de Hidrogênio , Metalocenos , Oxirredução , Espectrometria de Fluorescência , Sulfatos/química
10.
Nat Commun ; 10(1): 4196, 2019 09 13.
Artigo em Inglês | MEDLINE | ID: mdl-31519936

RESUMO

Nicotinamide adenine dinucleotide (NAD+)-dependent ADP-ribosylation plays important roles in physiology and pathophysiology. It has been challenging to study this key type of enzymatic post-translational modification in particular for protein poly-ADP-ribosylation (PARylation). Here we explore chemical and chemoenzymatic synthesis of NAD+ analogues with ribose functionalized by terminal alkyne and azido groups. Our results demonstrate that azido substitution at 3'-OH of nicotinamide riboside enables enzymatic synthesis of an NAD+ analogue with high efficiency and yields. Notably, the generated 3'-azido NAD+ exhibits unexpected high activity and specificity for protein PARylation catalyzed by human poly-ADP-ribose polymerase 1 (PARP1) and PARP2. And its derived poly-ADP-ribose polymers show increased resistance to human poly(ADP-ribose) glycohydrolase-mediated degradation. These unique properties lead to enhanced labeling of protein PARylation by 3'-azido NAD+ in the cellular contexts and facilitate direct visualization and labeling of mitochondrial protein PARylation. The 3'-azido NAD+ provides an important tool for studying cellular PARylation.


Assuntos
NAD/metabolismo , ADP Ribose Transferases/metabolismo , Cromatografia Líquida de Alta Pressão , Células HeLa , Humanos , Espectroscopia de Ressonância Magnética , Modelos Biológicos , Nicotinamida-Nucleotídeo Adenililtransferase/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Poli(ADP-Ribose) Polimerase-1/metabolismo , Poli ADP Ribosilação , Poli(ADP-Ribose) Polimerases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Sirtuína 2/metabolismo
11.
Nat Commun ; 10(1): 4291, 2019 09 20.
Artigo em Inglês | MEDLINE | ID: mdl-31541116

RESUMO

Supplementation with the NAD+ precursor nicotinamide riboside (NR) ameliorates and prevents a broad array of metabolic and aging disorders in mice. However, little is known about the physiological role of endogenous NR metabolism. We have previously shown that NR kinase 1 (NRK1) is rate-limiting and essential for NR-induced NAD+ synthesis in hepatic cells. To understand the relevance of hepatic NR metabolism, we generated whole body and liver-specific NRK1 knockout mice. Here, we show that NRK1 deficiency leads to decreased gluconeogenic potential and impaired mitochondrial function. Upon high-fat feeding, NRK1 deficient mice develop glucose intolerance, insulin resistance and hepatosteatosis. Furthermore, they are more susceptible to diet-induced liver DNA damage, due to compromised PARP1 activity. Our results demonstrate that endogenous NR metabolism is critical to sustain hepatic NAD+ levels and hinder diet-induced metabolic damage, highlighting the relevance of NRK1 as a therapeutic target for metabolic disorders.


Assuntos
Dieta Hiperlipídica/efeitos adversos , Hepatopatias/prevenção & controle , Niacinamida/análogos & derivados , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Substâncias Protetoras/metabolismo , Substâncias Protetoras/farmacologia , Animais , Glicemia , Dano ao DNA , Modelos Animais de Doenças , Técnicas de Inativação de Genes , Predisposição Genética para Doença/genética , Intolerância à Glucose , Hepatócitos/metabolismo , Resistência à Insulina , Metabolismo dos Lipídeos , Fígado/metabolismo , Hepatopatias/genética , Hepatopatias/patologia , Masculino , Síndrome Metabólica/genética , Síndrome Metabólica/prevenção & controle , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , NAD/metabolismo , Niacinamida/genética , Niacinamida/metabolismo , Niacinamida/farmacologia
12.
Org Biomol Chem ; 17(38): 8716-8720, 2019 10 14.
Artigo em Inglês | MEDLINE | ID: mdl-31538639

RESUMO

Nicotinamide adenine dinucleotide, NAD+, is an essential cofactor and substrate for many cellular enzymes. Its sustained intracellular levels have been linked to improved physiological end points in a range of metabolic diseases. Biosynthetic precursors to NAD+ include nicotinic acid, nicotinamide, the ribosylated parents and the phosphorylated form of the ribosylated parents. By combining solvent-assisted mechanochemistry and sealed reaction conditions, access to the ribosylated NAD+ precursors and to the isotopologues of NAD+ precursors was achieved in high yields and levels of purity. The latter is critical as it offers means to better trace biosynthetic pathways to NAD+, investigate the multifaceted roles of the intracellular NAD+ pools, and better exploit NAD+ biology.


