RESUMEN
Dry eye disease (DED) is a chronic multifactorial ocular surface disease mainly caused by the instability of tear film, characterized by a series of ocular discomforts and even visual disorders. Oxidative stress has been recognized as an upstream factor in DED development. Diquafosol sodium (DQS) is an agonist of the P2Y2 receptor to restore the integrity/stability of the tear film. With the ability to alternate between Ce3+ and Ce4+, cerium oxide nanozymes could scavenge overexpressed reactive oxygen species (ROS). Hence, a DQS-loaded cerium oxide nanozyme was designed to boost the synergistic treatment of DED. Cerium oxide with branched polyethylenimine-graft-poly(ethylene glycol) as nucleating agent and dispersant was fabricated followed with DQS immobilization via a dynamic phenylborate ester bond, obtaining the DQS-loaded cerium oxide nanozyme (defined as Ce@PBD). Because of the ability to mimic the cascade processes of superoxide dismutase and catalase, Ce@PBD could scavenge excessive accumulated ROS, showing strong antioxidant and anti-inflammatory properties. Meanwhile, the P2Y2 receptors in the conjunctival cells could be stimulated by DQS in Ce@PBD, which can relieve the incompleteness and instability of the tear film. The animal experiments demonstrated that Ce@PBD significantly restored the defect of the corneal epithelium and increased the number of goblet cells, with the promotion of tear secretion, which was the best among commercial DQS ophthalmic solutions.
Asunto(s)
Cerio , Síndromes de Ojo Seco , Cerio/química , Cerio/farmacología , Animales , Síndromes de Ojo Seco/tratamiento farmacológico , Síndromes de Ojo Seco/patología , Síndromes de Ojo Seco/metabolismo , Nucleótidos de Uracilo/química , Nucleótidos de Uracilo/farmacología , Especies Reactivas de Oxígeno/metabolismo , Humanos , Antioxidantes/química , Antioxidantes/farmacología , Estrés Oxidativo/efectos de los fármacos , Polifosfatos/química , Polifosfatos/farmacología , Ratones , ConejosRESUMEN
This study aimed to develop, optimize, and evaluate hot-melt-extruded ophthalmic inserts capable of sustained release of diquafosol tetrasodium (DQS) via a design of experiments approach. DQS, a tear stimulant for dry eye management, faces challenges of frequent administration and low bioavailability. The developed insert uses biodegradable polymers in varied proportions to achieve sustained release. Optimized through mixture design, the insert completely dissolved within 24 h and maintained a stable drug content, thickness, and surface pH over three months at room temperature. In vitro corneal permeation studies on excised rabbit corneas demonstrated increased bioavailability, suggesting a reduced dosing frequency compared with conventional eye drops. Therefore, this insert has potential to enhance treatment outcomes by improving patient compliance and providing sustained drug effects.
Asunto(s)
Córnea , Preparaciones de Acción Retardada , Polifosfatos , Nucleótidos de Uracilo , Conejos , Animales , Polifosfatos/química , Nucleótidos de Uracilo/administración & dosificación , Nucleótidos de Uracilo/química , Córnea/metabolismo , Córnea/efectos de los fármacos , Soluciones Oftálmicas/administración & dosificación , Soluciones Oftálmicas/química , Disponibilidad Biológica , Liberación de Fármacos , Administración Oftálmica , Composición de Medicamentos/métodos , Implantes de Medicamentos , Calor , Química Farmacéutica/métodosRESUMEN
Guanine-rich regions of the human genome can adopt non-canonical secondary structures. Their role in regulating gene expression has turned them into promising targets for therapeutic intervention. Ligands based on polyaromatic moieties are especially suitable for targeting G-quadruplexes utilizing their size complementarity to interact with the large exposed surface area of four guanine bases. A predictable way of (de)stabilizing specific G-quadruplex structures through efficient base stacking of polyaromatic functional groups could become a valuable tool in our therapeutic arsenal. We have investigated the effect of pyrene-modified uridine nucleotides incorporated at several positions of the thrombin binding aptamer (TBA) as a model system. Characterization using spectroscopic and biophysical methods provided important insights into modes of interaction between pyrene groups and the G-quadruplex core as well as (de)stabilization by enthalpic and entropic contributions. NMR data demonstrated that incorporation of pyrene group into G-rich oligonucleotide such as TBA may result in significant changes in 3D structure such as formation of novel dimeric topology. Site specific structural changes induced by stacking of the pyrene moiety on nearby nucleobases corelate with distinct thrombin binding affinities and increased resistance against nuclease degradation.
