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1.
Biochemistry ; 57(38): 5533-5543, 2018 09 25.
Artículo en Inglés | MEDLINE | ID: mdl-30183257

RESUMEN

Mobilization of iron from bacterioferritin (BfrB) requires specific interactions with a [2Fe-2S] ferredoxin (Bfd). Blocking the BfrB:Bfd interaction results in irreversible iron accumulation in BfrB and iron deficiency in the cytosol [Eshelman, K., et al. (2017) Metallomics 9, 646-659]. The only known Bfd structure, which was obtained in complex with BfrB (Protein Data Bank entry 4E6K ), indicated a new fold and suggested that the stability of Bfd is aided by an anion binding site consisting of R26, R29, and K46. We investigated the Bfd fold using site-directed mutagenesis, X-ray crystallography, and biochemistry in solution. The X-ray structure, which is nearly identical to that of Bfd in the BfrB:Bfd complex, shows that the [2Fe-2S] cluster preorganizes residues at the BfrB:Bfd interface into a structure complementary to the Bfd binding site on BfrB. Studies in solution showed rapid loss of the [2Fe-2S] cluster at a low ionic strength but higher stability with an increasing ionic strength, thus supporting a structural anion binding site. Structures of the R26E and R26E/K46Y mutants are nearly identical to that of Bfd, except for a new network of hydrogen bonds stabilizing the region encompassing the former anion binding site. The stability of the R26E and R26E/K46Y mutants, which is weakly and completely independent of solution ionic strength, respectively, corroborates that Bfd requires an anion binding site. The mutations, which caused only small changes to the strength of the BfrB:Bfd interaction and mobilization of iron from BfrB, indicate that the anion binding site in Bfd serves primarily a structural role.


Asunto(s)
Aniones/metabolismo , Proteínas Bacterianas/metabolismo , Grupo Citocromo b/metabolismo , Ferritinas/metabolismo , Homeostasis , Proteínas Hierro-Azufre/metabolismo , Hierro/metabolismo , Pseudomonas aeruginosa/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Sitios de Unión , Catálisis , Cristalografía por Rayos X , Grupo Citocromo b/química , Grupo Citocromo b/genética , Ferredoxinas/metabolismo , Ferritinas/química , Ferritinas/genética , Proteínas Hierro-Azufre/química , Proteínas Hierro-Azufre/genética , Modelos Moleculares , Mutagénesis Sitio-Dirigida , Mutación , Conformación Proteica , Dominios Proteicos
2.
Microb Pathog ; 117: 100-108, 2018 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-29432914

RESUMEN

Vibrio vulnificus, the causative agent of serious, often fatal, infections in humans, requires iron for its pathogenesis. As such, it obtains iron via both vulnibactin and heme-mediated iron-uptake systems. In this study, we identified the heme acquisition system in V. vulnificus M2799. The nucleotide sequences of the genes encoding heme receptors HupA and HvtA and the ATP-binding cassette (ABC) transport system proteins HupB, HupC, and HupD were determined, and then used in the construction of deletion mutants developed from a Δics strain, which could not synthesize vulnibactin. Growth experiments using these mutants indicated that HupA and HvtA are major and minor heme receptors, respectively. The expressions of two proteins were analyzed by the quantitative reverse-transcriptase polymerase chain reaction (qRT-PCR). Furthermore, complementation analyses confirmed that the HupBCD proteins are the only ABC transport system shared by both the HupA and HvtA receptors. This is the first genetic evidence that the HupBCD proteins are essential for heme acquisition by V. vulnificus. Further investigation showed that hupA, hvtA, and hupBCD are regulated by Fur. The qRT-PCR analysis of the heme receptor genes revealed that HupR, a LysR-family positive transcriptional activator, upregulates the expression of hupA, but not hvtA. In addition, ptrB was co-transcribed with hvtA, and PtrB had no influence on growth in low-iron CM9 medium supplemented with hemin, hemoglobin, or cytochrome C.


Asunto(s)
Proteínas de la Membrana Bacteriana Externa/metabolismo , Proteínas Bacterianas/metabolismo , Proteínas Portadoras/metabolismo , Hierro/metabolismo , Factores de Transcripción/metabolismo , Vibrio vulnificus/metabolismo , Transportadoras de Casetes de Unión a ATP/genética , Transportadoras de Casetes de Unión a ATP/metabolismo , Amidas/metabolismo , Proteínas de la Membrana Bacteriana Externa/genética , Proteínas Bacterianas/genética , Secuencia de Bases , Proteínas Portadoras/genética , Grupo Citocromo b/genética , Citocromos c/metabolismo , ADN Bacteriano , Perfilación de la Expresión Génica , Regulación Bacteriana de la Expresión Génica , Genes Bacterianos/genética , Hemina/metabolismo , Hemoglobinas/metabolismo , Humanos , Hidrogenasas/genética , Transferasas Intramoleculares/metabolismo , Metaloendopeptidasas/metabolismo , Oxazoles/metabolismo , Proteínas de Unión Periplasmáticas/genética , Proteínas de Unión Periplasmáticas/metabolismo , Proteínas Represoras/genética , Proteínas Represoras/metabolismo , Análisis de Secuencia , Eliminación de Secuencia , Factores de Transcripción/genética , Transcripción Genética , Vibrio vulnificus/genética , Vibrio vulnificus/crecimiento & desarrollo
3.
JCI Insight ; 2(2): e87094, 2017 01 26.
Artículo en Inglés | MEDLINE | ID: mdl-28138551

RESUMEN

A role for oxidative stress in the brain has been suggested in the pathogenesis of diet-induced obesity (DIO), although the underlying neural regions and mechanisms remain incompletely defined. We tested the hypothesis that NADPH oxidase-dependent oxidative stress in the paraventricular nucleus (PVN), a hypothalamic energy homeostasis center, contributes to the development of DIO. Cre/LoxP technology was coupled with selective PVN adenoviral microinjection to ablate p22phox , the obligatory subunit for NADPH oxidase activity, in mice harboring a conditional p22phox allele. Selective deletion of p22phox in the PVN protected mice from high-fat DIO independent of changes in food intake or locomotor activity. This was accompanied by ß3-adrenoceptor-dependent increases in energy expenditure, elevations in brown adipose tissue thermogenesis, and browning of white adipose tissue. These data reveal a potentially novel role for brain oxidative stress in the development of DIO by modulating ß3-adrenoceptor mechanisms and point to the PVN as an underlying neural site.


