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WHAT IS KNOWN ON THE SUBJECT?: Psychological distress is a major health concern, and university student populations are at a risk due to high academic, economic and interpersonal demands at this time. While a number of studies have been conducted looking at distress and service uptake amongst university students, there is a lack of comparative information in the student nurse population. Therefore, this study focuses on level of psychological distress and service uptake in student nurses during their training. Student nurses in training are acknowledged as particularly vulnerable to anxiety and depression. Anxiety and depression can interfere with educational achievement and interfere with working relationships, which are an important aspect of clinical practice. WHAT THIS PAPER ADDS TO EXISTING KNOWLEDGE?: This study investigates psychological distress and service uptake in student nurses, in a single study, by measuring the level of distress and comparing this with adult nonpatient norms. The key findings suggest high levels of anxiety and depression and poor uptake of support in student nurses. The main barrier to seeking support was the fear of disclosure about their anxiety and/or depression, and feared impact on their suitability to train as a nurse. WHAT ARE THE IMPLICATIONS FOR PRACTICE?: Due to the fear of disclosure and low levels of support seeking, mental health nurses and academics have a key role to play in mental health literacy. It seems reasonable, given the key findings, to suggest that interventions be directed at decreasing anxiety and depression such as mindfulness training and other evidence-based strategies. It is recommended that psychological distress needs due attention from higher education sector and those health professionals involved in mental health service provision to support evidence-based strategies that target these issues. ABSTRACT: Introduction This study adds to the existing international evidence on psychological distress in the student population by focusing on student nurses. It quantitatively assesses psychological distress with comparative norms and investigates service uptake in a single study. Aim The aim of this study was to investigate the level of psychological distress in students and compare this with population norms and highlight potential facilitators and barriers to help seeking. Methods This study recruited N = 121 student nurses from one university in a cross-sectional design. Data were analysed using descriptive statistics, independent t tests and one-way ANOVAs. Findings The key findings show high levels of psychological distress, which is above levels seen in the general population. The main barriers to seeking support were fear of disclosure and the perceived impact on their suitability as a student nurse. Discussion The study highlights that high levels of distress identified in the literature are seen in student nurses and that fear of disclosure may account for some not seeking support. Relevance The fear of disclosure and low levels of seeking support suggest there is a need for mental health nurses and academics to play a key role in mental health literacy and evidence-based interventions such as mindfulness to combat these issues.
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Ansiedad/psicología , Depresión/psicología , Estrés Laboral/psicología , Aceptación de la Atención de Salud/psicología , Autorrevelación , Estudiantes de Enfermería/psicología , Adolescente , Adulto , Habilitación Profesional , Estudios Transversales , Femenino , Humanos , Masculino , Persona de Mediana Edad , Enfermeras y Enfermeros , Adulto JovenRESUMEN
BACKGROUND/OBJECTIVES: Children with atopic dermatitis are at increased risk of both general behaviour problems, and those specific to the condition and its treatment. This can hamper the ability of parents to carry out treatment and manage the condition effectively. To date, there is no published instrument available to assess child behaviour difficulties in the context of atopic dermatitis management. Our aim was to develop a reliable and valid instrument to assess atopic dermatitis-specific child behaviour problems, and parents' self-efficacy (confidence) for managing these behaviours. METHODS: The Eczema Behaviour Checklist (EBC) was developed as a 25-item questionnaire to measure (i) extent of behaviour problems (EBC Extent scale), and (ii) parents' self-efficacy for managing behaviour problems (EBC Confidence scale), in the context of child atopic dermatitis management. A community-based sample of 292 parents completed the EBC, measures of general behaviour difficulties, self-efficacy with atopic dermatitis management and use of dysfunctional parenting strategies. RESULTS: There was satisfactory internal consistency and construct validity for EBC Extent and Confidence scales. There was a negative correlation between atopic dermatitis-specific behaviour problems and parents' self-efficacy for dealing with behaviours (r = -.53, p < .001). Factor analyses revealed a three-factor structure for both scales: (i) treatment-related behaviours; (ii) symptom-related behaviours; and (iii) behaviours related to impact of the illness. Variation in parents' self-efficacy for managing their child's atopic dermatitis was explained by intensity of illness-specific child behaviour problems and parents' self-efficacy for dealing with the behaviours. CONCLUSIONS: The new measure of atopic dermatitis-specific child behaviour problems was a stronger predictor of parents' self-efficacy for managing their child's condition than was the measure of general child behaviour difficulties. Results provide preliminary evidence of reliability and validity of the EBC, which has potential for use in clinical and research settings, and warrant further psychometric evaluation.
