| 1. |
- Kilk, Kalle, et al.
(författare)
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Evaluation of transportan 10 in PEI mediated plasmid delivery assay
- 2005
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Ingår i: Journal of Controlled Release. - : Elsevier BV. - 0168-3659 .- 1873-4995. ; 103:2, s. 511-23
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Tidskriftsartikel (refereegranskat)abstract
- Cell-penetrating peptides (CPPs) are novel high-capacity delivery vectors for different bioactive cargoes. We have evaluated the CPP transportan 10 (TP10) as a delivery vector in different in vitro plasmid delivery assays. Tested methods include: TP10 crosslinked to a plasmid via a peptide nucleic acid (PNA) oligomer, TP10 conjugation with polyethyleneimine (PEI), and addition of unconjugated TP10 to standard PEI transfection assay. We found that without additional DNA condensing agents, TP10 has poor transfection abilities. However, the presence of TP10 increases the transfection efficiency several folds compared to PEI alone. At as low concentrations as 0.6 nM, TP10–PNA constructs were found to enhance plasmid delivery up to 3.7-fold in Neuro-2a cells. Interestingly, the transfection efficiency was most significant at low PEI concentrations, allowing reduced PEI concentration without loss of gene delivery. No increase in cytotoxicity due to TP10 was observed and the uptake mechanism was determined to be endocytosis, as previously reported for PEI mediated transfection. In conclusion, TP10 can enhance PEI mediated transfection at relatively low concentrations and may help to develop future gene delivery systems with reduced toxicity.
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| 2. |
- Lundström, Linda, et al.
(författare)
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A galanin receptor subtype 1 specific agonist
- 2005
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Ingår i: International Journal of Peptide Research and Therapeutics. - : Springer Science and Business Media LLC. - 1573-3904 .- 1573-3149. ; 11:1, s. 17-27
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Tidskriftsartikel (refereegranskat)abstract
- The chimeric peptide M617, galanin(1–13)-Gln14-bradykinin(2–9)amide, is a novel galanin receptor ligand with increased subtype specificity for GalR1 and agonistic activity in cultured cells as well as in vivo. Displacement studies on cell membranes expressing hGalR1 or hGalR2 show the presence of a high affinity binding site for M617 on GalR1 (K i=0.23±.12 nM) while lower affinity was seen towards GalR2 (K i=5.71±1.28 nM) resulting in 25-fold specificity for GalR1. Activation of GalR1 upon stimulation with M617 is further confirmed by internalization of a GalR1-EGFP conjugate. Intracellular signaling studies show the ability of M617 to inhibit forskolin stimulated cAMP formation with 57% and to produce a 5-fold increase in inositol phosphate (IP) accumulation. Agonistic effects on signal transduction are shown on both receptors studied after treatment with M617 in the presence of galanin. In noradrenergic locus coeruleus neurons, M617 induces an outward current even in the presence of TTX plus Ca2+, high Mg2+, suggesting a postsynaptic effect. Intracerebroventricular (i.c.v.) administration of M617 dose-dependently stimulates food uptake in rats while, in contrast, M35 completely fails to affect the feeding behavior. Spinal cord flexor reflex is facilitated by intrathecal (i.t.) administration of M617 as well as galanin with no significant change upon pre-treatment with M617. M617 dose dependently antagonizes the spinal cord hyperexcitablility induced by C-fiber conditioning stimulus and does neither enhance nor antagonize the effect of galanin. These data demonstrate a novel galanin receptor ligand with subtype specificity for GalR1 and agonistic activity, both in vitro and in vivo.
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| 3. |
- Lundström, Linda, et al.
(författare)
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Important pharmacophores for binding to galanin receptor 2
- 2005
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Ingår i: Neuropeptides. - 0143-4179 .- 1532-2785. ; 39:3, s. 169-171
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Tidskriftsartikel (refereegranskat)abstract
- Galanin(2–11) has been introduced as a receptor subtype selective ligand for the GalR2 subtype of the galanin receptors, and has gained use in pharmacological studies of galaninergic signaling in the past two years. By introducing l-Ala substitutions in the galanin(2–11) sequence, we have examined the amino acid residues which are of importance for binding to the GalR2 receptor. Our study shows that Trp2, Asn5, Gly8 and Tyr9 are of great importance for high affinity binding. When placed in an α-helical conformation, the side chains of these residues are, with the exception of Tyr9, displayed on the same “side” of the peptide. This information is useful in the rational design of non-peptide type GalR2 receptor ligands.
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| 4. |
- Lundström, Linda, et al.
