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1.
Circ Res ; 116(8): 1312-23, 2015 Apr 10.
Artículo en Inglés | MEDLINE | ID: mdl-25711438

RESUMEN

RATIONALE: Matrix vesicles (MVs), secreted by vascular smooth muscle cells (VSMCs), form the first nidus for mineralization and fetuin-A, a potent circulating inhibitor of calcification, is specifically loaded into MVs. However, the processes of fetuin-A intracellular trafficking and MV biogenesis are poorly understood. OBJECTIVE: The objective of this study is to investigate the regulation, and role, of MV biogenesis in VSMC calcification. METHODS AND RESULTS: Alexa488-labeled fetuin-A was internalized by human VSMCs, trafficked via the endosomal system, and exocytosed from multivesicular bodies via exosome release. VSMC-derived exosomes were enriched with the tetraspanins CD9, CD63, and CD81, and their release was regulated by sphingomyelin phosphodiesterase 3. Comparative proteomics showed that VSMC-derived exosomes were compositionally similar to exosomes from other cell sources but also shared components with osteoblast-derived MVs including calcium-binding and extracellular matrix proteins. Elevated extracellular calcium was found to induce sphingomyelin phosphodiesterase 3 expression and the secretion of calcifying exosomes from VSMCs in vitro, and chemical inhibition of sphingomyelin phosphodiesterase 3 prevented VSMC calcification. In vivo, multivesicular bodies containing exosomes were observed in vessels from chronic kidney disease patients on dialysis, and CD63 was found to colocalize with calcification. Importantly, factors such as tumor necrosis factor-α and platelet derived growth factor-BB were also found to increase exosome production, leading to increased calcification of VSMCs in response to calcifying conditions. CONCLUSIONS: This study identifies MVs as exosomes and shows that factors that can increase exosome release can promote vascular calcification in response to environmental calcium stress. Modulation of the exosome release pathway may be as a novel therapeutic target for prevention.


Asunto(s)
Calcio/metabolismo , Exocitosis , Exosomas/metabolismo , Músculo Liso Vascular/metabolismo , Miocitos del Músculo Liso/metabolismo , Vesículas Secretoras/metabolismo , Calcificación Vascular/fisiopatología , Adolescente , Adulto , Estudios de Casos y Controles , Células Cultivadas , Citocinas/metabolismo , Exosomas/patología , Femenino , Humanos , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Masculino , Persona de Mediana Edad , Músculo Liso Vascular/patología , Miocitos del Músculo Liso/patología , Transporte de Proteínas , Proteómica/métodos , Interferencia de ARN , Vesículas Secretoras/patología , Esfingomielina Fosfodiesterasa/genética , Esfingomielina Fosfodiesterasa/metabolismo , Tetraspaninas/metabolismo , Factores de Tiempo , Transfección , Calcificación Vascular/genética , Calcificación Vascular/metabolismo , Calcificación Vascular/patología , Adulto Joven , alfa-2-Glicoproteína-HS/metabolismo
2.
J Surg Res ; 185(1): 27-35, 2013 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-23831229

RESUMEN

BACKGROUND: One-third of all breast cancers are present as clinically nonpalpable lesions. The current gold standard treatment is surgical excision by wire-guided localization. This technique has patient, technical, and scheduling drawbacks. Alternatives exist but depend on radioisotopes with their legislative and waste management issues. Magnetic nanoparticles (MNPs) have already been successfully used for sentinel lymph node biopsy in breast cancer. We therefore aimed to determine the feasibility of using iron oxide MNPs and a handheld magnetometer for the localization of nonpalpable breast cancers using a preclinical model. METHODS: We constructed phantom models to assess the relationship between the handheld magnetometer peak readings and the variation in volume of iron oxide MNPs and their depth of injection in a series of porcine and avian tissue models. We also radiolabeled the MNPs with (99m)Tc and alendronate to create the conjugate (99m)Tc-dipicolylamine-alendronate-MNP and used nano-single-photon emission computed tomography-computed tomography to perform imaging to demonstrate localization properties. RESULTS: The handheld magnetometer readings follow a linear relationship with variations in volume of magnetic tracer and a logarithmic relationship with variations in depth of injection of the magnetic tracer at a fixed volume. The application of derived quadratic equations from the R(2) curves of handheld magnetometer signal versus depth allowed calculation of the depth of injection of iron oxide MNPs from the handheld magnetometer readings and demonstrated the predictable behavior of the iron oxide MNPs and the handheld magnetometer. Satisfactory localization characteristics were confirmed in the phantoms and imaged using nano-single-photon emission computed tomography and computed tomography. CONCLUSIONS: Iron oxide MNPs demonstrate positive localization characteristics in phantom models with predictable behavior patterns. We suggest that the use of MNPs provides a potential technique for the localization of nonpalpable breast lesions and deserves further exploration in animal and human feasibility studies.


