Apolipoprotein A-I (ApoA-I) Mimetic Peptide P2a by Restoring CholesterolEsterification Unmasks ApoA-I Anti-Inflammatory Endogenous Activity In Vivo. CO-FIRST AUTHOR

The acute-phase protein haptoglobin (Hpt) binds apolipoprotein A-I (ApoA-I) and impairs its action on lecithin-cholesterol acyltransferase, an enzyme that plays a key role in reverse cholesterol transport. We have previously shown that an ApoA-I mimetic peptide, P2a, displaces Hpt from ApoA-I, restoring the enzyme activity in vitro. The aim of this study was to evaluate whether P2a displaces Hpt from ApoA-I in vivo and whether this event leads to anti-inflammatory activity. Mice received subplantar injections of carrageenan. Paw volume was measured before the injection and 2, 4, 6, 24, 48, 72, and 96 h thereafter. At the same time points, concentrations of HDL cholesterol (C) and cholesterol esters (CEs) were measured by high-performance liquid chromatography, and Hpt and ApoA-I plasma levels were evaluated by enzyme-linked immunosorbent assay. Western blotting analysis for nitric-oxide synthase and cyclooxygenase (COX) isoforms was also performed on paw homogenates. CEs significantly decreased in carrageenan-treated mice during edema development and negatively correlated with the Hpt/ApoA-I ratio. P2a administration significantly restored the CE/C ratio. In addition, P2a displayed an anti-inflammatory effect on the late phase of edema with a significant reduction in COX2 expression coupled to an inhibition of prostaglandin E2 synthesis, implying that, in the presence of P2a, CE/C ratio rescue and edema inhibition were strictly related. In conclusion, the P2a effect is due to its binding to Hpt with consequent displacement of ApoA-I that exerts anti-inflammatory activity. Therefore, it is feasible to design drugs that, by enhancing the physiological endogenous protective role of ApoA-I, may be useful in inflammation-based diseases

Bombesin peptide antagonist for target-selective delivery of liposomal doxorubicin on cancer cells

Purpose: This study addresses novel peptide modified liposomal doxorubicin to specifically target tissues overexpressing bombesin (BN) receptors. Methods: DOTA-(AEEA)2-peptides containing the [7–14]bombesin and the new BN-AA1 sequence have been synthesized to compare their binding properties and in serum stabilities. The amphiphilic peptide derivative (MonYBN- AA1) containing BN-AA1, a hydrophobic moiety, polyethylenglycole (PEG), and diethylenetriaminepentaacetate (DTPA), has been synthesized. Liposomes have been obtained by mixing of MonY-BN-AA1 with 1,2-distearoylsn-glycero-3-phosphocholine (DSPC). Results: Both 111In labeled peptide derivatives present nanomolar Kd to PC-3 cells. 177Lu labeled peptide DOTA- (AEEA)2-BN-AA1 is very stable (half-life 414.1 h), while DOTA-(AEEA)2-BN, shows a half-life of 15.5 h. In vivo studies on the therapeutic efficacy of DSPC/MonY-BN-AA1/Dox in comparison to DSPC/MonY-BN/Dox, were performed in PC-3 xenograft bearing mice. Both formulations showed similar tumor growth inhibition (TGI) compared to control animals treated with non-targeted DSPC/Dox liposomes or saline solution. For DSPC/MonY-BN-AA1/Dox the maximum effect was observed 19 days after treatment. Conclusions: DSPC/MonY-BN-AA1/Dox nanovectors confirm the ability to selectively target and provide therapeutic efficacy in mice. The lack of receptor activation and possible acute biological side effects provided by using the AA1 antagonist bombesin sequence should provide safe working conditions for further development of this class of drug delivery vehicles

Reply to Elmendorf and Ettinger: Photoperiod playsa dominantand irreplaceable role in triggering secondary growth resumption

In their Letter, Elmendorf and Ettinger (1) question the dominant role of photoperiod in driving secondary growth resumption (hereafter referred to as xylem formation onset) of the Northern Hemisphere conifers, recently reported by Huang et al. (2). Their opinions are grounded on the following three aspects, including 1) the seasonality of the photoperiod, 2) the dependence of the predictor variables (e.g., photoperiod, forcing, and chilling) on the response variable (the date of onset of xylem formation, day of the year [DOY]), and 3) the limited value of the obtained models for interannual forecasting. We think they bring up an interesting issue that deserves further discussion and clarification. Photoperiod is acknowledged to regulate spring bud swelling while wood formation starts (3, 4). Although photoperiod seasonality occurs at each site, its influence is marginal in our study given that the analysis involved comparisons among sites across the Northern Hemisphere. Our conclusion that photoperiod plays a dominant role was built upon the combination of several coherent pieces of evidence, rather than “the crux of Huang et al….” as they pointed out. First, we clearly stated that model 2, which modeled DOY as a function of the mean annual temperature of the site (MAT), forcing, chilling, and soil moisture, was considered the best model in terms of parsimony according to minimum Akaike information criterion and Bayesian information criterion, rather than R2 as referred to in their Letter. Second, photoperiod interacted with MAT and can explain 61.7% of the variance of MAT alone (2). Therefore, we concluded that secondary growth resumption was driven primarily by MAT and photoperiod or by their interaction, which is challenging to be disentangled without experimental data, we agree. In terms of biological functioning, they play an ..

Structural Analysis of a Helical Peptide Unfolding Pathway

The analysis of the folding mechanism in peptides adopting welldefined secondary structure is fundamental to understand protein folding. Herein, we describe the thermal unfolding of a 15-mer vascular endothelial growth factor mimicking α-helical peptide (QKL10A) through the combination of spectroscopic and computational analyses. In particular, on the basis of the temperature dependencies of QKL10A Hα chemical shifts we show that the first phase of the thermal helix unfolding, ending at around 320 K, involves mainly the terminal regions. A second phase of the transition, ending at around 333 K, comprises the central helical region of the peptide. The determination of high-resolution QKL10A conformational preferences in water at 313 K allowed us to identify, at atomic resolution, one intermediate of the folding-unfolding pathway. Molecular dynamics simulations corroborate experimental observations detecting a stable central helical turn, which represents the most probable site for the helix nucleation in the folding direction. The data presented herein allows us to draw a folding-unfolding picture for the small peptide QKL10A compatible with the nucleation-propagation model. This study, besides contributing to the basic field of peptide helix folding, is useful to gain an insight into the design of stable helical peptides, which could find applications as molecular scaffolds to target protein-protein interactions. © 2010 Wiley-VCH Verlag GmbH & Co. KGaA