(författare)
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Molecular characterization of the ligand binding site of the human galanin receptor type 2, identifying subtype selective interactions
- 2007
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Ingår i: Journal of Neurochemistry. - : Wiley. - 0022-3042 .- 1471-4159. ; 103:5, s. 1774-1784
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Tidskriftsartikel (refereegranskat)abstract
- To define the specific role of the galanin receptors when mediating the effect of galanin, effective tools for distinct activation and inhibition of the different receptor subtypes are required. Several of the physiological effects modulated by galanin are implicated to be mediated via the GalR2 subtype and have been distinguished from GalR1 effects by utilizing the Gal(2–11) peptide, recognizing only GalR2 and GalR3. In this study, we have performed a mutagenesis approach on the GalR2 subtype and present, for the first time, a molecular characterization of the interactions responsible for ligand binding and receptor activation at this receptor subtype. Our results identify four residues, His252 and His253 located in transmembrane domain 6 and Phe264 and Tyr271 in the extracellular loop 3, to be of great significance. We show evidence for the N-terminal tail of GalR2 to participate in ligand binding and that selective binding of Gal(2–11) includes interaction with the Ile256 residue, located at the very top of TM 6. In conclusion, we present a mutagenesis study on GalR2 and confer interactions responsible for ligand binding and receptor activation as well as selective recognition of the Gal(2–11) peptide at this receptor subtype. The presented observations could be of major importance for the design and development of new and improved peptide and non-peptide ligands, selectively activating the GalR2 subtype.
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| 5. |
- Quynh, Nguyen Thi Thu, et al.
(författare)
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Effects of galnon, a non-peptide galanin-receptor agonist, on insulin release from rat pancreatic islets
- 2005
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Ingår i: Biochemical and Biophysical Research Communications - BBRC. - : Elsevier BV. - 0006-291X .- 1090-2104. ; 328:1, s. 213-220
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Tidskriftsartikel (refereegranskat)abstract
- Galanin is a neurotransmitter peptide that suppresses insulin secretion. The present study aimed at investigating how a non-peptide galanin receptor agonist, galnon, affects insulin secretion from isolated pancreatic islets of healthy Wistar and diabetic Goto-Kakizaki (GK) rats. Galnon stimulated insulin release potently in isolated Wistar rat islets; 100 microM of the compound increased the release 8.5 times (p<0.001) at 3.3 mM and 3.7 times (p<0.001) at 16.7 mM glucose. Also in islet perifusions, galnon augmented several-fold both acute and late phases of insulin response to glucose. Furthermore, galnon stimulated insulin release in GK rat islets. These effects were not inhibited by the presence of galanin or the galanin receptor antagonist M35. The stimulatory effects of galnon were partly inhibited by the PKA and PKC inhibitors, H-89 and calphostin C, respectively, at 16.7 but not 3.3 mM glucose. In both Wistar and GK rat islets, insulin release was stimulated by depolarization of 30 mM KCl, and 100 microM galnon further enhanced insulin release 1.5-2 times (p<0.05). Cytosolic calcium levels, determined by fura-2, were increased in parallel with insulin release, and the L-type Ca2+-channel blocker nimodipine suppressed insulin response to glucose and galnon. In conclusion, galnon stimulates insulin release in islets of healthy rats and diabetic GK rats. The mechanism of this stimulatory effect does not involve galanin receptors. Galnon-induced insulin release is not glucose-dependent and appears to involve opening of L-type Ca2+-channels, but the main effect of galnon seems to be exerted at a step distal to these channels, i.e., at B-cell exocytosis.
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| 6. |
- Sollenberg, Ulla E., et al.
(författare)
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M871 – a novel peptide antagonist selectively recognizing the galanin receptor type 2
- 2006
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Ingår i: International Journal of Peptide Research and Therapeutics. - : Springer Science and Business Media LLC. - 1573-3149 .- 1573-3904. ; 12:2, s. 115-119
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Tidskriftsartikel (refereegranskat)abstract
- Galanin and its three receptors have been linked to a wide variety of physiological processes and are distributed in both the central and peripheral nervous systems. Further knowledge of the properties of galanin-activated signaling systems can best be obtained by the availability of peptide and non-peptide ligands that are selective for the different receptor subtypes. The current study describes binding and signaling data for the chimeric peptide, galanin-(2–13)-Glu-His-(Pro)3-(Ala-Leu)2-Ala-amide (M871). This compound binds to the galanin receptor type 2 with more than 30-fold higher affinity than to the galanin receptor type 1 and exhibits antagonist actions at galanin receptor type 2, blocking increased release of inositol phosphate produced by galanin in CHO cells. This peptide opens new possibilities for the study of galanin receptor physiology.
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