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1.
Acta Cir Bras ; 34(11): e201901102, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31859816

RESUMO

PURPOSE: To investigate the effect of Picroside II on testicular ischemia and reperfusion (l/R) injury and the underlying mechanism. METHODS: Sprague-Dawley rats were randomly divided into 4 groups: sham operated group (Sham), Sham with Picroside II treatment group (Sham+ Pic II), l/R group (l/R) and l/R with Picroside II treatment group (I/R+ Pic II). l/R model was established by rotating the left testis 720° in a clock-wise direction for 4 hours. The histopathologic and spermatogenetic evaluation was performed. The apoptosis changes and the levels of HO-1 (heme oxygenase-1), MPO (myeloperoxidase), NOX (NADPH oxidase), SOD (superoxide dismutase), XO (xanthine oxidase) and NOS (nitric oxide synthase) were measured. RESULTS: The seminiferous tubules were damaged in l/R rats, but Picroside II alleviated the changes induced by l/R. The increased level of apoptosis was decreased by Picroside II (P=0.01, 9.05±0.35 vs. 4.85±0.25). The activities of HO-1, MPO, NOX, XO and MDA content were increased and the SOD activity was decreased in l/R (P<0.05) and could be reversed by Picroside II (P=0.03, 405.5±7.5 vs. 304±17U/mgprot; P=0.02, 0.99±0.05 vs. 0.52±0.04 mgprot; P=0.01, 260+7 vs. 189±2 mgprot; P=0.04, 10.95+0.55 vs. 8.75+0.35 U/mgprot; P=0.045, 6.8+0.7 vs. 3.75+0.35 mgprot; P=0.04, 44.5+3.5 vs. 57.5+3.5 mgprot). Western blot showed that the expression of iNOS, nNOS and eNOS were increased in l/R (P<0.05); however, they were decreased after Picroside II treatment (P<0.05). CONCLUSION: Picroside II attenuated testicular I/R injury in rats mainly through suppressing apoptosis and oxidative stress through reduction of nitric oxide synthesis.


Assuntos
Apoptose/efeitos dos fármacos , Cinamatos/farmacologia , Glucosídeos Iridoides/farmacologia , Óxido Nítrico/biossíntese , Estresse Oxidativo/efeitos dos fármacos , Traumatismo por Reperfusão/prevenção & controle , Testículo/irrigação sanguínea , Animais , Western Blotting , Heme Oxigenase-1/análise , Marcação In Situ das Extremidades Cortadas , Masculino , Malondialdeído/análise , NADP/análise , Peroxidase/análise , Distribuição Aleatória , Ratos Sprague-Dawley , Traumatismo por Reperfusão/metabolismo , Traumatismo por Reperfusão/patologia , Reprodutibilidade dos Testes , Superóxido Dismutase/análise , Testículo/patologia , Xantina Oxidase/análise
2.
Acta cir. bras ; 34(11): e201901102, Nov. 2019. tab, graf
Artigo em Inglês | LILACS | ID: biblio-1054682

RESUMO

Abstract Purpose: To investigate the effect of Picroside II on testicular ischemia and reperfusion (l/R) injury and the underlying mechanism. Methods: Sprague-Dawley rats were randomly divided into 4 groups: sham operated group (Sham), Sham with Picroside II treatment group (Sham+ Pic II), l/R group (l/R) and l/R with Picroside II treatment group (I/R+ Pic II). l/R model was established by rotating the left testis 720° in a clock-wise direction for 4 hours. The histopathologic and spermatogenetic evaluation was performed. The apoptosis changes and the levels of HO-1 (heme oxygenase-1), MPO (myeloperoxidase), NOX (NADPH oxidase), SOD (superoxide dismutase), XO (xanthine oxidase) and NOS (nitric oxide synthase) were measured. Results: The seminiferous tubules were damaged in l/R rats, but Picroside II alleviated the changes induced by l/R. The increased level of apoptosis was decreased by Picroside II (P=0.01, 9.05±0.35 vs. 4.85±0.25). The activities of HO-1, MPO, NOX, XO and MDA content were increased and the SOD activity was decreased in l/R (P<0.05) and could be reversed by Picroside II (P=0.03, 405.5±7.5 vs. 304±17U/mgprot; P=0.02, 0.99±0.05 vs. 0.52±0.04 mgprot; P=0.01, 260+7 vs. 189±2 mgprot; P=0.04, 10.95+0.55 vs. 8.75+0.35 U/mgprot; P=0.045, 6.8+0.7 vs. 3.75+0.35 mgprot; P=0.04, 44.5+3.5 vs. 57.5+3.5 mgprot). Western blot showed that the expression of iNOS, nNOS and eNOS were increased in l/R (P<0.05); however, they were decreased after Picroside II treatment (P<0.05). Conclusion: Picroside II attenuated testicular I/R injury in rats mainly through suppressing apoptosis and oxidative stress through reduction of nitric oxide synthesis.