Asunto(s)
Grupo Citocromo b/genética , Dieta Alta en Grasa , Metabolismo Energético/genética , NADPH Oxidasas/genética , Obesidad/genética , Estrés Oxidativo , Núcleo Hipotalámico Paraventricular/metabolismo , Receptores Adrenérgicos beta 3/metabolismo , Termogénesis/genética , Tejido Adiposo Pardo/metabolismo , Tejido Adiposo Blanco/metabolismo , Animales , Encéfalo/metabolismo , Grupo Citocromo b/metabolismo , Ingestión de Alimentos , Hipotálamo/metabolismo , Locomoción , Ratones , NADPH Oxidasas/metabolismo , Obesidad/metabolismo
4.
Artículo en Inglés | MEDLINE | ID: mdl-25090397

RESUMEN

Pilos antler (Lu-Rong in Chinese) is a famous traditional medicine in China. Many adulterants have been discovered in Chinese markets in recent years. However, few DNA-based methods are effective for discrimination of this DNA-degraded animal medicine. Here, novel and deft amplification refractory mutation sequencing system (ARMSS), integrating the advantages of the amplification refractory mutation system (ARMS) and the short DNA barcode, was first developed to discriminate Pilos antler from its adulterants. We aimed to provide a new sight and inspiration for deft detection. The results showed that developed ARMS achieved strong specificity and high sensitivity in rapid identification, while the short Cytb gene was of excellent identification power in terms of accurate identification, which suggested that ARMSS successfully integrated the advantages of the ARMS and short DNA barcode, and that it was useful for deft detection. Our study determined that the deft ARMSS could be the well candidate for discrimination of Pilos antler, as well as be a valuable tool for deft identification of Chinese medicine.


Asunto(s)
Cuernos de Venado/química , Grupo Citocromo b/genética , Código de Barras del ADN Taxonómico/métodos , Mutación , Técnicas de Amplificación de Ácido Nucleico/métodos , Animales , Artiodáctilos/genética , Secuencia de Bases , Medicina Tradicional China , Datos de Secuencia Molecular
5.
Nutrients ; 7(11): 9683-96, 2015 Nov 23.
Artículo en Inglés | MEDLINE | ID: mdl-26610564

RESUMEN

Iron deficiency affects thousands of people worldwide. Biofortification of staple food crops aims to support the reduction of this deficiency. This study evaluates the effect of combinations of common beans and rice, targets for biofortification, with high carotenoid content crops on the iron bioavailability, protein gene expression, and antioxidant effect. Iron bioavailability was measured by the depletion/repletion method. Seven groups were tested (n = 7): Pontal bean (PB); rice + Pontal bean (R + BP); Pontal bean + sweet potato (PB + SP); Pontal bean + pumpkin (PB + P); Pontal bean + rice + sweet potato (PB + R + P); Pontal bean + rice + sweet potato (PB + R + SP); positive control (Ferrous Sulfate). The evaluations included: hemoglobin gain, hemoglobin regeneration efficiency (HRE), gene expression of divalente metal transporter 1 (DMT-1), duodenal citocromo B (DcytB), ferroportin, hephaestin, transferrin and ferritin and total plasma antioxidant capacity (TAC). The test groups, except the PB, showed higher HRE (p < 0.05) than the control. Gene expression of DMT-1, DcytB and ferroportin increased (p < 0.05) in the groups fed with high content carotenoid crops (sweet potato or pumpkin). The PB group presented lower (p < 0.05) TAC than the other groups. The combination of rice and common beans, and those with high carotenoid content crops increased protein gene expression, increasing the iron bioavailability and antioxidant capacity.


Asunto(s)
Carotenoides/análisis , Fabaceae/química , Alimentos Fortificados , Hierro/farmacocinética , Oryza/química , Animales , Disponibilidad Biológica , Carotenoides/administración & dosificación , Proteínas de Transporte de Catión/genética , Proteínas de Transporte de Catión/metabolismo , Grupo Citocromo b/genética , Grupo Citocromo b/metabolismo , Ferritinas/genética , Ferritinas/metabolismo , Regulación de la Expresión Génica , Hemoglobinas/metabolismo , Historia Antigua , Hierro/sangre , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Fenoles/análisis , Ácido Fítico/análisis , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ratas , Ratas Wistar , Transferrina/genética , Transferrina/metabolismo
6.
PLoS One ; 10(9): e0138479, 2015.
Artículo en Inglés | MEDLINE | ID: mdl-26381264

