RESUMEN
Background: Pomegranate granatum (molasses and peels) and its constituents showed protective effects against natural toxins such as phenylhydrazine (PHZ) as well as chemical toxicants such as arsenic, diazinon, and carbon tetrachloride. Aim: The current study aimed to assess the effect of pomegranate molasses (PM), white peel extract, and red peel extract on nephrotoxicity induced by PHZ. Methods: 80 male rats were divided into eight equal groups; a control group, PM pure group, white peel pomegranate pure group, red peel pomegranate pure group, PHZ group, PM + PHZ group, white peel pomegranate + PHZ group and red peel pomegranate + PHZ group. Kidney function, inflammation markers, antioxidant activities, and renal tissue histopathology were investigated. Results: The results revealed that PHZ group showed a significant increase in lactate Dehydrogenase (LDH), malondialdehyde (MDA), creatinine, uric acid, BUNBUN, C - reactive protein (CRP), tumor necrosis factor, thiobarbituric acid reactive substances (TBARSs), and total antioxidant capacity (TAC) with a significant decrease of catalase (CAT), glutathione peroxidase (GPx), and superoxide dismutase (SOD) as compared with a control group. Other pomegranate-treated and PHZ co-treated groups with pomegranate showed a significant decrease of LDH, MDA, creatinine, uric acid, BUN, tumor necrosis factor, TBARSs, and TAC with a significant increase of CAT, GPx, and SOD as compared with PHZ group. Conclusion: Collectively, our data suggest that red, white peels, and molasses have anti-toxic and anti-inflammatory effects on renal function and tissues.
Asunto(s)
Antioxidantes , Granada (Fruta) , Ratas , Masculino , Animales , Antioxidantes/farmacología , Antioxidantes/análisis , Antioxidantes/metabolismo , Granada (Fruta)/metabolismo , Frutas/química , Frutas/metabolismo , Ácido Úrico/análisis , Ácido Úrico/metabolismo , Creatinina/análisis , Creatinina/metabolismo , Extractos Vegetales/farmacología , Riñón/metabolismo , Superóxido Dismutasa/análisis , Superóxido Dismutasa/metabolismo , Factores de Necrosis Tumoral/análisis , Factores de Necrosis Tumoral/metabolismo , Fenilhidrazinas/análisis , Fenilhidrazinas/metabolismoRESUMEN
Amphibian metamorphosis results in drastic whole-body remodeling. Thyroid hormone (TH) drives most of these metamorphic changes. A prominent event during this remodeling is the red blood cell (RBC) transition from larval to adult forms, which exclusively contain larval and adult hemoglobin, respectively. However, the role of TH in RBC transition remains unclear. Here we reconfirmed that RBC transition of Xenopus laevis is completed much later than morphological metamorphosis. Further, larval and adult RBCs/erythroblasts proliferated both in the erythropoietic liver and in circulation during metamorphic climax. RBC transition was also confirmed in Rana ornativentris, but in contrast to X. laevis, adult RBC-specific proliferation was observed from the early climax stages. We also revealed in either species that RBC transition occurs in the liver prior to circulating RBCs. Moreover, anemia induction using phenylhydrazine during the prometamorphosis of X. laevis caused precocious RBC transition even when TH synthesis was blocked, resulting in metamorphosis-arrested larvae in which most of RBCs were of adult type. These results indicate that a decline in larval RBCs facilitates RBC transition during metamorphosis in a TH-independent manner. Further, combined administration of phenylhydrazine and TH induced precocious appearance of adult RBCs in early prometamorphic X. laevis tadpoles, whereas individual treatment with phenylhydrazine or TH did not cause precocious RBC transition; this suggests that TH is required to initiate RBC transition by promoting the differentiation of adult erythroblasts during early prometamorphosis in X. laevis. These results show that TH-dependent and independent processes are present in RBC transition in X. laevis.