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Trastornos de la Conducta Infantil/diagnóstico , Trastornos de la Conducta Infantil/etiología , Dermatitis Atópica/psicología , Adulto , Lista de Verificación , Niño , Conducta Infantil , Preescolar , Dermatitis Atópica/terapia , Fármacos Dermatológicos/efectos adversos , Femenino , Humanos , Masculino , Relaciones Padres-Hijo , Responsabilidad Parental/psicología , Psicometría , Reproducibilidad de los Resultados , Autoeficacia , Índice de Severidad de la Enfermedad , Cuidados de la Piel/métodos , Cuidados de la Piel/psicología , Factores Socioeconómicos , Encuestas y CuestionariosRESUMEN
We used two approaches for studying the relationships between wine consumption, wine composition and cancer In the first approach, a transgenic mouse model of human neurofibromatosis, combined with the use of well-defined, chemically purified diets, showed that red wine contains nonalcoholic components that can delay tumor onset. In additional studies, catechin, the main monomeric polyphenol of red wine, delayed tumor onset in this mouse model in a positive, linear relationship when incorporated into the diet at levels of 0.5-4 mmol/kg diet. In the second approach, low doses of the chemical carcinogen 2-amino-1-methyl-6-phenylimidazo(4, 5-b)pyridine (PhlP) were administered to rats, and formation of DNA adducts was evaluated by accelerator mass spectrometry. Consumption of red wine solids (the residue from red wine remaining after removal of alcohol and water) and the wine polyphenol quercetin did not influence PhlP-DNA adduct levels or induce liver enzymes (glutathione-S-transferase and quinone reductase). However, quercetin did alter distribution of PhlP in the rat tissues compared to control animals and animals fed other potential dietary chemopreventive agents, including phenylethyl isothiocyanate and sulforaphane. These studies demonstrate the feasibility of these approaches for studying the chemopreventive potential of dietary components at physiologic levels in
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Anticarcinógenos/farmacología , Carcinógenos/toxicidad , Imidazoles/toxicidad , Neurofibromatosis/prevención & control , Vino , Animales , Carcinógenos/metabolismo , Catequina/farmacología , Aductos de ADN/biosíntesis , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Virus Linfotrópico T Tipo 1 Humano/genética , Humanos , Imidazoles/metabolismo , Pruebas de Función Hepática , Masculino , Ratones , Ratones Transgénicos , Neurofibromatosis/genética , Quercetina/farmacología , Ratas , Ratas Endogámicas F344 , Vitis/químicaRESUMEN
Gram-negative infection is an important cause of morbidity and mortality after unrelated donor-bone marrow transplantation (UD-BMT). We performed a retrospective case-control study to examine the risk factors, prophylaxis, therapy and outcome of Gram-negative bacteraemia (GNB) in 428 patients undergoing UD-BMT. The incidence of GNB was 3.6% in children and 19% in adults. Of the adults, 11% developed GNB >60 days post UD-BMT. Predisposing risk factors for GNB included 'high-risk' disease status, chronic graft-versus-host disease and use of systemic steroids. Fever, a raised C-reactive protein (CRP) and hypotension were common findings at presentation. Patients were routinely given prophylactic ciprofloxacin: resistance to this antibiotic was seen in 33% of isolates. We identified an age-matched control group undergoing UD-BMT over the same time period as the study group. Gram-positive bacteraemia was significantly more common in cases than controls. Mortality from GNB was 17% in children and 24% in adults. We conclude that GNB is a common complication of UD-BMT with a high associated mortality. Patients should be educated further to present rapidly with symptoms suggestive of infection.