Asunto(s)
Aptámeros de Nucleótidos/química , G-Cuádruplex , Pirenos/química , Aptámeros de Nucleótidos/sangre , Aptámeros de Nucleótidos/metabolismo , Desoxirribonucleasas , Dimerización , Entropía , Humanos , Termodinámica , Trombina/metabolismo , Nucleótidos de Uracilo/químicaRESUMEN
Dinucleoside 5',5'-polyphosphates (DNPs) are endogenous substances that play important intra- and extracellular roles in various biological processes, such as cell proliferation, regulation of enzymes, neurotransmission, platelet disaggregation and modulation of vascular tone. Various methodologies have been developed over the past fifty years to access these compounds, involving enzymatic processes or chemical procedures based either on P(III) or P(V) chemistry. Both solution-phase and solid-support strategies have been developed and are reported here. Recently, green chemistry approaches have emerged, offering attracting alternatives. This review outlines the main synthetic pathways for the preparation of dinucleoside 5',5'-polyphosphates, focusing on pharmacologically relevant compounds, and highlighting recent advances.
Asunto(s)
Fosfatos de Dinucleósidos/síntesis química , Agonistas del Receptor Purinérgico P2Y/síntesis química , Nucleótidos de Desoxicitosina/agonistas , Nucleótidos de Desoxicitosina/química , Nucleótidos de Desoxicitosina/farmacología , Fosfatos de Dinucleósidos/química , Fosfatos de Dinucleósidos/aislamiento & purificación , Síndromes de Ojo Seco/tratamiento farmacológico , Tecnología Química Verde , Humanos , Soluciones Oftálmicas , Fosforilación , Polifosfatos/síntesis química , Polifosfatos/química , Agonistas del Receptor Purinérgico P2Y/química , Agonistas del Receptor Purinérgico P2Y/aislamiento & purificación , Receptores Purinérgicos/metabolismo , Nucleótidos de Uracilo/química , Uridina/agonistas , Uridina/análogos & derivados , Uridina/química , Uridina/farmacologíaRESUMEN
Message RNA poly(A) tails are vital for their function and regulation. However, the full-length sequence of mRNA isoforms with their poly(A) tails remains undetermined. Here, we develop a method at single-cell level sensitivity that enables quantification of poly(A) tails along with the full-length cDNA while reading non-adenosine residues within poly(A) tails precisely, which we name poly(A) inclusive RNA isoform sequencing (PAIso-seq). Using this method, we can quantify isoform specific poly(A) tail length. More interestingly, we find that 17% of the mRNAs harbor non-A residues within the body of poly(A) tails in mouse GV oocytes. We show that PAIso-seq is sensitive enough to analyze single GV oocytes. These findings will not only provide an accurate and sensitive tool in studying poly(A) tails, but also open a door for the function and regulation of non-adenosine modifications within the body of poly(A) tails.
