Bioactive peptides from food proteins: new opportunities and challenges

Food proteins such as milk and soy are a rich source of bioactive peptides. In the last decade, research into this area has intensified and new bioactive peptide sequences have been discovered with a range of apparent biological functions; for example, antihypertensive, antioxidant, and antimicrobial effects and opiate-like qualities have been reported. These peptides could therefore lead to the development of important functional food products and ingredients for the prevention and even treatment of chronic diseases such as cardiovascular disease and cancer. Peptides can be produced by fermentation with dairy starters for instance, and by enzymatic hydrolysis with pancreatic and microbial enzymes. Further purification is typically carried out by membrane filtration and/or chromatographic methods. The production of novel bioactive peptides and their incorporation into functional food products poses several technological challenges as well as regulatory and marketing issues. Proof of efficacy is of paramount importance; this should be verified by conducting appropriate tests in vivo in animals and in humans. In addition, tests for cytotoxicity and allergenicity must be conducted. Despite all of these hurdles, scientific evidence is increasingly demonstrating the health benefits of diet-based disease prevention, and therefore new developments in this area are likely to continue both at the research and the commercialisation level.

Permeation of bioactive constituents from Arnica montana preparations through human skin in-vitro

This study investigated and characterised transdermal permeation of bioactive agents from a topically applied Arnica montana tincture. Permeation experiments conducted over 48 h used polyclimethylsiloxane (silastic) and human epidermal membranes mounted in Franz-type diffusion cells with a methanol-water (50:50 v/v) receptor fluid. A commercially available tincture of A. montana L. derived from dried Spanish flower heads was a donor solution. Further donor solutions prepared from this stock tincture concentrated the tincture constituents 1, 2 and 10 fold and its sesquiterpene lactones 10 fold. Permeants were assayed using a high-performance liquid chromatography method. Five components permeated through silastic membranes providing peaks with relative retention factors to an internal standard (santonin) of 0.28, 1.18, 1.45, 1.98 and 2.76, respectively. No permeant was detected within 12 h of applying the Arnica tincture onto human epidermal membranes. However, after 12 h, the first two of these components were detected. These were,shown by Zimmermann reagent reaction to be sesquiterpene lactones and liquid chromatography/diode array detection/mass spectrometry indicated that these two permeants were 11,13-dihydrohelenalin (DH) analogues (methacrylate and tiglate esters). The same two components were also detected within 3 h of topical application of the 10-fold concentrated tincture and the concentrated sesquiterpene lactone extract.

Positioning Janus nanoparticles in block copolymer scaffolds

The periodic domains formed by block copolymer melts have been heralded as potential scaffolds for arranging nanoparticles in 3d space, provided we can control the positioning of the particles. Recent experiments have located particles at the domain interfaces by grafting mixed brushes to their surfaces. Here the underlying mechanism, which involves the transformation into Janus particles, is investigated with self-consistent field theory using a new multi-coordinate-system algorithm.

Lack of association between lipaemia and central adiposity in subjects with an atherogenic lipoprotein phenotype (ALP)

OBJECTIVE: To investigate the associations between indices of adiposity and cardiovascular risk factors in individuals with an atherogenic lipoprotein phenotype (ALP). SUBJECTS: Fifty-five men, aged 34-69 y, body mass index (BMI) 22-35 kg/m2, with an ALP lipid profile (triglycerides (TG) 1.5-4.0 mmol/l, HDL<1.1 mmol/l; %LDL-3>40% total LDL). DESIGN: Each participant provided a fasting blood sample and underwent an 8 h postprandial assessment and had anthropometric measurements taken. OUTCOME MEASURES: BMI, waist circumference (W), waist-to-hip ratio (W/H), sum of skinfolds (SSK), fasting and postprandial concentrations of glucose, insulin and plasma lipids, post-heparin lipase activity, and apoE genotype. RESULTS: The expected positive associations between BMI, W and SSK and fasting and postprandial insulin were observed (r=0.42-0.65). Little association between glucose responses and any measures of adiposity was evident. Unexpectedly, there were no positive associations between measures of central adiposity (W and W/H) and fasting and postprandial TG responses, with a trend towards negative associations in this study group (TG AUC vs W, r=-0.23, P=0.097; TG IAUC vs W/H, r=-0.26, P=0.068). Subgroup analysis indicated that lack of a positive association between central adiposity and postprandial TG values was more evident in those with one E4 allele (r=-0.42, P=0.077) relative to non-E4 carriers (r=-0.16, P=0.430). The expected positive associations between insulin and TG responses were not observed (r=-0.03 to -0.36). CONCLUSION: In this ALP group the expected positive association between TG responses and a centralized distribution of body fat was not observed, particularly in individuals with an apoE4 genotype. Our findings are not in line with the view that there is a clear causal relationship between insulin resistance and the lipid abnormalities associated with ALP.

Tuning the self-assembly of the bioactive dipeptide L-carnosine by incorporation of a bulky aromatic substituent

The dipeptide L-carnosine has a number of important biological properties. Here, we explore the effect of attachment of a bulky hydrophobic aromatic unit, Fmoc [N-(fluorenyl-9-methoxycarbonyl)] on the self-assembly of Fmoc-L-carnosine, i.e., Fmoc-Beta-alanine-histidine (Fmoc-BetaAH). It is shown that Fmoc-BetaAH forms well-defined amyloid fibril containing Beta sheets above a critical aggregation concentration, which is determined from pyrene and ThT fluorescence experiments. Twisted fibrils were imaged by cryogenic transmission electron microscopy. The zinc-binding properties of Fmoc-BetaAH were investigated by FTIR and Raman spectroscopy since the formation of metal ion complexes with the histidine residue in carnosine is well-known, and important to its biological roles. Observed changes in the spectra may reflect differences in the packing of the Fmoc-dipeptides due to electrostatic interactions. Cryo-TEM shows that this leads to changes in the fibril morphology. Hydrogelation is also induced by addition of an appropriate concentration of zinc ions. Our work shows that the Fmoc motif can be employed to drive the self-assembly of carnosine into amyloid fibrils.