Asunto(s)
Metamorfosis Biológica , Hormonas Tiroideas , Animales , Xenopus laevis , Larva/metabolismo , Hormonas Tiroideas/metabolismo , Eritrocitos/metabolismo , Hemoglobinas/metabolismo , Fenilhidrazinas/metabolismoRESUMEN
Ammonia-oxidizing archaea (AOA) and bacteria (AOB) perform key steps in the global nitrogen cycle, the oxidation of ammonia to nitrite. While the ammonia oxidation pathway is well characterized in AOB, many knowledge gaps remain about the metabolism of AOA. Hydroxylamine is an intermediate in both AOB and AOA, but homologues of hydroxylamine dehydrogenase (HAO), catalyzing bacterial hydroxylamine oxidation, are absent in AOA. Hydrazine is a substrate for bacterial HAO, while phenylhydrazine is a suicide inhibitor of HAO. Here, we examine the effect of hydrazines in AOA to gain insights into the archaeal ammonia oxidation pathway. We show that hydrazine is both a substrate and an inhibitor for AOA and that phenylhydrazine irreversibly inhibits archaeal hydroxylamine oxidation. Both hydrazine and phenylhydrazine interfered with ammonia and hydroxylamine oxidation in AOA. Furthermore, the AOA "Candidatus Nitrosocosmicus franklandus" C13 oxidized hydrazine into dinitrogen (N2), coupling this reaction to ATP production and O2 uptake. This study expands the known substrates of AOA and suggests that despite differences in enzymology, the ammonia oxidation pathways of AOB and AOA are functionally surprisingly similar. These results demonstrate that hydrazines are valuable tools for studying the archaeal ammonia oxidation pathway. IMPORTANCE Ammonia-oxidizing archaea (AOA) are among the most numerous living organisms on Earth, and they play a pivotal role in the global biogeochemical nitrogen cycle. Despite this, little is known about the physiology and metabolism of AOA. We demonstrate in this study that hydrazines are inhibitors of AOA. Furthermore, we demonstrate that the model soil AOA "Ca. Nitrosocosmicus franklandus" C13 oxidizes hydrazine to dinitrogen gas, and this reaction yields ATP. This provides an important advance in our understanding of the metabolism of AOA and expands the short list of energy-yielding compounds that AOA can use. This study also provides evidence that hydrazines can be useful tools for studying the metabolism of AOA, as they have been for the bacterial ammonia oxidizers.
Asunto(s)
Amoníaco , Archaea , Adenosina Trifosfato/metabolismo , Amoníaco/metabolismo , Archaea/metabolismo , Bacterias/metabolismo , Humanos , Hidrazinas/metabolismo , Hidrazinas/farmacología , Hidroxilaminas/metabolismo , Nitrificación , Fenilhidrazinas/metabolismo , Microbiología del SueloRESUMEN
NEW FINDINGS: What is the central question of this study? Can an anaemic state modify adiposity and metabolic parameters in hypothalamic obese rats? What is the main finding and its importance? Hypothalamic obese rats do not display iron deficiency. However, the pharmacological induction of anaemia in hypothalamic obese rats resulted in reduced adiposity, characterized by a decrease in subcutaneous white and brown adipose tissue depots. These findings suggest that iron imbalance in obesity may elevate lipolysis. ABSTRACT: Iron imbalance is frequent in obesity. Herein, we evaluated the impact of anaemia induced by phenylhydrazine on adiposity and metabolic state of hypothalamic obese rats. Hypothalamic obesity was induced by high doses of monosodium glutamate (MSG; 4 g/kg) administered to neonatal male rats (n = 20). Controls (CTL; non-obese rats) received equimolar saline (n = 20). Rats were weaned at 21 days of life. At 70 days, half of the rats received three intraperitoneal doses of phenylhydrazine (PHZ; 40 mg/kg/dose) or saline solution. Body weight and food intake were followed for 4 weeks after PHZ administration. At 92 days, rats were killed and blood was collected for microcapillary haematocrit (Hct) analysis and plasma quantification of glucose, triglycerides, total cholesterol and iron levels. The liver, the spleen, and the white (WAT) and brown (BAT) adipose tissues were excised, weighed and used for histology. MSG-treated rats developed obesity, hypertriglyceridaemia and insulin resistance, compared to CTL rats, without changes in iron levels and Hct. PHZ administration reduced plasma iron levels and promoted similar tissue injuries in the spleen and liver from MSG and CTL rats. However, in MSG-treated rats, PHZ decreased fasting glucose levels and Hct, as well as diminishing the subcutaneous WAT and BAT mass. Although MSG-obesity does not affect plasma iron levels and Hct by itself, PHZ-induced anaemia associated with obesity induces a marked drop in subcutaneous WAT and BAT mass, suggesting that iron imbalance may lead to increased lipolytic responses in obese rats, compared to lean rats.