Asunto(s)
Nucleótidos de Citosina/química , Nucleótidos de Guanina/química , Oocitos/metabolismo , Poli A/química , ARN Mensajero/química , Análisis de Secuencia de ARN/métodos , Nucleótidos de Uracilo/química , Animales , Proteínas de Ciclo Celular/genética , Ciclina B1/genética , Nucleótidos de Citosina/análisis , ADN (Citosina-5-)-Metiltransferasa 1/genética , Nucleótidos de Guanina/análisis , Ratones , Poli A/análisis , Biosíntesis de Proteínas , Isoformas de ARN , ARN Mensajero/análisis , Análisis de la Célula Individual , Activador de Tejido Plasminógeno/genética , Nucleótidos de Uracilo/análisisRESUMEN
Several health benefits, associated with human milk oligosaccharides (HMOS), have been revealed in the last decades. Further progress, however, requires not only the establishment of a simple "routine" method for absolute quantification of complex HMOS mixtures but also the development of novel synthesis strategies to improve access to tailored HMOS. Here, we introduce a combination of salvage-like nucleotide sugar-producing enzyme cascades with Leloir-glycosyltransferases in a sequential pattern for the convenient tailoring of stable isotope-labeled HMOS. We demonstrate the assembly of [13C6]galactose into lacto-N- and lacto-N-neo-type HMOS structures up to octaoses. Further, we present the enzymatic production of UDP-[15N]GlcNAc and its application for the enzymatic synthesis of [13C6/15N]lacto-N-neo-tetraose for the first time. An exemplary application was selected-analysis of tetraose in complex biological mixtures-to show the potential of tailored stable isotope reference standards for the mass spectrometry-based quantification, using matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF-MS) as a fast and straightforward method for absolute quantification of HMOS. Together with the newly available well-defined tailored isotopic HMOS, this can make a crucial contribution to prospective research aiming for a more profound understanding of HMOS structure-function relations.
Asunto(s)
Leche Humana/química , Oligosacáridos/química , Isótopos de Carbono/química , Catálisis , Glicosiltransferasas/química , Humanos , Isótopos de Nitrógeno/química , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Nucleótidos de Uracilo/químicaRESUMEN
Ectonucleotide pyrophosphatase/phosphodiesterase-1 (NPP1) hydrolyzes phosphodiester bonds of nucleotides such as ATP, resulting mainly in the formation of AMP and pyrophosphate. NPP1 activity plays a deleterious function in calcified aortic valve disease and calcium pyrophosphate deposition disease. Thus, inhibitors of NPP1 represent a medical need. We developed novel NPP1 inhibitors based on uridine 5'-Pα,α-dithiophosphate analogues, 9-12. All these analogues potently inhibited hNPP1 (80-100% inhibition) at 100 µM, with no, or minimal, inhibition of NPP3 and other ectonucleotidases (NTPDase1,2,3,8). These compounds showed nearly no activity at uracil-nucleotide sensitive P2Y2,4,6-receptors and thus represent highly selective NPP1 inhibitors. The most promising inhibitor was diuridine 5'-Pα,α,5â³-Pα,α-tetrathiotetraphosphate, 12, exhibiting Ki of 27 nM. Analogues 9-12 proved to be highly stable to air oxidation and to acidic and basic pH. Docking simulations suggested that the enhanced NPP1 inhibitory activity and selectivity of analogue 12 could be attributed to the simultaneous occupancy of two sites (the AMP site and an alternative site) of NPP1 by this compound.
Asunto(s)
Diseño de Fármacos , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Pirofosfatasas/antagonistas & inhibidores , Nucleótidos de Uracilo/química , Nucleótidos de Uracilo/farmacología , Estabilidad de Medicamentos , Inhibidores Enzimáticos/metabolismo , Humanos , Hidrólisis , Concentración 50 Inhibidora , Simulación del Acoplamiento Molecular , Hidrolasas Diéster Fosfóricas/química , Hidrolasas Diéster Fosfóricas/metabolismo , Conformación Proteica , Pirofosfatasas/química , Pirofosfatasas/metabolismo , Relación Estructura-Actividad , Especificidad por Sustrato , Nucleótidos de Uracilo/metabolismoRESUMEN
A conjugate of triphosphorylated 2',3'-dideoxyuridine (ddU) with SiO2 nanoparticles was obtained via the CuAAC click chemistry between a γ-alkynyl ddU triphosphate and azido-modified SiO2 nanoparticles. Assessment of cytotoxicity in human breast adenocarcinoma MCF7 cells demonstrated that ddU triphosphate conjugated to SiO2 nanoparticles exhibited a 50% decrease in cancer cell growth at a concentration of 183⯱â¯57⯵g/mL, which corresponds to 22⯱â¯7⯵M of the parent nucleotide, whereas the parent nucleoside, nucleotide and alkynyl triphosphate precursor do not show any cytotoxicity. The data provide an example of remarkable potential of novel conjugates of SiO2 nanoparticles with phosphorylated nucleoside analogues, even those, which have not been used previously as therapeutics, for application as new anticancer agents.