RESUMEN

Iron (Fe) deficiency is a highly prevalent micronutrient insufficiency predominantly caused by a lack of bioavailable Fe from the diet. The consumption of beans as a major food crop in some populations suffering from Fe deficiency is relatively high. Therefore, our objective was to determine whether a biofortified variety of cream seeded carioca bean (Phaseolus vulgaris L.) could provide more bioavailable-Fe than a standard variety using in-vivo (broiler chicken, Gallus gallus) and in-vitro (Caco-2 cell) models. Studies were conducted under conditions designed to mimic the actual human feeding protocol. Two carioca-beans, a standard (G4825; 58 µg Fe/g) and a biofortified (SMC; 106 µg Fe/g), were utilized. Diets were formulated to meet the nutrient requirements of Gallus gallus except for Fe (33.7 and 48.7 µg Fe/g, standard and biofortified diets, respectively). In-vitro observations indicated that more bioavailable-Fe was present in the biofortified beans and diet (P<0.05). In-vivo, improvements in Fe-status were observed in the biofortified bean treatment, as indicated by the increased total-body-Hemoglobin-Fe, and hepatic Fe-concentration (P<0.05). Also, DMT-1 mRNA-expression was increased in the standard bean treatment (P<0.05), indicating an upregulation of absorption to compensate for less bioavailable-Fe. These results demonstrate that the biofortified beans provided more bioavailable Fe; however, the in vitro results revealed that ferritin formation values were relatively low. Such observations are indicative of the presence of high levels of polyphenols and phytate that inhibit Fe absorption. Indeed, we identified higher levels of phytate and quercetin 3-glucoside in the Fe biofortified bean variety. Our results indicate that the biofortified bean line was able to moderately improve Fe-status, and that concurrent increase in the concentration of phytate and polyphenols in beans may limit the benefit of increased Fe-concentration. Therefore, specific targeting of such compounds during the breeding process may yield improved dietary Fe-bioavailability. Our findings are in agreement with the human efficacy trial that demonstrated that the biofortified carioca beans improved the Fe-status of Rwandan women. We suggest the utilization of these in vitro and in vivo screening tools to guide studies aimed to develop and evaluate biofortified staple food crops. This approach has the potential to more effectively utilize research funds and provides a means to monitor the nutritional quality of the Fe-biofortified crops once released to farmers.


Asunto(s)
Alimentos Fortificados , Deficiencias de Hierro , Phaseolus/metabolismo , Animales , Disponibilidad Biológica , Células CACO-2 , Proteínas de Transporte de Catión/genética , Proteínas de Transporte de Catión/metabolismo , Pollos , Grupo Citocromo b/genética , Grupo Citocromo b/metabolismo , Femenino , Ferritinas/metabolismo , Humanos , Necesidades Nutricionales , Oxidorreductasas/genética , Oxidorreductasas/metabolismo , Phaseolus/genética , Rwanda
7.
Biochemistry ; 54(40): 6162-75, 2015 Oct 13.
Artículo en Inglés | MEDLINE | ID: mdl-26368531

RESUMEN

Mobilization of iron stored in the interior cavity of BfrB requires electron transfer from the [2Fe−2S] cluster in Bfd to the core iron in BfrB. A crystal structure of the Pseudomonas aeruginosa BfrB:Bfd complex revealed that BfrB can bind up to 12 Bfd molecules at 12 structurally identical binding sites, placing the [2Fe−2S] cluster of each Bfd immediately above a heme group in BfrB [Yao, H., et al. (2012) J. Am. Chem. Soc., 134, 13470−13481]. We report here study aimed at characterizing the strength of the P. aeruginosa BfrB:Bfd association using surface plasmon resonance and isothermal titration calorimetry as well as determining the binding energy hot spots at the protein−protein interaction interface. The results show that the 12 Bfd-binding sites on BfrB are equivalent and independent and that the protein−protein association at each of these sites is driven entropically and is characterized by a dissociation constant (Kd) of approximately 3 µM. Determination of the binding energy hot spots was carried out by replacing certain residues that comprise the protein−protein interface with alanine and by evaluating the effect of the mutation on Kd and on the efficiency of core iron mobilization from BfrB. The results identified hot spot residues in both proteins [LB 68, EA 81, and EA 85 in BfrB (superscript for residue number and subscript for chain) and Y2 and L5 in Bfd] that network at the interface to produce a highly complementary hot region for the interaction. The hot spot residues are conserved in the amino acid sequences of Bfr and Bfd proteins from a number of Gram-negative pathogens, indicating that the BfrB:Bfd interaction is of widespread significance in bacterial iron metabolism.


Asunto(s)
Proteínas Bacterianas/metabolismo , Grupo Citocromo b/metabolismo , Ferredoxinas/metabolismo , Ferritinas/metabolismo , Pseudomonas aeruginosa/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Cristalografía por Rayos X , Grupo Citocromo b/química , Grupo Citocromo b/genética , Ferredoxinas/química , Ferredoxinas/genética , Ferritinas/química , Ferritinas/genética , Humanos , Modelos Moleculares , Mutación , Conformación Proteica , Mapas de Interacción de Proteínas , Infecciones por Pseudomonas/microbiología , Pseudomonas aeruginosa/química , Pseudomonas aeruginosa/genética , Termodinámica
8.
Plant Physiol ; 169(2): 986-95, 2015 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-26282237

RESUMEN

Trans-plasma membrane electron transfer is achieved by b-type cytochromes of different families, and plays a fundamental role in diverse cellular processes involving two interacting redox couples that are physically separated by a phospholipid bilayer, such as iron uptake and redox signaling. Despite their importance, no direct recordings of trans-plasma membrane electron currents have been described in plants. In this work, we provide robust electrophysiological evidence of trans-plasma membrane electron flow mediated by a soybean (Glycine max) cytochrome b561 associated with a dopamine ß-monooxygenase redox domain (CYBDOM), which localizes to the plasma membrane in transgenic Arabidopsis (Arabidopsis thaliana) plants and CYBDOM complementary RNA-injected Xenopus laevis oocytes. In oocytes, two-electrode voltage clamp experiments showed that CYBDOM-mediated currents were activated by extracellular electron acceptors in a concentration- and type-specific manner. Current amplitudes were voltage dependent, strongly potentiated in oocytes preinjected with ascorbate (the canonical electron donor for cytochrome b561), and abolished by mutating a highly conserved His residue (H292L) predicted to coordinate the cytoplasmic heme b group. We believe that this unique approach opens new perspectives in plant transmembrane electron transport and beyond.