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Trasplante de Médula Ósea/efectos adversos , Infecciones por Bacterias Gramnegativas/etiología , Adolescente , Adulto , Profilaxis Antibiótica , Bacteriemia/etiología , Trasplante de Médula Ósea/métodos , Estudios de Casos y Controles , Niño , Preescolar , Ciprofloxacina/uso terapéutico , Farmacorresistencia Bacteriana , Femenino , Infecciones por Bacterias Gramnegativas/epidemiología , Infecciones por Bacterias Gramnegativas/mortalidad , Humanos , Incidencia , Lactante , Masculino , Persona de Mediana Edad , Estudios Retrospectivos , Factores de Riesgo , Resultado del TratamientoAsunto(s)
Antineoplásicos Alquilantes/uso terapéutico , Astrocitoma/tratamiento farmacológico , Carboplatino/uso terapéutico , Ganglioglioma/tratamiento farmacológico , Neoplasias de la Médula Espinal/tratamiento farmacológico , Astrocitoma/complicaciones , Astrocitoma/patología , Astrocitoma/cirugía , Vértebras Cervicales , Quimioterapia Adyuvante , Preescolar , Terapia Combinada , Femenino , Trastornos Neurológicos de la Marcha/etiología , Ganglioglioma/complicaciones , Ganglioglioma/patología , Ganglioglioma/cirugía , Humanos , Imagen por Resonancia Magnética , Recurrencia Local de Neoplasia/tratamiento farmacológico , Pronóstico , Inducción de Remisión , Neoplasias de la Médula Espinal/complicaciones , Neoplasias de la Médula Espinal/patología , Neoplasias de la Médula Espinal/cirugía , Vértebras TorácicasRESUMEN
BACKGROUND: The purpose of this study was to examine the clinical and radiologic response to carboplatin by children with progressive optic/thalamic gliomas. PATIENTS AND METHODS: Between July 1997 and July 1999, 12 consecutive children were treated with monthly carboplatin for progressive optic/thalamic gliomas. RESULTS: Five children have completed 12 cycles of carboplatin and five children are currently receiving treatment. Two children had progressive disease noted both clinically and radiologically. Nine children have stable radiologic disease and one child has had a partial radiologic response to chemotherapy. Eight children have had regular visual assessments. Four children (three with stable radiology and one with a partial radiologic response) have had improvement in their vision. Three children with radiologically stable disease have had no change in vision. One child has had deterioration in vision despite radiologically stable disease. CONCLUSIONS: The results suggest that the clinical response of optic/thalamic gliomas to carboplatin, as measured by visual acuity and visual fields, may be better than predicted by radiologic assessment. These data suggest that a prospective clinical study is warranted of the role of carboplatin in children with progressive optic/thalamic gliomas and visual impairment.
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Antineoplásicos Alquilantes/uso terapéutico , Neoplasias Encefálicas/tratamiento farmacológico , Carboplatino/uso terapéutico , Glioma/tratamiento farmacológico , Quiasma Óptico , Neoplasias del Nervio Óptico/tratamiento farmacológico , Tálamo , Trastornos de la Visión/etiología , Adolescente , Anemia/inducido químicamente , Antineoplásicos Alquilantes/administración & dosificación , Antineoplásicos Alquilantes/efectos adversos , Neoplasias Encefálicas/complicaciones , Neoplasias Encefálicas/cirugía , Carboplatino/administración & dosificación , Carboplatino/efectos adversos , Niño , Preescolar , Terapia Combinada , Progresión de la Enfermedad , Femenino , Glioma/complicaciones , Glioma/cirugía , Humanos , Infusiones Intravenosas , Masculino , Neurofibromatosis 1/complicaciones , Neutropenia/inducido químicamente , Quiasma Óptico/cirugía , Neoplasias del Nervio Óptico/complicaciones , Neoplasias del Nervio Óptico/cirugía , Inducción de Remisión , Estudios Retrospectivos , Tálamo/cirugía , Trombocitopenia/inducido químicamente , Resultado del Tratamiento , Agudeza Visual , Campos VisualesRESUMEN
Typical volatile heterocyclic compounds found in brewed coffee extracts-pyrroles, furans, thiophenes, and thiazoles-were examined for antioxidative activity, which was determined by measuring the oxidative conversion of hexanal to hexanoic acid using gas chromatography. 2-Acetylpyrrole, 1-methylpyrrole, and pyrrole inhibited hexanal oxidation by 98, 87, and 78%, respectively, at a concentration of 500 microgram/mL over a period of 30 days. 2-Methylfuran, which inhibited hexanal oxidation by 90% at all concentrations tested (500, 200, and 100 microgram/mL) for a 30-day period, exhibited the greatest activity among furans tested. Similarly, 2-methylthiophene, which inhibited hexanal oxidation by almost 100% at a concentration of 500 microgram/mL over 30 days, exhibited the greatest activity among the thiophenes tested. In general, thiazoles were ineffective antioxidants at all concentrations tested. However, 4,5-dimethylthiazole was able to inhibit hexanal oxidation by 50% at the highest level tested (500 microgram/mL). 2-Acetylpyrrole, 2-methylfuran, and 2-methylthiophene at concentrations of 500, 200, and 100 microgram/mL and furan at a concentration of 500 microgram/mL exhibited antioxidative activities comparable to that of the synthetic antioxidant butylated hydroxytoluene at a concentration of 50 microgram/mL.
