RESUMEN

Sweet cherry is a valuable non-climacteric fruit with elevated phytonutrients, whose fruit quality attributes are prone to rapid deterioration after harvest, especially peel damage and water loss of stem. Here the metabolic and transcriptional response of exogenous melatonin was assessed in two commercial cultivars of sweet cherry (Santina and Royal Rainier) during cold storage. Gene expression profiling revealed that cuticle composition and water movement may underlie the effect of melatonin in delaying weight loss. An effect of melatonin on total soluble solids and lower respiration rate was observed in both cultivars. Melatonin induces overexpression of genes related to anthocyanin biosynthesis, which correlates with increased anthocyanin levels and changes in skin color (Chroma). Our results indicate that along with modulating antioxidant metabolism, melatonin improves fruit quality traits by triggering a range of metabolic and gene expression changes, which ultimately contribute to extend sweet cherry postharvest storability.

Asunto(s)

Regulación de la Expresión Génica/efectos de los fármacos , Melatonina/farmacología , Prunus avium/efectos de los fármacos , Antocianinas/metabolismo , Frutas/metabolismo , Prunus avium/metabolismo

RESUMEN

Accurate quantification depends on normalization of the measured gene expression data. In particular, gene expression studies with exposure to metals are challenging due their toxicity and redox-active properties. Here, we assessed the stability of potential reference genes in three cell lines commonly used to study metal cell metabolism: Caco-2 (colon), HepG2 (liver) and THP-1 (peripheral blood) under copper (Cu) or zinc (Zn) exposure. We used combined statistical tools to identify the best reference genes from a set of eleven candidates, which included traditional "housekeeping" genes such as GAPDH and B-ACTIN, in cell lines exposed to high and low, Zn and Cu concentrations. The expression stabilities of ATP5B (ATP synthase) and CYC1 (subunits of the cytochrome) were the highest considering the effect of Zn and Cu treatments whereas SDHA (succinate dehydrogenase) was found to be the most unstable gene. Even though the transcriptional effect of Zn and Cu is very different in term of redox properties, the same best reference genes were identified when Zn or Cu treatments were analyzed together. Our results indicate that ATP5B/CYC1 are the best candidates for reference genes after metal exposure, which can be used as a suitable starting point to evaluate gene expression with other metals or in different cell types in human models.

Asunto(s)

Cobre/farmacología , Grupo Citocromo c/genética , ATPasas de Translocación de Protón Mitocondriales/genética , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , Zinc/farmacología , Línea Celular , Grupo Citocromo c/metabolismo , Grupo Citocromo c/normas , Perfilación de la Expresión Génica , Humanos , ATPasas de Translocación de Protón Mitocondriales/metabolismo , ATPasas de Translocación de Protón Mitocondriales/normas , Reacción en Cadena en Tiempo Real de la Polimerasa/normas , Estándares de Referencia

RESUMEN

UNLABELLED: It is not clear how frequent is copper deficiency in humans. Current copper markers are not sensitive enough to detect early copper deficiency and new markers are needed. CCS is a candidate to become a copper biomarker. OBJECTIVE: Measuring CCS mRNA relative expression in malnourished children and compare results (a) with those of the same children after nutritional recovery and (b) with well-nourished children. METHOD: On admission to the protocol and after 15 day nutritional treatment, severely (G1=18) and moderately (G2=10) malnourished children were compared with well-nourished healthy controls (G3=15), measuring anthropometric indicators, blood biochemistry, Cu, Fe and Zn serum concentrations, ceruloplasmin, C Reactive protein and mRNA abundance of CCS, SOD and MT2 in peripheral mononuclear cells. RESULT: In malnourished groups, mean serum copper concentration was below the cut-off on admission to hospital and increased after 15 days (t-test, p<0.01). On admission to protocol, CCS mRNA abundance in G1 and G2 was higher than in G3 (one way ANOVA, p<0.001). After 15 days, CCS expression decreased as expected (t-test, p<0.001). Initial SOD mRNA relative abundance was higher in study groups than controls and also between G1 and G2 (One way ANOVA, both p<0.01); after 15 days, G1 and G2 were not different (t-test, NS). MT2A abundance of transcripts did not follow a clear change pattern. CONCLUSION: CCS mRNA abundance responded as expected, being higher in malnourished children than in controls; nutritional recovery in these latter resulted in decreasing expression of the chaperone, supporting the hypothesis that CCS may be a copper biomarker.