Assuntos
Animais , Masculino , Testículo/irrigação sanguínea , Traumatismo por Reperfusão/prevenção & controle , Cinamatos/farmacologia , Apoptose/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Glucosídeos Iridoides/farmacologia , Óxido Nítrico/biossíntese , Traumatismo por Reperfusão/metabolismo , Traumatismo por Reperfusão/patologia , Distribuição Aleatória , Western Blotting , Ratos Sprague-Dawley , Peroxidase/análise , Marcação In Situ das Extremidades Cortadas , Heme Oxigenase-1/análise , Malondialdeído/análise , NADP/análise
3.
Biosens Bioelectron ; 146: 111753, 2019 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-31600627

RESUMO

Nicotinamide adenine nucleotide phosphate (NADPH) has been known to be involved in the multiple pathways of cell metabolism. However, conventional quantification assays for NADPH have required breaking down the cell membranes of around one million cells per assay, and monitoring NADPH flux in living cells has been limited by a few available tools. Here, we visualized NADPH levels in human cervical cancer cells HeLa using metagenome-derived blue fluorescent protein (mBFP), which specifically binds to NADPH and enhances the intrinsic fluorescence of NADPH up to 10-fold when imaged by two-photon microscopy to reduce photodamage. Adding an oxidizing agent such as diamide to HeLa cells that expressed mBFP led to an immediate decrease of intracellular NADPH depending on glucose availability in culture media. Furthermore, inhibiting glucose-6-phosphate dehydrogenase (G6PD) in the pentose phosphate pathway with dehydroandrosterone (DHEA) and knockdown of G6PD transcripts gradually decreased NADPH when diamide was added to living cells. These results demonstrate that introducing a bacterial mBFP gene into mammalian cells is a straightforward approach to monitoring intracellular NADPH flux in real time at the single-cell level. Moreover, this strategy can be expanded to tracking the spatio-temporal changes in NADPH even in single-cell organelles such as mitochondria and chloroplasts, which will allow us to more precisely assess the efficacy of biochemically or biophysically metabolic perturbations in animal and plant cells.


Assuntos
Técnicas Biossensoriais/métodos , Corantes Fluorescentes/análise , Proteínas Luminescentes/análise , NADP/análise , Corantes Fluorescentes/metabolismo , Células HeLa , Humanos , Proteínas Luminescentes/metabolismo , Microscopia de Fluorescência por Excitação Multifotônica/métodos , NADP/metabolismo
4.
Methods Mol Biol ; 1996: 61-73, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31127548

RESUMO

Pyridine nucleotides which include NAD+, NADH, NADP, and NADPH play vital roles in many different biological processes. These metabolites can be accurately quantified in a wide variety of biological samples using LC-MS/MS. The quality and precision of these measurements was enhanced using heavy isotope-labeled internal standards and carefully crafted protocols for sample processing.


Assuntos
Metabolômica/métodos , NADP/análise , NAD/análise , Espectrometria de Massas em Tandem/métodos , Animais , Células Cultivadas , Cromatografia Líquida de Alta Pressão/métodos , Metabolômica/normas , NAD/química , NAD/metabolismo , NADP/química , NADP/metabolismo , Oxirredução , Isótopos de Oxigênio/química , Padrões de Referência
5.
BMC Biochem ; 20(1): 4, 2019 04 08.
Artigo em Inglês | MEDLINE | ID: mdl-30961528

RESUMO

BACKGROUND: The reduction of tetrazolium salts by NAD(P)H to formazan product has been widely used to determine the metabolic activity of cells, and as an indicator of cell viability. However, the application of a WST-8 based assay for the quantitative measurement of dehydrogenase enzyme activity has not been described before. In this study, we reported the application of an assay based on the tetrazolium salt WST-8 for the quantitative measurement of dehydrogenase activity. The assay is performed in a microplate format, where a single endpoint is measured at 450 nm. RESULTS: The optimized dehydrogenase-WST-8 assay conditions, the limit of detection (LOD), accuracy, and precision for measuring NAD(P)H, were demonstrated. The sensitivity of the WST-8 assay for detecting NAD(P)H was 5-fold greater than the spectrophotometric measurement of NAD(P)H absorption at 340 nm (LOD of 0.3 nmole vs 1.7 nmole, respectively). In the dehydrogenase assay, the colorimetric WST-8 method exhibits excellent assay reproducibility with a Z' factor of 0.9. The WST-8 assay was also used to determine dehydrogenase activity in biological samples, and for screening the substrate of uncharacterized short-chain dehydrogenase/oxidoreductase from Burkholderia pseudomallei. CONCLUSION: The results suggest that the WST-8 assay is a sensitive and rapid method for determining NAD(P)H concentration and dehydrogenase enzyme activity, which can be further applied for the high-throughput screening of dehydrogenases.