Asunto(s)
Membrana Celular/metabolismo , Grupo Citocromo b/metabolismo , Glycine max/metabolismo , Proteínas de Plantas/metabolismo , Animales , Arabidopsis/genética , Arabidopsis/metabolismo , Ácido Ascórbico/metabolismo , Grupo Citocromo b/genética , Dopamina beta-Hidroxilasa/genética , Dopamina beta-Hidroxilasa/metabolismo , Transporte de Electrón , Fenómenos Electrofisiológicos/fisiología , Ferricianuros/farmacología , Oocitos/efectos de los fármacos , Oocitos/metabolismo , Técnicas de Placa-Clamp , Proteínas de Plantas/genética , Plantas Modificadas Genéticamente , Estructura Terciaria de Proteína , Glycine max/genética , Xenopus laevis/metabolismo
9.
Br J Nutr ; 113(6): 901-8, 2015 Mar 28.
Artículo en Inglés | MEDLINE | ID: mdl-25745840

RESUMEN

Prebiotics may increase intestinal Fe absorption in anaemic growing rats. The present study evaluated the effects of high-performance (HP) inulin and oligofructose on factors that regulate Fe absorption in anaemic rats during the growth phase. Male Wistar rats aged 21 d of age were fed AIN-93G ration without Fe for 2 weeks to induce Fe-deficiency anaemia. The rats were fed on day 35 a control diet, or a diet with 10 % HP inulin, or a diet with 10 % oligofructose, without Fe supplementation. The animals were euthanised after 2 weeks, and segments of the duodenum, caecum, colon and liver were removed. The expression levels of proteins in the intestinal segments were assessed using Western blotting. The levels of serum, urine and liver hepcidin and the concentrations of IL-10, IL-6 and TNF-α in the caecum, colon and liver were measured using the ELISA test. HP inulin increased the expression of the divalent metal transporter 1 protein in the caecum by 162 % (P= 0·04), and the expression of duodenal cytochrome b reductase in the colon by 136 % (P= 0·02). Oligofructose decreased the expression of the protein ferroportin in the duodenum (P= 0·02), the concentrations of IL-10 (P= 0·044), IL-6 (P= 0·036) and TNF-α (P= 0·004) in the caecum, as well as the level of urinary hepcidin (P< 0·001). These results indicate that prebiotics may interfere with the expression of various intestinal proteins and systemic factors involved in the regulation of intestinal Fe absorption in anaemic rats during the growth phase.


Asunto(s)
Anemia Ferropénica/dietoterapia , Proteínas de Transporte de Catión/metabolismo , Grupo Citocromo b/metabolismo , Mucosa Intestinal/metabolismo , Prebióticos , Regulación hacia Arriba , Anemia Ferropénica/inmunología , Anemia Ferropénica/metabolismo , Anemia Ferropénica/patología , Animales , Proteínas de Transporte de Catión/agonistas , Ciego/inmunología , Ciego/metabolismo , Ciego/patología , Colon/enzimología , Colon/inmunología , Colon/metabolismo , Grupo Citocromo b/química , Grupo Citocromo b/genética , Duodeno/inmunología , Duodeno/metabolismo , Duodeno/patología , Hepcidinas/sangre , Hepcidinas/metabolismo , Hepcidinas/orina , Mediadores de Inflamación/metabolismo , Mucosa Intestinal/inmunología , Mucosa Intestinal/patología , Inulina/efectos adversos , Inulina/uso terapéutico , Hígado/inmunología , Hígado/metabolismo , Hígado/patología , Masculino , Oligosacáridos/efectos adversos , Oligosacáridos/uso terapéutico , Tamaño de los Órganos , Prebióticos/efectos adversos , Ratas Wistar , Aumento de Peso
10.
Nutr J ; 12: 3, 2013 Jan 04.
Artículo en Inglés | MEDLINE | ID: mdl-23286295

RESUMEN

BACKGROUND: Iron (Fe) deficiency is the most common micronutrient deficiency worldwide. Iron biofortification is a preventative strategy that alleviates Fe deficiency by improving the amount of absorbable Fe in crops. In the present study, we used an in vitro digestion/Caco 2 cell culture model as the guiding tool for breeding and development of two maize (Zea mays L.) lines with contrasting Fe bioavailability (ie. Low and High). Our objective was to confirm and validate the in vitro results and approach. Also, to compare the capacities of our two maize hybrid varieties to deliver Fe for hemoglobin (Hb) synthesis and to improve the Fe status of Fe deficient broiler chickens. METHODS: We compared the Fe-bioavailability between these two maize varieties with the presence or absence of added Fe in the maize based-diets. Diets were made with 75% (w/w) maize of either low or high Fe-bioavailability maize, with or without Fe (ferric citrate). Chicks (Gallus gallus) were fed the diets for 6 wk. Hb, liver ferritin and Fe related transporter/enzyme gene-expression were measured. Hemoglobin maintenance efficiency (HME) and total body Hb Fe values were used to estimate Fe bioavailability from the diets. RESULTS: DMT-1, DcytB and ferroportin expressions were higher (P<0.05) in the "Low Fe" group than in the "High Fe" group (no added Fe), indicating lower Fe status and adaptation to less Fe-bioavailability. At times, Hb concentrations (d 21,28,35), HME (d 21), Hb-Fe (as from d 14) and liver ferritin were higher in the "High Fe" than in the "Low Fe" groups (P<0.05), indicating greater Fe absorption from the diet and improved Fe status. CONCLUSIONS: We conclude that the High Fe-bioavailability maize contains more bioavailable Fe than the Low Fe-bioavailability maize, presumably due to a more favorable matrix for absorption. Maize shows promise for Fe biofortification; therefore, human trials should be conducted to determine the efficacy of consuming the high bioavailable Fe maize to reduce Fe deficiency.


