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In this study, we aimed to assess whether the composite of chitosan/ZnO‐doped bioglass can be applied as a suitable scaffold for the incorporation of bioactive peptides. Material of a porous composite with 1:1 ratio of bioglass:polymer was produced and used as a matrix for delivery of peptide. A peptide with the PEPTIDES sequence (Pro‐Glu‐Pro‐Thr‐Ile‐Asp‐Glu‐Ser) was chosen as a model peptide. Microstructure and pore sizes of chitosan/ZnO‐doped bioglass were assessed. Open porosity and pore sizes of the composite were suitable for enabling the migration of cells and ensuring the easy delivery of nutrients within the implant. In addition, composite showed bioactivity and bactericidal activity against Staphylococcus aureus and Pseudomonas aeruginosa strains. Peptide alone did not have any cytotoxic activity on human fibroblasts and keratinocytes. Also it did not show any antibacterial properties and did not cause hemolysis of red blood cells. The peptide incorporated in composite showed a rapid release in the kinetics profile. The obtained results indicate that there is the technological possibility to incorporate peptides in chitosan/ZnO‐doped bioglass scaffolds. Such biomaterials have potential application in bone tissue engineering. [ABSTRACT FROM AUTHOR]

Endoplasmic reticulum (ER) stress conditions promote a cellular adaptive mechanism called the unfolded protein response (UPR) that utilizes three stress sensors, inositol‐requiring protein 1, protein kinase RNA‐like ER kinase, and activating transcription factor 6. These sensors activate a number of pathways to reduce the stress and facilitate cell survival. While much is known about the mechanisms involved that modulate apoptosis during chronic stress, less is known about the transition between the prosurvival and proapoptotic factors that determine cell fate. Here, we employed a genetic screen that utilized three different pharmacological stressors to induce ER stress in a human‐immortalized airway epithelial cell line, immortalized human bronchial epithelial cells. We followed the stress responses over an 18‐h time course and utilized real‐time monitoring of cell survival, next‐generation sequencing, and quantitative real‐time PCR to identify and validate genes that were upregulated with all three commonly employed ER stressors, inhibitor of calpain 1, tunicamycin, and thapsigargin. growth arrest and DNA damage‐inducible alpha (GADD45A), a proapoptotic factor, and regulator of calcineurin 1 (RCAN1) mRNAs were identified and verified by showing that small interfering RNA (siRNA) knockdown of GADD45A decreased CCAAT‐enhancer‐binding protein homologous protein (a.k.a DDIT3), BCL2‐binding component 3 (a.k.a. BBC3), and phorbol‐12‐myristate‐13‐acetate‐induced protein 1 expression, 3 proapoptotic factors, and increased cell viability during ER stress conditions, whereas siRNA knockdown of RCAN1 dramatically decreased cell viability. These results suggest that the relative levels of these two genes regulate cell fate decisions during ER stress independent of the type of ER stressor. [ABSTRACT FROM AUTHOR]

This is the study on the effect of opiorphin, sialorphin and their analogs on antitumor activity. We demonstrated that conjugation of opiorphin and sialorphin with a proapoptotic, antimicrobial peptide klak (klaklakklaklak) led to compounds (opio-klak and sialo-klak) that were cytotoxic against cancer cells (LN18, PC3, A549, HCT116 and B10-F16) in the MTT test. The conjugated analogs were designed to increase the effectiveness of the peptide. The opio-klak derivative was the most effective in the in vitro assays and led to a decrease in viability of cancer cells over time as compared with that of untreated controls. In contrast, treatment with either the untargeted klak peptide or opiorphin as a negative control led to a negligible loss in viability. Antitumor effect of the opio-klak was also observed in vivo in murine melanoma tumor-bearing mice. Cessation of peptide administration resulted in tumor regrowth. Our results are seemingly valuable for the development of opiorphin analogs with potential clinical applications. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd. [ABSTRACT FROM AUTHOR]

