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C W Pouton

Publications and source records attributed to C W Pouton.

27 records · Page 2Linked to original sources
PubMed1994

The melanocortin (MC3) receptor from rat hypothalamus: photoaffinity labelling and binding of alanine-substituted alpha-MSH analogues.

Membrane preparations of cells expressing the cloned rat hypothalamus melanocortin receptor, MC3, have been photoaffinity labelled using a radiolabelled photoreactive analogue of alpha-MSH, [125I-Tyr2,Nle4,D-Phe7,ATB-Lys11]alpha-MSH. SDS-PAGE followed by autoradiography showed a single band at 53-56 kDa for the native receptor or 35 kDa after deglycosylated with PNGase F, consistent with the predicted cDNA sequence. Receptor binding studies with alpha-MSH, gamma-MSH and [Nle4,D-Phe7]alpha-MSH established that alpha-MSH and gamma-MSH had similar affinities while [Nle4,D-Phe7]alpha-MSH bound 100 times more strongly. These results suggest that the receptor recognises the conserved 'core sequence' (-Met-Glu/Gly-His-Phe-Arg-Trp-) of MSH/ACTH peptides. The binding affinities of alanine-substituted analogues of alpha-MSH were determined to investigate the role of individual residues in ligand-receptor interactions. While in the terminal regions only the replacement of Tyr2 reduced the affinity of the peptide, replacement of Met4, Phe7, Arg8 and Trp9 within the peptide core led to a significant loss of affinity. Glu5 appeared unimportant for receptor recognition.

Affinity Labels↗
PubMed1994

Synthesis and biological evaluation of alpha-MSH analogues substituted with alanine.

The influence of single amino acid replacements by alanine on the binding affinity and biological activity of alpha-MSH in B16 murine melanoma cells has been studied systematically. alpha-MSH analogues were synthesized by solid-phase peptide synthesis and their binding affinities to the melanocortin receptor expressed by B16 mouse melanoma cells were determined using a radioreceptor assay. Biological activity of the analogues was determined by measuring tyrosinase stimulation. Relative activity and affinity data were generally in agreement with earlier results using terminal deletion fragments of alpha-MSH, but the alanine scan revealed important new insights into the role of individual residues. The three terminal amino acids at either end were not necessary for binding or activity, with amino acids 4-9 forming a core sequence required for receptor binding and triggering of the biological response. It was observed that replacement of the glutamic acid residue in position 5 was possible without loss of affinity or activity, whereas replacement of Met4 resulted in a 100-fold loss of binding affinity and biological activity. Each residue within the conserved melanocortin sequence His-Phe-Arg-Trp was shown to be essential with Phe7, Arg8, and Trp9 being the most sensitive to replacement by alanine. Generally, there was a rank correlation between binding affinity and tyrosinase stimulation within the group of analogues studied. Tyrosinase activity was less affected by alanine substitution than binding affinity, which suggests that full receptor binding is not required for maximum biological response.

Alanine↗
PubMed1994

Influence of alpha-MSH terminal amino acids on binding affinity and biological activity in melanoma cells.

The influence of the terminal amino acids of alpha-MSH on its biological action in B16 murine melanoma cells has been systematically studied. Fragments of alpha-MSH lacking various sequences of terminal residues were synthesized by solid-phase peptide synthesis and their binding affinity to melanoma cells was measured using a radioreceptor assay. Biological activity was determined by measuring both tyrosinase activity and melanogenesis. The relative affinities and activities of the fragments generally followed the same pattern as found previously in other assay systems (frog and lizard bioassay and Cloudman S91 mouse melanoma), with the three amino acids at each terminal not being essential for binding and biological activity, although the C-terminal amino acids 11-13 are more important than those in the N-terminus. The differences in biological activity between the fragments can be explained by their relative binding affinities for the receptor.

Amino Acid Sequence↗
PubMed1992

Isolation and partial purification of a melanocyte-stimulating hormone receptor from B16 murine melanoma cells. A novel approach using a cleavable biotinylated photoactivated ligand and streptavidin-coated magnetic beads.