Assuntos
NAD/síntese química , Estrutura Molecular , NAD/química
13.
Korean J Parasitol ; 57(4): 423-427, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31533410

RESUMO

Coenurosis is an important zoonotic helminthic disease caused by the larval stage of the tapeworm Taenia multiceps. This parasite typically infects the brain of the intermediate hosts, including sheep, goat, cattle and even humans. We report a case of T. multiceps infection in a yak confirmed by clinical symptoms, morphological characteristics, and molecular and phylogenetic analyses. The coenurus was thin-walled, whitish, and spherical in shape with a diameter of 10 cm. The parasite species was identified as T. multiceps by PCR amplification and sequencing of the 18S rRNA, cox1 and nad1 genes. Three gene sequences all showed high homology (all above 97%) with the reference sequences from different hosts. Moreover, phylogenetic reconstructions with the 3 published Taenia gene sequences confirmed that the Qinghai yak isolate was closely related to T. multiceps. Although there are advanced diagnosis and treatment methods for coenurosis, early infection is difficult to diagnose. Importantly, the findings of yak infection case should not be ignored due to its zoonotic potential.


Assuntos
Doenças dos Bovinos/parasitologia , Neurocisticercose/veterinária , Taenia/genética , Animais , Bovinos , Ciclo-Oxigenase 1/genética , Eletroforese em Gel de Ágar/veterinária , Masculino , NAD/genética , Neurocisticercose/parasitologia , Filogenia , Reação em Cadeia da Polimerase/veterinária , RNA Ribossômico 18S/genética , Alinhamento de Sequência/veterinária , Análise de Sequência de DNA/veterinária , Taenia/classificação , Taenia/isolamento & purificação , Tibet
14.
Mater Sci Eng C Mater Biol Appl ; 104: 109938, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31499948

RESUMO

Surface based on polyelectrolytes functionalized with amino acids onto amino-terminated solid surfaces of silicon wafers was prepared, with the purpose of evaluate the chemical functionality of the polyelectrolyte films in adsorption and catalytic activity of an enzyme. In this work, the adsorption of the enzyme glucose 6-phosphate dehydrogenase from Leuconostoc mesenteroides (LmG6PD) was studied as model. The polyelectrolytes were obtained from poly (maleic anhydride-alt-vinylpyrrolidone) [poly(MA-alt-VP)] and functionalized with amino acids of different hydropathy index: glutamine (Gln), tyrosine (Tyr) and methionine (Met). The polyelectrolytes were adsorbed onto the amino-terminated silicon wafer at pH 3.5 and 4.5 and at low and high ionic strength. At low ionic strength and pH 3.5, the largest quantity of adsorbed polyelectrolyte was on the films containing glutamine moiety as the most hydrophilic amino acid in the side chain of polymer chain (5.88 mg/m2), whereas at high ionic strength and pH 4.5, the lowest quantity was in films containing tyrosine moiety in the side chain (1.88 mg/m2). The films were characterized by ellipsometry, contact angle measurements and atomic force microscopy (AFM). The polyelectrolyte films showed a moderate degree of hydrophobicity, the methionine derivative being the most hydrophobic film. With the aim of evaluate the effect of the amino acid moieties on the ability of the surface to adsorb enzymes, we study the activity of the enzyme on these surfaces. We observed that the polarity of the side chain of the amino acid in the polyelectrolyte affected the quantity of LmG6PD adsorbed, as well as its specific activity, showing that films prepared from poly(MA-alt-VP) functionalized with Met provide the best enzymatic performance. The results obtained demonstrated that the surfaces prepared from polyelectrolytes functionalized with amino acids could be an attractive and simple platform for the immobilization of enzymes, which could be of interest for biocatalysis applications.