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Tejido Adiposo Pardo , Anemia , Tejido Adiposo/metabolismo , Tejido Adiposo Pardo/metabolismo , Anemia/inducido químicamente , Anemia/metabolismo , Animales , Glucosa/metabolismo , Hierro , Masculino , Obesidad/metabolismo , Fenilhidrazinas/efectos adversos , Fenilhidrazinas/metabolismo , Ratas , Glutamato de SodioRESUMEN
Human aspartyl/asparaginyl beta-hydroxylase (HAAH) is a member of the superfamily of nonheme Fe2+/α-ketoglutarate (αKG) dependent oxygenase enzymes with a noncanonical active site. HAAH hydroxylates epidermal growth factor (EGF) like domains to form the ß-hydroxylated product from substrate asparagine or aspartic acid and has been suggested to have a negative impact in a variety of cancers. In addition to iron, HAAH also binds divalent calcium, although the role of the latter is not understood. Herein, the metal binding chemistry and influence on enzyme stability and activity have been evaluated by a combined biochemical and biophysical approach. Metal binding parameters for the HAAH active site were determined by use of isothermal titration calorimetry, demonstrating a high-affinity regulatory binding site for Ca2+ in the catalytic domain in addition to the catalytic Fe2+ cofactor. We have analyzed various active site derivatives, utilizing LC-MS and a new HPLC technique to determine the role of metal binding and the second coordination sphere in enzyme activity, discovering a previously unreported residue as vital for HAAH turnover. This analysis of the in vitro biochemical function of HAAH furthers the understanding of its importance to cellular biochemistry and metabolic pathways.
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Isoenzimas/metabolismo , Oxigenasas de Función Mixta/metabolismo , Calcio/metabolismo , Calorimetría/métodos , Dominio Catalítico , Cromatografía Líquida de Alta Presión/métodos , Compuestos Ferrosos/metabolismo , Humanos , Isoenzimas/química , Cinética , Oxigenasas de Función Mixta/química , Modelos Moleculares , Fenilhidrazinas/metabolismo , Espectrometría de Masas en Tándem/métodosRESUMEN
The aim of the study was to modify a simple and widely used spectrophotometric assay for MAO activity evaluation with 2,4-dinitrophenylhydrazine. A modified procedure includes molar absorption coefficients of 2,4-DNP-hydrazone benzaldehyde and 2,4-DNP-hydrazone 5-hydroxyindolylacetaldehyde as 2.3 × 104mol-1l cm-1 and 1.0 × 104 mol-1l cm-1, respectively. Such an approach allows to express specific enzyme activity as nmol product formed/min/mg protein.
Asunto(s)
Inhibidores de la Monoaminooxidasa/química , Monoaminooxidasa/metabolismo , Fenilhidrazinas/química , Acetaldehído/química , Benzaldehídos/química , Activación Enzimática , Hidrazonas/química , Inhibidores de la Monoaminooxidasa/metabolismo , Fenilhidrazinas/metabolismo , Unión Proteica , EspectrofotometríaRESUMEN
We report here an extensive structure-activity relationship study of balsalazide, which was previously identified in a high-throughput screening as an inhibitor of Sirt5. To get a closer understanding why this compound is able to inhibit Sirt5, we initially performed docking experiments comparing the binding mode of a succinylated peptide as the natural substrate and balsalazide with Sirt5 in the presence of NAD+. Based on the evidence gathered here, we designed and synthesized 13 analogues of balsalazide, in which single functional groups were either deleted or slightly altered to investigate which of them are mandatory for high inhibitory activity. Our study confirms that balsalazide with all its given functional groups is an inhibitor of Sirt5 in the low micromolar concentration range and structural modifications presented in this study did not increase potency. While changes on the N-aroyl-ß-alanine side chain eliminated potency, the introduction of a truncated salicylic acid part minimally altered potency. Calculations of the associated reaction paths showed that the inhibition potency is very likely dominated by the stability of the inhibitor-enzyme complex and not the type of inhibition (covalent vs. non-covalent). Further in-vitro characterization in a trypsin coupled assay determined that the tested inhibitors showed no competition towards NAD+ or the synthetic substrate analogue ZKsA. In addition, investigations for subtype selectivity revealed that balsalazide is a subtype-selective Sirt5 inhibitor, and our initial SAR and docking studies pave the way for further optimization.