Assuntos
Colorimetria/métodos , Oxirredutases/análise , Sais de Tetrazólio/química , Burkholderia pseudomallei/enzimologia , Glucosefosfato Desidrogenase , Humanos , Limite de Detecção , NAD/análise , NAD/metabolismo , NADP/análise , NADP/metabolismo , Oxirredutases/metabolismo , Espectrofotometria
6.
Anal Bioanal Chem ; 411(13): 2971-2979, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30923861

RESUMO

Chinese hamster ovary (CHO) cells are predominant in the production of therapeutic proteins to treat various diseases. Characterization and investigation of CHO cell metabolism in a quick and simple way could boost process and cell line development. Therefore, a method to simultaneously detect seven redox- and energy-related metabolites in CHO cells by capillary electrophoresis has been developed. An on-line focusing technique was applied to improve the peak shape and resolution by using a 50 µm × 44 cm uncoated fused silica capillary. Key parameters and their interactions were investigated by design of experiments (DoE) and optimized conditions were determined by desirability function as follows: 24 °C, 95 mM, and pH 9.4 of BGE. The method was validated to ensure sensitivity, linearity, and reproducibility. The limits of detection (LODs) ranged from 0.050 to 0.688 mg/L for seven metabolites, and correlation coefficients of linearity were all greater than 0.996. The relative standard deviations (RSD) of migration time and peak area were smaller than 0.872% and 5.5%, respectively, except for NADPH, and the recoveries were between 97.5 and 101.2%. The method was successfully applied to analyze the extracts from CHO cells under two different culture conditions. Graphical abstract.


Assuntos
Monofosfato de Adenosina/análise , Eletroforese Capilar/métodos , NADP/análise , NAD/análise , Difosfato de Adenosina/análise , Trifosfato de Adenosina/análise , Animais , Anticorpos Monoclonais/química , Células CHO , Técnicas de Cultura de Células , Cricetulus , Limite de Detecção , Oxirredução
7.
Med Sci Monit ; 25: 2132-2140, 2019 Mar 22.
Artigo em Inglês | MEDLINE | ID: mdl-30901320

RESUMO

BACKGROUND Endothelial injury is the main mechanism of atherosclerosis, and is caused by oxidized low-density lipoprotein (ox-LDL). Astragaloside IV (AS-IV) is the primary active ingredient of the Chinese herb Huangqi, and exhibits antioxidant and anti-inflammatory properties in cardiovascular diseases. This study investigated the protective effect of AS-IV in human umbilical vein endothelial cells (HUVECs). MATERIAL AND METHODS HUVEC cells were induced with ox-LDL to establish an in vitro atherosclerosis model. Then HUVECs were pretreated for 1 h with AS-IV at different concentrations (10, 20, and 50 µM) and then exposed to ox-LDL (100 µg/mL) for 48 h. The cell viability, lactate dehydrogenase (LDH) release, apoptosis, migration, intracellular reactive oxygen species (ROS), and NADPH oxidase activity of HUVECs were measured. qRT-PCR was performed to measure the mRNA expressions of Nrf2, HO-1, TNFalpha, and IL-6. Enzyme-linked immunosorbent assay (ELISA) was performed to measure the supernatant contents of TNFalpha and IL-6. RESULTS Exposure of HUVECs to ox-LDL reduced cell viability and migration, induced apoptosis, and increased intracellular ROS production and NADPH oxidase. Pretreatment with AS-IV (10, 20, and 50 µM) significantly enhanced the cell viability and migration, suppressed LDH release, apoptosis, ROS production, and NADPH oxidase in HUVECs, in a concentration-dependent manner. The AS-IV (50 µM) alone did not show significant differences from control. AS-IV increased mRNA expressions of Nrf2 and HO-1 and decreased mRNA expressions of TNFalpha and IL-6 in the ox-LDL-HUEVC cells. Furthermore, AS-IV reduced supernatant contents of TNFalpha and IL-6. CONCLUSIONS Astragaloside IV prevents ox-LDL-induced endothelial cell injury by reducing apoptosis, oxidative stress, and inflammatory response.


Assuntos
Células Endoteliais/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Saponinas/farmacologia , Triterpenos/farmacologia , Antioxidantes/farmacologia , Apoptose/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células Endoteliais/fisiologia , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Humanos , Inflamação/metabolismo , L-Lactato Desidrogenase/análise , Lipoproteínas LDL/metabolismo , NADP/análise , NADP/efeitos dos fármacos , NADPH Oxidases/metabolismo , Substâncias Protetoras/farmacologia , Espécies Reativas de Oxigênio/metabolismo , Saponinas/metabolismo , Triterpenos/metabolismo , Fator de Necrose Tumoral alfa/metabolismo
8.
PLoS One ; 14(2): e0212061, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30742684

RESUMO

The reduced form of nicotinamide adenine dinucleotide phosphate (NADPH) functions as a reducing agent involved in many biosynthetic and antioxidant reactions in cells. Therefore, a lots of detection or assaying method of this cofactor are developed and used broadly in various research and application fields. These detection or assay tools, however, have often some problems, such as the low sensitivity, susceptibility to environmental interference and time-consuming pretreatment steps, remaining hurdle to successful quantification of NADPH or its derivatives accurately and immediately. Herein, we present a rapid (assay time < 30 s) and sensitive (detection limit < 2 pmol) detection method of NADPH using metagenome-derived blue fluorescent protein (mBFP), a protein capable of significantly enhancing NADPH fluorescence upon binding to this cofactor. Our method takes advantage of the high specificity of mBFP to NADPH and the immediate fluorescence enhancement upon the addition of mBFP to a solution of interest containing NADPH. We can apply this detection scheme to directly quantitative assessment of NADP(H)-dependent enzyme activities in-vitro, and further accessed to quantitative assay of other nicotine amide cofactors, such as NAD+ and NADH, by coupling assay using NAD(H) kinase. Thus, our method enabled us to quantitatively assess the activity of nicotinamide cofactor-associated enzymes in both bacterial and human cell lysates.


