Conformational differences of ovine and human corticotrophin releasing hormone A CD,IR, NMR and dynamic light scattering study

The differences in the conformational properties of ovine (o) and human (h) CRH in aqueous solution, structure-inducing TFE and in the presence of detergent micelles and lipid vesicles have been investigated by circular dichroism, Fourier transform infrared spectroscopy, NMR and dynamic light scattering. o-CRH was found to exist as a monomer with little regular structure in dilute aqueous solution. Association at concentrations higher than 10^?3 mol/L results predominantly in dimers. The induction of a substantial amount of intermolecular β-structure seems to be the result of interactions of the C-terminal hexapeptide and the N-terminal region 6-12 of o-CRH chains in antiparallel orientation. In contrast, h-CRH exhibits a high tendency of association which is highly sensitive to the pH. The formation of tetramers at millimolar peptide concentration is related to a helical content of ca. 50%. The potentially helical, highly hydrophobic region 6-20 enlarged by more hydrophobic residues in position 23 and 25 is proposed to stabilize the h-CRH associates. In the presence of structure inducing TFE (<40%v) both CRH peptides exist as monomers. o-CRH reveals about 72% helicity, in h-CRH the formation of about 85% helix is observed. The differences in helicity of the two CRH molecules are located in the C-terminal heptapeptide, as concluded on the basis of NMR studies. Both peptides bind to detergent micelles at pH 4 as well as 7.4 associated with an increase in the α-helical content. Interaction of the two peptides with DMPC vesicles was found exclusively at pH 4. Above the phase transition temperature of DMPC the α-helical content in h-CRH increases slightly; however, o-CRH reveals a substantial amount of β-type structure. The intramolecular type of β-structure is associated with a deeper insertion of the o-CRH region 6-12 into the hydrophobic region of the lipid bilayer, whereas the corresponding region of h-CRH is kept in the bilayer surface. The higher helicity of h-CRH might explain to some extent its higher affinity to the CRH receptor, CRH antibodies and the CRH binding protein. © Munksgaard 1996.     

CIRCULAR DICHROISM STUDIES OF SUBSTANCE P AND ITS C-TERMINAL SEQUENCES

CD spectra of substance P (SP) and its C-terminal partial sequences have been measured in diluted aqueous solution including variation of hydrogen ion concentration. In the far u.v. region there is an overlapping of the amide CD absorption by the CD of the phenylalanine residue aromatic side chains (217–220 nm). This complex CD absorption is reduced during changes from acidic to alkaline pH, especially in those cases where a phenylalanine residue is at the N-terminus of the peptide chain. These CD changes dependent on pH are due more to charge effects on the aromatic chromophores than to substantial conformational changes. However, solvation effects on the conformational features of SP peptides caused by the deprotonation of the amino groups have to be taken into account. CD and potentiometric titrations indicate that the N-terminal α-amino groups of the SP peptides in general are freely accessible to the solvent. Our studies did not give any evidence of the occurrence of ordered structures of SP peptides in diluted aqueous solution.     

Conformation of a water-soluble β-sheet model peptide. A circular dichroism and Fourier-transform infrared spectroscopie study of double D-amino acid replacements

Among peptide secondary structures β-sheet domains have been much less intensively studied than α-helical conformations, mainly because of the lack of well characterized model peptides. In the present paper the secondary structure of a water-soluble de novo peptide consisting of 26 amino acids (DPKGDPKGVTVTVTVTVTGKGDPKPD-NH₂) and the corresponding double D-amino acid replacement set have been studied by circular dichroism and Fourier-transform infrared spectroscopy. The model peptide was found to be unstructured in aqueous solution at peptide concentrations < 10^?3 mol/L but to adopt a predominantly β-sheet structure in the presence of 15 mM sodium dodecyl sulfate or at apolar/water interfaces. Although the peptide is composed of amino acids with low helical propensity, it formed a single-stranded helical structure in aqueous trifluoroethanol. The D-amino acid replacement set was synthesized in order to study the conformational stability of the model peptide selectively in distinct regions. The data show that both the α-helix present in 50% trifluoroethanol as well as the β-sheet domain formed in the presence of sodium dodecyl sulfate or at apolar/water interfaces, are located in the region between Val⁹ and Thr¹⁸. Pairwise substitution of adjacent amino acids by their corresponding D-amino acids provides a pronounced β-sheet disturbance. These findings demonstrate that double D-amino acid replacements may be used to locate β-sheet domains in peptides.     

Long-term survival under maintenance gemcitabine chemotherapy for metastatic transitional cell carcinoma

We report a case of a 74-year-old patient who received 41 courses of maintenance therapy with gemcitabine over a length of 28 months for metastatic transitional cell carcinoma. One year earlier the patient had received three cycles of adjuvant cisplatin-based combination chemotherapy after nephro-ureterectomy for a locally advanced urothelial cancer of the right renal pelvis. This case demonstrates a paradigm shift in the palliative treatment of advanced urothelial cancer, with the implementation of more tolerable agents such as gemcitabine. Even elderly patients with impaired renal function may benefit in terms of tumor reduction and survival from systemic chemotherapy, which may be applied over a prolonged period of time.     

The effect of SC-15396, atropine and mepyramine on gastrin-, bethanechol- and histamine-stimulated gastric acid secretion in rats and guinea-pigs

The effect of SC-15396 (“antigastrin”; 2-phenyl-2-(2-pyridyl)thioacetamide), atropine and mepyramine on gastrin-, bethanechol- and histamine-stimulated gastric acid secretion was studied in rats and guinea-pigs. For all three stimulants parallel dose response curves were obtained except in guinea-pigs where bethanechol even in very high doses displays a poor activity in stimulating gastric acid secretion. The maximal secretory response was found to be 12·7 ± 5·0 μ-equiv HCl/10 min in rats and 53·2 ± 27£9 μ-equiv HCl/10 min in guinea-pigs. All stimulating effects on gastric acid secretion were reduced by SC-15396; atropine abolished the secretory responses to bethanechol. Mepyramine was ineffective. In accordance with these findings the mechanism of action of gastrin and a receptor model on the oxyntic cell are discussed.