Asunto(s)

Trastornos de la Nutrición del Niño/sangre , Trastornos de la Nutrición del Niño/enzimología , Cobre/deficiencia , Superóxido Dismutasa/metabolismo , Preescolar , Cobre/sangre , Femenino , Humanos , Lactante , Masculino , Superóxido Dismutasa/análisis

RESUMEN

Copper is an essential micronutrient that functions as an enzymatic cofactor in a wide range of cellular processes. Although adequate Cu levels are essential for normal metabolism, excess Cu can be toxic to cells. Cellular responses to copper deficiency and overload involve changes in the expression of genes directly and indirectly involved in copper metabolism. However little is known on the effect of physiological copper concentration on gene expression changes. In the current study we aimed to establish whether the expression of genes encoding enzymes related to cholesterol (hmgcs1, hmgcr, fdft) and fatty acid biosynthesis and LDL receptor can be induced by an iso-physiological copper concentration. The iso-physiological copper concentration was determined as the bioavailable plasmatic copper in a healthy adult population. In doing so, two blood cell lines (Jurkat and THP-1) were exposed for 6 or 24 h to iso- or supraphysiological copper concentrations. Our results indicated that in cells exposed to an iso-physiological copper concentration the early induction of genes involved in lipid metabolism was not mediated by copper itself but by the modification of the cellular redox status. Thus our results contributed to understand the involvement of copper in the regulation of cholesterol metabolism under physiological conditions.

Asunto(s)

Colesterol/biosíntesis , Cobre/farmacología , Expresión Génica/efectos de los fármacos , Histidina/análogos & derivados , Compuestos Organometálicos/farmacología , ARN Mensajero/genética , Colesterol/genética , Farnesil Difosfato Farnesil Transferasa/genética , Farnesil Difosfato Farnesil Transferasa/metabolismo , Acido Graso Sintasa Tipo I/genética , Acido Graso Sintasa Tipo I/metabolismo , Histidina/farmacología , Humanos , Hidroximetilglutaril-CoA Reductasas/genética , Hidroximetilglutaril-CoA Reductasas/metabolismo , Hidroximetilglutaril-CoA Sintasa/genética , Hidroximetilglutaril-CoA Sintasa/metabolismo , Células Jurkat , Metabolismo de los Lípidos/efectos de los fármacos , Oxidación-Reducción , ARN Mensajero/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Receptores de LDL/genética , Receptores de LDL/metabolismo

RESUMEN

UNLABELLED: Copper (Cu) deficiency has been reported to influence lipid metabolism, but the effects in humans are controversial. To evaluate the effects of 8 mg Cu/day supplementation (as copper sulfate) for 6 months on the lipid profile and hepatic function of apparently healthy men. The design was randomized double-blind placebo-controlled clinical trial. SUBJECTS AND METHODS: 60 apparently healthy males aged 18-51 years were randomly assigned to Cu supplementation (n = 30) or placebo (n = 30). There was a nonsignificant reduction of 17 % in total cholesterol in both groups after supplementation. A 23 % nonsignificant reduction was observed in LDL cholesterol levels in the supplemented group. There was a nonsignificant increase of HDL cholesterol of 47 and 66 % in the control and supplemented groups, respectively. Triglyceride levels over 150 mg/dl were found in 17 subjects supplemented and 13 controls at baseline and decreased after supplementation to seven and eight subjects, respectively. There were no effects on serum Cu concentration or ceruloplasmin (protein) and hepatic transaminases. Supplementation of 8 mg Cu for 6 months had no effect on lipid profile of apparently healthy Chilean men with adequate Cu status.

Asunto(s)

LDL-Colesterol/sangre , Cobre/administración & dosificación , Suplementos Dietéticos , Hígado/metabolismo , Adolescente , Adulto , Chile , Método Doble Ciego , Humanos , Masculino , Persona de Mediana Edad

RESUMEN

It has been suggested that calcium inhibits the absorption of dietary iron by directly affecting enterocytes. However, it is not clear if this effect is due to a decreased uptake of iron or its efflux from enterocytes. We studied the effect of calcium on the uptake, efflux, and net absorption of non-heme iron using the intestinal-like epithelial cell line Caco-2 as an in vitro model. Caco-2 cells were incubated for 60 min in a buffer supplemented with non-heme iron (as sulfate) and calcium to achieve calcium to iron molar ratios ranging from 50:1 to 1,000:1. The uptake, efflux, and net absorption of non-heme iron were calculated by following a radioisotope tracer of (55)Fe that had been added to the buffer. Administration of calcium and iron at molar ratios between 500 and 1,000:1 increased the uptake of non-heme iron and decreased efflux. Calcium did not have an effect on the net absorption of non-heme iron. At typical supplementary doses for calcium and non-heme iron, calcium may not have an effect on the absorption of non-heme iron. The effect of higher calcium to iron molar ratios on the efflux of non-heme iron may be large enough to explain results from human studies.

