RESUMEN

Antisense oligonucleotide (ASO) silencing of the expression of disease-associated genes is an attractive novel therapeutic approach, but treatments are limited by the ability to deliver ASOs to cells and tissues. Following systemic administration, ASOs preferentially accumulate in liver and kidney. Among the cell types refractory to ASO uptake is the pancreatic insulin-secreting ß-cell. Here, we show that conjugation of ASOs to a ligand of the glucagon-like peptide-1 receptor (GLP1R) can productively deliver ASO cargo to pancreatic ß-cells both in vitro and in vivo. Ligand-conjugated ASOs silenced target genes in pancreatic islets at doses that did not affect target gene expression in liver or other tissues, indicating enhanced tissue and cell type specificity. This finding has potential to broaden the use of ASO technology, opening up novel therapeutic opportunities, and presents an innovative approach for targeted delivery of ASOs to additional cell types.

Asunto(s)

Sistemas de Liberación de Medicamentos/métodos , Receptor del Péptido 1 Similar al Glucagón/metabolismo , Células Secretoras de Insulina/efectos de los fármacos , Oligonucleótidos Antisentido/administración & dosificación , Animales , Proteína Forkhead Box O1/genética , Proteína Forkhead Box O1/metabolismo , Silenciador del Gen , Receptor del Péptido 1 Similar al Glucagón/agonistas , Receptor del Péptido 1 Similar al Glucagón/genética , Células HEK293 , Humanos , Ratones Endogámicos C57BL , Ratones Noqueados , Oligonucleótidos Antisentido/química , Oligonucleótidos Antisentido/farmacocinética , ARN Largo no Codificante/genética

RESUMEN

Different O-glycosyl trichloroacetimidates bearing base sensitive Fmoc protected hydroxy groups were efficiently prepared with CCl(3)CN using a catalytic amount of sodium hydride. The resulting glycosyl donors were engaged in glycosylation reactions both in solution and on solid support with a new ester-type linker with good results. In both approaches, Fmoc groups were afterward quantitatively cleaved using mild basic conditions.

Asunto(s)

Oligosacáridos/síntesis química , Secuencia de Carbohidratos , Datos de Secuencia Molecular

RESUMEN

Upon agonist activation, the nicotinic acetylcholine receptor undergoes allosteric transitions leading to channel opening and sodium ion influx. The molecular structure of the agonist binding site has been mapped previously by photoaffinity labeling, but most photosensitive probes used for this purpose interact only with closed receptor states (resting or desensitized). We have synthesized two novel photoactivatable 4-diazocyclohexa-2,5-dienone derivatives as cholinergic agonist candidates, with the objective of identifying structural changes at the acetylcholine binding site associated with receptor activation. One of these ligands, 9b, is a functional agonist at muscle acetylcholine receptors in human TE 671 cells. In photolabeling experiments with 9b, up to 35% inactivation of agonist binding sites was observed at Torpedo acetylcholine receptors. Tritiated 9b was synthesized, and photolabeling was found to occur mainly on the alpha-subunit in a partially protectable manner. This novel radiolabeled photoprobe appears to be suitable for future investigation of the molecular dynamics of allosteric transitions occurring at the active acetylcholine receptor binding site.

Asunto(s)

Receptores Nicotínicos/química , Animales , Línea Celular , Humanos , Espectroscopía de Resonancia Magnética , Espectrometría de Masas , Sondas Moleculares , Agonistas Nicotínicos/farmacología , Técnicas de Placa-Clamp , Fotoquímica , Receptores Nicotínicos/efectos de los fármacos , Receptores Nicotínicos/metabolismo , Torpedo , Tritio

RESUMEN

Genetic analyses were conducted among 18 provisionary isozyme loci in Cucumis sativus L. Fourteen loci demonstrated simple Mendelian inheritance while observed variation at four loci (Gpi2, Gr2, Pgm3, Skdh2) was determined not to have a predictable genetic basis. Joint segregation analyses among the 14 genetically predictable polymorphic loci resulted in the assignment of 12 loci to four linkage groups. Linkage groups contain the following loci: (1) Gr1, Pgm1, Idh, Pgd1; (2) Pep-pap, Mdh2, Mdh3, Gpi1; (3) Pep-la, Per4; (4) Pgd2, G2dh. Mpi2 and Mdh1 segregated independently. Recombination fractions for linked loci ranged between 0.051 (Pgm1-Idh) to 0.385 (Pep-la-Per4). Some practical applications of isozyme marker loci for cucumer improvement are discussed.

RESUMEN

The genetic diversity of the U.S. Cucumis sativus L. germplasm collection [757 plant introductions (PI) representing 45 countries] was assessed using 40 enzymes which represented 74 biochemical loci. Polymorphisms were observed at 18 loci (G2dh-1, Gpi-1, Gpi-2, Gr-1, Gr-2, Idh, Mdh-1, Mdh-2, Mdh-3, Mpi-2, Pepla-2, Peppap-2, Per-4, Pgd-1, Pgd-2, Pgm-1, Pgm-3, and Skdh). Two PIs (285606 and 215589) contained alleles [G2dh-1(1) and Per-4(2), respectively] which did not occur in any other PI. Other alleles which occurred in low frequencies (in < 1% of the PIs) included Gpi-1(3), Gpi-2(3), Gr-1(3), Gr-2(1), Idh(1), Mdh-1(2), Mdh-2(1), Peppap-2(1), and Pgd-1(1). Individual loci containing more than one allele in greater than 20% of the PIs included Mpi-2, Pepla-2, Pgd-2, and Pgm-1. Multivariate analyses aided in the reduction of data (principle components), depicted relationships among PIs (cluster), and identified the most discriminating enzyme loci (Pgm-1, Pepla-2, Gr-1, Pgd-2, Mpi-2, and Skdh) (classification and regression tree).