Asunto(s)
Antineoplásicos/farmacología , Didesoxinucleótidos/farmacología , Nanopartículas/química , Dióxido de Silicio/farmacología , Nucleótidos de Uracilo/farmacología , Antineoplásicos/síntesis química , Antineoplásicos/química , Proliferación Celular/efectos de los fármacos , Didesoxinucleótidos/síntesis química , Didesoxinucleótidos/química , Relación Dosis-Respuesta a Droga , Ensayos de Selección de Medicamentos Antitumorales , Humanos , Células MCF-7 , Estructura Molecular , Dióxido de Silicio/química , Relación Estructura-Actividad , Nucleótidos de Uracilo/síntesis química , Nucleótidos de Uracilo/químicaRESUMEN
Arrays of singly labeled short oligonucleotides that hybridize to a specific target revolutionized RNA biology, enabling quantitative, single-molecule microscopy analysis and high-efficiency RNA/RNP capture. Here, we describe a simple and efficient method that allows flexible functionalization of inexpensive DNA oligonucleotides by different fluorescent dyes or biotin using terminal deoxynucleotidyl transferase and custom-made functional group conjugated dideoxy-UTP. We show that (i) all steps of the oligonucleotide labeling-including conjugation, enzymatic synthesis, and product purification-can be performed in a standard biology laboratory, (ii) the process yields >90%, often >95% labeled product with minimal carryover of impurities, and (iii) the oligonucleotides can be labeled with different dyes or biotin, allowing single-molecule FISH, RNA affinity purification, and Northern blot analysis to be performed.
Asunto(s)
ADN Nucleotidilexotransferasa/metabolismo , Hibridación Fluorescente in Situ/métodos , Sondas ARN/química , Animales , Biotina , Didesoxinucleótidos/química , Didesoxinucleótidos/metabolismo , Drosophila melanogaster/genética , Femenino , Colorantes Fluorescentes/química , Sondas de Oligonucleótidos/química , Oligonucleótidos/química , Ovario/fisiología , Sondas ARN/metabolismo , Nucleótidos de Uracilo/química , Nucleótidos de Uracilo/metabolismoRESUMEN
P2Y6 receptor (P2Y6-R) is involved in various physiological and pathophysiological events. With a view to set rules for the design of UDP-based reversible P2Y6-R antagonists as potential drugs, we established structure-activity relationship of UDP analogues, bearing modifications at the uracil ring, ribose moiety, and the phosphate chain. For instance, C5-phenyl- or 3-NMe-uridine-5'-α,ß-methylene-diphosphonate, 16 and 23, or lack of 2'-OH, in 12-15, resulted in loss of both agonist and antagonist activity toward hP2Y6-R. However, uridylyl phosphosulfate, 19, selectively inhibited hP2Y6-R (IC50 112 µM) versus P2Y2/4-Rs. In summary, we have established a comprehensive SAR for hP2Y6-R ligands towards the development of hP2Y6-R antagonists.
Asunto(s)
Receptores Purinérgicos P2/química , Nucleótidos de Uracilo/síntesis química , Humanos , Estructura Molecular , Relación Estructura-Actividad , Nucleótidos de Uracilo/químicaRESUMEN
RNA molecules are subjected to post-transcriptional modifications that might determine their maturation, activity, localization and stability. These alterations can occur within the RNA molecule or at its 5'- or 3'- extremities, and are essential for gene regulation and proper function of the RNA. One major type of modification is the 3'-end addition of nontemplated nucleotides. Polyadenylation is the most well studied type of 3'-RNA modification, both in eukaryotes and prokaryotes. The importance of 3'-oligouridylation has recently gained attention through the discovery of several types of uridylated-RNAs, by the existence of enzymes that specifically add poly(U) tails and others that preferentially degrade these tails. Namely, Dis3L2 is a 3'-5' exoribonuclease from the RNase II/RNB family that has been shown to act preferentially on oligo(U)-tailed transcripts. Our understanding of this process is still at the beginning, but it is already known to interfere in the regulation of diverse RNA species in most eukaryotes. Now that we are aware of the prevalence of RNA uridylation and the techniques available to globally evaluate the 3'-terminome, we can expect to make rapid progress in determining the extent of terminal oligouridylation in different RNA populations and unravel its impact on RNA decay mechanisms. Here, we sum up what is known about 3'-RNA modification in the different cellular compartments of eukaryotic cells, the conserved enzymes that perform this 3'-end modification and the effectors that are selectively activated by this process.
