RESUMEN
Dual-tracer molecular imaging is a powerful approach to quantify receptor expression in a wide range of tissues by using an untargeted tracer to account for any nonspecific uptake of a molecular-targeted tracer. This approach has previously required the pharmacokinetics of the receptor-targeted and untargeted tracers to be identical, requiring careful selection of an ideal untargeted tracer for any given targeted tracer. In this study, methodology capable of correcting for tracer differences in arterial input functions, as well as binding-independent delivery and retention, is derived and evaluated in a mouse U251 glioma xenograft model using an Affibody tracer targeted to epidermal growth factor receptor (EGFR), a cell membrane receptor overexpressed in many cancers. Simulations demonstrated that blood, and to a lesser extent vascular-permeability, pharmacokinetic differences between targeted and untargeted tracers could be quantified by deconvolving the uptakes of the two tracers in a region of interest devoid of targeted tracer binding, and therefore corrected for, by convolving the uptake of the untargeted tracer in all regions of interest by the product of the deconvolution. Using fluorescently labeled, EGFR-targeted and untargeted Affibodies (known to have different blood clearance rates), the average tumor concentration of EGFR in four mice was estimated using dual-tracer kinetic modeling to be 3.9 ± 2.4 nM compared to an expected concentration of 2.0 ± 0.4 nM. However, with deconvolution correction a more equivalent EGFR concentration of 2.0 ± 0.4 nM was measured.
Asunto(s)
Receptores ErbB/metabolismo , Imagen Molecular/métodos , Animales , Arterias Carótidas/metabolismo , Línea Celular Tumoral , Transformación Celular Neoplásica , Femenino , Humanos , Cinética , Ratones , Trazadores Radiactivos , Distribución TisularRESUMEN
Osteoblast-like cells were grown on a surface that presents cell membrane components to the cells in culture. The culture surface was a bimolecular layer formed by the interaction of osteoblast plasma membrane vesicles with an alkanethiol monolayer. The potential of these osteoblast-membrane hybrid bilayers for promoting osteoblast adhesion, growth and differentiation was examined. UMR-106 osteoblast-like cells cultured on these surfaces are normal in appearance, and in the presence of serum, proliferate as well or better than on control surfaces. The level of alkaline phosphatase production in the presence and absence of serum suggests that the osteoblast-like cells retain their differentiated phenotype, and appear to respond to the cell surface ligands presented by the osteoblast-membrane biomimetic surface. These observations suggest that biomimetic membrane films prepared from osteoblast cell membranes support osteoblast cell growth, allow the cells to maintain their differentiation state and may be suitable as a model system to probe cell-cell interactions.
RESUMEN
Six photoactivatable analogs of the human thrombin receptor activating peptide (TRAP), SFLLRN-NH2, were synthesized by substituting the photoactive amino acid, p-benzoylphenylalanine (Bpa), into each position of the peptide sequence. Platelet aggregation assays indicated that the peptides with Bpa substitutions at positions 3 to 6 retained agonist activity. These peptides were prepared in tritiated form as potential thrombin receptor photoaffinity labels. The [3H]Bpa-containing analogs were constructed by resynthesizing the peptides with the amino acid, 4-benzoyl-2',5'-dibromophenylalanine (Br2Bpa), and subjecting the purified peptides to Pd-catalyzed tritiodebromination. The radiochemical yields for the reductive tritiation were < 2% for peptides with [3H]Bpa in the third and fourth positions, and between 7 and 16% for the peptides with substitutions at the fifth and sixth positions. The low yields were due to over-reduction of the Bpa carbonyl group and nonspecific degradation during reductive tritiation. This report describes the first use of Br2Bpa for the preparation of tritiated photoactivatable peptides.
Asunto(s)
Fragmentos de Péptidos/farmacología , Receptores de Trombina/agonistas , Plaquetas/efectos de los fármacos , Reactivos de Enlaces Cruzados , Humanos , Ligandos , Luz , Fragmentos de Péptidos/síntesis química , Fenilalanina/análogos & derivados , Fenilalanina/química , Etiquetas de Fotoafinidad , Receptor PAR-1 , Tritio/químicaRESUMEN
Right ventricular (RV) dysfunction has adverse effects on long-term outcome in patients with repaired tetralogy of Fallot (TOF). We employed serial radionuclide angiography (RNA) to examine RV and left ventricular (LV) systolic function in adults late after TOF repair and its relation to clinical outcome. We reviewed 10-year records of 95 patients (53 men) with TOF followed in our clinic (mean age at repair 12.6 +/- 10.5 years, mean age at last follow-up 37.7 +/- 9.8 years) who underwent at least 2 RNAs between 1987 and 1997. Most patients were well by the end of the study (80% were New York Heart Association class I, 17% were class II, and 3% were in class III). Sixteen patients experienced sustained tachyarrhythmias (8 had atrial; 8 patients had ventricular). One patient died suddenly. Fifteen patients underwent RV outflow reoperations (15 underwent pulmonary valve replacement; 7 had relief of RV outflow obstruction); RV systolic function during exercise in these 15 patients was significantly impaired before and returned to similar levels after surgery, compared with the rest of the patients. Overall, RV and LV function remained stable in the whole group at a mean interval of 5.7 +/- 2.2 years between first and last RNA. This group of closely followed adults with TOF remained well over 10 years with a low incidence of sudden death and stable RV and LV systolic function, despite a relatively large number of RV outflow reoperations. Aggressive intervention for right-sided hemodynamic abnormalities may have contributed to this outcome. Preserved ventricular function may herald a favorable long-term outlook in this group.