Asunto(s)

Calcio/farmacología , Mucosa Intestinal/metabolismo , Hierro/metabolismo , Transporte Biológico , Células CACO-2 , Calcio/administración & dosificación , Humanos

RESUMEN

Amyloid precursor protein (APP) is a transmembrane glycoprotein widely expressed in mammalian tissues and plays a central role in Alzheimer's disease. However, its physiological function remains elusive. Cu(2+) binding and reduction activities have been described in the extracellular APP135-156 region, which might be relevant for cellular copper uptake and homeostasis. Here, we assessed Cu(2+) reduction and (64)Cu uptake in two human HEK293 cell lines overexpressing APP. Our results indicate that Cu(2+) reduction increased and cells accumulated larger levels of copper, maintaining cell viability at supra-physiological levels of Cu(2+) ions. Moreover, wild-type cells exposed to both Cu(2+) ions and APP135-155 synthetic peptides increased copper reduction and uptake. Complementation of function studies in human APP751 transformed Fre1 defective Saccharomyces cerevisiae cells rescued low Cu(2+) reductase activity and increased (64)Cu uptake. We conclude that Cu(2+) reduction activity of APP facilitates copper uptake and may represent an early step in cellular copper homeostasis.

Asunto(s)

Precursor de Proteína beta-Amiloide/biosíntesis , Cobre/metabolismo , Cobre/toxicidad , Línea Celular , Cobre/análisis , Homeostasis/efectos de los fármacos , Humanos , Oxidación-Reducción/efectos de los fármacos , Fragmentos de Péptidos/farmacología

RESUMEN

Copper is an essential micronutrient for all biological systems. Multiple proteins require one or more atoms of copper for proper structure and function, but excess of copper is toxic. To prevent the consequences of copper deficiency and overload, living organisms have evolved molecular mechanisms that regulate its uptake, intracellular traffic, storage, and efflux. Underlying some of the cellular responses to variations in copper levels are changes in the expression of genes encoding molecular components of copper metabolism. In recent years, genome-scale expression analysis in several eukaryotic models has allowed the identification of copper-responsive genes involved in copper homeostasis. Characterization of the transcriptional changes in response to varying copper levels include both genes directly involved in copper homeostasis and genes involved in different cellular process that, even though they are not directly connected to copper metabolism, change their expression during the cellular adaptation to copper availability. Evaluation of these gene expression patterns could aid in the identification of biologically relevant markers to monitor copper status in humans.

Asunto(s)

Cobre/metabolismo , Regulación de la Expresión Génica , Proteínas de Transporte de Catión/genética , Perfilación de la Expresión Génica , Humanos , Análisis de Secuencia por Matrices de Oligonucleótidos , ARN Mensajero/genética , Transcripción Genética

RESUMEN

The limits of copper homeostatic regulation in humans are not known, making it difficult to define the milder effects of early copper excess. Furthermore, a robust assay to facilitate the detection of early stages of copper excess is needed. To address these issues, we assessed changes in relative mRNA abundance of methallothionein 2A (MT2A), prion (PrP), amyloid precursor-like protein 2 (APLP2), Cu/Zn superoxide dismutase (SOD1) and its copper chaperone (CCS) in peripheral mononuclear cells (PMNCs) from healthy adults representing the 5% highest and lowest extremes in the distribution curve of serum ceruloplasmin (Cp) concentrations of 800 individuals. The intracellular Cu content was also determined. PMNCs were isolated from individuals before and after exposure to a single daily dose of 10 mg Cu (as CuSO(4)) for 2 months. Results showed that although there were fluctuations in serum Cp values of the samples assessed before copper exposure, no significant differences were observed in cell copper content or in the relative abundance of MT2A, PrP and APLP2 transcripts in PMNCs. Also, these values were not modified after copper supplementation. However, CCS and SOD1 mRNA levels were reduced in PMNCs after copper supplementation in the individuals with the high Cp values, suggesting that they should be further explored as biomarkers of moderate copper overload in humans.

Asunto(s)

Ceruloplasmina/análisis , Cobre/administración & dosificación , Chaperonas Moleculares/metabolismo , Superóxido Dismutasa/metabolismo , Precursor de Proteína beta-Amiloide/genética , Precursor de Proteína beta-Amiloide/metabolismo , Cobre/metabolismo , Humanos , Leucocitos Mononucleares/metabolismo , Metalotioneína/genética , Metalotioneína/metabolismo , Chaperonas Moleculares/genética , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Priones/genética , Priones/metabolismo , ARN Mensajero/metabolismo , Superóxido Dismutasa/genética , Superóxido Dismutasa-1