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Antioxidantes/análisis , Café/química , Culinaria , Compuestos Heterocíclicos/análisis , Aldehídos/química , Antioxidantes/química , Caproatos/química , Cromatografía de Gases , Compuestos Heterocíclicos/química , Oxidación-ReducciónRESUMEN
Aroma extracts from fresh soybeans, mung beans, kidney beans, and azuki beans were prepared using simultaneous steam distillation and solvent extraction (SDE) under mild conditions (55 degrees C and 95 mmHg). Extracts were examined for antioxidative activities in two different assays. The aroma extracts isolated from all beans inhibited the oxidation of hexanal for nearly one month at a level of 250 microL/mL. Mung bean and soybean extracts inhibited malonaldehyde (MA) formation from cod-liver oil by 86% and 88%, respectively, at the 250 microL/mL level. Azuki and kidney bean extracts inhibited MA formation from cod-liver oil by 76% and 53%, respectively, at the 250 microL/mL level. The antioxidative activities of mung bean and soybean extracts were comparable with that of the natural antioxidant, alpha-tocopherol (vitamin E).
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Antioxidantes/química , Fabaceae/química , Odorantes/análisis , Plantas Medicinales , Cromatografía de GasesRESUMEN
Acute exposure to naphthalene produces severe bronchiolar epithelial cell necrosis in mice, whereas subchronic exposure to naphthalene (200 mg/kg/7 days) fails to produce epithelial necrosis and renders the animals tolerant to subsequent challenge doses of naphthalene. Mechanisms responsible for the development of tolerance have not been delineated. The few studies exploring naphthalene tolerance focus on expression of microsomal enzymes and have yet to delve into expression of the hepatic detoxification enzymes such as glutathione S-transferases (GSTs; EC 2.5.1.18). Glutathione conjugation catalyzed by GSTs accounts for one of the two primary routes of naphthalene detoxification. In this study, we rigorously quantify levels of individual GST isozymes expressed within the livers and lungs of mice with acquired tolerance to naphthalene. Subchronic exposure to naphthalene increases the abundance of some hepatic GSTs to levels as much as 68% greater than controls. Naphthalene-tolerant mice displayed increases in mGSTM1 (51%), mGSTM2 (58%), and mGSTP1 (66%), whereas no significant difference in mGSTA3 was observed between exposed and control mice. Extracts of pulmonary tissues from naphthalene-tolerant mice showed minor increases in levels of mGSTP1 (7%) and Peak 8 isozyme (27%) and decreases in levels of mGSTM1 (31%), mGSTM2 (17%), and mGSTA3 (8%). The total enzymatic activity for the conjugation of 1-chloro-2,4-dinitrobenzene (CDNB) was 22% lower in lung extracts from naphthalene-tolerant animals than in controls. These results indicate that induction of hepatic GSTs is substantial and may be an important factor in the development of tolerance to naphthalene.
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Glutatión Transferasa/análisis , Hígado/enzimología , Pulmón/enzimología , Naftalenos/toxicidad , Animales , Cromatografía Líquida de Alta Presión , Aceite de Maíz , Relación Dosis-Respuesta a Droga , Tolerancia a Medicamentos , Glutatión Transferasa/biosíntesis , Glutatión Transferasa/metabolismo , Isoenzimas/análisis , Isoenzimas/biosíntesis , Isoenzimas/metabolismo , Hígado/efectos de los fármacos , Pulmón/efectos de los fármacos , Masculino , Ratones , Espectrometría de Masa por Ionización de ElectrosprayRESUMEN
Lysyl oxidase activity is critical for the assembly and cross-linking of extracellular matrix proteins, such as collagen and elastin. Moreover, lysyl oxidase activity is sensitive to changes in copper status and genetic perturbations in copper transport, e.g., mutations in the P-type ATPase gene, ATP7A, associated with cellular copper transport. Lysyl oxidase may also serve as a vehicle for copper transport from extracellular matrix cells. Herein, we demonstrate that sufficient lysyl oxidase functional activity is present in the rat embryo at gestation day (GD) 9 to be detected in conventional enzyme assays. Estimation of embryonic lysyl oxidase functional activity, however, required partial purification in order to remove inhibitors. From GD 9 to GD 15, lysyl oxidase activity was relatively constant when expressed per unit of protein or DNA. In contrast, the steady-state levels of lysyl oxidase and ATP7A mRNA, measured by RT-PCR and expressed relative to total RNA and cyclophilin mRNA, increased approximately fourfold from GD 9 to 15. The pattern of temporal expression for ATP7A was consistent with its possible role in copper delivery to lysyl oxidase.