Asunto(s)
Inhibidores de Histona Desacetilasas/química , Inhibidores de Histona Desacetilasas/farmacología , Mesalamina/química , Mesalamina/farmacología , Simulación del Acoplamiento Molecular , Fenilhidrazinas/química , Fenilhidrazinas/farmacología , Sirtuinas/antagonistas & inhibidores , Diseño de Fármacos , Inhibidores de Histona Desacetilasas/metabolismo , Mesalamina/metabolismo , Fenilhidrazinas/metabolismo , Conformación Proteica , Ácido Salicílico/química , Sirtuinas/química , Sirtuinas/metabolismo , Relación Estructura-ActividadRESUMEN
The polymicrobial dysbiotic subgingival biofilm microbes associated with periodontal disease appear to contribute to developing pathologies in distal body sites, including the brain. This study examined oxidative stress, in the form of increased protein carbonylation and oxidative protein damage, in the tumor necrosis factor-α (TNF-α) transgenic mouse that models inflammatory TNF-α excess during bacterial infection; and in the apolipoprotein knockout (ApoE-/-) mouse brains, following Porphyromonas gingivalis gingival monoinfection. Following 2,4-dinitrophenylhydrazine derivatization, carbonyl groups were detected in frontal lobe brain tissue lysates by immunoblotting and immunohistochemical analysis of fixed tissue sections from the frontotemporal lobe and the hippocampus. Immunoblot analysis confirmed the presence of variable carbonyl content and oxidative protein damage in all lysates, with TNF-α transgenic blots exhibiting increased protein carbonyl content, with consistently prominent bands at 25âkDa (pâ=â0.0001), 43âkDa, and 68âkDa, over wild-type mice. Compared to sham-infected ApoE-/- mouse blots, P. gingivalis-infected brain tissue blots demonstrated the greatest detectable protein carbonyl content overall, with numerous prominent bands at 25âkDa (pâ=â0.001) and 43âkDa (pâ=â0.0001) and an exclusive band to this group between 30-43âkDa* (pâ=â0.0001). In addition, marked immunostaining was detected exclusively in the microvasculature in P. gingivalis-infected hippocampal tissue sections, compared to sham-infected, wild-type, and TNF-α transgenic mice. This study revealed that the hippocampal microvascular structure of P. gingivalis-infected ApoE-/- mice possesses elevated oxidative stress levels, resulting in the associated tight junction proteins being susceptible to increased oxidative/proteolytic degradation, leading to a loss of functional integrity.
Asunto(s)
Apolipoproteínas E/deficiencia , Infecciones por Bacteroidaceae/fisiopatología , Microvasos/patología , Estrés Oxidativo/genética , Porphyromonas gingivalis/patogenicidad , Factor de Necrosis Tumoral alfa/metabolismo , Animales , Apolipoproteínas E/genética , Infecciones por Bacteroidaceae/genética , Infecciones por Bacteroidaceae/virología , Modelos Animales de Enfermedad , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Microvasos/virología , Fenilhidrazinas/metabolismo , Carbonilación Proteica/genética , Carbonilación Proteica/fisiología , Factor de Necrosis Tumoral alfa/genéticaRESUMEN
A spectrophotometric screening method for avermectin oxidizing microbes by determination of 4â³-oxo-avermectin was established based on the reaction between 4â³-oxo-avermectin and 2,4-dinitrophenylhydrazine. Combined with a gradient HPLC assay, microorganisms capable of regioselectively oxidizing avermectin to 4â³-oxo-avermectin were successfully obtained by this method.
Asunto(s)
Ivermectina/análogos & derivados , Oxidantes , Espectrofotometría/métodos , Bacterias/genética , Bacterias/metabolismo , Cromatografía Líquida de Alta Presión/métodos , ADN Bacteriano , Enzimas , Ivermectina/análisis , Ivermectina/metabolismo , Oxidación-Reducción , Fenilhidrazinas/metabolismo , ARN Ribosómico 16S/genética , Streptomyces/genética , Streptomyces/metabolismoRESUMEN
Ammonia-oxidizing bacteria (AOB), ubiquitous chemoautotrophic bacteria, convert ammonia (NH3) to nitrite (NO2(-)) via hydroxylamine as energy source. Excessive growth of AOB, enhanced by applying large amounts of ammonium-fertilizer to the farmland, leads to nitrogen leaching and nitrous oxide gas emission. To suppress these unfavorable phenomena, nitrification inhibitors, AOB specific bactericides, are widely used in fertilized farmland. However, new nitrification inhibitors are desired because of toxicity and weak-effects of currently used inhibitors. Toward development of novel nitrification inhibitors that target hydroxylamine oxidoreductase (HAO), a key enzyme of nitrification in AOB, we established inhibitor evaluation systems that include simplified HAO purification procedure and high-throughput HAO activity assays for the purified enzymes and for the live AOB cells. The new assay systems allowed us to observe distinct inhibitory responses of HAOs from beta-proteobacterial AOB (ßAOB) Nitrosomonas europaea (NeHAO) and gamma-proteobacterial AOB (γAOB) Nitrosococcus oceani (NoHAO) against phenylhydrazine, a well-known suicide inhibitor for NeHAO. Consistently, the live cells of N. europaea, Nitrosomonas sp. JPCCT2 and Nitrosospira multiformis of ßAOB displayed higher responses to phenylhydrazine than those of γAOB N. oceani. Our homology modeling studies suggest that different inhibitory responses of ßAOB and γAOB are originated from different local environments around the substrate-binding sites of HAOs in these two classes of bacteria due to substitutions of two residues. The results reported herein strongly recommend inhibitor screenings against both NeHAO of ßAOB and NoHAO of γAOB to develop HAO-targeting nitrification inhibitors with wide anti-AOB spectra.