Assuntos
Medições Luminescentes/métodos , Proteínas Luminescentes/metabolismo , NADP/análise , Catálise , Células Cultivadas , Fluorescência , Glucosefosfato Desidrogenase/metabolismo , Humanos , Proteínas Luminescentes/química , Proteínas Luminescentes/genética , Metagenoma , NADP/metabolismo , Oxirredução , Fosfotransferases/metabolismo , Sensibilidade e Especificidade , Fatores de Tempo
9.
Cytometry A ; 95(1): 110-121, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30604477

RESUMO

Redox changes in live HeLa cervical cancer cells after doxorubicin treatment can either be analyzed by a novel fluorescence lifetime microscopy (FLIM)-based redox ratio NAD(P)H-a2%/FAD-a1%, called fluorescence lifetime redox ratio or one of its components (NAD(P)H-a2%), which is actually driving that ratio and offering a simpler and alternative metric and are both compared. Auto-fluorescent NAD(P)H, FAD lifetime is acquired by 2- photon excitation and Tryptophan by 3-photon, at 4 time points after treatment up to 60 min demonstrating early drug response to doxorubicin. Identical Fields-of-view (FoV) at each interval allows single-cell analysis, showing heterogeneous responses to treatment, largely based on their initial control redox state. Based on a discrete ROI selection method, mitochondrial OXPHOS and cytosolic glycolysis are discriminated. Furthermore, putative FRET interaction and energy transfer between tryptophan residue carrying enzymes and NAD(P)H correlate with NAD(P)H-a2%, as does the NADPH/NADH ratio, highlighting a multi-parametric assay to track metabolic changes in live specimens. © 2019 International Society for Advancement of Cytometry.


Assuntos
Mitocôndrias/metabolismo , NADP/análise , NAD/análise , Triptofano/química , Citosol/efeitos dos fármacos , Citosol/metabolismo , Doxorrubicina/farmacologia , Metabolismo Energético/efeitos dos fármacos , Flavina-Adenina Dinucleotídeo/análise , Fluorescência , Transferência Ressonante de Energia de Fluorescência/métodos , Células HeLa , Humanos , Microscopia de Fluorescência por Excitação Multifotônica/métodos , Mitocôndrias/efeitos dos fármacos , NAD/efeitos dos fármacos , NADP/efeitos dos fármacos , Imagem Óptica , Oxirredução , Fosforilação Oxidativa/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo , Análise de Célula Única/métodos
10.
Int J Mol Sci ; 19(12)2018 Dec 06.
Artigo em Inglês | MEDLINE | ID: mdl-30563212

RESUMO

Nicotinamide adenine dinucleotide (NAD) and its phosphorylated form, NADP, are the major coenzymes of redox reactions in central metabolic pathways. Nicotinamide adenine dinucleotide is also used to generate second messengers, such as cyclic ADP-ribose, and serves as substrate for protein modifications including ADP-ribosylation and protein deacetylation by sirtuins. The regulation of these metabolic and signaling processes depends on NAD availability. Generally, human cells accomplish their NAD supply through biosynthesis using different forms of vitamin B3: Nicotinamide (Nam) and nicotinic acid as well as nicotinamide riboside (NR) and nicotinic acid riboside (NAR). These precursors are converted to the corresponding mononucleotides NMN and NAMN, which are adenylylated to the dinucleotides NAD and NAAD, respectively. Here, we have developed an NMR-based experimental approach to detect and quantify NAD(P) and its biosynthetic intermediates in human cell extracts. Using this method, we have determined NAD, NADP, NMN and Nam pools in HEK293 cells cultivated in standard culture medium containing Nam as the only NAD precursor. When cells were grown in the additional presence of both NAR and NR, intracellular pools of deamidated NAD intermediates (NAR, NAMN and NAAD) were also detectable. We have also tested this method to quantify NAD+ in human platelets and erythrocytes. Our results demonstrate that ¹H NMR spectroscopy provides a powerful method for the assessment of the cellular NAD metabolome.