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Adenosina Trifosfatasas/genética , Proteínas Portadoras/genética , Proteínas de Transporte de Catión , Embrión de Mamíferos/enzimología , Regulación del Desarrollo de la Expresión Génica/genética , Proteína-Lisina 6-Oxidasa/genética , Proteínas Recombinantes de Fusión , Animales , Transporte Biológico , Cobre/metabolismo , ATPasas Transportadoras de Cobre , Desarrollo Embrionario y Fetal , Regulación Enzimológica de la Expresión Génica , Edad Gestacional , Isomerasa de Peptidilprolil/genética , ARN Mensajero/metabolismo , Ratas , Ratas Sprague-Dawley , Espectrometría de Masa por Láser de Matriz Asistida de Ionización DesorciónRESUMEN
The observation of accelerated collagen glycation in association with enhanced progression of many age-associated diseases in hyperglycemic subjects has led researchers to propose a role of glycation in the aging process. Although short-term studies in healthy animals suggest that feeding a diet high in fructose may increase serum glucose concentrations and increase glycemic stress, the effects of a long-term feeding, i.e., life span, are unknown. This study was designed to evaluate the long-term effects of dietary carbohydrates on serum and tissue markers of glycemic stress. Three-month-old male Fischer 344 rats were given free access to or restricted to 60% caloric intake of one of five isocaloric diets that contained as their carbohydrate source either cornstarch, glucose, sucrose, fructose or equimolar amounts of fructose and glucose. Rats were killed at 9-, 18- or 26-mo of age. Glycated hemoglobin, serum glucose and fructosamine levels were measured as markers of serum glycemic stress. Collagen-associated fluorescence and pentosidine concentrations were measured in skin, aortic, tracheal and tail tendon collagen as markers of advanced glycation endproducts (AGE). The source of dietary carbohydrate had little effect on markers of glycemic stress and the accumulation of AGE. Restricting the amount of calories consumed resulted in lower serum glucose concentrations, glycated hemoglobin levels and pentosidine concentrations in tail tendon collagen. Our data suggest that the rate of collagen glycation is tissue-specific. These results suggest that long-term feeding of specific dietary carbohydrates does not alter serum glucose concentrations or the rate of collagen glycation. Rather, age-related accumulation of AGE is more closely related to caloric intake.
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Envejecimiento/metabolismo , Carbohidratos de la Dieta/farmacología , Productos Finales de Glicación Avanzada/metabolismo , Animales , Arginina/análogos & derivados , Arginina/metabolismo , Glucemia/efectos de los fármacos , Colágeno/metabolismo , Reactivos de Enlaces Cruzados/metabolismo , Carbohidratos de la Dieta/administración & dosificación , Sacarosa en la Dieta/administración & dosificación , Ingestión de Energía , Fructosamina/sangre , Fructosa/administración & dosificación , Glucosa/administración & dosificación , Lisina/análogos & derivados , Lisina/metabolismo , Masculino , Ratas , Ratas Endogámicas F344 , Piel/metabolismo , Almidón/administración & dosificaciónRESUMEN
O-quinone cofactors derived from tyrosine and tryptophan are involved in novel biological reactions that range from oxidative deaminations to free-radical redox reactions. The formation of each of these cofactors appears to involve post-translational modifications of either tyrosine or tryptophan residues. The modifications result in cofactors, such as topaquinone (TPQ), tryptophan tryptophylquinone (TTQ), lysine tyrosylquinone (LTQ) or the copper-complexed cysteinyl-tyrosyl radical from metal-catalyzed reactions. Pyrroloquinoline quinone (PQQ) appears to be formed from the annulation of peptidyl glutamic acid and tyrosine residues stemming from their modification as components of a precursor peptide substrate. PQQ, a primary focus of this review, has invoked considerable interest because of its presence in foods, antioxidant properties and role as a growth-promoting factor. Although no enzymes in animals have been identified that exclusively utilize PQQ, oral supplementation of PQQ in nanomolar amounts increases the responsiveness of B- and T-cells to mitogens and improves neurologic function and reproductive outcome in rodents. Regarding TPQ and LTQ, a case may be made that the formation of TPQ and LTQ is also influenced by nutritional status, specifically dietary copper. For at least one of the amine oxidases, lysyl oxidase, enzymatic activity correlates directly with copper intake. TPQ and LTQ are generated following the incorporation of copper by a process that involves the two-step oxidation of a specified tyrosyl residue to first peptidyl dopa and then peptidyl topaquinone to generate active enzymes, generally classed as "quinoenzymes." Limited attention is also paid to TTQ and the copper-complexed cysteinyl-tyrosyl radical, cofactors important to fungal and bacterial redox processes.