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Compuestos de Amonio/metabolismo , Pruebas de Enzimas/métodos , Gammaproteobacteria/efectos de los fármacos , Gammaproteobacteria/enzimología , Oxidorreductasas/antagonistas & inhibidores , Oxidorreductasas/metabolismo , Agroquímicos/metabolismo , Secuencia de Aminoácidos , Inhibidores Enzimáticos/metabolismo , Gammaproteobacteria/química , Modelos Moleculares , Nitrificación/efectos de los fármacos , Oxidación-Reducción/efectos de los fármacos , Oxidorreductasas/química , Fenilhidrazinas/metabolismoRESUMEN
Human diamine oxidase (hDAO) efficiently degrades polyamines and histamine. Reduced enzyme activities might cause complications during pregnancy and be involved in histamine intolerance. So far hDAO has been characterized after isolation from either native sources or the heterologous production in insect cells. Accessibility to human enzyme is limited and insect cells produce non-human glycosylation patterns that may alter its biochemical properties. We present the heterologous expression of hDAO in Chinese Hamster Ovary (CHO) cells and a three step purification protocol. Analysis of metal content using ICP-MS revealed that 93% of the active sites were occupied by copper. Topaquinone (TPQ) cofactor content was determined using phenylhydrazine titration. Ninety-four percent of DAO molecules contained TPQ and therefore the copper content at the active site was indirectly confirmed. Mass spectrometric analysis was conducted to verify sequence integrity of the protein and to assess the glycosylation profile. Electronic circular dichroism and UV-vis spectra data were used to characterize structural properties. The substrate preference and kinetic parameters were in accordance with previous publications. The establishment of a recombinant production system for hDAO enables us to generate decent amounts of protein with negligible impurities to address new scientific questions.
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Amina Oxidasa (conteniendo Cobre)/biosíntesis , Proteínas Recombinantes/biosíntesis , Amina Oxidasa (conteniendo Cobre)/química , Amina Oxidasa (conteniendo Cobre)/aislamiento & purificación , Secuencia de Aminoácidos , Animales , Western Blotting , Células CHO , Cromatografía Liquida , Dicroismo Circular , Coenzimas/metabolismo , Cricetinae , Cricetulus , Dihidroxifenilalanina/análogos & derivados , Dihidroxifenilalanina/metabolismo , Electroforesis en Gel de Poliacrilamida , Expresión Génica , Glicosilación , Humanos , Cinética , Metales/metabolismo , Péptidos/química , Fenilhidrazinas/metabolismo , Polisacáridos/química , Estructura Secundaria de Proteína , Proteínas Recombinantes/aislamiento & purificación , Espectrometría de Masa por Ionización de Electrospray , Espectrofotometría Ultravioleta , Especificidad por SustratoRESUMEN
The design and construction of efficient artificial enzymes is highly desirable. Recent studies have demonstrated that a series of carbon nanomaterials possess intrinsic peroxidase activity. Among them, graphene quantum dots (GQDs) have a high enzymatic activity. However, the catalytic mechanism remains unclear. Therefore, in this report, we chose to decipher their peroxidase activity. By selectively deactivating the ketonic carbonyl, carboxylic, or hydroxy groups and investigating the catalytic activities of these GQD derivatives, we obtained evidence that the -C=O groups were the catalytically active sites, whereas the O=C-O- groups acted as substrate-binding sites, and -C-OH groups can inhibit the activity. These results were corroborated by theoretical studies. This work should not only enhance our understanding of nanocarbon-based artificial enzymes, but also facilitate the design and construction of other types of target-specific artificial enzymes.