Assuntos
Técnicas de Cultura de Células/métodos , Metabolômica/métodos , NAD/análise , Plaquetas/química , Eritrócitos/química , Células HEK293 , Humanos , Redes e Vias Metabólicas , NADP/análise , Niacina/análise , Niacinamida/análise , Espectroscopia de Prótons por Ressonância Magnética
11.
J Microbiol Biotechnol ; 28(7): 1141-1146, 2018 Jul 28.
Artigo em Inglês | MEDLINE | ID: mdl-29926704

RESUMO

2'-Fucosyllactose (2'-FL) is one of the most important human milk oligosaccharides and has several health benefits for infants. The levels of 2'-FL in breast milk or samples from other sources can bequantified by high-performance liquid chromatography. However, this method cannot be used for simultaneous detection of the target compound in numerous samples. Here, we developed a simple method for quantifying 2'-FL in a microplate format. The method involves two steps: (1) release of L-fucose from 2'-FL by α-(1-2,3,4,6)-L-fucosidase and (2) measurement of NADPH formed during the oxidation of L-fucose by L-fucose dehydrogenase. This method enables measurement of up to 5 g/l 2'-FL in 50 min using a 96-well microplate. The efficiency and simplicity of the proposed method make it suitable for the analyses of a large number of samples simultaneously.


Assuntos
Ensaios Enzimáticos/métodos , Leite Humano/química , Trissacarídeos/metabolismo , alfa-L-Fucosidase/metabolismo , Desidrogenases de Carboidrato/metabolismo , Fucose/metabolismo , Humanos , NADP/análise , Temperatura , Trissacarídeos/química
12.
Elife ; 72018 05 29.
Artigo em Inglês | MEDLINE | ID: mdl-29809136

RESUMO

We introduce a new class of semisynthetic fluorescent biosensors for the quantification of free nicotinamide adenine dinucleotide (NAD+) and ratios of reduced to oxidized nicotinamide adenine dinucleotide phosphate (NADPH/NADP+) in live cells. Sensing is based on controlling the spatial proximity of two synthetic fluorophores by binding of NAD(P) to the protein component of the sensor. The sensors possess a large dynamic range, can be excited at long wavelengths, are pH-insensitive, have tunable response range and can be localized in different organelles. Ratios of free NADPH/NADP+ are found to be higher in mitochondria compared to those found in the nucleus and the cytosol. By recording free NADPH/NADP+ ratios in response to changes in environmental conditions, we observe how cells can react to such changes by adapting metabolic fluxes. Finally, we demonstrate how a comparison of the effect of drugs on cellular NAD(P) levels can be used to probe mechanisms of action.


Assuntos
Técnicas Biossensoriais , Transferência Ressonante de Energia de Fluorescência/métodos , Mitocôndrias/metabolismo , NADP/metabolismo , NAD/metabolismo , Animais , Linhagem Celular Tumoral , Núcleo Celular/metabolismo , Citosol/metabolismo , Corantes Fluorescentes/química , Corantes Fluorescentes/metabolismo , Células HEK293 , Células HeLa , Humanos , Concentração de Íons de Hidrogênio , Cinética , Camundongos , NAD/análise , NADP/análise , Células NIH 3T3 , Osteoblastos/metabolismo , Osteoblastos/ultraestrutura , Oxirredução , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Rodaminas/química , Rodaminas/metabolismo , Sulfametoxazol/metabolismo , Sulfapiridina/metabolismo
13.
BMC Complement Altern Med ; 18(1): 149, 2018 May 08.
Artigo em Inglês | MEDLINE | ID: mdl-29739458

RESUMO

BACKGROUND: Human placenta hydrolysate (hPH) has been utilized to improve menopausal, fatigue, liver function. Its high concentration of bioactive substances is known to produce including antioxidant, anti-inflammatory and anti-nociceptive activities. However, its mechanisms of stress-induced depression remain unknown. METHODS: The present study examined the effect of hPH on stress-induced depressive behaviors and biochemical parameters in rats. hPH (0.02 ml, 0.2 ml or 1 ml/rat) was injected intravenously 30 min before the daily stress session in male Sprague-Dawley rats exposed to repeated immobilization stress (4 h/day for 7 days). The depressive-like behaviors of all groups were measured by elevated plus maze (EPM) and forced swimming test (FST). After the behavior tests, brain samples of all groups were collected for the analysis of glutathione peroxidase (GPx) and nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d) staining. RESULTS: Treatment with hPH produced a significant decrease of immobility time in the FST compared to the controls. Additionally, hPH treatment elicited a slightly decreasing trend in anxiety behavior on the EPM. Furthermore, hPH increased the level of GPx protein in the hippocampus, and decreased the expression of NADPH-d in the paraventricular nucleus (PVN). CONCLUSION: This study demonstrated that hPH has anti-stress effects via the regulation of nitric oxide (NO) synthase and antioxidant activity in the brain. These results suggest that hPH may be useful in the treatment of stress-related diseases such as chronic fatigue syndrome.


