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N1.5-Glycosyl enkephalins are potent analgesics. Two enkephalin-related glycosyl peptides: [DMet2, Pro5] enkephalin (N1.5 β-glucopyranosyl] amide and [DMet2, Pro5] enkephalin [N1.5 β-galactosyl] amide have been prepared following the liquid phase procedure. The chromatographic behavior of both the final glycosyl enkephalins and their synthetic intermediates on reversed-phase columns has been tested. Semipreparative, reversed-phase, HPLC conditions have been developed in order to obtain a simple and rapid procedure for the purification of the two synthetic pentapeptides. Thus, using isocratic elution, injections of 10mg of crude material after gel filtration gave homogeneous (99.9%) glycosyl enkephalins.

The miscibility of phosphatidylserine, phosphatidylcholine, and cholesterol in monolayers were studied. The influence of sodium and calcium ions in this system was determined. The compression isotherms of mixed monolayers of the above cited three components spread on subphases containing opiate molecules are elucidated. Moreover, the penetration kinetics of opiate molecules in these mixed monolayers was also recorded. The results show that the presence of cholesterol always lowers the penetration of opioid molecules; this effect is weaken for meperidine, the most hydrophobic of the molecules assayed.

By using both β-N-glycosylation and β-O-glycosylation procedures, different methods for the incorporation of glucose moieties to proline, hydroxyproline or glutamic acid containing protected neuropeptides have been examined. As far as glycosylation of Glu and Hyp containing fragments is concerned, the Incorporation of either γ-β-N-glucosylated glutamic acid or 4-β-O-glucosylated hydroxyproline to the rest of the peptide have been chosen. However, in the case of C-termlnal proline containing peptide fragments, direct β-N-glucosylation of the full peptide has been preferred. Acetyl protecting groups on the sugar moiety led to better yields than the bulkier benzyl groups.

The relative potencies of a series of substance P (6–11) analogues have been determined for spasmogenic activity in the guinea pig ileum in vitro and for potentiation of electrically evoked contractions in the rat vas deferens in vitro. ED 50 values were determined for the new analogues. Substance P and its methyl ester were used as standard agonists. Substitution of Gly 9 by Pro on [Glu 6 ]SP (6–11) increased four times the activity on the NK-1 receptor. The glycosilation of [Glu 6 ]SP (6–11) by the incorporation of a β-D-glucopyranosyl amide residue on the γ-carboxyl group of Glu 6 reduced both the activity and selectivity. The simultaneous substitution of Gly 9 by Pro and the incorporation of a monosaccharide moiety on the γ-carboxyl of Glu 6 on [Glu 6 ]SP (6–11) yielded an analogue with 60-fold enhanced selectivity relative to substance P for the NK-1 receptor. These results may indicate that the critical factor providing potency to SP (6–11) analogues is mostly related to conformational rather than hydrophilicity aspects of the molecular structure.

The peptide sequence (175–192) RFPFHRCGAGPKLTKDLE (P59) of the E2 envelope protein of GB virus C (GBV-C) has been proved to decrease cellular membrane fusion and interfere with the HIV-1 infectivity in a dose-dependent manner. Based on these previous results, the main objective of this study was to deepen in the physicochemical aspects involved in this interaction. First, we analyzed the surface activity of P59 at the air–water interface as well as its interaction with zwitterionic or negatively charged lipid monolayers. Then we performed the same experiments with mixtures of P59/gp41-FP. Studies on lipid monolayers helped us to understand the lipid–peptide interaction and the influence of phospholipids on peptide penetration into lipid media. On another hand, studies with lipid bilayers showed that P59 decreased gp41-FP binding to anionic Large Unilamellar Vesicles. Results can be attributed to the differences in morphology of the peptides, as observed by Atomic Force Microscopy. When P59 and gp41-FP were incubated together, annular structures of about 200 nm in diameter appeared on the mica surface, thus indicating a peptide–peptide interaction. All these results confirm the gp41-FP–P59 interaction and thus support the hypothesis that gp41-FP is inhibited by P59., This work was supported by Grants CTQ2009-13969-C02-01/02 from the Ministerio de Ciencia e Innovación and by 2009SGR560 from the Generalitat de Catalunya, Spain. We thank Mrs. Tania Ballesteros for her technical assistance in the monolayer section. We are grateful to Robin Rycroft and Tanya Yates of the Serveis Lingüístics (UB) for their careful correction of grammar and stile. Finally, we would like to thank the referees for their suggestions to improve the manuscript.