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Materiales Biomiméticos/química , Grafito/química , Puntos Cuánticos/química , Benzoatos/química , Benzoatos/metabolismo , Sitios de Unión , Materiales Biomiméticos/metabolismo , Catálisis , Dominio Catalítico , Organotiofosfatos/química , Peroxidasa/química , Peroxidasa/metabolismo , Fenilhidrazinas/química , Fenilhidrazinas/metabolismo , Puntos Cuánticos/metabolismo , Especificidad por SustratoRESUMEN
The metabolism of gentiopicroside in vivo was studied by LC/MS following 2,4-dinitrophenylhydrazine derivatization for the first time. The ionization efficiency of the major metabolites erythrocentaurin and gentiopicral were greatly enhanced by the new analytical method developed, and they were successfully detected in rat plasma after oral administration of gentiopicroside. Methyl 4-formylbenzoate was used as the internal standard to quantify erythrocentaurin and gentiopicral in rat plasma in negative mode by UPLC-TOF-MS. It was found that erythrocentaurin reached the maximum plasma concentration of 625.2±246.3 ng/mL at about 2 h and gentiopicral reached the maximum plasma concentration of 157.6±86.6 ng/mL at about 4 h after oral administration of gentiopicroside at a dose of 200 mg/kg. The metabolic pathway of gentiopicroside to erythrocentaurin and gentiopicral was proposed. The monoterpene compound gentiopicroside was found to be metabolized to dihydroisocoumarin in vivo which may be responsible for the pharmacological effect of gentiopicroside. The results may shed light on clinical efficacy of gentiopicroside and the new analytical method developed may assist in studies for the metabolism of other natural iridoids and secoiridoids in vivo.
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Glucósidos Iridoides/sangre , Glucósidos Iridoides/química , Fenilhidrazinas/química , Plasma/química , Animales , Cromatografía Líquida de Alta Presión/métodos , Glucósidos Iridoides/metabolismo , Iridoides/química , Isocumarinas/química , Espectrometría de Masas/métodos , Fenilhidrazinas/metabolismo , Ratas , Ratas WistarRESUMEN
Tyrosinase was found to catalyze the oxidation of phenylhydrazine to phenol in a reaction that did not resemble those typically performed by tyrosinase. The kinetics of this reaction was investigated by measuring the initial velocity of the formation of phenol (25 °C). The values of k cat and K M for the oxidation of phenylhydrazine were obtained as 11.0 s(-1) and 0.30 mM, respectively. The generation of superoxides during the oxidation of phenylhydrazine by tyrosinase was monitored by nitroblue tetrazolium (NBT) assay. In the phenylhydrazine-tyrosinase reaction, 1 mol O2 was required for the production of 1 mol phenol and 1/6 mol superoxide. The decomposition of superoxide by superoxide dismutase enhanced the rate constant of the oxidation of phenylhydrazine. Phenol formed in the oxidation of phenylhydrazine by tyrosinase was further oxidized by tyrosinase to an o-quinone, after the oxidation of phenylhydrazine by tyrosinase was almost completed.
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Monofenol Monooxigenasa/metabolismo , Fenilhidrazinas/metabolismo , Cinética , Nitroazul de Tetrazolio/metabolismo , Oxidación-Reducción , Quinonas/metabolismo , Superóxido Dismutasa/metabolismo , Superóxidos/metabolismoRESUMEN
Aging of retinal pigment epithelial (RPE) cells of the eye is marked by accumulations of bisretinoid fluorophores; two of the compounds within this lipofuscin mixture are A2E and all-trans-retinal dimer. These pigments are implicated in pathological mechanisms involved in some vision-threatening disorders including age-related macular degeneration (AMD). Studies have shown that bisretinoids are photosensitive compounds that undergo photooxidation and photodegradation when irradiated with short wavelength visible light. Utilizing ultra performance liquid chromatography (UPLC) with electrospray ionization mass spectrometry (ESI-MS) we demonstrate that photodegradation of A2E and all-trans-retinal dimer generates the dicarbonyls glyoxal (GO) and methylglyoxal (MG), that are known to modify proteins by advanced glycation endproduct (AGE) formation. By extracellular trapping with aminoguanidine, we established that these oxo-aldehydes are released from irradiated A2E-containing RPE cells. Enzyme-linked immunosorbant assays (ELISA) revealed that the substrate underlying A2E-containing RPE was AGE-modified after irradiation. This AGE deposition was suppressed by prior treatment of the cells with aminoguanidine. AGE-modification causes structural and functional impairment of proteins. In chronic diseases such as diabetes and atherosclerosis, MG and GO modify proteins by non-enzymatic glycation and oxidation reactions. AGE-modified proteins are also components of drusen, the sub-RPE deposits that confer increased risk of AMD onset. These results indicate that photodegraded RPE bisretinoid is likely to be a previously unknown source of MG and GO in the eye.