Asunto(s)
Barajamiento de ADN , Alimentos Fortificados , Hierro de la Dieta/farmacocinética , Zea mays/química , Anemia Ferropénica , Animales , Disponibilidad Biológica , Células CACO-2 , Proteínas de Transporte de Catión/genética , Proteínas de Transporte de Catión/metabolismo , Pollos , Grupo Citocromo b/genética , Grupo Citocromo b/metabolismo , Dieta , Compuestos Férricos/farmacocinética , Expresión Génica , Hemoglobinas/análisis , Humanos , Hígado/efectos de los fármacos , Hígado/metabolismo , Oxidorreductasas/genética , Oxidorreductasas/metabolismo , Ácido Fítico/administración & dosificación , Ácido Fítico/análisis , ARN Mensajero/genética , ARN Mensajero/metabolismo , Zea mays/genética
11.
Am J Physiol Endocrinol Metab ; 302(1): E77-86, 2012 Jan 01.
Artículo en Inglés | MEDLINE | ID: mdl-21917632

RESUMEN

Iron is an essential trace metal for most organisms. However, excess iron causes oxidative stress through production of highly toxic hydroxyl radicals via the Fenton/Haber-Weiss reaction. Iron storage in the body is reported to be associated with fat accumulation and type 2 diabetes mellitus. We investigated the role of iron in adiposity by using KKAy mice and obese and diabetic model mice. Eight-week-old KKAy mice were divided into two groups and treated with deferoxamine (DFO), an iron chelator agent, or a vehicle for 2 wk. DFO treatment diminished fat iron concentration and serum ferritin levels in KKAy mice. Fat weight and adipocyte size were reduced significantly in DFO-treated mice compared with vehicle-treated mice. Macrophage infiltration into fat was also decreased in DFO-treated mice compared with vehicle-treated mice. Superoxide production and NADPH oxidase activity in fat, as well as urinary 8-hydroxy-2'-deoxyguanosine excretion, were decreased in KKAy mice after DFO treatment while p22(phox) expression in adipose tissue was diminished in such mice. Ferritin expression in the fat of DFO-treated KKAy mice was decreased. In addition, F4/80-positive cells also presented through both p22(phox) and ferritin expression. The mRNA expression levels of inflammatory cytokines were also reduced in fat tissue of DFO-treated mice. These findings suggest that reduction of iron levels ameliorates adipocyte hypertrophy via suppression of oxidative stress, inflammatory cytokines, and macrophage infiltration, thereby breaking a vicious cycle in obesity.


Asunto(s)
Adiposidad/efectos de los fármacos , Terapia por Quelación , Deferoxamina/uso terapéutico , Diabetes Mellitus Tipo 2/tratamiento farmacológico , Quelantes del Hierro/uso terapéutico , Obesidad/tratamiento farmacológico , Estrés Oxidativo/efectos de los fármacos , Tejido Adiposo Blanco/química , Tejido Adiposo Blanco/efectos de los fármacos , Tejido Adiposo Blanco/metabolismo , Tejido Adiposo Blanco/patología , Animales , Tamaño de la Célula/efectos de los fármacos , Grupo Citocromo b/genética , Grupo Citocromo b/metabolismo , Citocinas/genética , Citocinas/metabolismo , Diabetes Mellitus Tipo 2/complicaciones , Diabetes Mellitus Tipo 2/inmunología , Diabetes Mellitus Tipo 2/metabolismo , Ferritinas/sangre , Regulación de la Expresión Génica/efectos de los fármacos , Hierro/análisis , Macrófagos/efectos de los fármacos , Macrófagos/inmunología , Masculino , Ratones , Ratones Obesos , NADPH Oxidasas/genética , NADPH Oxidasas/metabolismo , Obesidad/complicaciones , Obesidad/inmunología , Obesidad/metabolismo , Subunidades de Proteína/genética , Subunidades de Proteína/metabolismo , ARN Mensajero/metabolismo
12.
Free Radic Res ; 44(7): 821-9, 2010 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-20528577

RESUMEN

Oxidative stress contributes to lipid peroxidation and decreases nitric oxide (NO) bioavailability in atherosclerosis. While long-chain (n-3) polyunsaturated fatty acids (PUFA) are easily oxidized in vitro, they improve endothelial function. Hence, this study postulates that long-chain (n-3) PUFA decrease atherogenic oxidative stress in vivo. To test this, apoE(-/-) mice were fed a corn oil- or a fish oil (FO)-rich diet for 8, 14 or 20 weeks and parameters related to NO and superoxide (O(2)(.-)) plus markers of lipid peroxidation and protein oxidative damage in the aortic root were evaluated. The FO-rich diet increased NO production and endothelial NO synthase (NOS) expression and lowered inducible NOS, p22(phox) expression and O(2)(.-)production after 14 and 20 weeks of diet. Protein lipoxidative damage (including 4-hydroxynonenal) was decreased after a long-term FO-diet. This supports the hypothesis that a FO-rich diet could counteract atherogenic oxidative stress, showing beneficial effects of long-chain (n-3) PUFA.


Asunto(s)
Apolipoproteínas E/deficiencia , Endotelio Vascular/efectos de los fármacos , Aceites de Pescado/uso terapéutico , Hiperlipoproteinemia Tipo II/dietoterapia , Estrés Oxidativo/efectos de los fármacos , Aldehídos/análisis , Animales , Aorta/química , Aorta/efectos de los fármacos , Aorta/enzimología , Apolipoproteínas E/genética , Aterosclerosis/genética , Aterosclerosis/prevención & control , Aceite de Maíz/administración & dosificación , Aceite de Maíz/farmacología , Grupo Citocromo b/biosíntesis , Grupo Citocromo b/genética , Aceites de Pescado/administración & dosificación , Aceites de Pescado/farmacología , Hiperlipoproteinemia Tipo II/genética , Hiperlipoproteinemia Tipo II/metabolismo , Peroxidación de Lípido/efectos de los fármacos , Masculino , Ratones , Ratones Noqueados , NADPH Oxidasas/biosíntesis , NADPH Oxidasas/genética , Óxido Nítrico/metabolismo , Óxido Nítrico Sintasa de Tipo II/biosíntesis , Óxido Nítrico Sintasa de Tipo II/genética , Óxido Nítrico Sintasa de Tipo III/biosíntesis , Óxido Nítrico Sintasa de Tipo III/genética
13.
Poult Sci ; 89(3): 514-21, 2010 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-20181868