RESUMEN

The amyloid precursor protein (APP) contains a Cu binding domain (CuBD) localized between amino acids 135 and 156 (APP135-156), which can reduce Cu2+ to Cu1+ in vitro. The physiological function of this APP domain has not yet being established; nevertheless several studies support the notion that the CuBD of APP is involved in Cu homeostasis. We used APP synthetic peptides to evaluate their protective properties against Cu2+ neurotoxicity in a bilateral intra-hippocampal injection model. We found that human APP135-156 protects against Cu2+-induced neurotoxic effects, such as, impairment of spatial memory, neuronal cell loss, and astrogliosis. APP135-156 lacking two histidine residues showed protection against Cu2+; however, APP135-156 mutated in cysteine 144, a key residue in the reduction of Cu2+ to Cu1+, did not protect against Cu2+ neurotoxicity. In accordance with recent reports, the CuBD of the Caenorhabditis elegans, APL-1, protected against Cu2+ neurotoxicity in vivo. We also found that Cu2+ neurotoxicity is associated with an increase in nitrotyrosine immunofluorescence as well as with a decrease in Cu2+ uptake. The CuBD of APP therefore may play a role in the detoxification of brain Cu.

Asunto(s)

Precursor de Proteína beta-Amiloide/química , Precursor de Proteína beta-Amiloide/fisiología , Cobre/metabolismo , Cobre/toxicidad , Tirosina/análogos & derivados , Precursor de Proteína beta-Amiloide/uso terapéutico , Animales , Sitios de Unión , Proteínas de Caenorhabditis elegans/química , Cobre/antagonistas & inhibidores , Cisteína/química , Gliosis/inducido químicamente , Gliosis/prevención & control , Humanos , Transporte Iónico , Proteínas de la Membrana/química , Memoria/efectos de los fármacos , Neuronas/citología , Síndromes de Neurotoxicidad/prevención & control , Péptidos/química , Péptidos/uso terapéutico , Estructura Terciaria de Proteína , Proteínas/química , Ratas , Tirosina/análisis

RESUMEN

MTs (metallothioneins) increase the resistance of cells to exposure to high Cu (copper) levels. Characterization of the MT-Cu complex suggests that MT has an important role in the cellular storage and/or delivery of Cu ions to cuproenzymes. In this work we investigate how these properties contribute to Cu homoeostasis by evaluating the uptake, accumulation and efflux of Cu in wild-type and MT I/II null rat fibroblast cell lines. We also assessed changes in the expression of Cu metabolism-related genes in response to Cu exposure. At sub-physiological Cu levels (0.4 microM), the metal content was not dependent on MT; however, when extracellular Cu was increased to physiological levels (10 microM), MTs were required for the cell's ability to accumulate the metal. The subcellular localization of the accumulated metal in the cytoplasm was MT-dependent. Following supra-physiological Cu exposure (>50 microM), MT null cells had a decreased capacity for Cu storage and an elevated sensitivity to a minor increment in intracellular metal levels, suggesting that intracellular Cu toxicity is due not to the metal content but to the interactions of the metal with cellular components. Moreover, MT null cells failed to show increased levels of mRNAs encoding MT I, SOD1 (superoxide dismutase 1) and Ccs1 (Cu chaperone for SOD) in response to Cu exposure. These results support a role for MT in the storage of Cu in a safe compartment and in sequestering an intracellular excess of Cu in response to supra-physiological Cu exposure. Gene expression analysis suggests the necessity of having MT as part of the signalling pathway that induces gene expression in response to Cu.

Asunto(s)

Cobre/metabolismo , Metalotioneína/fisiología , Animales , Transporte Biológico , Línea Celular , Supervivencia Celular , Cobre/análisis , Cobre/toxicidad , Fibroblastos/química , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Regulación de la Expresión Génica , Metalotioneína/biosíntesis , Metalotioneína/genética , Ratones , Chaperonas Moleculares/biosíntesis , Chaperonas Moleculares/genética , Mutación , Biosíntesis de Proteínas , ARN Mensajero/metabolismo , Superóxido Dismutasa/biosíntesis , Superóxido Dismutasa/genética , Superóxido Dismutasa-1

RESUMEN

With this work, we have determined the cellular content of Cu, Fe and Zn in different cell lines, by using total reflection X-ray fluorescence spectrometry (TXRF). In addition, we examined whether cellular exposure to 100 micromoles l(-1) of Cu-His modifies the intracellular content and distribution of these trace metals. Our results indicate that all the cell lines displayed the same pattern of relative intracellular abundance of trace metals (Cu

Asunto(s)

Cobre/farmacología , Oligoelementos/metabolismo , Células 3T3 , Animales , Transporte Biológico/efectos de los fármacos , Línea Celular , Células HeLa , Humanos , Hierro/metabolismo , Cinética , Ratones , Células Tumorales Cultivadas , Zinc/metabolismo