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Glioxal/metabolismo , Degeneración Macular/metabolismo , Piruvaldehído/metabolismo , Retina/metabolismo , Retina/patología , Células Cultivadas , Cromatografía Liquida , Ensayo de Inmunoadsorción Enzimática , Productos Finales de Glicación Avanzada/metabolismo , Guanidinas/metabolismo , Humanos , Lipofuscina/metabolismo , Fenilhidrazinas/metabolismo , Compuestos de Piridinio/metabolismo , Drusas Retinianas/metabolismo , Retinaldehído/análogos & derivados , Retinaldehído/metabolismo , Retinoides/metabolismo , Espectrometría de Masa por Ionización de ElectrosprayRESUMEN
A new method for the synthesis of ß-N-(γ-L(+)-glutamyl)phenylhydrazine is presented. This compound was prepared from L-glutamine and phenylhydrazine through a transpeptidation reaction of Escherichia coli γ-glutamyltranspeptidase although phenylhydrazine has been reported to be an inhibitor of the enzyme. The optimum reaction conditions were 60 mM L-glutamine, 300 mM phenylhydrazine, 40 U γ-glutamyltranspeptidase/ml, and pH 9 in approx. 800 ml. After 6 h at 37 °C, the product was obtained with a conversion rate of 93 % (mol/mol). γ-Glutamyltranspeptidase was reversibly inhibited only when phenylhydrazine was above 300 mM.
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Proteínas de Escherichia coli/metabolismo , Glutamatos/metabolismo , Fenilhidrazinas/metabolismo , Proteínas Recombinantes/metabolismo , gamma-Glutamiltransferasa/metabolismo , Escherichia coli/enzimología , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/aislamiento & purificación , Glutamatos/análisis , Glutamatos/química , Concentración de Iones de Hidrógeno , Fenilhidrazinas/análisis , Fenilhidrazinas/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Temperatura , gamma-Glutamiltransferasa/genética , gamma-Glutamiltransferasa/aislamiento & purificaciónRESUMEN
Fetal liver hematopoiesis in mice disappears approximately 2 weeks after birth; however, under experimental acute anemia extramedullary hematopoiesis occurs in the livers of adult mice. The hematopoietic foci in the liver during extramedullary hematopoiesis contain erythroblasts and macrophages. In this study, the extent of involvement of macrophages in the development and involutional process of the hematopoietic foci in adult mice livers was clarified by experimentally inducing extramedullary hematopoiesis. Hematopoietic cells appeared in the livers 2 days after phenylhydrazine (PHZ) injections. The number and area of the foci increased rapidly, reaching peak values on the sixth day. F4/80-positive macrophages were observed in the sinusoids as well as the hematopoietic foci, and were tightly surrounded by erythroblasts. Sinusoidal macrophages in normal adult livers were positive for F4/80 but negative for ER-HR3. However, in extramedullary hematopoiesis-induced livers, sinusoidal macrophages became positive for ER-HR3 antibodies. The number of ER-HR3-positive macrophages was 9.2 ± 2.9/mm(2) on the second day after PHZ was administered, and increased to 200.3 ± 4.2/mm(2) on the sixth day. On the seventh day after the PHZ injections, the number decreased and they were no longer detected at 30 days after PHZ was injected. The present study revealed that erythroblasts accumulate around sinusoidal macrophages to form an erythroblastic island with a central macrophage similar to erythropoiesis in the fetal liver. Furthermore, in line with development and regression of extramedullary hematopoiesis, macrophages express ER-HR3, a hematopoiesis related antigen.
Asunto(s)
Hematopoyesis Extramedular/fisiología , Hígado/citología , Macrófagos/citología , Animales , Enfermedades Hematológicas/metabolismo , Enfermedades Hematológicas/patología , Inmunohistoquímica , Hígado/metabolismo , Hígado/patología , Macrófagos/metabolismo , Macrófagos/patología , Masculino , Ratones , Ratones Endogámicos ICR , Fenilhidrazinas/metabolismoRESUMEN
OBJECTIVES: Redox-reactive antibodies, mainly of the IgG class, gained a wide area of interest after their autoimmune reactivity was revealed following the application of chemical and physiological oxidants. In this study, we examined the susceptibility of IgMs to oxidation and evaluated their binding to the autoantigens important in some autoimmune diseases. METHODS: IgM and IgG fractions, isolated from healthy individuals' sera, were oxidized using direct electric current or physiological oxidant hemin. Specificities towards beta-2-glycoprotein I (ß(2)-GPI), cardiolipin (CL), and rheumatoid factor were evaluated with the enzyme-linked immunosorbent assays (ELISAs). Post-translational modification was investigated by 2,4-dinitrophenylhydrazine reaction. RESULTS: Electrochemically oxidized IgM fractions exhibited altered immunoreactivity - low to medium titers in anti-CL and low positive titers in anti-ß(2)-GPI ELISA but exhibited no rheumatoid factor reactivity. Oxidized IgG and IgM fractions exhibited 2.5- and 5-fold increase in the carbonyl content, respectively. DISCUSSION: An increase in the carbonyl content along with increased immunoreactivity after oxidation suggests modifications of the IgM paratopes. These results point towards possible modifications of native IgMs to their autoimmune state despite the fact that IgMs were less susceptible to oxidation than IgGs. The importance of an individual's redox status in maintenance of autoimmune reactions was emphasized by in vitro diagnostic tests.