RESUMEN

Iron fortification of foods and biofortification of staple food crops are strategies that can help to alleviate Fe deficiency. The broiler chicken may be a useful model for initial in vivo screening of Fe bioavailability in foods due to its growth rate, anatomy, size, and low cost. In this study, we assess the broiler as a model for hemoglobin (Hb) maintenance studies and present a unique duodenal loop technique for direct measurement of intestinal Fe absorption. One-week-old chicks were allocated into Fe-deficient versus Fe-adequate treatment groups. For 6 wk, blood Hb, feed consumption, and BW were measured. At wk 7, birds were anesthetized and their duodenal loops were exposed. The loop was isolated and a nonocclusive catheter was inserted into the duodenal vein for blood sampling. A stable isotope solution containing (58)Fe (1 mg of Fe in 10 mM ascorbic acid) was injected into the loop. Blood samples were collected every 5 min and for 120 min postinjection and analyzed by inductively coupled argon-plasma mass spectrometry for (58)Fe concentrations. In the low-Fe group, Hb concentrations, total body Hb Fe, and BW were lower and Hb maintenance efficiency (indicator for dietary Fe availability) was higher than in the high-Fe group (P < 0.05). Iron absorption was higher in the Fe-deficient birds (P < 0.05). In addition, expression of proteins involved in Fe uptake and transfer [i.e., divalent metal transporter 1 (Fe uptake transporter), ferroportin (involved in Fe transport across the enterocyte), and duodenal cytochrome B reductase (reduces Fe at brush border membrane)] were elevated in the low-Fe group. These results indicate that this model exhibits the appropriate responses to Fe deficiency and has potential to serve as a model for Fe bioavailability. Such a model should be most useful as an intermediate test of in vivo Fe bioavailability observations in preparation for subsequent human studies.


Asunto(s)
Disponibilidad Biológica , Pollos , Hierro/farmacocinética , Absorciometría de Fotón , Animales , Células CACO-2 , Proteínas de Transporte de Catión/genética , Proteínas de Transporte de Catión/metabolismo , Pollos/crecimiento & desarrollo , Grupo Citocromo b/genética , Grupo Citocromo b/metabolismo , ADN Complementario/genética , ADN Complementario/metabolismo , Análisis de los Alimentos , Regulación de la Expresión Génica/fisiología , Hemoglobinas , Humanos , Intestino Delgado/metabolismo , Hierro/administración & dosificación
14.
Bioorg Med Chem ; 16(9): 5331-44, 2008 May 01.
Artículo en Inglés | MEDLINE | ID: mdl-18343120

RESUMEN

The auxins, plant hormones, regulate many aspects of the growth and development of plants. Terfestatin A (TrfA), a novel auxin signaling inhibitor, was identified in a screen of Streptomyces sp. F40 extracts for inhibition of the expression of an auxin-inducible gene. However, the mode of action of TrfA has not been elucidated. To identify the active core structure, 25 derivatives of TrfA were synthesized and their inhibitory activities against auxin-inducible gene expression were evaluated. The structure-activity relationships revealed the essential active core structure of TrfA, 3-butoxy-4-methylbiphenyl-2,6-diol, which will lead to the design of biotin-tagged active TrfA.


Asunto(s)
Glucósidos/farmacología , Ácidos Indolacéticos/antagonistas & inhibidores , Transducción de Señal/efectos de los fármacos , Compuestos de Terfenilo/farmacología , Sitios de Unión , Grupo Citocromo b/efectos de los fármacos , Grupo Citocromo b/genética , Diseño de Fármacos , Evaluación Preclínica de Medicamentos , Complejo IV de Transporte de Electrones/efectos de los fármacos , Complejo IV de Transporte de Electrones/genética , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Glucósidos/síntesis química , Glucósidos/química , Glucuronidasa/efectos de los fármacos , Glucuronidasa/genética , Ácidos Indolacéticos/farmacología , Modelos Moleculares , Estructura Molecular , Estereoisomerismo , Streptomyces/química , Relación Estructura-Actividad , Compuestos de Terfenilo/síntesis química , Compuestos de Terfenilo/química
15.
Biochim Biophys Acta ; 1777(3): 260-8, 2008 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-18194661

RESUMEN

Duodenal cytochrome b (Dcytb or Cybrd1) is an iron-regulated protein, highly expressed in the duodenal brush border membrane. It has ferric reductase activity and is believed to play a physiological role in dietary iron absorption. Its sequence identifies it as a member of the cytochrome b(561) family. A His-tagged construct of human Dcytb was expressed in insect Sf9 cells and purified. Yields of protein were increased by supplementation of the cells with 5-aminolevulinic acid to stimulate heme biosynthesis. Quantitative analysis of the recombinant Dcytb indicated two heme groups per monomer. Site-directed mutagenesis of any of the four conserved histidine residues (His 50, 86, 120 and 159) to alanine resulted in much diminished levels of heme in the purified Dcytb, while mutation of the non-conserved histidine 33 had no effect on the heme content. This indicates that those conserved histidines are heme ligands, and that the protein cannot stably bind heme if any of them is absent. Recombinant Dcytb was reduced by ascorbate under anaerobic conditions, the extent of reduction being 67% of that produced by dithionite. It was readily reoxidized by ferricyanide. EPR spectroscopy showed signals from low-spin ferriheme, consistent with bis-histidine coordination. These comprised a signal at gmax=3.7 corresponding to a highly anisotropic species, and another at gmax=3.18; these species are similar to those observed in other cytochromes of the b561 family, and were reducible by ascorbate. In addition another signal was observed in some preparations at gmax=2.95, but this was unreactive with ascorbate. Redox titrations indicated an average midpoint potential for the hemes in Dcytb of +80 mV+/-30 mV; the data are consistent with either two hemes at the same potential, or differing in potential by up to 60 mV. These results indicate that Dcytb is similar to the ascorbate-reducible cytochrome b561 of the adrenal chromaffin granule, though with some differences in midpoint potentials of the hemes.