Assuntos
Ansiolíticos/farmacologia , Produtos Biológicos/farmacologia , Estresse Oxidativo/efeitos dos fármacos , Placenta/química , Estresse Psicológico/metabolismo , Animais , Comportamento Animal , Química Encefálica/efeitos dos fármacos , Feminino , Glutationa Peroxidase/análise , Humanos , Masculino , Aprendizagem em Labirinto/efeitos dos fármacos , NADP/análise , NADP/metabolismo , Gravidez , Ratos , Ratos Sprague-Dawley
14.
Protein J ; 37(2): 180-193, 2018 04.
Artigo em Inglês | MEDLINE | ID: mdl-29508210

RESUMO

The bioconversion of vitamin D3 catalyzed by cytochrome P450 (CYP) requires 25-hydroxylation and subsequent 1α-hydroxylation to produce the hormonal activated 1α,25-dihydroxyvitamin D3. Vitamin D3 25-hydroxylase catalyses the first step in the vitamin D3 biosynthetic pathway, essential in the de novo activation of vitamin D3. A CYP known as CYP107CB2 has been identified as a novel vitamin D hydroxylase in Bacillus lehensis G1. In order to deepen the understanding of this bacterial origin CYP107CB2, its detailed biological functions as well as biochemical characteristics were defined. CYP107CB2 was characterized through the absorption spectral analysis and accordingly, the enzyme was assayed for vitamin D3 hydroxylation activity. CYP-ligand characterization and catalysis optimization were conducted to increase the turnover of hydroxylated products in an NADPH-regenerating system. Results revealed that the over-expressed CYP107CB2 protein was dominantly cytosolic and the purified fraction showed a protein band at approximately 62 kDa on SDS-PAGE, indicative of CYP107CB2. Spectral analysis indicated that CYP107CB2 protein was properly folded and it was in the active form to catalyze vitamin D3 reaction at C25. HPLC and MS analysis from a reconstituted enzymatic reaction confirmed the hydroxylated products were 25-hydroxyitamin D3 and 1α,25-dihydroxyvitamin D3 when the substrates vitamin D3 and 1α-hydroxyvitamin D3 were used. Biochemical characterization shows that CYP107CB2 performed hydroxylation activity at 25 °C in pH 8 and successfully increased the production of 1α,25-dihydroxyvitamin D3 up to four fold. These findings show that CYP107CB2 has a biologically relevant vitamin D3 25-hydroxylase activity and further suggest the contribution of CYP family to the metabolism of vitamin D3.


Assuntos
Bacillus/enzimologia , Proteínas de Bactérias/química , Colestanotriol 26-Mono-Oxigenase/química , Sistema Enzimático do Citocromo P-450/química , Bacillus/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Colestanotriol 26-Mono-Oxigenase/genética , Colestanotriol 26-Mono-Oxigenase/metabolismo , Sistema Enzimático do Citocromo P-450/genética , Sistema Enzimático do Citocromo P-450/metabolismo , Estabilidade Enzimática , Escherichia coli/genética , NADP/análise , NADP/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Vitamina D/análogos & derivados , Vitamina D/metabolismo
15.
Biochemistry ; 57(7): 1178-1189, 2018 02 20.
Artigo em Inglês | MEDLINE | ID: mdl-29341594

RESUMO

The development of genetically encoded fluorescent probes for analyte-specific imaging has revolutionized our understanding of intracellular processes. Current classes of intracellular probes depend on the selection of binding domains that either undergo conformational changes on analyte binding or can be linked to thiol redox chemistry. Here we have designed novel probes by fusing a flavoenzyme, whose fluorescence is quenched on reduction by the analyte of interest, with a GFP domain to allow for rapid and specific ratiometric sensing. Two flavoproteins, Escherichia coli thioredoxin reductase and Saccharomyces cerevisiae lipoamide dehydrogenase, were successfully developed into thioredoxin and NAD+/NADH specific probes, respectively, and their performance was evaluated in vitro and in vivo. A flow cell format, which allowed dynamic measurements, was utilized in both bacterial and mammalian systems. In E. coli the first reported intracellular steady-state of the cytoplasmic thioredoxin pool was measured. In HEK293T mammalian cells, the steady-state cytosolic ratio of NAD+/NADH induced by glucose was determined. These genetically encoded fluorescent constructs represent a modular approach to intracellular probe design that should extend the range of metabolites that can be quantitated in live cells.


Assuntos
Escherichia coli/enzimologia , Flavoproteínas/metabolismo , Substâncias Luminescentes/metabolismo , Imagem Óptica/métodos , Saccharomyces cerevisiae/enzimologia , Di-Hidrolipoamida Desidrogenase/análise , Di-Hidrolipoamida Desidrogenase/metabolismo , Proteínas de Fluorescência Verde/metabolismo , Células HEK293 , Humanos , Modelos Moleculares , NADP/análise , NADP/metabolismo , Oxirredução , Proteínas Recombinantes de Fusão/metabolismo , Tiorredoxina Dissulfeto Redutase/análise , Tiorredoxina Dissulfeto Redutase/metabolismo , Tiorredoxinas/análise , Tiorredoxinas/metabolismo
16.
Microb Cell Fact ; 17(1): 10, 2018 Jan 22.
Artigo em Inglês | MEDLINE | ID: mdl-29357933