Asunto(s)
Neoplasias de la Mama/diagnóstico por imagen , Neoplasias de la Mama/cirugía , Compuestos Férricos , Nanopartículas del Metal , Alendronato , Aminas , Animales , Pollos , Estudios de Factibilidad , Femenino , Campos Magnéticos , Modelos Animales , Compuestos de Organotecnecio , Fantasmas de Imagen , Ácidos Picolínicos , Radiofármacos , Porcinos , Tomografía Computarizada de Emisión de Fotón Único/métodos
3.
Nat Chem Biol ; 5(12): 882-4, 2009 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-19915535

RESUMEN

Here we report the de novo design and NMR structure of a four-helical bundle di-iron protein with phenol oxidase activity. The introduction of the cofactor-binding and phenol-binding sites required the incorporation of residues that were detrimental to the free energy of folding of the protein. Sufficient stability was, however, obtained by optimizing the sequence of a loop distant from the active site.


Asunto(s)
Biología Computacional , Proteínas de Unión a Hierro/química , Modelos Moleculares , Monofenol Monooxigenasa/química , Ingeniería de Proteínas , Secuencia de Aminoácidos , Catálisis , Dominio Catalítico , Proteínas de Unión a Hierro/genética , Datos de Secuencia Molecular , Monofenol Monooxigenasa/genética , Resonancia Magnética Nuclear Biomolecular , Conformación Proteica , Especificidad por Sustrato
4.
Dalton Trans ; 44(11): 4963-75, 2015 Mar 21.
Artículo en Inglés | MEDLINE | ID: mdl-25559039

RESUMEN

The first (99m)Tc and (188)Re complexes containing two pendant bisphosphonate groups have been synthesised, based on the mononuclear M(v) nitride core with two dithiocarbamate ligands each with a pendant bisphosphonate. The structural identity of the (99)Tc and stable rhenium analogues as uncharged, mononuclear nitridobis(dithiocarbamate) complexes was determined by electrospray mass spectrometry. The (99m)Tc complex showed greater affinity for synthetic and biological hydroxyapatite, and greater stability in biological media, than the well-known but poorly-characterised and inhomogeneous bone imaging agent (99m)Tc-MDP. It gave excellent SPECT images of both bone calcification (mice and rats) and vascular calcification (rat model), but the improved stability and the availability of two pendant bisphosphonate groups conferred no dramatic advantage in imaging over the conventional (99m)Tc-MDP agent in which the bisphosphonate group is bound directly to Tc. The (188)Re complex also showed preferential uptake in bone. These tracers and the biological model of vascular calcification offer the opportunity to study the biological interpretation and clinical potential of radionuclide imaging of vascular calcification and to deliver radionuclide therapy to bone metastases.


Asunto(s)
Calcinosis/diagnóstico por imagen , Difosfonatos/química , Compuestos de Organotecnecio/química , Renio/química , Tecnecio , Tomografía Computarizada de Emisión de Fotón Único/métodos , Animales , Proteínas Sanguíneas/metabolismo , Durapatita/metabolismo , Femenino , Humanos , Hidrazinas/química , Ligandos , Masculino , Arterias Mesentéricas/diagnóstico por imagen , Compuestos de Organotecnecio/metabolismo , Compuestos de Organotecnecio/farmacocinética , Radioisótopos , Ratas , Distribución Tisular
5.
Chem Commun (Camb) ; (1): 76-7, 2004 Jan 07.
Artículo en Inglés | MEDLINE | ID: mdl-14737340

RESUMEN

The presence of second-sphere -NH(2) groups in the proximity of a zinc(ii)-bound water molecule enhances its acidity by ca. 2 pK(a) units.

