RESUMEN
In recent years, significant progress has been made in the development of fluorescent contrast agents for clinical applications. For the development of a fluorescent probe, it is crucial to evaluate its safety profile, including biodistribution. Specific methods need to be developed for the absolute quantification of fluorescent probes in tissue specimens from animals administered with test compounds in the framework of biodistribution/efficacy/toxicity studies. Here, we describe a new method for the absolute quantification of fluorescent probes in tissue specimens from animals administered with compounds that have absorption and emission wavelength in the Near-Infrared region (600-800 nm). The protocol is based on the standard addition approach in order to minimize the interference of the matrix on the analyte signal causing inaccuracy in the absolute determination of the concentration. The measurement of the fluorescence intensity is done via a microplate reader. The method has been fully validated and applied for the quantification of a fluorescence-guided surgery targeted contrast agent in a Good Laboratory Practice (GLP) biodistribution study. Results clearly demonstrate that this procedure is fully applicable in a preclinical setting and that it overcomes common issues associated with fluorescence signal quantification in tissue extracts.
Asunto(s)
Colorantes Fluorescentes , Extractos de Tejidos , Animales , Fluorescencia , Distribución TisularRESUMEN
The strong molecular interaction between biotin and streptavidin is widely used in the growing field of nucleic acid nanotechnology. Several biotin labeled oligonucleotide tools have been developed for the detection of biological molecules as well as for protein purification. For these reasons, biotinylation can be considered one of the main chemical reactions for nucleic acid labeling. However, despite its widespread application and the presence on the market of many reagents for the conjugation of biotin to oligonucleotides, it is not yet available a cheap, easy and sensitive system able to assess the effectiveness and reproducibility of this reaction. Here, we present an accurate and reliable method to achieve a qualitative and quantitative analysis of oligonucleotide biotinylation. The protocol employs basic laboratory instruments and standard software for molecular biology applications and does not require advanced expertise for its execution. Most importantly, our method is independent from complex kinetic equilibrium parameters and shows a limit of detection more than one order of magnitude lower than the current fluorometric gold standard assay. Therefore, this method could become a standard, inexpensive and routinely used quality test for post-synthesis evaluation of biotin conjugation reactions.
Asunto(s)
Biotinilación/métodos , Oligonucleótidos/química , ARN/química , Coloración y Etiquetado/métodos , Biotina/química , CinéticaRESUMEN
An increased prevalence of liver diseases such as hepatitis C and nonalcoholic fatty liver results in an augmented incidence of the most common form of liver cancer, hepatocellular carcinoma (HCC). HCC is most often found in the cirrhotic liver and it can therefore be challenging to rely on anatomical information alone when diagnosing HCC. Valuable information on specific cellular metabolism can be obtained with high sensitivity thanks to an emerging magnetic resonance (MR) technique that uses 13C labeled hyperpolarized molecules. Our interest was to explore potential new high contrast metabolic markers of HCC using hyperpolarized 13C-MR. This work led to the identification of a class of substrates, low molecular weight ethyl-esters, which showed high specificity for carboxyl esterases and proved in many cases to possess good properties for signal enhancement. In particular, hyperpolarized [1,3-13C2 ]ethyl acetoacetate (EAA) was shown to provide a metabolic fingerprint of HCC. Using this substrate a liver cancer implanted in rats was diagnosed as a consequence of an â¼4 times higher metabolic substrate-to-product ratio than in the surrounding healthy tissue, (p=0.009). Unregulated cellular uptake as well as cosubstrate independent enzymatic conversion of EAA, made this substrate highly useful as a hyperpolarized 13C-MR marker. This could be appreciated by the signal-to-noise (SNR) obtained from EAA, which was comparable to the SNR reported in a literature liver cancer study with state-of-the-art hyperpolarized substrate, [1-13C]pyruvate. Also, the contrast-to-noise (CNR) in the EAA based metabolic ratio images was significantly improved compared with the CNR in equivalent images reported using [1-13C]pyruvate.