© 2014 Elsevier B.V. All rights reserved. The interaction between a peptide sequence from GB virus C E1 protein (E1P8) and its structural analogs (E1P8-12), (E1P8-13), and (E1P8-21) with anionic lipid membranes (POPG vesicles and POPG, DPPG or DPPC/DPPG (2:1) monolayers) and their association with HIV-1 fusion peptide (HIV-1 FP) inhibition at the membrane level were studied using biophysical methods. All peptides showed surface activity but leakage experiments in vesicles as well as insertion kinetics in monolayers and lipid/peptide miscibility indicated a low level of interaction: neither E1P8 nor its analogs induced the release of vesicular content and the exclusion pressure values (πe) were clearly lower than the biological membrane pressure (24-30 mN m- 1) and the HIV-1 FP (35 mN m- 1). Miscibility was elucidated in terms of the additivity rule and excess free energy of mixing (GE). E1P8, E1P8-12 and E1P8-21 (but not E1P8-13) induced expansion of the POPG monolayer. The mixing process is not thermodynamically favored as the positive GE values indicate. To determine how E1 peptides interfere in the action of HIV-1 FP at the membrane level, mixed monolayers of HIV-1 FP/E1 peptides (2:1) and POPG were obtained. E1P8 and its derivative E1P8-21 showed the greatest HIV-1 FP inhibition. The LC-LE phase lipid behavior was morphologically examined via fluorescence microscopy (FM) and atomic force microscopy (AFM). Images revealed that the E1 peptides modify HIV-1 FP-lipid interaction. This fact may be attributed to a peptide/peptide interaction as indicated by AFM results. Finally, hemolysis assay demonstrated that E1 peptides inhibit HIV-1 FP activity.

A new series of O-glycosyl enkephalins has been prepared, following a convergent strategy, with high chemical yields. The galactosyl analogue, O1.5-(beta-D-galactopyranosyl) [DMet2, Hyp5] enkephalin amide proved to be one of the most potent in vivo opioid agonists synthesized up to now.

Antibodies directed against citrullinated proteins and peptides (ACPAs) are the most specific serological markers available for diagnosing rheumatoid arthritis (RA). ACPAs may be detected several years before symptoms of RA appear, and their presence at disease onset is a good predictor of the development of erosive joint lesions. RA patients can be classified into two major groups: those who have ACPAs and those who do not. The presence of ACPAs at early stages of RA predicts the development of earlier and more widespread joint erosions, and low remission rates.Synthetic peptides can replace cognate proteins in solid-phase assays for specific autoantibody recognition in RA patients. The use of synthetic peptides instead of proteins represents an advantage in terms of the reproducibility of such immunoassays. Proteins also contain non-citrullinated epitopes that are recognized by non-RA sera and this could reduce the specificity of the test. The use of synthetic citrullinated peptides gives absolute control over the exact epitopes presented. Furthermore, it is difficult to prepare sufficient amounts of high-quality antigenic proteins with a well-defined degree of citrullination. Synthetic citrullinated peptides, in contrast, are easily obtained in a pure form with a well-defined chemical structure and the epitopes can be precisely oriented in the plate by covalent binding of the peptides.Chimeric peptides bearing different citrullinated protein domains have recently been used in the design of RA diagnosis systems. The results of the application of those systems indicate that more than one serological test is required to classify RA patients based on the presence or absence of ACPAs. Each of the target molecules reported (fibrin, vimentin and filaggrin) helps to identify a particular subset of RA patients.