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Coenzimas/metabolismo , Enzimas/metabolismo , Indolquinonas , Quinonas/metabolismo , Animales , Dihidroxifenilalanina/análogos & derivados , Dihidroxifenilalanina/química , Dihidroxifenilalanina/metabolismo , Humanos , Lisina/análogos & derivados , Lisina/química , Lisina/metabolismo , Cofactor PQQ , Quinolonas/química , Quinolonas/metabolismo , Quinonas/química , Triptófano/análogos & derivados , Triptófano/química , Triptófano/metabolismoRESUMEN
Monomeric and oligomeric procyanidins present in cocoa liquors and chocolates were separated and quantified in four different laboratories using a normal-phase high-performance liquid chromatography (HPLC) method with fluorescence detection. Procyanidin standards through decamers were obtained by extraction from cocoa beans, enrichment by Sephadex LH-20 gel permeation chromatography, and final purification by preparative normal-phase HPLC. The purity of each oligomeric fraction was assessed using HPLC coupled to mass spectrometry. A composite standard was then prepared, and calibration curves were generated for each oligomeric class using a quadratic fit of area sum versus concentration. Results obtained by each of the laboratories were in close agreement, which suggests this method is reliable and reproducible for quantification of procyanidins. Furthermore, the procyanidin content of the samples was correlated to the antioxidant capacity measured using the ORAC assay as an indicator for potential biological activity.
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Antioxidantes/análisis , Biflavonoides , Cacao , Catequina/análisis , Cromatografía Líquida de Alta Presión , Proantocianidinas , Cromatografía Líquida de Alta Presión/métodos , Fluorescencia , Humanos , Espectrometría de Masas , Extractos Vegetales/químicaRESUMEN
Monomeric and oligomeric procyanidins present in cocoa and chocolate were separated and identified using a modified normal-phase high-performance liquid chromatography (HPLC) method coupled with on-line mass spectrometry (MS) analysis using an atmospheric pressure ionization electrospray chamber. The chromatographic separation was achieved using a silica stationary phase in combination with a gradient ascending in polarity. This qualitative report confirms the presence of a complex series of procyanidins in raw cocoa and certain chocolates using HPLC/MS techniques. Although both cocoa and chocolate contained monomeric and oligomeric procyanidin units 2-10, only use of negative mode provided MS data for the higher oligomers (i.e., >pentamer). Application of this method for qualitative analysis of proanthocyanidins in other food products and confirmation of this method as a reliable quantitative tool for determining levels of procyanidins in cocoa, chocolate, and other food products are currently being investigated.
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Biflavonoides , Cacao/química , Catequina/química , Proantocianidinas , Cromatografía Líquida de Alta Presión , Espectrometría de MasasRESUMEN
Lysyl oxidase (EC 1.4.3.13), a cuproenzyme, can account for 10-30% of the copper present in connective tissue. Herein, we assess the extent to which tissue copper concentrations and lysyl oxidase activity are related because the functional activity of lysyl oxidase and the copper content of chick tendon are both related to dietary copper intake. Chicks (1-d old) were fed diets (basal copper concentration, 0.4 microg/g diet) to which copper was added from 0 to 16 microg/g diet. Liver and plasma copper levels tended to normalize in chickens that consumed from 1 to 4 microg copper/g of diet, whereas tendon copper concentrations suggested an unusual accumulation of copper in chickens that consumed 16 microg copper/g diet. The molecular weight of lysyl oxidase was also estimated using matrix-assisted laser desorption ionization/time-of-flight/mass spectrometry (MALDI/TOF/MS). A novel aspect of these measurements was estimation of protein mass directly from the surface of chick tendons and aortae. Whether copper deficiency (0 added copper) or copper supplementation (16 microg copper/g of diet) caused changes in the molecular weight of protein(s) in tendon corresponding to lysyl oxidase was addressed. The average molecular weight of the peak corresponding to lysyl oxidase in tendon and aorta from copper-deficient birds was 28,386 Da +/- 86, whereas the average molecular weight of corresponding protein in tendon from copper-supplemented birds was 28,639 Da +/- 122. We propose that the shift in molecular weight is due in part to copper binding and the formation of lysyl tyrosyl quinone, the cofactor at the active site of lysyl oxidase.