Asunto(s)
Acetoacetatos , Medios de Contraste , Neoplasias Hepáticas Experimentales/diagnóstico , Acetoacetatos/farmacocinética , Animales , Biomarcadores de Tumor , Carboxilesterasa/metabolismo , Medios de Contraste/farmacocinética , Células Hep G2 , Humanos , Hígado/metabolismo , Neoplasias Hepáticas Experimentales/metabolismo , Trasplante de Neoplasias , Ratas Endogámicas BUF , Relación Señal-RuidoRESUMEN
A set of molecules in which a glucose moiety is bound to a hydrogenable synthon has been synthesized and evaluated for hydrogenation reactions and for the corresponding para-hydrogen-induced polarization (PHIP) effects, in order to select suitable candidates for an in vivo magnetic resonance imaging (MRI) method for the assessment of glucose cellular uptake. It has been found that amidic derivatives do not yield any polarization enhancement, probably due to singlet-triplet state mixing along the reaction pathway. In contrast, ester derivatives are hydrogenated in high yield and afford enhanced (1)H and (13)C NMR spectra after para-hydrogenation. The obtained PHIP patterns are discussed and explained on the basis of the calculated spin level populations in the para-hydrogenated products. These molecules may find interesting applications in (13)C MRI as hyperpolarized probes for assessing the activity of glucose transporters in cells.
Asunto(s)
Glucosa/química , Imagen por Resonancia Magnética/métodos , Transporte Biológico , Isótopos de Carbono , Glucosa/metabolismo , Humanos , Hidrógeno/química , Hidrogenación , Células K562 , Espectroscopía de Resonancia MagnéticaRESUMEN
A Cu(I) metallochaperone, Atx1, interacts with the amino-terminal domain of a Cu(I)-transporting ATPase, PacS(N), but not with a domain of related Zn-transporting ATPase, ZiaA(N) in Synechocystis PCC 6803. This is thought to prevent ZiaA(N) from acquiring Cu(I), which it binds more tightly than Zn. Solution structures of Atx1, PacS(N), and the heterodimer were previously described. Here we report solution structural studies of the ZiaA(N) soluble domain. Apo-ZiaA(N) has a typical ferredoxin-like fold followed by an atypical 34 residues of unstructured polypeptide containing a His(7) motif. ZiaA(N) competes with the metallochromic indicator 4-(2-pyridylazo)resorcinol for 1 equiv of Zn, which can be displaced by thiol-modifying p-mercuriphenylsulfonic acid, establishing that a high-affinity site involves thiols of the CXXC motif within the ferredoxin-like fold. A single equivalent of Zn affects nuclear magnetic resonance signals arising from the CXXC motif as well as all seven His residues. The presence of NMR-line broadening in both sites implies that Zn(1)-ZiaA(N) undergoes exchange phenomena, consistent with CXXC-bound Zn coincidentally sampling various His ligands. These Zn-dependent dynamic changes could either aid metal transfer or alter intramolecular interactions. No formation of Atx1-Cu(I)-ZiaA(N) heterodimers was observed, and in the presence of equimolar ZiaA(N) and PacS(N), only Atx1-Cu(I)-PacS(N) complexes were detected. Residues flanking the CXXC motif of PacS(N) (R(13)-ASS(20)) differ in charge and bulk from those of ZiaA(N) (D(18)-KLK(25)) and make contacts in the Atx1-Cu(I)-PacS(N) complex. Crucially, swapping these residues flanking the CXXC motifs of ZiaA(N) and PacS(N) reciprocally swaps partner choice by Atx1. These few residues of the two ATPases have diverged during evolution to bias Atx1 interactions in favor of PacS(N) rather than ZiaA(N.).
Asunto(s)
Adenosina Trifosfatasas/química , Adenosina Trifosfatasas/metabolismo , Proteínas Bacterianas/metabolismo , Cobre/metabolismo , Chaperonas Moleculares/metabolismo , Synechocystis/enzimología , Adenosina Trifosfatasas/genética , Secuencia de Aminoácidos , Apoenzimas/química , Apoenzimas/genética , Apoenzimas/metabolismo , Proteínas de Transporte de Catión/metabolismo , ATPasas Transportadoras de Cobre , Modelos Moleculares , Datos de Secuencia Molecular , Mutación , Resonancia Magnética Nuclear Biomolecular , Estructura Terciaria de Proteína , Solubilidad , Especificidad por Sustrato , Synechocystis/metabolismo , Zinc/metabolismoRESUMEN
A dinuclear gadolinium(III) chelate containing two moieties of diethylenetriaminepentaacetic acid (DTPA), covalently conjugated to an analogue of deoxycholic acid, was synthesized and thoroughly characterized. A full relaxometric analysis was carried out, consisting of 1)â the acquisition of nuclear magnetic resonance dispersion (NMRD) profiles in various media; 2)â the study of binding affinity to serum albumin; 3)â the measurement of 17 O transverse relaxation rate versus temperature, and 4)â a transmetallation assay. In vivo biodistribution MRI studies at 1â T and blood pharmacokinetics assays were carried out in comparison with Gd-DTPA (Magnevist) and gadocoletic acid trisodium salt (B22956/1), two well-known Gd complexes that share the same chelating cage and the same deoxycholic acid residue of the Gd complex investigated herein ((GdDTPA)2 -Chol). High affinity for plasma protein and, in particular, the availability of more than one binding site, allows the complex to reach a fairly high relaxivity value in plasma (â¼20â mm-1 s-1 , 20â MHz, 310â K) as well as to show unexpectedly enhanced properties of blood pooling, with an elimination half-life in rats approximately seven times longer than that of B22956/1.