Asunto(s)
Procesamiento de Término de ARN 3' , ARN/química , ARN/metabolismo , Animales , Compartimento Celular , Exorribonucleasas/química , Exorribonucleasas/metabolismo , Humanos , Redes y Vías Metabólicas , Modelos Biológicos , Modelos Moleculares , Oligorribonucleótidos/química , Oligorribonucleótidos/metabolismo , Poli U/química , Poli U/metabolismo , Conformación Proteica , Estabilidad del ARN , Nucleótidos de Uracilo/química , Nucleótidos de Uracilo/metabolismoRESUMEN
The pluripotency factor Lin28 inhibits the biogenesis of the let-7 family of mammalian microRNAs. Lin28 is highly expressed in embryonic stem cells and has a fundamental role in regulation of development, glucose metabolism and tissue regeneration. Overexpression of Lin28 is correlated with the onset of numerous cancers, whereas let-7, a tumour suppressor, silences several human oncogenes. Lin28 binds to precursor let-7 (pre-let-7) hairpins, triggering the 3' oligo-uridylation activity of TUT4 and TUT7 (refs 10-12). The oligoU tail added to pre-let-7 serves as a decay signal, as it is rapidly degraded by Dis3l2 (refs 13, 14), a homologue of the catalytic subunit of the RNA exosome. The molecular basis of Lin28-mediated recruitment of TUT4 and TUT7 to pre-let-7 and its subsequent degradation by Dis3l2 is largely unknown. To examine the mechanism of Dis3l2 substrate recognition we determined the structure of mouse Dis3l2 in complex with an oligoU RNA to mimic the uridylated tail of pre-let-7. Three RNA-binding domains form an open funnel on one face of the catalytic domain that allows RNA to navigate a path to the active site different from that of its exosome counterpart. The resulting path reveals an extensive network of uracil-specific interactions spanning the first 12 nucleotides of an oligoU-tailed RNA. We identify three U-specificity zones that explain how Dis3l2 recognizes, binds and processes uridylated pre-let-7 in the final step of the Lin28-let-7 pathway.
Asunto(s)
Exorribonucleasas/química , Exorribonucleasas/metabolismo , MicroARNs/metabolismo , Proteínas de Unión al ARN/metabolismo , Animales , Biocatálisis , Dominio Catalítico , Cristalografía por Rayos X , Complejo Multienzimático de Ribonucleasas del Exosoma/química , Ratones , MicroARNs/química , MicroARNs/genética , Modelos Moleculares , Oligorribonucleótidos/química , Oligorribonucleótidos/metabolismo , Proteínas de Unión al ARN/química , Proteínas de Schizosaccharomyces pombe/química , Especificidad por Sustrato , Nucleótidos de Uracilo/química , Nucleótidos de Uracilo/metabolismoRESUMEN
P2Y receptors are activated by nucleotides and involved in numerous physiological/pathophysiological processes. However, investigations of specific P2Y receptor functions have been hampered by lack of suitable receptor agonists-antagonists. Recently, we identified the nucleotide 5-OMe-UDP as potent and selective agonist for human P2Y6 receptors. We studied a series of derivatives of this analog with a Pα-borano group substituting a non-bridging oxygen and found increased potency and receptor specificity. Rp-5-OMe-UDPαB (Rp-5-OMe-uridine 5'-O-α-boranodiphosphate) was most potent and selective in inducing intracellular calcium signaling in 1321N1 astrocytoma cells expressing the human P2Y6 receptor. Here, we investigated whether Rp-5-OMe-UDPαB evokes cell protection through human P2Y6 receptors. We tested a well-established model, tumor necrosis factor α (TNFα)-induced cell death in 1321N1 astrocytoma cells. Rp-5-OMe-UDPαB inhibited TNFα-induced cell death even stronger than UDP. These first data of a neuro-protective activity of the human P2Y6 receptor emphasize the potential of the stable, selective, and potent Rp-5-OMe-UDPαB analog for exploiting P2Y6 receptor-mediated cellular functions, like cytoprotection in human tissues, with suitability for future neuro-protective drug development.