Peptide-based drugs are promising group of compounds which are characterized by specificity to their in vivo targets and high potency of action (antineoplastic, immunoregulatory, antibacterial). The peptides, however, involve a relatively high risk of allergic reactions that are not predictable on the basis of their sequence and chemical properties. In this study, peripheral blood was obtained from 53 patients including 38 hypersensitive patients and 15 control patients. Basophil activation stimulated by two antibacterial peptides (camel, citropin 1.1), and acetylsalicylic acid was assessed by means of BAT (basophil activation test). Basophil activation stimulated by camel occurred in 7 of 38 patients with hypersensitivity (18.42%) as well as in 2 of 15 control patients (13.33%). Basophils were activated by citropin 1.1 in 7 of 38 hypersensitive patients (18.42%) and in none of the control patients. Using the Structural Database of Allergenic Proteins, we confirmed that the examined peptides share some structural similarities with common environmental allergens. Therefore, the cross-reactivity between potentially present anti-allergen IgE with examined peptides cannot be excluded. Our study proved that BAT, together with other biological tests and specific databases of allergenic compounds, may serve as an initial selection of new active peptides and proteins. [ABSTRACT FROM AUTHOR]

Poly( l-lysine) dendrimer end-functionalized with arginine-6-oligomer conjugated biodegradable poly(3-hydroxybutyrate) (PHB)-poly(ethylene glycol) (PEG)-PHB copolymer may be considered a promising macromolecular drug delivery vehicle. To prepare amphiphilic triblock copolymers, bulk ring-opening polymerization (ROP) of ( R, S)-β-butyrolactone initiated with poly(ethylene glycol) is carried out. The copolymers are synthesized with a high yield of 90%. The structure and physical-chemical properties of the obtained products are confirmed by hydrogen and carbon nuclear magnetic resonance (1H and 13C NMR), viscosity method, gel permeation chromatography (GPC), matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF), and differential scanning calorimetry (DSC) technique. Furthermore, the cytotoxicity of the synthesized copolymers is evaluated with a bacterial luminescence test and protozoan assay, which show that the obtained copolymers are not cytotoxic. Poly( l-lysine) dendrimer end-functionalized with arginine-6-oligomer is further covalently bonded to a copolymer using the 1,1'-carbonyldiimidazole/4-(dimethylamino)pyridine (CDI/DMAP) coupling method. The presence of a newly formed urethane bond between the peptide dendrimer and the copolymer is confirmed by 1H NMR studies. This novel peptide dendrimer functionalized with an amphiphilic triblock copolymer can be applied as a compatible biodegradable material for drug carrier applications.β-butyrolactone; biodegradable polymers; drug delivery system; peptide dendrimers; ring-opening polymerization [ABSTRACT FROM AUTHOR]

Hypoxia-inducible factors (HIFs) 1 and 2 are dimeric α/β transcription factors that regulate cellular responses to low oxygen. HIF-1 is induced first, whereas HIF-2 is associated with chronic hypoxia. To determine how HIF1AmRNA, the inducible subunit of HIF-1, is regulated during hypoxia, we followed HIF1A mRNA levels in primary HUVECs over 24 hours using quantitative PCR. HIF1A and VEGF A (VEGFA) mRNA, a transcriptional target of HIF-1, increased ~2.5- and 8-fold at 2-4 hours, respectively. To determine how the mRNAs were regulated, we identified a microRNA (miRNA), miR-429, that destabilized HIF1A message and decreased VEGFA mRNA by inhibiting HIF1A Target protector analysis, which interferes with miRNA-mRNA complex formation, confirmed that miR-429 targeted HIF1A message. Desferox-amine treatment, which inhibits the hydroxylases that promote HIF-1 a protein degradation, stabilized HIF-1 activity during normoxic conditions and elevated miR-429 levels, demonstrating that HIF-1 promotes miR-429 expression. RNA-sequencing-based transcriptome analysis indicated that inhibition of miRNA-429 in HUVECs up-regulated 209 mRNAs, a number of which regulate angio-genesis. The results demonstrate that HIF-1 is in a negative regulatory loop with miR-429, that miR-429 attenuates HIF-1 activity by decreasing HIF1A message during the early stages of hypoxia before HIF-2 is activated, and this regulatory network helps explain the HIF-1 transition to HIF-2 during chronic hypoxia in endothelial cells. [ABSTRACT FROM AUTHOR]

