No description available.

No description available.

No description available.

No description available.

A crucial aspect of drug delivery is efficient transport to the site of action. Thus, there is a need to design and evaluate new delivery vehicles. In this thesis two delivery vehicles, cell-penetrating peptides and bacterial ghosts, were evaluated. The understanding of the internalization and degradation kinetics of cell-penetrating peptides is important for the practical aspects of cargo delivery since peptides have a notorious reputation of being rapidly degraded. If the cell-penetrating peptide remains intact inside the cellular environment, there is a possibility that the peptide-cargo conjugate leaks back to the extracellular environment. However, if it is degraded outside the cell, the cargo will never be delivered. In order to improve uptake efficiency and to be able to foresee side effects, the translocation mechanism needs to be fully elucidated. Data gathered from the first two papers led to the proposal of a new me-chanism involved in cell-penetrating peptide uptake: the membrane repair response, a resealing mechanism rapidly patching up broken membranes. This mechanism could explain the divergence in perception concerning the uptake pathways. Furthermore a new assay to produce the second delivery vehicle, bacterial ghosts, was developed based on data from the cell-penetrating peptide investigations. Bacterial ghosts are dead bacteria devoid of cytoplasmic contents but still retaining their structural and morphological characteristics, after protein E lysis of the bacterial cell membrane. By using a cell-penetrating peptide with antimicrobial effects, a new rapid peptide-based strategy to produce ghosts was developed and the capability to deliver plasmid DNA into the cell for expression was evaluated.

Showing an extensive distribution in the nervous system, and often in co-localization with the classical neurotransmitters, neuropeptides are functioning as important modulators of neuronal signaling. Subsequently, compelling evidence has implicated a modulatory role for the neuropeptide galanin in several physiological functions. The effect of galanin is trancduced intracellularly by three different receptors, and defining the explicit effect from these receptor subtypes is of outmost interest, and likely to result in future therapeutic utilization of the galanin system. The main aim of this thesis was to improve the development of subtype selective ligands utilized to differentiate between the galanin receptor subtypes. To achieve this, we have designed and developed novel galanin receptor ligands and characterized the molecular interactions necessary for ligand bindig at the GalR2 subtype. The major findings include the introduction and characterization of two galanin receptor ligands, selectively activating GalR1 or inhibiting GalR2. Although having moderate selectivity, the two ligands have been utilized in a number of studies, pursuing their initial presentation, in order to differentiate between the galanin receptors and to establish their specific function. Further optimization is likely to improve the selectivity and utilization of these ligands. By identifying the major pharmacophores in the Gal(2-11) ligand and the residues in the GalR2 subtype participating in ligand binding, we have been able to characterize the binding site in this receptor subtype and interactions that are of significance for recognition of subtype specific ligands. Together, these findings on GalR2 and Gal(2-11) are of importance for future design of ligands acting on this receptor.

Procedures for the preparative purification of human acetylcholinesterase (AChE; EC 3.1.1.7) have been examined. Chromatofocussing showed caudate nucleus and cerebrospinal fluid (CSF) AChE eluting between isoelectric points (pi) of 5.2 - 5.57, whilst isoelectric focussing (IEF) revealed heterogeneous AChE isoforms migrating between pI 4.5 - 6. Affinity chromatography proved to be better in yielding highly pure samples. The ligands used were either acridinium or procainamide, which yielded brain AChE recoveries of 10.1% and 42.8%, respectively. Various other methods were used such as gel filtration and ion-exchange chromatography on a Pharmacia SMART System. Purified brain AChE was used as antigen to generate anti-AChE monoclonal antibodies (mAbs) by in vivo immunization. BMS-3E4, -7G10, and -9F4 reacted with erythrocyte (G2) AChE, whilst BMS-6D6 bound to brain (G4) AChE in dot blot, titration, and sedimentation experiments. Conversely, the four mAbs recognized only the G4 form and not the G₂ form on immunoblotted IEF and non-denaturing Clarke gels. Deglycosylation studies suggest that the four mAbs recognize a carbohydrate epitope linked to AChE. As a preparative step, tissue culture media containing these mAbs were ammonium sulphate precipitated and purified on a gel filtration column. Under reducing conditions, two bands migrating at 80 and 25 kDa were seen, corresponding to the heavy and light chains of Immunoglobulin M (IgM). Four additional mAbs were raised by the novel method of in vitro immunization, using a synthetically-produced C-terminus peptide as antigen. BMS-5, -6, -7, and -8 are all IgMs which recognize soluble brain and not erythrocyte nor membrane-bound AChE in dot blots, IEF, and sedimentation analyses. Although immunocytochemistry of the above-mentioned mAbs did not demonstrate any positive cholinergic binding, the present mAbs may still offer potential clinical and biochemical applications.

No description available.

No description available.

No description available.