Asunto(s)
Sístole/fisiología , Tetralogía de Fallot/cirugía , Función Ventricular Izquierda/fisiología , Función Ventricular Derecha/fisiología , Adulto , Distribución de Chi-Cuadrado , Muerte Súbita Cardíaca/etiología , Ecocardiografía , Electrocardiografía , Femenino , Estudios de Seguimiento , Humanos , Incidencia , Análisis de los Mínimos Cuadrados , Modelos Lineales , Masculino , Esfuerzo Físico/fisiología , Válvula Pulmonar/cirugía , Angiografía por Radionúclidos , Ventriculografía con Radionúclidos , Reoperación , Estudios Retrospectivos , Estadísticas no Paramétricas , Taquicardia/etiología , Taquicardia Ventricular/etiología , Tetralogía de Fallot/fisiopatología , Resultado del Tratamiento , Disfunción Ventricular Derecha/etiología , Obstrucción del Flujo Ventricular Externo/cirugíaRESUMEN
Two maleimide-containing diacylglycerol derivatives were synthesized to permit the anchoring of short peptides and longer polypeptides to phospholipid bilayers and membranes. The maleimide was introduced at the site normally occupied by a phospholipid headgroup. The first lipid, the dipalmitoyl ester of 1-maleimido-2,3-propanediol, was developed as a membrane anchor for extracellular domains of transmembrane proteins. The second anchoring lipid, in which the 3-position contained a 6-aminohexanoate, was designed for convenient modification with amine-reactive reporter groups. Specifically, the NBD fluorophore, 7-nitrobenzo-2-oxa-1, 3-diazole-aminohexanoic-N-hydroxysuccinimide ester, was attached to give an fluorescent anchoring reagent. Next, these reagents were applied to the anchoring of a C-terminally cysteamine-modified 8 kDa polypeptide that comprises the extracellular N-terminal domain of the human thrombin receptor, a transmembrane protease-activated receptor (PAR-1). Gel filtration and fluorescence analysis showed that the fluorescent lipopolypeptide spontaneously inserted into preformed phospholipid vesicles, but it did not insert into whole cell membranes. In contrast, the dipalmitoyl derivative could only be reconstituted into artificial membranes by mixing the lipopolypeptide and phospholipid before vesicle formation. These results suggest that biophysical interactions governing the lipopolypeptide insertion into artificial and cellular membranes may differ. The thiol-reactive lipidating reagents should be valuable materials for studying the structure and function of peptides and polypeptides at phospholipid bilayer surfaces.
Asunto(s)
Membrana Dobles de Lípidos , Maleimidas/química , Fosfolípidos/química , Secuencia de Aminoácidos , Membrana Celular/química , Cromatografía en Gel , Colorantes Fluorescentes , Humanos , Receptores de Trombina/químicaRESUMEN
Phospholipase C (PLC)-beta1 and PLC-beta2 are regulated by the Gq family of heterotrimeric G proteins and contain C2 domains. These domains are Ca2+-binding modules that serve as membrane-attachment motifs in a number of signal transduction proteins. To determine the role that C2 domains play in PLC-beta1 and PLC-beta2 function, we measured the binding of the isolated C2 domains to membrane bilayers. We found, unexpectedly, that these modules do not bind to membranes but they associate strongly and specifically to activated [guanosine 5'-[gamma-thio]triphosphate (GTP[gammaS])-bound] Galphaq subunits. The C2 domain of PLC-beta1 effectively suppressed the activation of the intact isozyme by Galphaq(GTP[gammaS]), indicating that the C2-Galphaq interaction may be physiologically relevant. C2 affinity for Galphaq(GTP[gammaS]) was reduced when Galphaq was deactivated to the GDP-bound state. Binding to activated Galphai1 subunits or to Gbetagamma subunits was not detected. Also, Galphaq(GTP[gammaS]) failed to associate with the C2 domain of PLC-delta, an isozyme that is not activated by Galphaq. These results indicate that the C2 domains of PLC-beta1 and PLC-beta2 provide a surface to which Galphaq subunits can dock, leading to activation of the native protein.