Assuntos
Aminoácidos/metabolismo , Enzimas Imobilizadas/metabolismo , Polieletrólitos/metabolismo , Adsorção , Espectroscopia de Ressonância Magnética Nuclear de Carbono-13 , Glucosefosfato Desidrogenase/metabolismo , Leuconostoc/enzimologia , NAD/biossíntese , Polieletrólitos/química , Espectroscopia de Infravermelho com Transformada de Fourier , Molhabilidade
15.
Chemistry ; 25(67): 15288-15294, 2019 Dec 02.
Artigo em Inglês | MEDLINE | ID: mdl-31483908

RESUMO

Nanoscale assemblies of DNA strands are readily designed and can be generated in a wide range of shapes and sizes. Turning them into solids that bind biomolecules reversibly, so that they can act as active material in flow cells, is a challenge. Among the biomolecular ligands, cofactors are of particular interest because they are often the most expensive reagents of biochemical transformations, for which controlled release and recycling are desirable. We have recently described DNA triplex motifs that bind adenine-containing cofactors, such as NAD, FAD and ATP, reversibly with low micromolar affinity. We sought ways to convert the soluble DNA motifs into a macroporous solid for cofactor binding. While assemblies of linear and branched DNA motifs produced hydrogels with undesirable properties, long DNA triplexes treated with protamine gave materials suitable for flow cells. Using exchangeable cells in a flow system, thermally controlled loading and discharge were demonstrated. Employing a flow cell loaded with ATP, bioluminescence was induced through thermal release of the cofactor. The results show that materials generated from functional DNA structures can be successfully employed in macroscopic devices.


Assuntos
Adenina/química , DNA/química , Nanopartículas/química , Trifosfato de Adenosina/química , Sítios de Ligação , Flavina-Adenina Dinucleotídeo/química , Ligantes , NAD/química , Motivos de Nucleotídeos , Espectrometria de Fluorescência/métodos , Termodinâmica
16.
Nat Commun ; 10(1): 4068, 2019 09 06.
Artigo em Inglês | MEDLINE | ID: mdl-31492851

RESUMO

The aldehyde dehydrogenase (ALDH) family of metabolic enzymes converts aldehydes to carboxylates. Here, we find that the reductive consequence of ALDH7A1 activity, which generates NADH (nicotinamide adenine dinucleotide, reduced form) from NAD, underlies how ALDH7A1 coordinates a broad inhibition of the intracellular transport pathways. Studying vesicle formation by the Coat Protein I (COPI) complex, we elucidate that NADH generated by ALDH7A1 targets Brefeldin-A ADP-Ribosylated Substrate (BARS) to inhibit COPI vesicle fission. Moreover, defining a physiologic role for the broad transport inhibition exerted by ALDH7A1, we find that it acts to reduce energy consumption during hypoxia and starvation to promote cellular energy homeostasis. These findings advance the understanding of intracellular transport by revealing how the coordination of multiple pathways can be achieved, and also defining circumstances when such coordination is needed, as well as uncovering an unexpected way that NADH acts in cellular energetics.


Assuntos
Oxirredutases do Álcool/metabolismo , Aldeído Desidrogenase/metabolismo , Proteínas de Ligação a DNA/metabolismo , Metabolismo Energético , Homeostase , Espaço Intracelular/metabolismo , Oxirredutases do Álcool/genética , Aldeído Desidrogenase/genética , Transporte Biológico , Vesículas Revestidas pelo Complexo de Proteína do Envoltório/metabolismo , Hipóxia Celular , Proteínas de Ligação a DNA/genética , Células HEK293 , Células HeLa , Humanos , NAD/metabolismo , Transdução de Sinais , Inanição
17.
Invest Ophthalmol Vis Sci ; 60(10): 3538-3546, 2019 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-31415077

