RESUMEN
OBJECTIVE: Noninvasive determination of pancreatic beta-cell mass in vivo has been hampered by the lack of suitable beta-cell-specific imaging agents. This report outlines an approach for the development of novel ligands homing selectively to islet cells in vivo. RESEARCH DESIGN AND METHODS: To generate agents specifically binding to pancreatic islets, a phage library was screened for single-chain antibodies (SCAs) on rat islets using two different approaches. 1) The library was injected into rats in vivo, and islets were isolated after a circulation time of 5 min. 2) Pancreatic islets were directly isolated, and the library was panned in the islets in vitro. Subsequently, the identified SCAs were extensively characterized in vitro and in vivo. RESULTS: We report the generation of SCAs that bind highly selective to either beta- or alpha-cells. These SCAs are internalized by target cells, disappear rapidly from the vasculature, and exert no toxicity in vivo. Specific binding to beta- or alpha-cells was detected in cell lines in vitro, in rats in vivo, and in human tissue in situ. Electron microscopy demonstrated binding of SCAs to the endoplasmatic reticulum and the secretory granules. Finally, in a biodistribution study the labeling intensity derived from [(125)I]-labeled SCAs after intravenous administration in rats strongly predicted the beta-cell mass and was inversely related to the glucose excursions during an intraperitoneal glucose tolerance test. CONCLUSIONS: Our data provide strong evidence that the presented SCAs are highly specific for pancreatic beta-cells and enable imaging and quantification in vivo.
Asunto(s)
Diabetes Mellitus Experimental/fisiopatología , Células Secretoras de Glucagón/ultraestructura , Células Secretoras de Insulina/ultraestructura , Animales , Anticuerpos/análisis , Especificidad de Anticuerpos , Apoptosis , Línea Celular , Supervivencia Celular , Diabetes Mellitus Experimental/patología , Retículo Endoplásmico/inmunología , Femenino , Células Secretoras de Glucagón/inmunología , Células Secretoras de Glucagón/patología , Prueba de Tolerancia a la Glucosa , Humanos , Inmunohistoquímica , Células Secretoras de Insulina/inmunología , Células Secretoras de Insulina/patología , Microscopía Electrónica , Ratas , Vesículas Secretoras/inmunología , Vesículas Secretoras/patologíaRESUMEN
Human beta-glucuronidase, due to low intrinsic immunogenicity in humans, is an attractive enzyme for tumor-specific prodrug activation, but its utility is hindered by low activity at physiological pH. Here we describe the development of a high-throughput screening procedure for enzymatic activity based on the stable retention of fluorescent reaction product in mammalian cells expressing properly folded glycoproteins on their surface. We utilized this procedure on error-prone PCR and saturation mutagenesis libraries to isolate beta-glucuronidase tetramers that were up to 60-fold more active (k(cat)/K(m)) at pH 7.0 and were up to an order of magnitude more effective at catalyzing the conversion of two structurally disparate glucuronide prodrugs to anticancer agents. The screening procedure described here can facilitate investigation of eukaryotic enzymes requiring posttranslational modifications for biological activity.
Asunto(s)
Glucuronidasa/genética , Lisosomas/enzimología , Proteínas de la Membrana/genética , Ingeniería de Proteínas , Animales , Variación Genética , Glucuronidasa/metabolismo , Glucuronidasa/farmacología , Humanos , Concentración de Iones de Hidrógeno , Lisosomas/genética , Proteínas de la Membrana/metabolismo , Modelos Moleculares , Estructura Molecular , Profármacos/metabolismo , Regulación hacia ArribaRESUMEN
UNLABELLED: Preeclampsia (PE) is a significant cause of fetal and maternal mortality around the world and there is evidence that insulin resistance has been implicated in the pathophysiology of PE. The Akt/PKB pathway is stimulated by insulin and performs several vital functions relative to growth, survival and cellular metabolism. OBJECTIVE: To investigate the basal expression of Akt/PKB, HSP90 expression, proteins that regulate Akt/PKB activity and substrate in the placenta, skeletal muscle and adipocytes of normal and PE parturient. METHOD: Samples were collected from 17 normal patients and 17 PE patients, and analyzed by Western blot to quantify the protein expression involved in signaling cascade of Akt/PKB. RESULTS: Total Akt/PKB expression for normal placentas was 1.85 (1.07-3.12) and 1.53 (1.27-3.08) in PE (p = 1.00); in the adipose tissue of normal placentas it was 1.10 (0.53-1.73) and 1.66 (0.83-2.00) in PE (p = 0.37). CONCLUSIONS: There was no difference in the Akt/PKB pathway, in basal state, in placentas and skeletal muscle of normal and PE patients. However, defects in this signaling pathway as pathophysiology of PE cannot be excluded because it is necessary to analyze this pathway during stimulation.