RESUMO

BACKGROUND: Azotobacter vinelandii is a bacterium that produces alginate and polyhydroxybutyrate (P3HB); however, the role of NAD(P)H/NAD(P)+ ratios on the metabolic fluxes through biosynthesis pathways of these biopolymers remains unknown. The aim of this study was to evaluate the NAD(P)H/NAD(P) + ratios and the metabolic fluxes involved in alginate and P3HB biosynthesis, under oxygen-limiting and non-limiting oxygen conditions. RESULTS: The results reveal that changes in the oxygen availability have an important effect on the metabolic fluxes and intracellular NADPH/NADP+ ratio, showing that at the lowest OTR (2.4 mmol L-1 h-1), the flux through the tricarboxylic acid (TCA) cycle decreased 27.6-fold, but the flux through the P3HB biosynthesis increased 6.6-fold in contrast to the cultures without oxygen limitation (OTR = 14.6 mmol L-1 h-1). This was consistent with the increase in the level of transcription of phbB and the P3HB biosynthesis. In addition, under conditions without oxygen limitation, there was an increase in the carbon uptake rate (twofold), as well as in the flux through the pentose phosphate (PP) pathway (4.8-fold), compared to the condition of 2.4 mmol L-1 h-1. At the highest OTR condition, a decrease in the NADPH/NADP+ ratio of threefold was observed, probably as a response to the high respiration rate induced by the respiratory protection of the nitrogenase under diazotrophic conditions, correlating with a high expression of the uncoupled respiratory chain genes (ndhII and cydA) and induction of the expression of the genes encoding the nitrogenase complex (nifH). CONCLUSIONS: We have demonstrated that changes in oxygen availability affect the internal redox state of the cell and carbon metabolic fluxes. This also has a strong impact on the TCA cycle and PP pathway as well as on alginate and P3HB biosynthetic fluxes.


Assuntos
Azotobacter vinelandii/metabolismo , Análise do Fluxo Metabólico , NADP/análise , NAD/análise , Oxigênio/metabolismo , Alginatos/metabolismo , Biomassa , Vias Biossintéticas/efeitos dos fármacos , Carbono/metabolismo , Ciclo do Ácido Cítrico/efeitos dos fármacos , Meios de Cultura/química , NAD/efeitos dos fármacos , NAD/metabolismo , NADP/efeitos dos fármacos , NADP/metabolismo , Oxirredução , Oxigênio/farmacologia , Via de Pentose Fosfato/efeitos dos fármacos
17.
Electrophoresis ; 39(3): 540-547, 2018 02.
Artigo em Inglês | MEDLINE | ID: mdl-28880404

RESUMO

Here, we present a multifunctional microfluidic device whose integrative design enables to combine cell culture studies and quantitative single cell biomolecule analysis. The platform consists of 32 analysis units providing two key features; first, a micrometer-sized trap for hydrodynamic capture of a single Saccharomyces cerevisiae (S. cerevisiae) yeast cell; second, a convenient double-valve configuration surrounding the trap. Actuating of the outer valve with integrated opening results in a partial isolation in a volume of 11.8 nL, i.e. the cell surrounding fluid can be exchanged diffusion-based without causing shear stress or cell loss. Actuation of the inner ring-shaped valve isolates the trapped cell completely in a small analysis volume of 230 pL. The device was used to determine the growth rate of yeast cells (S. cerevisiae) under under optimum and oxidative stress conditions. In addition, we successfully quantified the cofactor beta-nicotinamide adenine dinucleotide phosphate (NAD(P)H) in single and few cells exposed to the different microenvironments. In conclusion, the microdevice enables to analyze the influence of an external stress factor on the cellular fitness in a fast and more comprehensive way as cell growth and intracellular biomolecule levels can be investigated.


Assuntos
Dispositivos Lab-On-A-Chip , Saccharomyces cerevisiae/isolamento & purificação , Análise de Célula Única/métodos , Técnicas de Cultura de Células , Rastreamento de Células/métodos , Dimetilpolisiloxanos/química , Hidrodinâmica , Técnicas Analíticas Microfluídicas/instrumentação , NADP/análise , Oxirredução
18.
Redox Rep ; 23(1): 47-56, 2018 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-29088980

RESUMO

Nicotinamide adenine dinucleotide (NAD+/NADH) along with its phosphorylated form (NADP+/NADPH) are two molecules ubiquitously present in all organisms, and they play key roles as cofactors in fundamental catabolic and anabolic processes, respectively. The oxidation of NADPH to NADP+ initiates a cascade of reactions, where a network of molecules is implicated. The molecules of this cascade form a network with eminent translational potential in redox metabolism. A special point of interest is that spectrophotometric assays have been developed both for NADH/NADPH and the molecules directly regulated by them. Therefore, crucial molecules of the NADPH-dependent redox network can be measured, and the results can be used to assess the bioenergetic and/or oxidative stress status. The main aim of this review is to collectively present the NADPH-related molecules, namely NADPH, NADH, NAD+ kinase, NADPH oxidase, peroxiredoxin, thioredoxin, thioredoxin reductase, and nitric oxide synthase, that can be measured in blood and tissues with the use of a spectrophotometer, which is probably the most simple, inexpensive and widely used tool in biochemistry. We are providing the researchers with reliable and valid spectrophotometric assays for the measurement of the most important biomarkers of the NADPH network in blood and other tissues, thus allowing the opportunity to follow the redox changes in response to a stimulus.