Asunto(s)
Medios de Contraste/química , Medios de Contraste/farmacocinética , Ácido Desoxicólico/análogos & derivados , Ácido Desoxicólico/farmacocinética , Gadolinio DTPA/análogos & derivados , Gadolinio DTPA/farmacocinética , Animales , Medios de Contraste/síntesis química , Medios de Contraste/metabolismo , Ácido Desoxicólico/síntesis química , Ácido Desoxicólico/metabolismo , Femenino , Gadolinio DTPA/síntesis química , Gadolinio DTPA/metabolismo , Imagen por Resonancia Magnética , Masculino , Ratas , Ratas Wistar , Albúmina Sérica/metabolismo , Distribución TisularRESUMEN
PhotoAcoustic Imaging (PAI) is a biomedical imaging modality currently under evaluation in preclinical and clinical settings. In this work, ICG is coupled to an integrin binding vector (ICG-RGD) to combine the good photoacoustic properties of ICG and the favourable αvß3-binding capabilities of a small RGD cyclic peptidomimetic. ICG-RGD is characterized in terms of physicochemical properties, biodistribution and imaging performance. Tumor uptake was assessed in subcutaneous xenograft mouse models of human glioblastoma (U-87MG, high αvß3 expression) and epidermoid carcinoma (A431, low αvß3 expression). ICG and ICG-RGD showed high PA signal in tumors already after 15 min post-injection. At later time points the signal of ICG rapidly decreased, while ICG-RGD showed sustained uptake in U-87MG but not in A431 tumors, likely due to the integrin-mediated retention of the probe. In conclusion, ICG-RGD is a novel targeted contrast agents for PAI with superior biodistribution, tumor uptake properties and diagnostic value compared to ICG.
RESUMEN
The presence of a paramagnetic center may disturb both coherent and incoherent communication between nuclear spins that are affected, to some extent, by the hyperfine interaction. This is a limiting factor to an extensive use of paramagnetic probes in NMR spectroscopy to enhance partial alignment and to exploit cross correlation effects and pseudocontact shifts. We propose here an HCCH-TOCSY experiment tailored to identify spin systems involving resonances that are partly or completely affected by hyperfine interaction. The efficiency of polarization transfer steps when fast relaxing nuclei are involved is discussed. The sequence is tested for the protein Calbindin D(9k), in which one of the two native Ca2+ ions is replaced by the paramagnetic Ce3+ ion as well as for the oxidized form of cytochrome b(562).
Asunto(s)
Proteínas de Escherichia coli , Espectroscopía de Resonancia Magnética/métodos , Proteína G de Unión al Calcio S100/análisis , Calbindinas , Calcio/química , Isótopos de Carbono , Cesio/química , Grupo Citocromo b/química , Relación Estructura-ActividadRESUMEN
Trypanosoma brucei is the causative agent of African sleeping sickness. Current work for the development of new drugs against this pathology includes evaluation of enzymes of the pentose phosphate pathway (PPP), which first requires a clear understanding of their function and mechanism of action. In this context, we focused on T. brucei 6-phosphogluconolactonase (Tb6PGL), which converts delta-6-phosphogluconolactone into 6-phosphogluconic acid in the second step of the PPP. We have determined the crystal structure of Tb6PGL in complex with two ligands, 6-phosphogluconic acid and citrate, at 2.2 A and 2.0 A resolution, respectively. We have performed molecular dynamics (MD) simulations on Tb6PGL in its empty form and in complex with delta-6-phosphogluconolactone, its natural ligand. Analysis of the structural data and MD simulations allowed us to propose a detailed enzymatic mechanism for 6PGL enzymes.