The occurrence of resistance or side effects in patients receiving antifungal agents leads to failure in the treatment of mycosis. The aim of this experimental study was to investigate the in vitro effects of IB-367 alone and in combination with three standard antifungal drugs, fluconazole ( FLU), itraconazole ( ITRA) and terbinafine ( TERB), against 20 clinical isolates of dermatophytes belonging to three species. Minimum inhibitory concentrations ( MICs), minimal fungicidal concentrations ( MFCs), synergy test, time-kill curves, fungal biomass ( FB) and hyphal damage using 2,3-bis-(2-methoxy-4-nitro-5-sulfenylamino carbonil)-2H-tetrazolium hydroxide assay ( XTT) were performed to study the efficacy of IB-367. In this study, we observed that TERB and ITRA had MICs lower values for all the strains compared to IB-367 and FLU. Synergy was found in 35%, 30% and 25% of IB-367/ FLU, IB-367/ ITRA and IB-367/ TERB interactions respectively. IB-367 exerted a fungicidal activity against Trichophyton mentagrophytes, T. rubrum and Microsporum canis at concentrations starting from 1x MIC. At a concentration of 5x MIC, IB-367 showed the highest rates of hyphae damage for M. canis 53% and T. mentagrophytes 50%; against the same isolates it caused a reduction of 1 log of the total viable count cell hyphae damage. We propose IB-367 as a promising candidate for the future design of antifungal drugs. [ABSTRACT FROM AUTHOR]

Opiorphin and sialorphin are two recently discovered endogenous enkephalin-degrading enzyme inhibitors. Our aim was to characterize their effect on the mouse ileum motility and to investigate the role of glutamine in position 1. Opiorphin, sialorphin, and their analogs substituted in position 1 with pyroglutamic acid (pGlu) were synthesized by the solid-phase method using Fmoc chemistry. The effect of peptides on gastrointestinal (GI) motility was characterized using in vitro assays and in mouse model of upper GI transit. Opiorphin and sialorphin, but not their analogs, significantly increased electrical field-stimulated contractions in the mouse ileum in a δ-opioid receptor-dependent manner. Opiorphin, sialorphin, and their analogs did not influence the effect of [Met5]enkephalin on smooth muscle contractility in the mouse ileum in vitro. [Met5]enkephalin and sialorphin, but not opiorphin injected intravenously (1 mg/kg), significantly inhibited the upper GI transit. The intraperitoneal administration of peptides (3 mg/kg) did not change the mouse upper GI transit. In conclusion, this is the first study investigating the effect of opiorphin and sialorphin on the mouse ileum motility and demonstrating that glutamine in position 1 is crucial for their pharmacological action. Our results may be important for further structure-activity relationship studies on opiorphin and sialorphin and future development of potent clinical therapeutics aiming at the enkephalinergic system. Copyright © 2013 European Peptide Society and John Wiley & Sons, Ltd. [ABSTRACT FROM AUTHOR]

The utility of three various analytical techniques [ion chromatography (IC), capillary electrophoresis (CE) and isotachophoresis (ITP)] was tested in the determination of counter-ions in synthetic peptides. The analyzed ions were acetates, trifluoroacetates and chlorides. IC provided the best results; CE, except limit of detection and limit of quantification, exhibited the comparable results. ITP was classified as the less useful because of the problem with the determination of the chloride ions. Nevertheless, all the three techniques were able to analyze trifluoroacetates and acetates ions with satisfactory results. Except analytical methods, three procedures using hydrochloric acid (HCl) (at two different concentrations) and acetic acid as sample solvents processed by lyophilization were tested. It has been found that the lyophilization not only by HCl but also by acetic acid is a simple and cheap procedure for removal of toxic trifluoroacetic counter-ions from peptides on satisfactory levels. Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd. [ABSTRACT FROM AUTHOR]

The article examines the effects of perturbations imposed on the amphipatic moments of Gramicidin S (GS) cyclo-decapeptide analogues. Previous studies indicate that better antimicrobial selectivity is attained when the amphipatic moments of the analogues of GS-14 is perturbed. Structural studies of GS by molecular dynamics show a two-stranded antiparallel Β-sheets imposed by two Β-turns. The study concluded that the idea of perturbation on the amphipatic moment does not work for GS-10-0 analogues.

