Journal:

Article Title: CGRP receptors mediating CGRP-, adrenomedullin- and amylin-induced relaxation in porcine coronary arteries. Characterization with 'Compound 1' (WO98/11128), a non-peptide antagonist

doi: 10.1038/sj.bjp.0704210

Figure Lengend Snippet: αCGRP, βCGRP, AM and amylin concentration-reponse relationship in porcine coronary arteries. Points represent mean values and vertical lines indicate s.e.mean. Relative responses are given as percentage fraction of the initial vessel response to U46619 (10−7 M) just before they were challenged with the relaxant peptides.

Article Snippet: Drugs Human αCGRP, βCGRP, AM, amylin and the fragments αCGRP 8–37 , βCGRP 8–37 and AM 22–52 were all obtained from Bachem AG, Switzerland.

Techniques: Concentration Assay

Journal:

Article Title: CGRP receptors mediating CGRP-, adrenomedullin- and amylin-induced relaxation in porcine coronary arteries. Characterization with 'Compound 1' (WO98/11128), a non-peptide antagonist

doi: 10.1038/sj.bjp.0704210

Figure Lengend Snippet: (a–d) Vasorelaxant effect of αCGRP (10−10–10−7 M) and βCGRP (10−10–10−7 M) in the porcine LAD contracted with U46619 (10−7 M). The antagonists αCGRP8–37 (10−7–10−5 M), βCGRP8–37 (10−7–10−5 M) were added 15 min and U46619 10 min prior to the αCGRP/βCGRP challenge. Points represent mean values and vertical lines indicate s.e.mean. Relative responses are given as percentage fraction of the initial vessel response to U46619 (10−7 M) just before they were challenged with αCGRP/βCGRP.

Article Snippet: Drugs Human αCGRP, βCGRP, AM, amylin and the fragments αCGRP 8–37 , βCGRP 8–37 and AM 22–52 were all obtained from Bachem AG, Switzerland.

Techniques:

Journal:

Article Title: CGRP receptors mediating CGRP-, adrenomedullin- and amylin-induced relaxation in porcine coronary arteries. Characterization with 'Compound 1' (WO98/11128), a non-peptide antagonist

doi: 10.1038/sj.bjp.0704210

Figure Lengend Snippet: Schild plots for αCGRP8–37 and βCGRP8–37 tested with human αCGRP and βCGRP as agonists in isolated porcine LAD. The Schild plot curve for αCGRP8–37 (10−7–10−5 M) tested with αCGRP was equal to 1.48× +10.3 (r=0.62; P=0.0011). The pA2 value=7.0 (6.4–8.6). The Schild plot curve for αCGRP8–37 (10−7–10−5 M) tested with βCGRP was equal to 1.23× +7.8 (r=0.63; P=0.0007). The pA2 value=6.9 (6.2–8.7). The Schild plot curve for βCGRP8–37 (10−7–10−5 M) tested with αCGRP was equal to 1.05× +7.2 (r=0.61; P=0.0031). The pA2 value=6.3 (5.9–7.0). The Schild plot curve for βCGRP8–37 (10−6–10−5 M) tested with βCGRP was equal to 1.68× +10.0 (r=0.65; P=0.0002). The pA2 value=5.9 (5.7–6.5). Each point represents mean values and vertical lines indicate s.e.mean.

Article Snippet: Drugs Human αCGRP, βCGRP, AM, amylin and the fragments αCGRP 8–37 , βCGRP 8–37 and AM 22–52 were all obtained from Bachem AG, Switzerland.

Techniques: Isolation

Journal:

Article Title: CGRP receptors mediating CGRP-, adrenomedullin- and amylin-induced relaxation in porcine coronary arteries. Characterization with 'Compound 1' (WO98/11128), a non-peptide antagonist

doi: 10.1038/sj.bjp.0704210

Figure Lengend Snippet: (a,b) Vasorelaxant effect of αCGRP (10−10–10−7 M) and βCGRP (10−10–10−7 M) in the porcine LAD contracted with U46619 (10−7 M). The antagonist AM22–52 (10−6 M) was added 15 min and U46619 10 min prior to the αCGRP/βCGRP challenge. Points represent mean values and vertical lines indicate s.e.mean. Relative responses are given as percentage fraction of the initial vessel response to U46619 (10−7 M) just before they were challenged with αCGRP/βCGRP.

Article Snippet: Drugs Human αCGRP, βCGRP, AM, amylin and the fragments αCGRP 8–37 , βCGRP 8–37 and AM 22–52 were all obtained from Bachem AG, Switzerland.