Assuntos
Biomarcadores/análise , NADP/metabolismo , Espectrofotometria/métodos , Biomarcadores/sangue , Biomarcadores/metabolismo , Humanos , NAD/análise , NAD/sangue , NAD/metabolismo , NADP/análise , NADP/sangue , NADPH Oxidases/sangue , NADPH Oxidases/metabolismo , Óxido Nítrico Sintase/análise , Óxido Nítrico Sintase/sangue , Óxido Nítrico Sintase/metabolismo , Oxirredução , Peroxirredoxinas/análise , Peroxirredoxinas/sangue , Peroxirredoxinas/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/análise , Fosfotransferases (Aceptor do Grupo Álcool)/sangue , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Tiorredoxina Dissulfeto Redutase/sangue , Tiorredoxina Dissulfeto Redutase/metabolismo , Tiorredoxinas
19.
Biochemistry ; 57(5): 772-780, 2018 02 06.
Artigo em Inglês | MEDLINE | ID: mdl-29261301

RESUMO

Thioredoxin 1 (Trx1) and glutaredoxin 1 (Grx1) are two ubiquitous redox enzymes that are central for redox homeostasis but also are implicated in many other processes, including stress sensing, inflammation, and apoptosis. In addition to their enzymatic redox activity, a growing body of evidence shows that Trx1 and Grx1 play regulatory roles via protein-protein interactions with specific proteins, including Ask1. The currently available inhibitors of Trx1 and Grx1 are thiol-reactive electrophiles or disulfides that may suffer from low selectivity because of their thiol reactivity. In this report, we used a phage peptide library to identify a 7-mer peptide, 2GTP1, that binds to both Trx1 and Grx1. We further showed that a cell-permeable derivative of 2GTP1, TAT-2GTP1, disrupts the Trx1-Ask1 interaction, which induces Ask1 phosphorylation with subsequent activation of JNK, stabilization of p53, and reduced viability of cancer cells. Notably, as opposed to a disulfide-derived Trx1 inhibitor (PX-12), TAT-2GTP1 was selective for activating the Ask1 pathway without affecting other stress signaling pathways, such as endoplasmic reticulum stress and AMPK activation. Overall, 2GTP1 will serve as a useful probe for investigating protein interactions of Trx1.


Assuntos
MAP Quinase Quinase Quinase 5/antagonistas & inibidores , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Oligopeptídeos/farmacologia , Biblioteca de Peptídeos , Estresse Fisiológico/fisiologia , Antineoplásicos/química , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Permeabilidade da Membrana Celular , Sobrevivência Celular/efeitos dos fármacos , Ensaios de Seleção de Medicamentos Antitumorais , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Ativação Enzimática/efeitos dos fármacos , Enzimas Imobilizadas , Glutarredoxinas , Células HEK293 , Humanos , MAP Quinase Quinase Quinase 5/química , MAP Quinase Quinase Quinase 5/fisiologia , NADP/análise , Oligopeptídeos/isolamento & purificação , Oxirredução , Fosforilação/efeitos dos fármacos , Ligação Proteica/efeitos dos fármacos , Mapeamento de Interação de Proteínas , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo , Proteínas Recombinantes/metabolismo
20.
Adv Exp Med Biol ; 1035: 19-30, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-29080128

RESUMO

TCSPC FLIM/PLIM is based on a multi-dimensional time-correlated single-photon counting process. The sample is scanned by a high-frequency-pulsed laser beam which is additionally modulated on/off synchronously with the pixels of the scan. FLIM is obtained by building up the distribution of the photons over the scanning coordinates and the times of the photons in the excitation pulse sequence, PLIM is obtained by building up the photon distribution over the scanning coordinates and the photon times in the modulation period. FLIM and PLIM data are thus obtained simultaneously within the same imaging process. Since the technique uses not only one but many excitation pulses for every phosphorescence signal period the sensitivity is much higher than for techniques that excite with a single pulse only. TCSPC FLIM/PLIM works both with one-photon and two-photon excitation, does not require a reduction of the laser pulse repetition rate by a pulse picker, and eliminates the need of high pulse energy for phosphorescence excitation.


Assuntos
Substâncias Luminescentes/química , Medições Luminescentes/métodos , Sondas Moleculares/química , Imagem Óptica/métodos , Compostos Organometálicos/química , Fótons , Animais , Células HEK293 , Humanos , Lasers , Medições Luminescentes/instrumentação , NADP/análise , NADP/metabolismo , Nanopartículas/química , Imagem Óptica/instrumentação , Oxigênio/análise , Oxigênio/metabolismo , Consumo de Oxigênio/fisiologia , Óxido de Zinco/química
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