The alpha-melanocyte-stimulating hormone (alpha-MSH) receptor of B16 mouse melanoma cells was characterized by photoaffinity labelling using radiolabelled photoactive derivatives of alpha-MSH. A doublet band of 43-46 kDa representing a ligand-receptor complex was identified. A novel adaptation of the streptovadin/biotin-based affinity system was used to isolate the alpha-MSH receptor. A probe was synthesized which contained biotin connected to a photolabelled alpha-MSH analogue via a cleavable disulphide linker and which displayed high affinity for the alpha-MSH receptor. Streptavidin-coated magnetic beads were used as a solid support instead of an affinity column. Covalently linked probe-receptor complexes solubilized in Triton X-100 were equilibrated with the beads, and after magnetic separation and washing, specifically bound complexes were treated with dithiothreitol to cleave the disulphide bridge in the biotin-peptide spacer arm and so release the receptor-ligand complex. The identity of the isolated protein was established by SDS/PAGE analysis. Methods to achieve purification to homogeneity and to allow quantitative isolation of the receptor are discussed.

Affinity Labels↗
PubMed1992

Self-emulsifying drug delivery systems: formulation and biopharmaceutic evaluation of an investigational lipophilic compound.

Self-emulsifying drug delivery systems (SEDDSs) represent a possible alternative to traditional oral formulations of lipophilic compounds. In the present study, a lipophilic compound, WIN 54954, was formulated in a medium chain triglyceride oil/nonionic surfactant mixture which exhibited self-emulsification under conditions of gentle agitation in an aqueous medium. The efficiency of emulsification was studied using a laser diffraction sizer to determine particle size distributions of the resultant emulsions. An optimized formulation which consisted of 25% (w/w) surfactant, 40% (w/w) oil, and 35% (w/w) WIN 54954 emulsified rapidly with gentle agitation in 0.1 N HCl (37 degrees C), producing dispersions with mean droplet diameters of less than 3 microns. The self-emulsifying preparation was compared to a polyethylene glycol 600 (PEG 600) solution formulation by administering each as prefilled soft gelatin capsules to fasted beagle dogs in a parallel crossover study. Pharmacokinetic parameters were determined and the absolute bioavailability of the drug was calculated by comparison to an i.v. injection. The SEDDS improved the reproducibility of the plasma profile in terms of the maximum plasma concentration (Cmax) and the time to reach the maximum concentration (tmax). There was no significant difference in the absolute bioavailability of WIN 54954 from either the SEDDS or the PEG formulations.

Administration, Oral↗
PubMed1985

Drug targeting--current aspects and future prospects.

A variety of materials have been suggested as carriers for the delivery of drugs to specific sites of action. Drugs may be covalently bound to carriers or physically trapped within particulate carriers. Likely mechanisms of action of targeting agents are described. The results of in vitro and in vivo experiments are reviewed and the prospective clinical uses of each type of carrier are discussed. In particular, monoclonal antibodies are promising agents for the targeting of cytotoxic agents to malignant cells. Radiolabelled monoclonal antibodies are likely to develop as agents for the radio-imaging of tumours which will prove useful in the diagnosis and treatment of cancer.

Antibodies, Monoclonal↗
PubMedDate not supplied

8-(omega-aminoalkyl)theophyllines and their use in preparing fluorescently labeled derivatives for applications in immunoassay.

Reaction of alkane-1, omega-diamines with 6-chloro-1,3-dimethylpyrimidine-2,4-dione under carefully controlled conditions gives 6-(omega-aminoalkylamino)-1,3-dimethylpyrimidine-2,4-diones, which can be readily separated from traces of products of disubstitution after benzyloxycarbonyl protection. A sequence of nitrosation at the pyrimidine 5-position, thermal cyclization, and deprotection affords 8-(omega-aminoalkyl) derivatives of theophylline, an important drug in the treatment of asthma and related diseases. These 8-(omega-aminoalkyl)theophyllines can be coupled to fluorescein-5-isothiocyanate and to dansyl chloride, giving fluorescent derivatives of theophylline with applications in automated immunoassay of the drug in biofluids using the fluorescence capillary fill device.

Fluorescent Dyes↗