Techniques:

Journal:

Article Title: CGRP receptors mediating CGRP-, adrenomedullin- and amylin-induced relaxation in porcine coronary arteries. Characterization with 'Compound 1' (WO98/11128), a non-peptide antagonist

doi: 10.1038/sj.bjp.0704210

Figure Lengend Snippet: Vasorelaxant effect of αCGRP in the porcine LAD contracted with U46619 (10−7 M). The non-peptide CGRP antagonist ‘Compound 1' (10−7–10−5 M) was added 15 min and U46619 10 min prior to the αCGRP (10−10–10−7 M) challenge. Points represent mean values and vertical lines indicate s.e.mean. Relative responses are given as percentage fraction of the initial vessel response to U46619 (10−7 M) just before they were challenged with αCGRP.

Article Snippet: Drugs Human αCGRP, βCGRP, AM, amylin and the fragments αCGRP 8–37 , βCGRP 8–37 and AM 22–52 were all obtained from Bachem AG, Switzerland.

Techniques:

Journal: Protein Science : A Publication of the Protein Society

Article Title: Atomic structures of IAPP (amylin) fusions suggest a mechanism for fibrillation and the role of insulin in the process

doi: 10.1002/pro.145

Figure Lengend Snippet: The crystal structure of the maltose binding protein (MBP)-IAPP fusions protein reveals the helical propensity of IAPP. (A) A helix-helix homodimerization interface (green arrow between IAPP molecules) is observed between fusion molecules. The two MBP molecules are shown in blue and yellow, respectively. Residues of the fusion attributable to IAPP are shown in red, each attached to MBP molecules. (B) Space filling representation showing the tight interface between parallel IAPP helices, centered at Phe 15. The view is in the direction of the black arrow in A. (C) By rotating the IAPP homodimer, it can be seen that the 8–18 helices interact at a 55° angle. (D and E) A MBP-IAPP1-22 fusion reveals the cyclic disulfide bond between Cys 2 and Cys 7 and the helical propensity within the N-terminus. MBP is shown in light blue and IAPP is shown in red. (F) View looking down the N-terminal helix of IAPP shows the 310 helix between residues Gln10 and Val17.

Article Snippet: IAPP 8–37 peptide (CSbio) was solubilized into 20 m M NaAc pH 6.5 to a final concentration of 100 μ M . Glutaraldehyde was immediately added to a final concentration of 0.8% (w/v) along with either 100 μ M insulin (Sigma Aldrich), 100 μ M myoglobin or buffer alone.

Techniques: Binding Assay

Journal: Protein Science : A Publication of the Protein Society

Article Title: Atomic structures of IAPP (amylin) fusions suggest a mechanism for fibrillation and the role of insulin in the process

doi: 10.1002/pro.145

Figure Lengend Snippet: The homodimerization of IAPP suggests an analogous structure for IAPP-insulin heterodimerization. (A) Sequence alignment of residues 8–23 from IAPP with residues 9–24 of the insulin B chain. (B) The putative IAPP-interacting segment from insulin (helical segment composed of residues 9–20 shown in gray) is shown overlaid on the IAPP 8–19 helix. This interaction is centered on the stacked aromatic sidechains (Phe 15 from IAPP and Tyr 16 from insulin) between the two helices. (C) Computationally energy-minimized packing between insulin 9–19 and IAPP 8–18 helices. Notice that the energy-minimized helices are more parallel than the helices in B. (D) Crosslinking of IAPP and Insulin reveals an IAPP dimer (red arrow), an insulin dimer (blue arrow) as well as an IAPP-insulin heterodimer (white arrow). IAPP also crosslinks to higher-order insulin multimers. Myoglobin is included as a negative control for nonspecific crosslinking with IAPP.

Article Snippet: IAPP 8–37 peptide (CSbio) was solubilized into 20 m M NaAc pH 6.5 to a final concentration of 100 μ M . Glutaraldehyde was immediately added to a final concentration of 0.8% (w/v) along with either 100 μ M insulin (Sigma Aldrich), 100 μ M myoglobin or buffer alone.