Asunto(s)
Hidrolasas de Éster Carboxílico/química , Simulación por Computador , Trypanosoma brucei brucei/enzimología , Animales , Hidrolasas de Éster Carboxílico/genética , Hidrolasas de Éster Carboxílico/metabolismo , Ácido Cítrico/química , Ácido Cítrico/metabolismo , Cristalografía por Rayos X , Gluconatos/química , Gluconatos/metabolismo , Humanos , Modelos Moleculares , Datos de Secuencia Molecular , Estructura Molecular , Tripanosomiasis Africana , Difracción de Rayos XRESUMEN
The Lewis X (LeX) determinant, a trisaccharide with the carbohydrate sequence Galbeta(1-->4)[Fucalpha(1-->3)]GlcNAcbeta, is believed to be responsible for Ca2+-mediated cell-cell interactions. In partly oriented phases composed of mixtures of penta(ethyleneglycol)monododecyl ether HO(CH2CH2O)5C12H25 and n-hexanol in the presence of Ca2+ ions, the variation of the residual dipolar couplings 1DCH of various CiHi vectors in LeX as a function of the concentration of the trisaccharide demonstrates the existence of very weak LeX-Ca2+-LeX complexes in solution. Synthetic 3-, 4-, and 6-deoxy-LeX variants were also shown to form complexes in the presence of calcium ions, despite the replacement of one of their hydroxyl groups by hydrogen atoms. This is the first direct observation in solution of a calcium-mediated interaction between LeX molecules.
Asunto(s)
Calcio/química , Antígeno Lewis X/química , Trisacáridos/química , Acetilglucosamina/química , Secuencia de Carbohidratos , Óxido de Deuterio , Espectroscopía de Resonancia Magnética , Datos de Secuencia Molecular , Protones , SolucionesRESUMEN
The relative intensity of Calpha-C' cross-peaks in homonuclear 13C COSY spectra depends on the relaxation properties of Calpha and C' spins, which, in the proximity of a paramagnetic center, are related to the metal-to-carbon distance. Their quantitative analysis has lead, for the cerium-substituted dicalcium protein, calbindin D9k, to the straightforward identification of peaks arising from metal-coordinating groups. The monodentate or bidentate metal binding mode of carboxylates was identified directly via NMR.
Asunto(s)
Algoritmos , Resonancia Magnética Nuclear Biomolecular/métodos , Proteínas/química , Isótopos de Carbono/química , LigandosRESUMEN
Major urinary protein (MUP) is a pheromone-carrying protein of the lipocalin family. Previous studies by isothermal titration calorimetry (ITC) show that the affinity of MUP for the pheromone 2-methoxy-3-isobutylpyrazine (IBMP) is mainly driven by enthalpy, with a small unfavourable entropic contribution. Entropic terms can be attributed in part to changes in internal motions of the protein upon binding. Slow internal motions can lead to correlated or anti-correlated modulations of the isotropic chemical shifts of carbonyl C' and amide N nuclei. Correlated chemical shift modulations (CSM/CSM) in MUP have been determined by measuring differences of the transverse relaxation rates of zero- and double-quantum coherences ZQC{C'N} and DQC{C'N}, and by accounting for the effects of correlated fluctuations of dipole-dipole couplings (DD/DD) and chemical shift anisotropies (CSA/CSA). The latter can be predicted from tensor parameters of C' and N nuclei that have been determined in earlier work. The effects of complexation on slow time-scale protein dynamics can be determined by comparing the temperature dependence of the relaxation rates of APO-MUP (i.e., without ligand) and HOLO-MUP (i.e., with IBMP as a ligand).
Asunto(s)
Feromonas/química , Proteínas/química , Aminoácidos , Cristalografía por Rayos X , Ligandos , Espectroscopía de Resonancia Magnética , Modelos Moleculares , Unión Proteica , Conformación Proteica , TemperaturaRESUMEN
Early steps of unfolding of P43M Calbindin D(9k) have been evaluated by NMR spectroscopy on the native dicalcium and on the paramagnetic monocerium-substituted derivative. Although at 2 M GdmHCl the protein core maintains its overall folding and structure, amide (15)N R(2) measurements and cross correlation rates between N-H dipole-dipole relaxation and (15)N CSA relaxation reveal a closer and stronger packing of the hydrophobic interactions in the protein as a response to the presence of denaturing agents in solution. A complete reorientation of the Met43 side chain toward the hydrophobic core is accomplished by the disappearance of the millisecond dynamics observed on the native form of Calbindin D(9k), while cross correlation rates provide evidence that the two-way hydrogen bond between Leu23 and Val61 is broken or substantially weakened. The substitution of the calcium ion in site II with the paramagnetic Ce(3+) ion allowed us to obtain a number of long-range nonconventional constraints, namely, pseudocontact shifts, which were used, together with the NOEs collected on the native state, to monitor subtle structural variations occurring in the non-native state of the protein. Although the average rmsd between the structures of native and non-native states is small (0.48 A), structural rearrangements could be reliably identified. Our results provide unprecedented information about the behavior of Calbindin D(9k) during the early steps of unfolding. Furthermore, they constitute strong evidence of the efficiency of paramagnetism-based constraints in monitoring subtle structural changes that are beyond the sensitivity of an approach based only on NOE.