6.
Dalton Trans ; (17): 2800-7, 2004 Sep 07.
Artículo en Inglés | MEDLINE | ID: mdl-15514768

RESUMEN

Tripodal ligands N(CH2Py)3-n(CH2Py-6-NHR)n(R=H, n=1-3 L1-3, n=0 tpa; R=CH2tBu, n=1-3 L'1-3) are used to investigate the effect of different hydrogen bonding microenvironments on structural features of their LZnX complexes (X=Cl-, NO3-, OH-). The X-ray structures of [(L2)Zn(Cl)](BPh4)2.0.5(H2O.CH3CN), [(L3)Zn(Cl)](BPh4)3.CH3CN, [(L'1)Zn(Cl)](BPh4) 1', [(L'2)Zn(Cl)](BPh4)2'.CH3OH, and [(L'3)Zn(Cl)](BPh4)3' have been determined and exhibit trigonal bipyramidal geometries with intramolecular (internal) N-HCl-Zn hydrogen bonds. The structure of [(L'2)Zn(ONO2)]NO3 4'.H2O with two internal N-HO-Zn hydrogen bonds has also been determined. The axial Zn-Cl distance lengthens from 2.275 A in [(tpa)Zn(Cl)](BPh4) to 2.280-2.347 A in 1-3, 1'-3'. Notably, the average Zn-N(py) distance is also progressively lengthened from 2.069 A in [(tpa)Zn(Cl)](BPh4) to 2.159 and 2.182 A in the triply hydrogen bonding cavity of 3 and 3', respectively. Lengthening of the Zn-Cl and Zn-N(py) bonds is accompanied by a progressive shortening of the trans Zn-N bond from 2.271 A in [(tpa)Zn(Cl)](BPh4) to 2.115 A in 3 (2.113 A in 3'). As a result of the triply hydrogen bonding microenvironment the Zn-Cl and Zn-N(py) distances of 3 are at the upper end of the range observed for axial Zn-Cl bonds, whereas the axial Zn-N distance is one of shortest among N4 ligands that induce a trigonal bipyramidal geometry. Despite the rigidity of these tripodal ligands, the geometry of the intramolecular RN-HX-Zn hydrogen bonds (X=Cl-, OH-, NO3-) is strongly dependent on the nature of X, however, on average, similar for R=H, CH2tBu.

7.
Dalton Trans ; (1): 172-7, 2004 Jan 07.
Artículo en Inglés | MEDLINE | ID: mdl-15356757

RESUMEN

Polydentate ligands (6-R1-2-pyridylmethyl)-R2(R1= NHCOtBu, R2= bis(2-pyridylmethyl)amine L1, bis(2-(methylthio)ethyl)amine L2 and N(CH2CH2)2S L3) form mononuclear zinc(II) complexes with intramolecular amide oxygen coordination and a range of coordination environments. Thus, the reaction of Zn(ClO4)2.6H2O with L1-3 in acetonitrile affords [(L)Zn](ClO4)2(L=L1, 1; L2, 2) and [(L3)Zn(H2O)(NCCH3)](ClO4)2 3. The simultaneous amide/water binding in resembles the motif that has been proposed to be involved in the double substrate/nucleophile Lewis acidic activation and positioning mechanism of amide bond hydrolysis in metallopeptidases. X-ray diffraction, 1H and 13C NMR and IR data suggests that the strength of amide oxygen coordination follows the trend 1>2 >3. L1-3 and undergo cleavage of the tert-butylamide upon addition of Me4NOH.5H2O (1 equiv.) in methanol at 50(1)degrees C. The rate of amide cleavage follows the order 1> 2>> 3, L1-3. The extent by which the amide cleavage reaction is accelerated in 1-3 relative to the free ligands, L1-3, is correlated with the strength of amide oxygen binding and Lewis acidity of the zinc(II) centre in deduced from the X-ray, NMR and IR studies.


Asunto(s)
Amidas/química , Oxígeno/química , Zinc/química , Amidohidrolasas/química , Cristalografía por Rayos X , Modelos Químicos , Conformación Molecular , Estructura Molecular
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