Techniques: Sequencing, Negative Control

Journal: Protein Science : A Publication of the Protein Society

Article Title: Atomic structures of IAPP (amylin) fusions suggest a mechanism for fibrillation and the role of insulin in the process

doi: 10.1002/pro.145

Figure Lengend Snippet: Computational docking experiments yielded an ensemble of solutions that suggest how IAPP might dimerize and the effect on the interaction of residue replacements at Phe15. In (A), a docked dimer between wild-type IAPP segments 8–19 reveals a relative rotation of the helices and tighter association than observed in the crystal structure of IAPP. This association disrupts the aromatic stacking between Phe15 residues in exchange for more contact area between the molecules. This association led us to believe that small residue substitutions for Phe15, such as alanine or serine may allow the helices to pack even closer and bury more surface area, as shown in (B) for F15S. The tighter association places Phe15 in closer proximity to His18 and Arg11 of the adjacent molecule, suggesting that acidic residues could potentially stabilize the interaction while large, positively charged residues, such as lysine or arginine could disrupt helical packing. In (C), the F15D dimer reveals how the negative charge may help stabilize the dimer by interacting with Arg11 in the neighboring molecule. In (D), a top scoring interaction model of the IAPP F15D mutant and insulin suggests an alternative interaction mode between IAPP and insulin as compared to that in Figure ​Figure2.2. The rotated IAPP helix exposes Asp15 to solvent and buries several hydrophobic residues from both IAPP and insulin. This packing was not observed in the MBP-fusion structure presumably because the Phe at position 15 preferred to be buried at the helical interface.

Article Snippet: IAPP 8–37 peptide (CSbio) was solubilized into 20 m M NaAc pH 6.5 to a final concentration of 100 μ M . Glutaraldehyde was immediately added to a final concentration of 0.8% (w/v) along with either 100 μ M insulin (Sigma Aldrich), 100 μ M myoglobin or buffer alone.

Techniques: Residue, Mutagenesis, Solvent

Journal: Protein Science : A Publication of the Protein Society

Article Title: Atomic structures of IAPP (amylin) fusions suggest a mechanism for fibrillation and the role of insulin in the process

doi: 10.1002/pro.145

Figure Lengend Snippet: Mutations at position 15 of human IAPP affect the rate of fibril formation and the ability of insulin to inhibit this fibrillation. Residue substitutions computationally predicted to affect IAPP homodimerization and Insulin-IAPP heterodimerization were tested by a fibril formation assay. (A and B) Substitutions predicted to enhance helical association (F15A, F15S, F15D) of IAPP formed amyloid fibrils more rapidly than wild-type IAPP, as determined by Thioflavin T binding. The F15K mutation was predicted to disrupt the helical interface and a marked delay in fibril formation is observed. The 8–37 construct was used for both wild-type and Phe15 mutants. Mouse IAPP serves as a negative control for aggregation and never achieves a fluorescence value higher than baseline. (C) Including equimolar insulin in the assay delays the fibril formation of each mutant tested but has comparatively less of an inhibitory effect on the F15K substitution. (D) Representative EM images taken at 30 min of WT, F15S, and F15K to confirm the presence or absence of fibrils. Images of additional time points are included in the supporting Information. Scale bar represents 100 nm.

Article Snippet: IAPP 8–37 peptide (CSbio) was solubilized into 20 m M NaAc pH 6.5 to a final concentration of 100 μ M . Glutaraldehyde was immediately added to a final concentration of 0.8% (w/v) along with either 100 μ M insulin (Sigma Aldrich), 100 μ M myoglobin or buffer alone.

Techniques: Residue, Tube Formation Assay, Binding Assay, Mutagenesis, Construct, Negative Control, Fluorescence

Journal: Protein Science : A Publication of the Protein Society

Article Title: Atomic structures of IAPP (amylin) fusions suggest a mechanism for fibrillation and the role of insulin in the process

doi: 10.1002/pro.145

Figure Lengend Snippet: Model of nucleus formation of IAPP fibrils and the inhibition of fibrillation by insulin. (A) IAPP monomers are assumed to be in equilibrium between disordered state and helical, shown in (B) as it appears in the crystal. Phe15 is shown in stick view for reference. (C and D) Two IAPP dimers align their C-terminal amyloid domains in an antiparallel orientation. The C-terminus of each IAPP molecule is labeled. The position of residue S20 is indicated by the green arrow. When this residue is mutated to Glycine, fibrils form more rapidly. (E) This region, residues 23–37, forms the steric zipper spine of the fibril, by interdigitating sidechains with a second IAPP dimer. (F) Formation of the N-terminal strand occurs subsequent to spine formation. (G) Insulin prevents homodimeric helical association by binding to monomeric IAPP and blocking IAPP homodimerization. The insulin A-chain is shown in gold and the B chain is shown in gray.

Article Snippet: IAPP 8–37 peptide (CSbio) was solubilized into 20 m M NaAc pH 6.5 to a final concentration of 100 μ M . Glutaraldehyde was immediately added to a final concentration of 0.8% (w/v) along with either 100 μ M insulin (Sigma Aldrich), 100 μ M myoglobin or buffer alone.

Techniques: Inhibition, Labeling, Residue, Binding Assay, Blocking Assay