Asunto(s)
Apoptosis/efectos de los fármacos , Compuestos de Boro/farmacología , Neuroglía/efectos de los fármacos , Neuroglía/metabolismo , Fármacos Neuroprotectores/farmacología , Agonistas del Receptor Purinérgico P2/farmacología , Receptores Purinérgicos P2/metabolismo , Nucleótidos de Uracilo/farmacología , Uridina Difosfato/análogos & derivados , Señalización del Calcio/efectos de los fármacos , Línea Celular Tumoral , Humanos , Factor de Necrosis Tumoral alfa/farmacología , Nucleótidos de Uracilo/química , Uridina Difosfato/farmacologíaRESUMEN
Jakeman et al. recently reported the inability to distinguish the diastereomers of uridine 5'-ß,γ-fluoromethylenetriphosphate (ß,γ-CHF-UTP, 1) by (19)F NMR under conditions we previously prescribed for the resolution of the corresponding ß,γ-CHF-dGTP spectra, stating further that 1 decomposed under these basic conditions. Here we show that the (19)F NMR spectra of 1 (~1:1 diastereomer mixture prepared by coupling of UMP-morpholidate with fluoromethylenebis(phosphonic acid)) in D2O at pH 10 are indeed readily distinguishable. 1 in this solution was stable for 24 h at rt.
Asunto(s)
Nucleótidos de Desoxiguanina/química , Flúor/química , Nucleótidos de Uracilo/química , Uridina/química , Concentración de Iones de Hidrógeno , Espectroscopía de Resonancia Magnética , Modelos Moleculares , EstereoisomerismoRESUMEN
5-(hydroxymethyl)cytosine (5-hmC) is a newly identified oxidative product of 5-methylcytosine (5-mC) in the mammalian genome, and is believed to be an important epigenetic marker influencing a variety of biological processes. In addition to its relatively low abundance, the fluctuation of 5-hmC levels over time during cell development poses a formidable challenge for its accurate mapping and quantification. Here we describe a specific chemoenzymatic approach to 5-hmC detection in DNA samples by using new uridine 5'-diphosphoglucosamine (UDP-GlcN) probes. Our approach requires modification of the glucose moiety of UDP-Glc with small amino groups and transfer of these glucose derivatives to the hydroxy moiety of 5-hmC by using T4 phage glucosyltransferases. We evaluated the transfer efficiencies of three glucosyltransferases (wild-type α- and ß-GTs and a Y261L mutant ß-GT) with five different UDP-Glc derivatives containing functionalized groups for subsequent bioconjugation and detection. Our results indicate that UDP-6-N3 -Glc, UDP-6-GlcN, and UDP-2-GlcN can be transferred by ß-GT with efficiencies similar to that seen with the native UDP-Glc cofactor. 6-N3 -Glc- and 6-GlcN-containing oligonucleotides were selectively labeled with reactive fluorescent probes. In addition, a 2 kb DNA fragment modified with 2-GlcN groups was specifically detected by use of a commercially available antiglucosamine antibody. Alternative substrates for ß-GT and correlated glycosyltransferases might prove useful for the study of the function and dynamics of 5-hmC and other modified nucleotides, as well as for multiplex analysis.