Superantigens are proteins comprising a group of molecules produced by various microorganisms. They are involved in pathogenesis of several human diseases. The aim of the study was the comparison of susceptibility to antibiotics and antimicrobial peptides (AMPs) of Staphylococcus aureus (SA) strains producing staphylococcal enterotoxins SEA, SEB, SEC, SED, and TSST-1 and nonproducing ones. In the group of the total 28 of the patients with erythrodermia the presence of SA was confirmed in 24 cases. The total of 14 strains of SA excreted enterotoxins SEA, SEC, SED, and TSST-1. We did not observe that strains producing mentioned superantigens were less susceptible to AMPs (aurein 1.2, citropin 1.1, lipopeptide, protegrin 1, tachyplesin 3, temporin A, and uperin 3.6). The opposite situation was observed in conventional antibiotics. SA strains excreting tested superantigens had higher MICs and MBCs than nonproducing ones. The interesting finding considering the high efficacy of AMPs, against all examined strains of SA, makes them attractive candidates for therapeutic implication. [ABSTRACT FROM AUTHOR]

The natural antimicrobial cationic peptide protegrin-1 displays a broad spectrum of antimicrobial activity and rapidly kills pathogens by interacting with their cell membrane. We investigated the structure–activity relationships of three protegrin-1 analogues: IB-367 (RGGLCYCRGRFCVCVGR-NH2), BM-1 (RGLCYCRGRFCVCVG-NH2) and BM-2 (RGLCYRPRFVCVG-NH2). Our antimicrobial and antifungal activity studies of these peptides showed that BM-1 was much more active than IB-367 against Gram-positive bacteria and fungi, whereas BM-2 was inactive. The BM-1 peptide showed fourfold reduced haemolysis relative to IB-367, an additional advantage of this peptide. In addition, BM-1 was about 15% cheaper than IB-367 to synthesize. The absence of two cysteine residues in the BM-2 sequence could be the main reason for its unstable conformation and antimicrobial inactivity. The solution structures of these peptides were determined in dimethyl sulphoxide using two-dimensional NMR and restrained molecular dynamics calculations. IB-367 and BM-1 formed short, antiparallel, β-hairpin structures connected by a type II′β-turn. The shorter, inactive BM-2 analogue exhibited major and minor conformations (predominantly unordered) in the NMR spectra and was much more flexible. [ABSTRACT FROM AUTHOR]

Temporin A (TA) is a small, basic and highly hydrophobic peptide, isolated from the skin of the European red frog, Rana temporaria. The TA (FLPLIGRVLSGIL-NH2) displays a broad spectrum of anti-microbial activity against Gram-positive bacteria and fungi Candida albicans. In this study we investigate the solution structure of two TA retro-analogues, (6-1)(7-13)-TA (GILPLFRVLSGIL-NH2) and retro-TA (LIGSLVRGILPLF-NH2) by using nuclear magnetic resonance (NMR). The 3D solution structure of the analogues was established by using inter-proton distances and vicinal coupling constants in the Simulated Annealing (SA) calculations (XPLOR program). The NMR conformational studies show the existence of the helical structure in the middle part of the (6-1)(7-13)-TA peptide and an unordered structure of the retro-TA analogue under the D3-TFE/H2O (3:7, v/v) conditions. Our investigations have shown that the hydrophobic cluster at N-terminus with the Pro amino acid residue in position 3 or 4, the helical structure and the amphipathic character of the peptide are responsible for the anti-microbial activity of the TA analogues. Copyright © 2007 European Peptide Society and John Wiley & Sons, Ltd. [ABSTRACT FROM AUTHOR]

