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Notes: Rationale Asthmatic airway remodelling is characterized by myofibroblast hyperplasia and subbasement membrane collagen deposition. We hypothesized that cytokines and growth factors implicated in asthmatic airway remodelling are increased in bronchoalveolar lavage (BAL) fluid of asthmatics after segmental allergen challenge (SAC), and that these growth factors and cytokines increase α-smooth muscle actin (α-SMA) and collagen III synthesis by human lung fibroblasts (HLFs).Methods Transforming growth factor (TGF)-β1, TGF-β2, IL-4 and IL-13 levels were measured in BAL fluid from 10 asthmatics and 9 non-asthmatic controls at baseline and then 1 day, 1 week and 2 weeks after SAC. Confluent cultures of HLFs were stimulated by exogenous addition of TGF-β1, TGF-β2, IL-4 or IL-13 (concentration range 0.01–10 ng/mL) over 48 h. Collagen III was measured in culture supernates and α-SMA in cell lysates by Western blot.Results At baseline, there was no difference in BAL fluid concentrations of TGF-β1, IL-4 and IL-13 between asthmatics and controls; however, non-asthmatics had higher concentrations of total TGF-β2. In asthmatics, BAL fluid concentrations of all four factors increased significantly 1 day after SAC. TGF-β1, TGF-β2 and IL-13 concentrations returned to baseline by 1 week after SAC, but BAL fluid IL-4 concentration remained elevated for at least 2 weeks. TGF-β1, TGF-β2 and IL-4 significantly increased α-SMA in fibroblasts, but only IL-4 caused corresponding increases in collagen III synthesis. IL-13 had no direct effects on collagen III synthesis and α-SMA expression.Conclusions Because IL-4 caused a dose-dependent increase in α-SMA and collagen III synthesis, it may be an important cytokine mediating asthmatic airway remodelling. TGF-β1 and TGF-β2 may also play a role in airway remodelling by stimulating phenotypic change of fibroblasts to myofibroblasts. Additionally, collagen III synthesis appears to be independent of myofibroblast phenotype and is apparently regulated by different growth factors and cytokines.

Notes: Synthetic analogues of the C-terminal portion of C5a were designed and found to be agonists of the C5a receptor [J. A. Ember et al. (1992) Journal of Immunology, Vol. 148, p. 3165]. Nuclear magnetic resonance experiments were carried out to determine the solution conformation of the most potent analogue, the peptide C5a 65-74 (Tyr65, Phe67) (Tyr65-Ser66-Phe67-Lys68-Asp69-Met70-Gln71-Leu72-Gly73-Arg74). Medium-range nuclear Overhauser effects (NOEs) were observed for residues 65-70 of this C5a peptide, suggesting that this region adopts a folded conformation in a significant population of the solution conformational ensemble. Quantitative analyses of 3JNH-aH coupling constants and sequential NOE cross peaks gave an estimated helical population of 65% in the region Ser66-Met70. Additional evidence supporting the presence of a helical turn includes reduced amide-proton temperature coefficients and lowered3JHN-aH coupling constants in the region of Phe67-Met70. Conformational behavior of this C5a analogue peptide was studied using molecular modeling incorporating observed NOEs as constraints. The side chains of Tyr65, Phe67, and Met70 consistently form a hydrophobic cluster in all the model structures. The side chains of residues Ser66 and Asp69 can form reciprocal hydrogen bonds with the backbone NH groups of these two residues, indicating that residues Ser66-Phe67-Lys68-Asp69 (or SFKD) form a helix-stablizing capping box [E. T. Harper and G. D. Rose (1993) Biochemistry, Vol. 32, p. 7605: H. X. Zhou et al. (1994) Proteins: Structure, Function and Genetics, Vol. 18. p. 1] even within the single turn of helical structure found in the analogue C5a peptide. A comparison of the nmr results obtained for the analogue peptide and the natural decapeptide C5a 65-74 (He65-Ser66-His67-Lys68-Asp69-Met70-Gln71-Leu72-Gly73-Arg74) indicated that incorporation of residues Tyr65 and Phe67 helps stabilize an isolated capping box involving residues Ser66-Asp69 in the C5a peptides through more extensive hydrophobic/aromatic interactions between residues Tyr65, Phe67, and Met70 in the analogue peptide C5a 65-74 (Tyr65, Phe67). These results constitute the first experimental demonstration of hydrophobic determinants in helical capping-box interactions, proposed recently by a statistical analysis of protein structures [J. W. Seale et al. (1994) Protein Science, Vol. 3. pp. 1741-1745]. The stabilized helical turn may also account for the greater potency of the analogue peptide C5a65-74(Tyr65, Phe67) in receptor-binding assays. © 1996 John Wiley & Sons, Inc.

Notes: In the present paper we investigate the influence of sample pH on the conformational and dynamical properties of the pseudotripeptide H-Tyr-TicΨ[CH2NH]Phe-OH(TIP[Ψ]:Tic: l, 2, 3, 4,-tetrahydroisoquinoline-3-carboxylic acid) using various one- and two-dimensional nmt techniques in conjunction with molecular modeling. Studies were conducted at three different pH levels-corresponding to the zwitterionic peptide containing a formal positive charge(pH 3. 1).the deprotonated molecule(pH 9. 1), and a situation at neutral pH(pH 7. 2) involving both protonated and deprotonated states of the reduced peptide bond. Analysis of the one-dimensional1H-nmr spectra reveals that in solution TIP[Ψ]is in slow dynamic exchange between conformations containing cis and trans configurations of the Tyr-Tic bond. An nmr pH dependence study of the cis:trans ratio indicated that the exchange process was governed by the protonation state of the reduced bond amine. From the nmr data, reduced peptide bond pKavalues of 6. 5 and 7. 5 were determined for the cis and trans conformers, respectively. It was concluded that conformations containing a trans Tyr-Tic bond are stabilized at law pH by an intramolecular hydrogen bond between the Tyr carbonyl and the reduced peptide bond protonated amine. This observation was corroborated by molecular mechanics investigations that revealed low energy trans structures compatible with nmr structural data, and furthermore, were consistently characterized by the existence of a strong N+ H-O= C interaction closing a seven-membered cycle. The dynamics of cis-trans isomerization about the Tyr-Tic peptide bond were probed by nmr exchange experiments. The selective presaturation of exchanging resonances carried out at several temperatures between 50 and 70°C allowed the determination of isomerization rate constants as well as thermodynamic activation parameters. ΔG≠ values were in close agreement with the cis → trans energy barrier found in X-Pro peptide fragments (∼83 kJ/mol).A large entropic barrier determined for the trans → cis conversion of TIP[Ψ](5. 7 JK-1 mol-1 at pH 3. 1; 6. 5 JK-1 mol-1 at pH 9. 1) is discussed in terms of decreased solvent molecular ordering around the conformers possessing a trans Tyr-Tic bond. Evidence that the neutral form of the reduced peptide bond gains rigidity upon protonation was obtained from relaxation measurements in the rotating frame. TJp measurements of several protons in the vicinity of the reduced peptide bond were made as a function of spin-lock field. Quantitative analysis of the relaxation data indicated that chemical shift fluctuations in the 10-4-10-5s range were more pronounced in the case of deprotonated TIP[Ψ]. Results of molecular dynamics simulations in addition to 3Jαβ coupling constant measurements support the experimentally observed greater flexibility in the C-terminal region of TIP[Ψ]. © 1995 John Wiley & Sons, Inc.