16 research outputs found
Ectopic synthesis of high-Mr calcitonin by the BEN lung carcinoma cell line reflects aberrant proteolytic processing
No full textAbstractCloning and nucleotide sequence analysis of the human calcitonin mRNA from the BEN lung carcinoma cell line, a cell line known to secrete high-Mr forms of calcitonin, showed no difference in the Coding region at the nucleotide level compared with calcitonin mRNA isolated from medullary thyroid carcinoma which secretes calcitonin monomer. Therefore, the secretion of high-Mr forms of calcitonin reflects the absence or limited activity of proteolytic processing enzymes within the secretory pathway of this cell line. In all other respects, as judged by RNA blotting and S1 mapping, calcitonin/α-CGRP expression was identical to that found in medullary thyroid carcinoma, including the differential use of an alternative splice donor site within intron 1. The BEN cell line also produces low levels of α-CGRP mRNA and secretes CGRP antigenic determinants. Analysis of plasma CGRP levels in 12 patients with anaplastic lung carcinoma showed elevated levels in 11 of these, suggesting that CGRP may be an important diagnostic marker for this disease
Constitutive and NF-κB—like proteins in the regulation of the serum amyloid a gene by interleukin 1
No full textPartial nucleotide sequence of human calcitonin precursor mRNA identifies flanking cryptic peptides
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Field trip guide to Oligocene Limestones and Caves in the Waitomo District
No full textThe field guide runs from Hamilton to Waitomo to Te Anga and return in limestone-dominated country developed in transgressive sedimentary deposits of the Oligocene Te Kuiti Group – a world class example of a temperate shelf carbonate depositional system. Attention focuses on the nature, distribution and paleoenvironmental controls of the main limestone facies and some of the mixed terrigenous-carbonate facies in the Group. Along the way features of the Waitomo karst landscape are noted and the trip concludes by going underground in the Ruakuri Cave to discuss cave origins and the evidence for paleoenvironmental changes locked up in speleothems
Lipid-peptide nanocomplexes for mRNA delivery in vitro and in vivo
No full textDespite recent advances in the field of mRNA therapy, the lack of safe and efficacious delivery vehicles with pharmaceutically developable properties remains a major limitation. Here, we describe the systematic optimisation of lipid-peptide nanocomplexes for the delivery of mRNA in two murine cancer cell types, B16-F10 melanoma and CT26 colon carcinoma as well as NCI-H358 human lung bronchoalveolar cells. Different combinations of lipids and peptides were screened from an original lipid-peptide nanocomplex formulation for improved luciferase mRNA transfection in vitro by a multi-factorial screening approach. This led to the identification of key structural elements within the nanocomplex associated with substantial improvements in mRNA transfection efficiency included alkyl tail length of the cationic lipid, the fusogenic phospholipid, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), and cholesterol. The peptide component (K16GACYGLPHKFCG) was further improved by the inclusion of a linker, RVRR, that is cleavable by the endosomal enzymes cathepsin B and furin, and a hydrophobic motif (X-S-X) between the mRNA packaging (K16) and receptor targeting domains (CYGLPHKFCG). Nanocomplex transfections of a murine B16-F10 melanoma tumour supported the inclusion of cholesterol for optimal transfection in vivo as well as in vitro. In vitro transfections were also performed with mRNA encoding interleukin-15 as a potential immunotherapy agent and again, the optimised formulation with the key structural elements demonstrated significantly higher expression than the original formulation. Physicochemical characterisation of the nanocomplexes over time indicated that the optimal formulation retained biophysical properties such as size, charge and mRNA complexation efficiency for 14 days upon storage at 4 °C without the need for additional stabilising agents. In summary, we have developed an efficacious lipid-peptide nanocomplex with promising pharmaceutical development properties for the delivery of therapeutic mRNA
Abstract 2713: Discovery and characterization of AZD8701, a high affinity antisense oligonucleotide targeting FOXP3 to relieve immunosuppression in cancer
No full textPharmacological Characterization of a Novel ENaCα siRNA (GSK2225745) With Potential for the Treatment of Cystic Fibrosis.
No full textLung pathology in cystic fibrosis is linked to dehydration of the airways epithelial surface which in part results from inappropriately raised sodium reabsorption through the epithelial sodium channel (ENaC). To identify a small-interfering RNA (siRNA) which selectively inhibits ENaC expression, chemically modified 21-mer siRNAs targeting human ENaCα were designed and screened. GSK2225745, was identified as a potent inhibitor of ENaCα mRNA (EC(50) (half maximal effective concentration) = 0.4 nmol/l, maximum knockdown = 85%) and protein levels in A549 cells. Engagement of the RNA interference (RNAi) pathway was confirmed using 5' RACE. Further profiling was carried out in therapeutically relevant human primary cells. In bronchial epithelial cells, GSK2225745 elicited potent suppression of ENaCα mRNA (EC(50) = 1.6 nmol/l, maximum knockdown = 82%). In human nasal epithelial cells, GSK2225745 also produced potent and long-lasting (≥72 hours) suppression of ENaCα mRNA levels which was associated with significant inhibition of ENaC function (69% inhibition of amiloride-sensitive current in cells treated with GSK2225745 at 10 nmol/l). GSK2225745 showed no evidence for potential to stimulate toll-like receptor (TLR)3, 7 or 8. In vivo, topical delivery of GSK2225745 in a lipid nanoparticle formulation to the airways of mice resulted in significant inhibition of the expression of ENaCα in the lungs. In conclusion, GSK2225745 is a potent inhibitor of ENaCα expression and warrants further evaluation as a potential novel inhaled therapeutic for cystic fibrosis.Molecular Therapy - Nucleic Acids (2013) 2, e65; doi:10.1038/mtna.2012.57; published online 15 January 2013