RESUMO

Purpose: To determine if trigeminal innervations of the corneal epithelium maintains its integrity and homeostasis through controlling the nicotinamide adenine dinucleotide (NAD) content of this tissue. Methods: Corneal denervation of C57BL/6 mice was induced by squeezing the nerve bundles that derive from the trigeminal ganglion and was confirmed by whole-mount corneal nerve staining and the sensation test. The apoptosis of the corneal epithelium was examined by TUNEL assay and annexin V/propidium iodide staining. NAD biosynthesis-related enzymes were analyzed by quantitative PCR, immunofluorescence staining, and Western blotting. FK866, an inhibitor of nicotinamide phosphoribosyltransferase (NAMPT), exogenous nicotinamide mononucleotide (NMN), and NAD+ were used to evaluate the effect of NAD+ on the apoptosis of cultured corneal epithelial cells and epithelial detachment in denervated mice. Protein expression that related to apoptosis and phosphorylation were analyzed by Western blotting. Results: The denervated mice showed spontaneous corneal epithelial detachment and cell apoptosis accompanied with impaired epithelial NAD+ contents due to low levels of NAMPT. Similarly, inhibition of NAMPT recapitulated epithelial detachment as in denervated mice and induced apoptosis in cultured corneal epithelial cells. The replenishment of NMN or NAD+ partially slowed down corneal nerve fiber degeneration, reduced the epithelial defect in denervated mice, and improved apoptosis induction in FK866-treated cells by restoring the activation levels of SIRT1, AKT, and CREB. Conclusions: Corneal denervation lowered epithelial NAD+ contents through reducing the expression of NAMPT and caused cell apoptosis and epithelial defects, suggesting that corneal innervations contribute to epithelial homeostasis by regulating NAD+ biosynthesis.


Assuntos
Apoptose , Córnea/inervação , Denervação , Epitélio Anterior/patologia , NAD/metabolismo , Nervo Trigêmeo/fisiologia , Animais , Anexina A5/metabolismo , Western Blotting , Proteína de Ligação a CREB/metabolismo , Córnea/metabolismo , Doenças da Córnea/diagnóstico , Doenças da Córnea/metabolismo , Epitélio Anterior/metabolismo , Técnica Indireta de Fluorescência para Anticorpo , Marcação In Situ das Extremidades Cortadas , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Sirtuína 1/metabolismo , Doenças do Nervo Trigêmeo/diagnóstico , Doenças do Nervo Trigêmeo/metabolismo , Proteína X Associada a bcl-2/metabolismo
18.
J Microbiol Biotechnol ; 29(8): 1288-1298, 2019 Aug 28.
Artigo em Inglês | MEDLINE | ID: mdl-31370116

RESUMO

Bacterial ATP synthases drive ATP synthesis by a rotary mechanism, and play a vital role in physiology and cell metabolism. Corynebacterium glutamicum is well known as an industrial workhorse for amino acid production, and its ATP synthase operon contains eight structural genes and two adjacent genes, cg1360 and cg1361. So far, the physiological functions of Cg1360 (GenBank CAF19908) and Cg1361 (GenBank CAF19909) remain unclear. Here, we showed that Cg1360 was a hydrophobic protein with four transmembrane helices (TMHs), while no TMH was found in Cg1361. Deletion of cg1360, but not cg1361, led to significantly reduced cell growth using glucose and acetic acid as carbon sources, reduced F1 portions in the membrane, reduced ATP-driven proton-pumping activity and ATPase activity, suggesting that Cg1360 plays an important role in ATP synthase function. The intracellular ATP concentration in the Δcg1360 mutant was decreased to 72% of the wild type, while the NADH and NADPH levels in the Δcg1360 mutant were increased by 29% and 26%, respectively. However, the Δcg1361 mutant exhibited comparable intracellular ATP, NADH and NADPH levels with the wild-type strain. Moreover, the effect of cg1360 deletion on L-valine production was examined in the L-valine-producing V-10 strain. The final production of L-valine in the V-10-Δcg1360 mutant reached 9.2 ± 0.3 g/l in shake flasks, which was 14% higher than that of the V-10 strain. Thus, Cg1360 can be used as an effective engineering target by altering energy metabolism for the enhancement of amino acid production in C. glutamicum.