Asunto(s)
Proteínas de Unión al GTP/química , Proteínas de Unión al GTP/metabolismo , Isoenzimas/química , Isoenzimas/metabolismo , Fragmentos de Péptidos/química , Fosfolipasas de Tipo C/química , Fosfolipasas de Tipo C/metabolismo , Animales , Línea Celular , Guanosina 5'-O-(3-Tiotrifosfato)/metabolismo , Cinética , Sustancias Macromoleculares , Fosfolipasa C beta , Proteína Quinasa C/química , Proteína Quinasa C/metabolismo , Proteína Quinasa C-delta , Proteolípidos/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Transducción de Señal , Spodoptera , TransfecciónRESUMEN
Photoactivatable analogs of the human thrombin receptor (PAR-1) antagonist, N-trans-cinnamoyl-p-fluoroPhe-p-guanidinoPhe-Leu-Arg-NH2 (BMS-197525), were prepared with benzophenone substitutions in the N-terminal, Leu, or Arg position. The analogs retained antagonist activity (with reduced potency); the tritium-labeled isotopomers are potential photoaffinity labels for the receptor. C-Terminal extension of the analogs with ornithine(biotin) did not significantly alter antagonist potency.
Asunto(s)
Guanidinas/farmacología , Luz , Oligopéptidos/farmacología , Receptores de Trombina/antagonistas & inhibidores , Cromatografía Líquida de Alta Presión , Reactivos de Enlaces Cruzados , Humanos , Concentración 50 Inhibidora , Receptor PAR-1RESUMEN
A photoaffinity analog of Helicoverpa zea pheromone biosynthesis activating neuropeptide (Hez-PBAN) was used to identify PBAN binding proteins in various tissues of the corn earworm moth, H. zea. Synthetic Hez-PBAN was derivatized on Lys-27 with p-benzoyldihydrocinnamoyl-N-hydroxysuccinimide ester (BZDC-NHS ester). The resulting BZDC-PBAN stimulated pheromone production in H. zea isolated abdomens at levels comparable to those of the unmodified peptide. Photoaffinity labeling experiments using [3H]BZDC-PBAN with female moth tissues revealed soluble 100 and 115 kDa proteins in the brain-subesophageal ganglia complex, ventral nerve cord, and thoracic muscle that were specifically labeled with the PBAN analog.
Asunto(s)
Proteínas Portadoras/análisis , Proteínas de Insectos/metabolismo , Neuropéptidos/metabolismo , Atractivos Sexuales/metabolismo , Marcadores de Afinidad , Secuencia de Aminoácidos , Animales , Electroforesis en Gel de Poliacrilamida , Femenino , Proteínas de Insectos/biosíntesis , Proteínas de Insectos/fisiología , Datos de Secuencia Molecular , Mariposas Nocturnas , Neuropéptidos/biosíntesis , Neuropéptidos/fisiología , Fotofluorografía , Atractivos Sexuales/biosíntesis , Atractivos Sexuales/fisiologíaRESUMEN
Benzophenones (BP) and related aryl ketone photophores have become established as the photoactivatable group of choice for high-efficiency covalent modification of hydrophobic regions of binding proteins, including enzymes and receptors that recognize peptide hormones, (oligo) nucleotides and nucleosides, phosphoinositides, inositol polyphosphates and a wide variety of therapeutic molecules. This review presents the advantages of BP as photoaffinity labels and provides specific examples from the last 3 years of applications of BP-containing ligands used in biochemistry.