Asunto(s)
Ácido Ascórbico/metabolismo , Grupo Citocromo b/metabolismo , Duodeno/enzimología , Hierro/metabolismo , Oxidorreductasas/metabolismo , Ácido Aminolevulínico/metabolismo , Animales , Baculoviridae/genética , Línea Celular , Clonación Molecular , Grupo Citocromo b/química , Grupo Citocromo b/genética , Espectroscopía de Resonancia por Spin del Electrón , Ferricianuros/metabolismo , Vectores Genéticos , Hemo/metabolismo , Histidina/metabolismo , Humanos , Ligandos , Mutagénesis Sitio-Dirigida , Oxidación-Reducción , Oxidorreductasas/química , Oxidorreductasas/genética , Potenciometría , Proteínas Recombinantes/metabolismo , Transducción Genética
16.
Gut ; 51(5): 648-53, 2002 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-12377801

RESUMEN

BACKGROUND: Although much progress has been made recently in characterising the proteins involved in duodenal iron trafficking, regulation of intestinal iron transport remains poorly understood. It is not known whether the level of mRNA expression of these recently described molecules is genetically regulated. This is of particular interest however as genetic factors are likely to determine differences in iron status among mouse strains and probably also contribute to the phenotypic variability seen with disruption of the haemochromatosis gene. AIMS: To investigate this issue, we examined concomitant variations in duodenal cytochrome b (Dcytb), divalent metal transporter 1 (DMT1), ferroportin 1 (FPN1), hephaestin, stimulator of Fe transport (SFT), HFE, and transferrin receptor 1 (TfR1) transcripts in response to different dietary iron contents in the four mouse strains C57BL/6, DBA/2, CBA, and 129/Sv. SUBJECTS: Six mice of each strain were fed normal levels of dietary iron, six were subjected to the same diet supplemented with 2% carbonyl iron, and six were fed an iron deficient diet. METHODS: Quantification of mRNAs isolated from the duodenum was performed using real time reverse transcription-polymerase chain reaction. RESULTS: There was a significant increase in mRNA expression of Dcytb, DMT1, FPN1, and TfR1 when mice were fed an iron deficient diet, and a significant decrease in mRNA expression of these molecules when mice were fed an iron supplemented diet. Strain to strain differences were observed not only in serum transferrin saturations, with C57BL/6 mice having the lowest values, but also in hepatic iron stores and in duodenal mRNA expression of Dcytb, DMT1, FPN1, hephaestin, HFE, and TfR1. CONCLUSIONS: The results favour some degree of genetic control of mRNA levels of these molecules.


Asunto(s)
Proteínas Portadoras/genética , Duodeno/metabolismo , Mucosa Intestinal/metabolismo , Hierro de la Dieta/administración & dosificación , ARN Mensajero/análisis , Enzimas Ubiquitina-Conjugadoras , Animales , Proteínas de Transporte de Catión/genética , Grupo Citocromo b/genética , Proteína de la Hemocromatosis , Antígenos de Histocompatibilidad Clase I/genética , Deficiencias de Hierro , Proteínas de Unión a Hierro/genética , Hígado/metabolismo , Proteínas de la Membrana/genética , Ratones , Ratones Endogámicos , Receptores de Transferrina/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Especificidad de la Especie , Transferrina/análisis
17.
Evolution ; 56(5): 888-98, 2002 May.
Artículo en Inglés | MEDLINE | ID: mdl-12093025

RESUMEN

Orchids of the genus Chiloglottis are pollinated through the sexual deception of male wasps mainly from the genus Neozeleboria (Tiphiidae: Thynninae). The orchids mimic both the appearance and sex pheromones of wingless female thynnines but provide no reward to the deceived males. Despite the asymmetry of this interaction, strong pollinator specificity is typical. Such plant-pollinator interactions would seem to be relatively flexible in the plant's adaptive response to variation in the local pollinator resource. However, we present DNA sequence data on both orchids and wasps that demonstrate a pattern of pollinator conservatism operating at a range of taxonomic levels. Sequence data from the wasps indicate 15 of 16 Chiloglottis pollinators are closely related members of one clade of Thynninae. A pattern of congruence between orchid and wasp phylogenies is also demonstrated below the generic level, such that related orchids tend to use related thynnine wasps as specific pollinators. Comparative physiological data on the wasp responses to the floral scents of two Chiloglottis species and one outgroup, Arthrochilus, indicate similar attractive volatile chemicals are used by related orchid taxa. By extension, we infer a similarity of sex pheromone signals among related thynnines. Thus, the conservative pattern of pollinator change in sexually deceptive orchids may reflect phylogenetic patterns in the sex pheromones of their pollinators.


Asunto(s)
ADN de Plantas/genética , Orchidaceae/clasificación , Orchidaceae/fisiología , Secuencia de Bases , Grupo Citocromo b/genética , Cartilla de ADN , Flores/fisiología , Orchidaceae/genética , Filogenia , Polen/fisiología , ARN Ribosómico 5.8S/genética , Reproducción
18.
J Mol Biol ; 320(4): 727-39, 2002 Jul 19.
Artículo en Inglés | MEDLINE | ID: mdl-12095251

RESUMEN

We report evidence of extensive substitutional editing of mitochondrial mRNAs in the dinoflagellate species Pfiesteria piscicida, Prorocentrum minimum and Crypthecodinium cohnii, based on a comparison of genomic and corresponding cDNA sequences determined for two mitochondrial DNA-encoded genes, cox1 (cytochrome oxidase subunit 1) and cob (apocytochrome b). In the cox1 mRNA, we identify 72 substitutions at 40 sites in 39 codons, whereas in cob mRNA, we infer 86 editing events at 51 sites in 48 codons. Editing, which takes place in distinct clusters, changes approximately 2% of the total sequence, occurs predominantly at first and second positions of codons, and involves mostly (but not exclusively) A-->G (47%), U-->C (23%) and C-->U (17%) substitutions. In all but four of the 158 cases, editing changes the identity of the specified amino acid. At 21 (cox1) and 26 (cob) sites, the same nucleotide change is observed at the same position in at least two of the species investigated. At about one-third of the sites, editing results in an amino acid change that increases similarity between the dinoflagellate Cox1 and Cob sequences and their homologs in other organisms; presumably editing at these sites is of particular functional significance. Overall, about half of the editing events either maintain or increase similarity between the dinoflagellate protein sequences and their non-dinoflagellate homologs, while a further one-third of the alterations are "dinoflagellate-specific" (i.e. they involve a change to an amino acid residue selectively conserved in at least two of the dinoflagellate species at a given position). The nature, pattern and phylogenetic distribution of the inferred edits implies either that more than one type of previously described editing process operates on a given transcript in dinoflagellate mitochondria, or that a mechanistically unique type of mitochondrial mRNA editing has evolved within the dinoflagellate lineage.