Asunto(s)
Citosina/análogos & derivados , Glucosamina/química , Coloración y Etiquetado , Nucleótidos de Uracilo/química , 5-Metilcitosina/análogos & derivados , Animales , Anticuerpos/inmunología , Bacteriófago T4/enzimología , Citosina/química , ADN/química , ADN/metabolismo , Células Madre Embrionarias/citología , Células Madre Embrionarias/metabolismo , Colorantes Fluorescentes/química , Glucosamina/inmunología , Glucosiltransferasas/antagonistas & inhibidores , Glucosiltransferasas/genética , Glucosiltransferasas/metabolismo , Glicosilación , Ratones , Oligonucleótidos/síntesis química , Oligonucleótidos/química , Oligonucleótidos/metabolismo , Especificidad por SustratoRESUMEN
The secretion of angiogenic factors by vascular endothelial cells is one of the key mechanisms of angiogenesis. Here we report on the isolation of a new potent angiogenic factor, diuridine tetraphosphate (Up4U) from the secretome of human endothelial cells. The angiogenic effect of the endothelial secretome was partially reduced after incubation with alkaline phosphatase and abolished in the presence of suramin. In one fraction, purified to homogeneity by reversed phase and affinity chromatography, Up4U was identified by MALDI-LIFT-fragment-mass-spectrometry, enzymatic cleavage analysis and retention-time comparison. Beside a strong angiogenic effect on the yolk sac membrane and the developing rat embryo itself, Up4U increased the proliferation rate of endothelial cells and, in the presence of PDGF, of vascular smooth muscle cells. Up4U stimulated the migration rate of endothelial cells via P2Y2-receptors, increased the ability of endothelial cells to form capillary-like tubes and acts as a potent inducer of sprouting angiogenesis originating from gel-embedded EC spheroids. Endothelial cells released Up4U after stimulation with shear stress. Mean total plasma Up4U concentrations of healthy subjects (N=6) were sufficient to induce angiogenic and proliferative effects (1.34 ± 0.26 nmol L(-1)). In conclusion, Up4U is a novel strong human endothelium-derived angiogenic factor.
Asunto(s)
Inductores de la Angiogénesis/metabolismo , Endotelio Vascular/metabolismo , Adulto , Inductores de la Angiogénesis/química , Inductores de la Angiogénesis/farmacología , Animales , Movimiento Celular/efectos de los fármacos , Membrana Corioalantoides/efectos de los fármacos , Membrana Corioalantoides/embriología , Embrión de Mamíferos/metabolismo , Células Endoteliales/citología , Células Endoteliales/efectos de los fármacos , Células Endoteliales/enzimología , Endotelio Vascular/citología , Endotelio Vascular/efectos de los fármacos , Humanos , Técnicas In Vitro , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Peso Molecular , Músculo Liso Vascular/citología , Miocitos del Músculo Liso/citología , Miocitos del Músculo Liso/efectos de los fármacos , Miocitos del Músculo Liso/metabolismo , Fosforilación/efectos de los fármacos , Factor de Crecimiento Derivado de Plaquetas/farmacología , Ratas , Ratas Wistar , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Nucleótidos de Uracilo/química , Nucleótidos de Uracilo/metabolismo , Nucleótidos de Uracilo/farmacologíaRESUMEN
Derivatives of UMP (uridine monophosphate) with a fluorogenic substituent in position 5 represent a small but unique class of fluorophores, which has found important applications in chemical biology and biomolecular chemistry. In this study, we have synthesised a series of derivatives of the uracil nucleotides UMP, UDP and UTP with different aromatic and heteroaromatic substituents in position 5, in order to systematically investigate the influence of the 5-substituent on fluorescence emission. We have determined relevant photophysical parameters for all derivatives in this series, including quantum yields for the best fluorophores. The strongest fluorescence emission was observed with a 5-formylthien-2-yl substituent in position 5 of the uracil base, while the corresponding 3-formylthien-2-yl-substituted regioisomer was significantly less fluorescent. The 5-(5-formylthien-2-yl) uracil fluorophore was studied further in solvents of different polarity and proticity. In conjunction with results from a conformational analysis based on NMR data and computational experiments, these findings provide insights into the steric and electronic factors that govern fluorescence emission in this class of fluorophores. In particular, they highlight the interplay between fluorescence emission and conformation in this series. Finally, we carried out ligand-binding experiments with the 5-(5-formylthien-2-yl) uracil fluorophore and a UDP-sugar-dependent glycosyltransferase, demonstrating its utility for biological applications. The results from our photophysical and biological studies suggest, for the first time, a structural explanation for the fluorescence quenching effect that is observed upon binding of these fluorophores to a target protein.