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Cobre/administración & dosificación , Proteína-Lisina 6-Oxidasa/metabolismo , Tendones/enzimología , Animales , Aorta/enzimología , Pollos , Cobre/deficiencia , Cobre/farmacología , Dieta , Relación Dosis-Respuesta a Droga , Activación Enzimática , Masculino , Peso Molecular , Proteína-Lisina 6-Oxidasa/química , Proteína-Lisina 6-Oxidasa/efectos de los fármacosRESUMEN
We describe a HPLC method coupled to electrospray ionization mass spectrometry (ESI/MS) for quantification and identification of pyrroloquinoline quinone (PQQ) and condensation products formed upon incubation of PQQ with amino acids (IPQ; imidazolopyrroloquinoline and I/OPQ/R; imidazolopyrroloquinoline with attached R-group). More importantly, using these methods we demonstrate the presence of both PQQ and IPQ in human milk in nanomolar to micromolar concentrations. PQQ was incubated with amino acids and condensation products were separated by HPLC. Fractions corresponding to each product were collected and molecular masses were determined using ESI/MS. Ala, Asp, Arg, Cys, Gly, Glu, Ser, Thr, Trp, and Tyr form IPQ upon incubation with PQQ. Yields of IPQ were low (<5%) for Asp and Glu, yet high (>60%) for Thr. In addition to IPQ, Ala, Arg, Cys, Ser, Trp, and Tyr formed IPQ/R derivatives. His, Ile, Leu, Glu, Leu, Lys, Met, and Phe form only IPQ/R derivatives. Proline did not react with PQQ. Mass spectra indicate that PQQ forms stable hydrated carbonyls and decarboxylates easily. Although mass spectra were complicated by the oxidation state of the quinone and decarboxylation of PQQ, these methods are invaluable for the rapid detection of the full range of PQQ adducts in biological matrices.
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Aminoácidos/análisis , Aminoácidos/química , Espectrometría de Masas/métodos , Leche Humana/química , Quinolinas/análisis , Cromatografía Líquida de Alta Presión , Femenino , Humanos , Estructura Molecular , Quinolinas/químicaRESUMEN
Mass spectrometric analysis of proteolysis products of haloenol lactone-modified glutathione S-transferase isozyme mGSTP1 indicates that the haloenol lactone 3-cinnamyl-5(E)-bromomethylidenetetrahydro-2-furanone is covalently attached to the protein at Cys-47. Comparisons of the extent of adduct formation with losses in enzymatic activity indicate that mGSTP1 exhibits greatest reactivity toward the haloenol lactone, followed by mGSTM1 and mGSTA3. Activities of mGSTP1 and mGSTM1 decrease in inverse proportion to haloenol lactone concentration, whereas modification had no apparent effect on catalytic activity of mGSTA3. Decreases in activity agree with the extent of protein modification observed in ESI mass spectra for mGSTP1 and mGSTM1 but not for mGSTA3. Kinetic studies employing recombinant human proteins with replacement of cysteine by serine at Cys-47 and Cys-101 indicate that rapid inactivation (t1/2 = 2 min) occurs only when residue 47 is cysteine. Mass spectra of C47S-hGSTP1 incubated with haloenol lactone demonstrate covalent attachment of a haloenol lactone-glutathione conjugate and suggest that an ester forms between the lactone and Ser-47. Therefore, we propose that initial opening of the lactone ring is promoted by Cys-47 through thioester formation between the lactone carbonyl and the Cys-47 sulfhydryl. Enol-keto tautomerization and enzyme-mediated hydrolytic cleavage of the thioester produces a reactive alpha-bromoketone which reacts a second time with Cys-47 and inactivates the enzyme. These results suggest that Pi class GSTs have thioesterase activity and that haloenol lactone inactivation occurs through an enzyme-mediated process.
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4-Butirolactona/farmacología , Inhibidores Enzimáticos/farmacología , Glutatión Transferasa/antagonistas & inhibidores , Secuencia de Aminoácidos , Animales , Activación Enzimática/efectos de los fármacos , Activación Enzimática/genética , Glutatión Transferasa/genética , Glutatión Transferasa/aislamiento & purificación , Glutatión Transferasa/metabolismo , Humanos , Isoenzimas/antagonistas & inhibidores , Isoenzimas/genética , Masculino , Ratones , Ratones Endogámicos , Datos de Secuencia Molecular , Fragmentos de Péptidos/aislamiento & purificación , Fragmentos de Péptidos/metabolismo , Proteínas Recombinantes/antagonistas & inhibidores , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Relación Estructura-Actividad , Factores de TiempoRESUMEN
Protein-lysine 6-oxidase (lysyl oxidase) is a cuproenzyme that is essential for stabilization of extracellular matrixes, specifically the enzymatic cross-linking of collagen and elastin. A hypothesis is proposed that links dietary copper levels to dynamic and proportional changes in lysyl oxidase activity in connective tissue. Although nutritional copper status does not influence the accumulation of lysyl oxidase as protein or lysyl oxidase steady state messenger RNA concentrations, the direct influence of dietary copper on the functional activity of lysyl oxidase is clear. The hypothesis is based on the possibility that copper efflux and lysyl oxidase secretion from cells may share a common pathway. The change in functional activity is most likely the result of posttranslational processing of lysyl oxidase. Copper is essential for organic cofactor formation in amine oxidases such as lysyl oxidase. Copper-containing amine oxidases have peptidyl 2,4,5 tri(oxo)phenylalanine (TOPA) at their active centers. TOPA is formed by copper-catalyzed oxidation of tyrosine, which takes place as part of Golgi or trans-Golgi processing. For lysyl oxidase, recent evidence (Science 1996;273:1078-84) indicates that as an additional step, a lysyl group at the active center of lysyl oxidase reacts with TOPA or its precursor to form lysyl tyrosylquinone.