Asunto(s)
Especificidad de Anticuerpos , Reacciones Antígeno-Anticuerpo , Autoantígenos/inmunología , Inmunoglobulina M/inmunología , Adulto , Autoantígenos/metabolismo , Sitios de Unión de Anticuerpos , Cardiolipinas/inmunología , Electroquímica , Electroforesis en Gel de Poliacrilamida , Ensayo de Inmunoadsorción Enzimática , Femenino , Hemina/farmacología , Humanos , Inmunoglobulina G/sangre , Inmunoglobulina G/inmunología , Inmunoglobulina G/metabolismo , Inmunoglobulina M/sangre , Inmunoglobulina M/metabolismo , Masculino , Oxidación-Reducción , Estrés Oxidativo , Fenilhidrazinas/metabolismo , Carbonilación Proteica , Procesamiento Proteico-Postraduccional , Factor Reumatoide/inmunología , beta 2 Glicoproteína I/inmunologíaRESUMEN
Tegafur (FT) is a 5-fluorouracil (5-FU) prodrug that has been clinically used for various cancer chemotherapies. The following metabolites of FT were identified in patients: 5-FU, fluoro-beta-alanine, and gamma-butyrolactone (GBL) and its acidic form, gamma-hydroxybutyrate (GHB). GBL/GHB, which is probably generated from the furan ring of FT, inhibits tumor cell angiogenesis, contributing to the antitumor effect of FT-based therapies. In the present study, we identified the metabolites formed from the furan ring of FT by CYP2A6 and thymidine phosphorylase (TPase) using 2,4-dinitrophenylhydrazine derivatization procedures and clarified the metabolic pathway of FT to GBL/GHB. Succinaldehyde (SA) and 4-hydroxybutanal (4-OH-BTL) were produced as the metabolites because of the cleavage of the furan ring of FT during its conversion to 5-FU in cDNA-expressed CYP2A6 and purified TPase, respectively; however, GBL/GHB was hardly detected in cDNA-expressed CYP2A6 and purified TPase. GBL/GHB was formed after human hepatic microsomes or cDNA-expressed CYP2A6 mixed with cytosol were incubated with FT. Furthermore, 4-OH-BTL was converted to GBL/GHB in the microsomes and cytosol. These results suggest that GBL/GHB is generated from FT through the formation of SA and 4-OH-BTL but not directly from FT. Furthermore, the amount of 5-FU and GBL/GHB formed in the hepatic S9 was markedly decreased in the presence of a CYP2A6 inhibitor, suggesting that GBL/GHB may be mainly generated through the CYP2A6-mediated formation of SA.
Asunto(s)
4-Butirolactona/metabolismo , Fluorouracilo/metabolismo , Furanos/metabolismo , Tegafur/metabolismo , Aldehídos/metabolismo , Hidrocarburo de Aril Hidroxilasas/genética , Hidrocarburo de Aril Hidroxilasas/metabolismo , Butanoles/metabolismo , Citocromo P-450 CYP2A6 , Citosol/metabolismo , ADN Complementario/metabolismo , Humanos , Hígado/enzimología , Microsomas Hepáticos/enzimología , Fenilhidrazinas/metabolismo , Oxibato de Sodio/metabolismo , Timidina Fosforilasa/genética , Timidina Fosforilasa/metabolismoRESUMEN
Azoreductase 1 from Pseudomonas aeruginosa strain PAO1 (paAzoR1) catalyses the activation of the prodrug balsalazide and reduces the azo dye methyl red using reduced nicotinamide adenine dinucleotide cofactor as an electron donor. To investigate the mechanism of the enzyme, a Y131F mutation was introduced and the enzymic properties of the mutant were compared with those of the wild-type enzyme. The crystallographic structure of the mutant with methyl red bound was solved at 2.1 A resolution and compared with the wild-type structure. Tyr131 is important in the architecture of the active site but is not essential for enzymic activity.