Asunto(s)
Colorantes Fluorescentes/química , Nucleótidos de Uracilo/química , Simulación por Computador , Conformación Molecular , Estructura Molecular , Espectrometría de Fluorescencia , Factores de Tiempo , Nucleótidos de Uracilo/síntesis químicaRESUMEN
A new, straightforward, reliable, and convenient protection-free one-pot method for the synthesis of 2'-deoxynucleoside-5'-tetraphosphate and ribonucleoside-5'-tetraphosphate is reported. The present synthetic strategy involves the monophosphorylation of a nucleoside followed by reaction with tris-(tri-n-butylammonium) triphosphate and subsequent hydrolysis of the putative cyclic tetrametaphosphate intermediate to provide nucleoside-5'-tetraphosphate in moderate yield with high purity. A plausible mechanism is proposed to account for the formation of product.
Asunto(s)
Nucleótidos de Adenina/síntesis química , Fosfatos de Dinucleósidos/síntesis química , Guanosina Tetrafosfato/síntesis química , Nucleótidos de Uracilo/síntesis química , Nucleótidos de Adenina/química , Fosfatos de Dinucleósidos/química , Guanosina Tetrafosfato/química , Hidrólisis , Fosforilación , Polifosfatos/química , Nucleótidos de Uracilo/químicaRESUMEN
Considerable progress has been made in understanding the origins of genomic uracil and its role in genome stability and host defense; however, the main question concerning the basal level of uracil in DNA remains disputed. Results from assays designed to quantify genomic uracil vary by almost three orders of magnitude. To address the issues leading to this inconsistency, we explored possible shortcomings with existing methods and developed a sensitive LC/MS/MS-based method for the absolute quantification of genomic 2'-deoxyuridine (dUrd). To this end, DNA was enzymatically hydrolyzed to 2'-deoxyribonucleosides and dUrd was purified in a preparative HPLC step and analyzed by LC/MS/MS. The standard curve was linear over four orders of magnitude with a quantification limit of 5 fmol dUrd. Control samples demonstrated high inter-experimental accuracy (94.3%) and precision (CV 9.7%). An alternative method that employed UNG2 to excise uracil from DNA for LC/MS/MS analysis gave similar results, but the intra-assay variability was significantly greater. We quantified genomic dUrd in Ung(+/+) and Ung(-/-) mouse embryonic fibroblasts and human lymphoblastoid cell lines carrying UNG mutations. DNA-dUrd is 5-fold higher in Ung(-/-) than in Ung(+/+) fibroblasts and 11-fold higher in UNG2 dysfunctional than in UNG2 functional lymphoblastoid cells. We report approximately 400-600 dUrd per human or murine genome in repair-proficient cells, which is lower than results using other methods and suggests that genomic uracil levels may have previously been overestimated.
Asunto(s)
ADN/química , Nucleótidos de Uracilo/química , Animales , Línea Celular , Cromatografía Líquida de Alta Presión , Cromatografía de Fase Inversa , ADN/genética , ADN/aislamiento & purificación , Contaminación de ADN , Genoma Humano , Humanos , Hidrólisis , Límite de Detección , Ratones , Ratones Noqueados , Estándares de Referencia , Espectrometría de Masas en Tándem/normas , Nucleótidos de Uracilo/genética , Nucleótidos de Uracilo/aislamiento & purificación , Uracil-ADN Glicosidasa/genética , Uracil-ADN Glicosidasa/metabolismoRESUMEN
The general acid/base catalyzed cleavage of a number of alkyl esters of uridine-3'- (and -5'-)phosphate has been studied by utilizing a cleaving agent, in which the catalytic moiety (a substituted 1,3,5-triazine) is tethered to an anchoring Zn(II):cyclen moiety. Around pH 7, formation of a strong ternary complex between uracil, Zn(II) and cyclen brings the general acid/base catalyst close to the scissile phosphodiester linkage, resulting in rate acceleration of 1-2 orders of magnitude with the uridine-3'-phosphodiesters. Curiously, no acceleration was observed with their 5'-counterparts. A ß(lg) value of -0.7 has been determined for the general acid/base catalyzed cleavage, consistent with a proton transfer to the leaving group in the rate-limiting step.