Asunto(s)
Sustitución de Aminoácidos , Calcio/química , Cerio/química , Metionina , Prolina , Pliegue de Proteína , Proteína G de Unión al Calcio S100/química , Amidas/química , Animales , Calbindinas , Cationes Bivalentes , Bovinos , Guanidina/química , Resonancia Magnética Nuclear Biomolecular , Conformación Proteica , Desnaturalización Proteica , Protones , Soluciones , TermodinámicaRESUMEN
Calbindin D(9k) is a dicalcium binding protein consisting of two helix-loop-helix EF-hand motifs joined together by a flexible linker region where one metal ion can bind to each of the two loops. A proline residue at position 43 in the linker region displays cis-trans isomerism in the wild-type (WT) protein. Such isomerism appeared to be removed by substituting the proline with a glycine or a methionine in the P43G or P43M mutant. We have extended the available mobility studies on the P43M mutant through amide (15)N R(1), R(2), and R(1)(rho)() measurements. This has revealed unexpected conformational equilibria on the millisecond time scale involving residues 38, 42-44, and 46 in the linker region and residues 18 and 19 in calcium binding site I with similar energy barriers. These data are discussed in comparison with those available for the WT, as well as the apo-, mono-, and disubstituted P43G mutant. Quantification of water-amide proton exchange rates using saturation transfer and qualitative application of (15)N-(CLEANEX-PM)-FHSQC shows the values are in agreement with high mobility for the above-mentioned residues. Cross correlation between N-H dipole-dipole relaxation and (15)N CSA relaxation indicates that some of these mobility differences may extend to the sub-nanosecond time scale. Similar data were also obtained for the derivative where the calcium ion in the C-terminal loop was replaced with lanthanum. The results presented here show that, contrary to expectations, there are significant differences in dynamics between the dicalcium state of P43G and P43M and that these differences are not confined to the flexible linker region containing the point mutation. They also demonstrate that substitution of a lanthanide ion for calcium, which is a common procedure, does not significantly alter the mobility of the native protein.
Asunto(s)
Calcio/química , Lantano/química , Resonancia Magnética Nuclear Biomolecular , Proteína G de Unión al Calcio S100/química , Proteína G de Unión al Calcio S100/genética , Amidas/química , Sustitución de Aminoácidos/genética , Animales , Calbindinas , Cationes Bivalentes/química , Bovinos , Metionina/genética , Isótopos de Nitrógeno , Resonancia Magnética Nuclear Biomolecular/métodos , Prolina/genética , Conformación Proteica , Protones , Temperatura , TermodinámicaRESUMEN
Cross correlation rates between Curie spin relaxation and H-N dipole-dipole coupling (gamma(HM,HN)CS,DD) have been determined for a calcium binding protein, Calbindin D9k, in which one of the two calcium ions is substituted with cerium(III). Gamma(HM,HN)CS,DD values depend on both the metal-to-proton distances and the M-H-N angles and can be used as an additional constraint in order to refine the solution structure of paramagnetic metalloproteins. For this purpose, we have implemented a new module (CCR-DYANA) in a version of the program DYANA (PARAMAGNETIC-DYANA), which can be used together with other paramagnetism-based constraints such as pseudocontact shifts, residual dipolar couplings and hyperfine based Karplus relationships. This integrated structure calculation protocol has the advantage that different paramagnetic-based constraints are treated by the same algorithm in a way that the efficiency of each class of constraints can be analyzed and compared.
Asunto(s)
Cerio/química , Proteína G de Unión al Calcio S100/química , Algoritmos , Calbindinas , Espectroscopía de Resonancia por Spin del Electrón , Enlace de Hidrógeno , Resonancia Magnética Nuclear Biomolecular , Conformación Proteica , Protones , SolucionesRESUMEN
In the case of oxidized plastocyanin from Synechocystis sp. PCC6803, an NMR approach based on classical two and three dimensional experiments for sequential assignment leaves unobserved 14 out of 98 amino acids. A protocol which simply makes use of tailored versions of 2D HSQC and 3D CBCA(CO)NH and CBCANH leads to the identification of nine of the above 14 residues. The proposed protocol differs from previous approaches in that it does not involve the use of unconventional experiments designed specifically for paramagnetic systems, and does not exploit the occurrence of a corresponding diamagnetic species in chemical exchange with the blue copper form. This protocol is expected to extend the popularity of NMR in the structural studies of copper (II) proteins, allowing researchers to increase the amount of information available via NMR on the neighborhood of a paramagnetic center without requiring a specific expertise in the field. The resulting 3D spectra are standard spectra that can be handled by any standard software for protein NMR data analysis.