Smoking is a known predictor of rheumatoid arthritis (RA), but its effect in radiographic progression is still controversial. To investigate the effects of cigarette smoking on radiographic progression in an early RA cohort two years after the beginning of DMARD therapy. Method: One hundred and fifty six patients (83% female) with early RA (disease duration < 2 years) treated with a similar therapeutic strategy with DMARDs and low-dose glucocorticoids were included. Several demographic, genetic (DRB genotype), clinical, laboratory and radiographic data were obtained at baseline and after one and two years of follow-up. Smoking was evaluated at study entry and patients were classified as past smokers, current smokers and non-smokers. Radiographic progression after one and two years of follow-up was evaluated in hand and feet radiographs using the erosion joint count (EJC) and Larsen-Scott score. Multivariate lineal regression analysis was performed. Result: The frequency of current, past and non-smokers were 30.1%, 12.2% and 57.7% respectively. Only 14% of patients were heavy smokers (> 20 pack/year). Radiographic damage at one and two years was higher in current smokers compared with non-smokers, although only in the EJC at two years the difference was statistically significant (1.2±1.7 vs. 0.7±1.7, p=0.04). The Larsen score at two years was higher in current smokers than in non-smokers although the difference was not significant (7.4±12.9 vs. 4.2±6.7, p>0.05). In the univariate analysis taking Larsen-Scott score as the measure of radiographic damage, female sex, higher HAQ at disease onset, the presence of anticitrullinated antibodies, the shared epitope (SE) and the HLA-DRB*04 had a significant effect on radiological score. In the multivariate regression analysis adjusted for baseline radiographic damage, female sex, HLA-DRB*04 genotype and current smoking were independently associated with two year radiographic damage measured by the Larsen Score as well as with the EJC (Table). No significant differences were seen in clinical disease activity or rates of EULAR response at one and two years of follow-up between smokers and non smokers. Conclusion: In our early RA cohort, current smoking emerges as an independent factor for radiographic progression after two years of DMARDs.

Miscibility of dipalmitoylphosphatidylcholine (DPPC) with dipalmitoylphosphatidylglycerol (DPPG), cardiolipin (CL), and dipalmitoylphosphatidylethanolamine (DPPE) was studied using mono- and bilayers of different compositions. The influence of MAP4-VP1 peptide construct (related to hepatitis A virus protein) in mixed monolayers of the lipids above cited was determined by studying penetration kinetics and compression isotherms. Moreover, liposomes with the same composition than monolayers were saturated with sodium anilinonaphthalene sulfonate or diphenylhexatriene and incubated with MAP4-VP1; polarization values as well as transition temperatures were determined. Results in isotherm and polarization studies showed some degree of interaction between the peptide and the phospholipids assayed. DPPC/DPPG composition showed the maximum interaction followed by DPPC/CL. The interaction was not strong, suggesting that the entrapment of MAP4-VP1 in liposomes in order to increase its immunogenic activity is possible.

Novel strategies in the design of HIV-1 fusion/entry inhibitors are based on the construction of dual-targeting fusion proteins and peptides with synergistic antiviral effects. In this work we describe the design of dual-targeting peptides composed of peptide domains of E2 and E1 envelope proteins from Human Pegivirus with the aim of targeting both the loop region and the fusion peptide domains of HIV-1 gp41. In a previous work, we described the inhibitory role of a highly conserved fragment of the E1 protein (domain 139–156) which interacts with the HIV-1 fusion peptide at the membrane level. Here, two different dual-targeting peptides, where this E1 peptide is located on the N- or the C-terminus respectively, have been chemically synthesized and their antiviral activities have been evaluated with HIV pseudotyped viruses from different clades. The study of the functional behaviour of peptides in a membranous environment attending to the peptide recognition of the target sites on gp41, the peptide conformation as well as the peptide affinity to the membrane, demonstrate that antiviral activity of the dual-targeting peptides is directly related to the peptide affinity and its subsequent assembly into the model membrane. The overall results point out to the necessity that fusion inhibitor peptides that specifically interfere with the N-terminal region of gp41 are embedded within the membrane in order to properly interact with their viral target. © 2019, The Author(s)., Financial support from the Spanish Ministry of Economy, Industry and Competitiveness (MINECO) and the European Regional Development Fund (Grant CTQ2015-63919-R) is gratefully acknowledged. JM and AM were supported by a grant from the Spanish Ministry of Economy, Industry and Competitive-ness and FEDER grant SAF2016-75505-R (AEI/MINECO/FEDER, UE) and through the “María de Maeztu” Programme for Units of Excellence in R&D (MDM-2014-0370). Partial financial support from the Spanish National Research Council (CSIC) is also gratefully acknowledged.