Asunto(s)
Marcadores de Afinidad/química , Benzofenonas/química , Péptidos/análisis , Preparaciones Farmacéuticas/análisis , Fosfatidilinositoles/análisis , Fotobiología/métodos , Fármacos Fotosensibilizantes/química , Marcadores de Afinidad/síntesis química , Benzofenonas/síntesis química , Fármacos Fotosensibilizantes/síntesis químicaRESUMEN
Synaptotagmin (Syt) II, a synaptic vesicle protein containing two copies of highly conserved protein kinase C homology regions known as the C2A and C2B domains, acts as a Ca2+ sensor and provides both phospholipid and inositol polyphosphate (IPn) recognition domains important in endo- and exocytosis. Four photoaffinity analogues of IP3, IP4, and IP6 containing a P-1- or P-2-linked 4-benzoyldihydrocinnamidyl (BZDC) photophore were used to label glutathione S-transferase (GST) fusion constructs of the Syt II-C2A and C2B domains. The P-2-linked [3H]BZDC-IP6 showed efficient, IP6-displaceable labeling of the GST-Syt II-C2B. The rank order of photocovalent modification paralleled the order of competitive displacement: IP6 (P-2-linked) > IP4 > IP3. The P-1-linked [3H]BZDC-IP6 failed to label the C2B domains. The GST-Syt III-C2B domain, which lacks IP6 binding affinity, also failed to undergo labeling by P-2-linked [3H]BZDC-IP6. When mixtures of the 32-amino acid basic peptide corresponding to the essential IPn binding region of the Syt II-C2B domain and GST-Syt II-C2B were labeled by a stoichiometric amount of P-2-linked [3H]BZDC-IP6, the two polypeptides showed equivalent affinity for the photolabel. Although the CD spectrum of this 32-mer at two pH values showed a random coil, the photoaffinity analogue of IP6 appeared to induce a binding-compatible structure in the short peptide.
Asunto(s)
Fosfatos de Inositol/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Marcadores de Afinidad , Secuencia de Aminoácidos , Animales , Benzofenonas/química , Unión Competitiva , Fosfatos de Inositol/química , Cinética , Ratones , Datos de Secuencia Molecular , Péptidos/metabolismo , Fotólisis , Unión Proteica , Sinaptotagmina II , TritioRESUMEN
A new radiolabeled, bifunctional photoaffinity cross-linking reagent, N-succinimidyl p-benzoyl-[2,3-3H2]dihydrocinnamate, has been synthesized in high yield and with high specific activity. This reagent can be used to append the benzophenone photophore to amino groups of small molecules, such as O-aminoalkylinositol polyphosphates and polypeptides. The resulting tritiated photoaffinity labels can be purified and manipulated in ambient light and can be activated at 360 nm.
Asunto(s)
Marcadores de Afinidad/síntesis química , Benzofenonas , Benzofenonas/síntesis química , Reactivos de Enlaces Cruzados/síntesis química , Succinimidas/síntesis química , Marcadores de Afinidad/química , Benzofenonas/química , Reactivos de Enlaces Cruzados/química , Indicadores y Reactivos , Fosfatos de Inositol , Marcaje Isotópico , Ligandos , Espectroscopía de Resonancia Magnética , Péptidos , Fotoquímica , Espectrofotometría Ultravioleta , Succinimidas/química , TritioRESUMEN
The molecular structure of human liver fructose-1,6-bisphosphatase complexed with AMP was determined by x-ray diffraction using molecular replacement, starting from the pig kidney enzyme AMP complex. Of the 34 amino acid residues which differ between these two sequences, only one interacts with AMP; Met30 in pig kidney is Leu30 in human liver. From this analysis, six sites in which side chains of amino acid residues are in contact with AMP, Ala24, Leu30, Thr31, Tyr113, Arg140, and Met177, were mutated by polymerase chain reaction. The wild-type and mutant forms were expressed in Escherichia coli, purified, and their kinetic properties determined. Circular dichroism spectra of the mutants were indistinguishable from that of the wild-type enzyme. Kinetic analyses revealed that all forms had similar turnover numbers, Km values for fructose 2,6-bisphosphate, and inhibition constants for fructose 2,6-bisphosphate. Apparent Ki values for AMP inhibition of the Leu30 --> Phe and Met177 --> Ala mutants were similar to those of the wild-type enzyme, but the apparent Ki values for the Arg140 --> Ala and Ala24 --> Phe mutants were 7-to 20-fold higher, respectively. The Thr31 --> Ser mutant exhibited a 5-fold increase in apparent Ki for AMP, while mutation of Thr31 to Ala increased the apparent Ki 120-fold. AMP inhibition of the Tyr113 --> Phe mutant was undetectable even at millimolar AMP concentrations. Fructose 2,6-bisphosphate potentiated AMP inhibition of the mutants to the same extent as for the wild-type enzyme, except in the case of the Thr31 --> Ala and Tyr113 --> Phe mutants. Thus, the Met177 --> Ala mutant suggests that the side chain beyond C alpha is not needed for AMP binding, and that the Leu30 --> Phe mutant preserves the AMP contacts with these side chains. Thr31, Tyr113, and Arg140 form key hydrogen bonds to AMP consistent with strong side chain interactions in the wild-type enzyme. Finally, the absence of any effect of fructose 2,6-bisphosphate on AMP inhibition observed in the Thr31 --> Ala mutant may be an important clue relating to the mechanism of synergism of these two inhibitors.