Human infection by the vector-borne protozoan Leishmania is responsible for substantial worldwide morbidity and mortality. The surface-metalloprotease (leishmanolysin) of Leishmania is a virulence factor which contributes to a variety of functions including evasion of complement-mediated parasite-killing and host intramacrophage survival. We tested the hypothesis that leishmanolysin serves to protect parasites from the cytolytic effects of various antimicrobial peptides (AMPs) which are important components of the innate immune system. We found that members of the α- and θ-defensins, magainins and cathelicidins had substantially higher leishmanicidal activity against leishmanolysin-knock out mutants of L. major. Using the magainin analogue, pexiganan, as a model peptide we show that AMP evasion is due to rapid and extensive peptide degradation by wild-type parasites. Pexiganan-treatment of knock out mutants induced disruption of surface-membrane permeability and expression of features of apoptosis including smaller cell size, loss of mitochondrial membrane potential, exposure of surface phosphatidyl serine as well as induction of caspase 3/7 activity. These results demonstrate leishmanolysin as a virulence factor preventing AMP-mediated apoptotic killing. This study serves as a platform for the dissection of the AMP-mediated death pathways of Leishmania and demonstrates the potential that AMP evasion plays during host infection by this parasite. [ABSTRACT FROM AUTHOR]

Edeines are pentapeptide amide antibiotics composed of four nonprotein amino acids, glycine, and polyamine. They exhibit antimicrobial and immunosuppressive activities and are universal inhibitors of translation. Moreover, it was proven that the free ionizable carboxy group in the (2 R, 6 S, 7 R)-2,6-diamino-7-hydroxyazelaic acid moiety is not essential for biological activity of these compounds. In this paper we describe the synthesis of four novel edeine A and D analogues in which the above-mentioned acid residue was replaced with the (3 R, 4 S)- or (3 S, 4 S)-4,5-diamino-3-hydroxypentanoic acid moiety. In one compound we also introduced into molecule the 3- N, N-dimethyl derivative of ( S)-2,3-diaminopropanoic acid to prevent the transpeptidation process, which results in the loss of biological activity of α-isomers of edeines. All peptides were synthesized applying the active ester and azide methods and on the basis of the coupling of suitable N-terminal tripeptides with proper C-terminal dipeptide amides. The activities of the newly obtained edeine analogues against selected strains of bacteria and fungi are also presented. Copyright © 2006 European Peptide Society and John Wiley & Sons, Ltd. [ABSTRACT FROM AUTHOR]

Temporin A (TA) is a hydrophobic peptide isolated from the skin of the European red frog Rana temporaria. Strong antimicrobial activity against gram-positive cocci and Candida, as well as its small molecular weight (10-13 aa residues), makes TA an interesting antimicrobial compound. However, its synthesis is rather problematic. Here, the synthesis of two retro-analogues of TA - retro-TA and (6-1)(7-13)-TA-is reported. The synthesis of retro-TA was performed without any problems, while during the synthesis of (6-1)(7-13)-TA problems similar to those encountered during the synthesis of TA were faced. Antimicrobial assays showed minimal inhibitory concentration (MIC) values of retro-TA to be, in most cases, only one dilution higher than those of original TA, but still remained relatively low. An analysis of the circular dichroism spectra of the peptides shows that TA and (6-1)(7-13)-TA adopt an α-helical structure in a hydrophobic environment, while retro-TA forms mainly unordered conformation under both hydrophobic and hydrophilic conditions. One can postulate that differences in conformation of the peptide chain might be responsible for the lower antimicrobial activity of retro-TA as compared to that of the parent molecule. In any case, retro-TA can be interesting owing to its simple and nonproblematic synthesis. Copyright © 2006 European Peptide Society and John Wiley & Sons, Ltd. [ABSTRACT FROM AUTHOR]