Assuntos
Trifosfato de Adenosina/metabolismo , Corynebacterium glutamicum/genética , Corynebacterium glutamicum/metabolismo , Deleção de Genes , ATPases Mitocondriais Próton-Translocadoras/metabolismo , Valina/biossíntese , Ácido Acético/metabolismo , Adenosina Trifosfatases , Carbono/metabolismo , Corynebacterium glutamicum/crescimento & desenvolvimento , Metabolismo Energético , Fermentação , Ordem dos Genes , Glucose/metabolismo , NAD/metabolismo , NADP/metabolismo , Alinhamento de Sequência
19.
Biochim Biophys Acta Bioenerg ; 1860(10): 148062, 2019 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-31419395

RESUMO

The hydrogen-oxidizing "Knallgas" bacterium Ralstonia eutropha can thrive in aerobic and anaerobic environments and readily switches between heterotrophic and autotrophic metabolism, making it an attractive host for biotechnological applications including the sustainable H2-driven production of hydrocarbons. The soluble hydrogenase (SH), one out of four different [NiFe]-hydrogenases in R. eutropha, mediates H2 oxidation even in the presence of O2, thus providing an ideal model system for biological hydrogen production and utilization. The SH reversibly couples H2 oxidation with the reduction of NAD+ to NADH, thereby enabling the sustainable regeneration of this biotechnologically important nicotinamide cofactor. Thus, understanding the interaction of the SH with the cellular NADH/NAD+ pool is of high interest. Here, we applied the fluorescent biosensor Frex to measure changes in cytoplasmic [NADH] in R. eutropha cells under different gas supply conditions. The results show that Frex is well-suited to distinguish SH-mediated changes in the cytoplasmic redox status from effects of general anaerobiosis of the respiratory chain. Upon H2 supply, the Frex reporter reveals a robust fluorescence response and allows for monitoring rapid changes in cellular [NADH]. Compared to the Peredox fluorescence reporter, Frex displays a diminished NADH affinity, which prevents the saturation of the sensor under typical bacterial [NADH] levels. Thus, Frex is a valuable reporter for on-line monitoring of the [NADH]/[NAD+] redox state in living cells of R. eutropha and other proteobacteria. Based on these results, strategies for a rational optimization of fluorescent NADH sensors are discussed.


Assuntos
Técnicas Biossensoriais/métodos , Cupriavidus necator/metabolismo , Hidrogênio/metabolismo , NAD/análise , Anaerobiose , Técnicas Biossensoriais/normas , Cupriavidus necator/citologia , Hidrogenase , NAD/metabolismo , Oxirredução
20.
Biosens Bioelectron ; 143: 111598, 2019 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-31442753

RESUMO

Herein, we report the anionic surfactant, ethylene diamine tetraacetic acid (EDTA), mediated synthesis of WO3 nanoparticles and its subsequent modification through gamma irradiation (GI) and electrochemical immobilization with nicotinamide adenine dinucleotide (NAD). Glassy carbon electrode (GCE) modified with GI-WO3 NPs and the enzyme NAD exhibited strong electro-oxidation of three important biomolecules such as norepinephrine (NEP), melatonin (MEL) and nicotine (NIC) in 0.1 M phosphate buffer saline (PBS) at physiological pH of 7. Square wave voltammetry (SWV) studies exhibited three well-defined peaks at potentials of 120, 570 and 840 mV, corresponding to the oxidation of NEP, MEL and NIC respectively, indicating that simultaneous determination of these compounds is feasible at the NAD/GI EDTA-WO3/GCE. The proposed sensor displayed a wide linear range of 0.010-1000 µM with the lowest detection limit of 1.4 nM for NEP, 2.6 nM for MEL and 1.7 nM for NIC respectively. Furthermore, the modified electrode was successfully applied to detect NEP, MEL and NIC in pharmaceutical and cigarette samples with excellent selectivity and reproducibility.


Assuntos
Técnicas Biossensoriais , Melatonina/isolamento & purificação , Nicotina/isolamento & purificação , Norepinefrina/isolamento & purificação , Técnicas Eletroquímicas , Limite de Detecção , Melatonina/química , NAD/química , Nanopartículas/química , Nanotubos de Carbono/química , Nicotina/química , Norepinefrina/química , Óxidos/química , Tungstênio/química
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