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Cobre/metabolismo , Proteínas de la Matriz Extracelular/metabolismo , Proteína-Lisina 6-Oxidasa/metabolismo , Animales , Proteínas de la Matriz Extracelular/química , Aparato de Golgi/metabolismo , Humanos , Proteína-Lisina 6-Oxidasa/biosíntesis , Proteína-Lisina 6-Oxidasa/químicaRESUMEN
Cytosolic glutathione S-transferase (GST) isoenzymes from brain, heart, lung, liver, kidney and gonads of male and female CD-1 mice were identified and quantified with a combination of affinity purification, electrospray ionization MS, Edman microsequencing, Western blot analysis and reverse-phase HPLC. The three principal hepatic GST subunits, mGSTA3 (25271 Da), mGSTP1 (23478 Da), and mGSTM1 (25839 Da), were isolated from liver, lung, kidney, testes and female heart, whereas brain, ovaries and male heart contained mGSTM1 and mGSTP1. Additional isoenzymes were detected in tissues, including mu class subunits mGSTM2 (25580 Da) and mGSTM3 (25570 Da), an N-terminally blocked Alpha subunit (25480 Da) assigned as mGSTA4, and proteins of molecular masses 25490, 22540, 24493, 24378 and 25383 Da. Distinct gender differences in expression of GST subunits were observed for liver, heart, kidney and gonads, whereas GST expression was similar in brain and lung for both genders. In contrast with patterns of expression in liver (high ratio of mGSTA3 to mGSTP1 in females relative to males), mGSTP1 was the most abundant subunit in female gonads, whereas mGSTA3 was not present in detectable quantities. The profile of GST expression in kidney was similar to that in liver; however, male kidneys expressed 30% more soluble GST than female kidneys. A striking gender-related difference in GST expression was found in cardiac tissue, where female animals expressed 50% more soluble GST than male tissues, and the GST isoenzyme with the greatest documented activity towards lipid hydroperoxides, mGSTA3, was present in female tissue yet absent from male tissue. These results point to complex gender- and tissue-dependent expression of individual mouse GST isoenzymes.
Asunto(s)
Regulación Enzimológica de la Expresión Génica , Glutatión Transferasa/biosíntesis , Isoenzimas/biosíntesis , Animales , Encéfalo/enzimología , Cromatografía Líquida de Alta Presión , Femenino , Glutatión Transferasa/química , Glutatión Transferasa/aislamiento & purificación , Isoenzimas/química , Isoenzimas/aislamiento & purificación , Riñón/enzimología , Hígado/enzimología , Sustancias Macromoleculares , Masculino , Ratones , Miocardio/enzimología , Especificidad de Órganos , Ovario/enzimología , Caracteres Sexuales , Espectrometría de Masa de Ion Secundario , Testículo/enzimologíaRESUMEN
A haloenol lactone derivative has been synthesized and found to be an isozyme-selective and active site-directed inactivator of glutathione S-transferase (GST). Preincubation of the haloenol lactone (100 microM) with murine Alpha, Mu, or Pi GST isozyme (1.0 microM) at pH 6.5, 37 degrees C resulted in time-dependent loss of enzyme activity with highly selective inhibition of the Pi isozyme (t1/2, approximately 2 min). In a separate experiment, a 10-fold excess of the lactone was incubated with GST-Pi isozyme at 37 degrees C for 3 h, followed by dialysis against Nanopure water. GST activity lost upon incubation with the lactone could not be restored by exhaustive dialysis, and only 8% of enzyme activity for the modified GST remained relative to the control that was treated identically except the lactone was omitted from the incubation. Both control and modified GST were characterized using electrospray ionization mass spectrometry. No native GST (23,478 Da) was observed in the spectrum of modified GST. Instead, protein incubated with the lactone exhibited an increase in molecular mass of 230 Da relative to control GST. The lactone (100 microM) was incubated with GST Pi isozyme (1.0 microM) in the presence of the competitive inhibitor S-hexylglutathione (10 microM), which suppressed time-dependent inhibition of GST by the lactone. The results suggest that this haloenol lactone is an irreversible and active site-directed inhibitor of GST that appears to inhibit the enzyme through two consecutive steps of nucleophilic attack.