Asunto(s)
Apoproteínas/genética , Grupo Citocromo b/genética , Dinoflagelados/genética , Complejo IV de Transporte de Electrones/genética , Pfiesteria piscicida/genética , Prostaglandina-Endoperóxido Sintasas , ARN Mensajero , ARN Protozoario , ARN , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Codón , Citocromos b , ADN Complementario , Genes Protozoarios , Humanos , Isoenzimas , Proteínas de la Membrana , Datos de Secuencia Molecular , Sistemas de Lectura Abierta , ARN Mitocondrial , Homología de Secuencia de Aminoácido
19.
Am J Physiol Gastrointest Liver Physiol ; 282(3): G527-33, 2002 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-11842003

RESUMEN

The influence of copper status on Caco-2 cell apical iron uptake and transepithelial transport was examined. Cells grown for 7-8 days in media supplemented with 1 microM CuCl(2) had 10-fold higher cellular levels of copper compared with control. Copper supplementation did not affect the integrity of differentiated Caco-2 cell monolayers grown on microporous membranes. Copper-repleted cells displayed increased uptake of iron as well as increased transport of iron across the cell monolayer. Northern blot analysis revealed that expression of the apical iron transporter divalent metal transporter-1 (DMT1), the basolateral transporter ferroportin-1 (Fpn1), and the putative ferroxidase hephaestin (Heph) was upregulated by copper supplementation, whereas the recently identified ferrireductase duodenal cytochrome b (Dcytb) was not. These results suggest that DMT1, Fpn1, and Heph are involved in the iron uptake process modulated by copper status. Although a clear role for Dcytb was not identified, an apical surface ferrireductase was modulated by copper status, suggesting that its function also contributes to the enhanced iron uptake by copper-repleted cells. A model is proposed wherein copper promotes iron depletion of intestinal Caco-2 cells, creating a deficiency state that induces upregulation of iron transport factors.


Asunto(s)
Cobre/metabolismo , Células Epiteliales/metabolismo , FMN Reductasa , Mucosa Intestinal/metabolismo , Hierro/metabolismo , Transporte Biológico/efectos de los fármacos , Northern Blotting , Células CACO-2 , Proteínas de Transporte de Catión/genética , Diferenciación Celular , Medios de Cultivo , Grupo Citocromo b/genética , Expresión Génica , Humanos , Mucosa Intestinal/citología , Radioisótopos de Hierro/metabolismo , Proteínas de la Membrana/genética , NADH NADPH Oxidorreductasas/metabolismo , Oxidorreductasas/genética
20.
J Biol Chem ; 277(7): 5490-7, 2002 Feb 15.
Artículo en Inglés | MEDLINE | ID: mdl-11739376

RESUMEN

A bacterial two-hybrid assay revealed interaction between a protein now designated bacterial Atx1 and amino-terminal domains of copper-transporting ATPases CtaA (cellular import) and PacS (thylakoid import) but not the related zinc (ZiaA) or cobalt (CoaT) transporters from the same organism (Synechocystis PCC 6803). The specificity of metallochaperone interactions coincides with metal specificity. After reconstitution in a N(2) atmosphere, bacterial Atx1 bound 1 mol of copper mol(-1), and apoPacS(N) acquired copper from copper-Atx1. Copper was displaced from Atx1 by p-(hydroxymercuri)phenylsulfonate, indicative of thiol ligands, and two cysteine residues were obligatory for two-hybrid interaction with PacS(N). This organism contains compartments (thylakoids) where the copper proteins plastocyanin and cytochrome oxidase reside. In copper super-supplemented mutants, photooxidation of cytochrome c(6) was greater in Deltaatx1DeltactaA than in DeltactaA, showing that Atx1 contributes to efficient switching from iron in cytochrome c(6) to copper in plastocyanin for photosynthetic electron transport. Cytochrome oxidase activity was also less in membranes purified from low [copper]-grown Deltaatx1 or DeltapacS, compared with wild-type, but the double mutant Deltaatx1DeltapacS was non-additive, consistent with Atx1 acting via PacS. Conversely, activity in Deltaatx1DeltactaA was less than in either respective single mutant, revealing that Atx1 can function without the major copper importer and consistent with a role in recycling endogenous copper.


Asunto(s)
Transportadoras de Casetes de Unión a ATP , Proteínas Bacterianas , Proteínas Portadoras/química , Proteínas Portadoras/metabolismo , Proteínas de Transporte de Catión/metabolismo , Cobre/química , Fotosíntesis , Proteínas de Saccharomyces cerevisiae , Tilacoides/química , Adenosina Trifosfatasas/metabolismo , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Sitios de Unión , Southern Blotting , Proteínas Portadoras/genética , Membrana Celular/metabolismo , Clonación Molecular , Cianobacterias/metabolismo , Cisteína/química , Grupo Citocromo b/química , Grupo Citocromo b/genética , Citocromos/metabolismo , Citocromos f , ADN/metabolismo , Complejo IV de Transporte de Electrones/metabolismo , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Cinética , Ligandos , Proteínas de la Membrana/química , Proteínas de la Membrana/genética , Modelos Moleculares , Datos de Secuencia Molecular , Mutación , Oxígeno/metabolismo , Fenotipo , Plastocianina/metabolismo , Unión Proteica , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Proteínas Recombinantes/metabolismo , Homología de Secuencia de Aminoácido , Factores de Tiempo , Técnicas del Sistema de Dos Híbridos , beta-Galactosidasa/metabolismo
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