Self-assembly of three bacterially-derived bioactive lipopeptides

The self-assembly in aqueous solution of three lipopeptides obtained from Bacillus subtilis has been investigated. The lipopeptides surfactin, plipastatin and mycosubtilin contain distinct cyclic peptide headgroups as well as differences in alkyl chain length, branching and chain length distribution. Cryogenic transmission electron microscopy and X-ray scattering reveal that surfactin and plipastatin aggregate into 2 nm-radius spherical micelles, whereas in complete contrast mycosubtilin self-assembles into extended nanotapes based on bilayer ordering of the lipopeptides. Circular dichroism and FTIR spectroscopy indicate the presence of turn structures in the cyclic peptide headgroup. The unexpected distinct mode of self-assembly of mycosubtilin compared to the other two lipopeptides is ascribed to differences in the surfactant packing parameter. This in turn is due to specific features of the conformation of the peptide headgroup and alkyl chain branching.

Bioactive films produced from self-assembling peptide amphiphiles as versatile substrates for tuning cell adhesion and tissue architecture in serum-free conditions

The development of versatile bioactive surfaces able to emulate in vivo conditions is of enormous importance to the future of cell and tissue therapy. Tuning cell behaviour on two-dimensional surfaces so that the cells perform as if they were in a natural three-dimensional tissue represents a significant challenge, but one that must be met if the early promise of cell and tissue therapy is to be fully realised. Due to the inherent complexities involved in the manufacture of biomimetic three-dimensional substrates, the scaling up of engineered tissue-based therapies may be simpler if based upon proven two-dimensional culture systems. In this work, we developed new coating materials composed of the self-assembling peptide amphiphiles (PAs) C16G3RGD (RGD) and C16G3RGDS (RGDS) shown to control cell adhesion and tissue architecture while avoiding the use of serum. When mixed with the C16ETTES diluent PA at 13 : 87 (mol mol-1) ratio at 1.25 times 10-3 M, the bioactive {PAs} were shown to support optimal adhesion, maximal proliferation, and prolonged viability of human corneal stromal fibroblasts ({hCSFs)}, while improving the cell phenotype. These {PAs} also provided stable adhesive coatings on highly-hydrophobic surfaces composed of striated polytetrafluoroethylene ({PTFE)}, significantly enhancing proliferation of aligned cells and increasing the complexity of the produced tissue. The thickness and structure of this highly-organised tissue were similar to those observed in vivo, comprising aligned newly-deposited extracellular matrix. As such, the developed coatings can constitute a versatile biomaterial for applications in cell biology, tissue engineering, and regenerative medicine requiring serum-free conditions.

The bioactivity of composite Fmoc-RGDS-collagen gels

The incorporation of small bioactive peptide motifs within robust hydrogels constitutes a facile procedure to chemically functionalise cell and tissue scaffolds. In this study, a novel approach to utilise Fmoc-linked peptide amphiphiles comprising the bio-functional cell-adhesion RGDS motif within biomimetic collagen gels was developed. The composite scaffolds thus created were shown to maintain the mechanical properties of the collagen gel while presenting additional bio-activity. In particular, these materials enhanced the adhesion and proliferation of viable human corneal stromal fibroblasts by 300% compared to nonfunctionalised gels. Furthermore, the incorporation of Fmoc-RGDS nanostructures within the collagen matrix significantly suppressed gel shrinkage resulting from the contractile action of encapsulated fibroblasts once activated by serum proteins. These mechanical and biological properties demonstrate that the incorporation of peptide amphiphiles provides a suitable and easy method to circumvent specific biomaterial limitations, such as cell-derived shrinkage, for improved performance in tissue engineering and regenerative medicine applications.

Self-assembly of a dual functional bioactive peptide amphiphile incorporating both matrix metalloprotease substrate and cell adhesion motifs

We describe a bioactive lipopeptide that combines the capacity to promote the adhesion and subsequent self-detachment of live cells, using template-cell-environment feedback interactions. This self-assembling peptide amphiphile comprises a diene-containing hexadecyl lipid chain (C16e) linked to a matrix metalloprotease-cleavable sequence, Thr-Pro-Gly-Pro-Gln-Gly-Ile-Ala-Gly-Gln, and contiguous with a cell-attachment and signalling motif, Arg-Gly-Asp-Ser. Biophysical characterisation revealed that the PA self-assembles into 3 nm diameter spherical micelles above a critical aggregation concentration (cac). In addition, when used in solution at 5–150 nM (well below the cac), the PA is capable of forming film coatings that provide a stable surface for human corneal fibroblasts to attach and grow. Furthermore, these coatings were demonstrated to be sensitive to metalloproteases expressed endogenously by the attached cells, and consequently to elicit the controlled detachment of cells without compromising their viability. As such, this material constitutes a novel class of multi-functional coating for both fundamental and clinical applications in tissue engineering.

Key bioactive reaction products of the NO/H2S interaction are S/N-hybrid species, polysulfides, and nitroxyl

Experimental evidence suggests that nitric oxide (NO) and hydrogen sulfide (H2S) signaling pathways are intimately intertwined, with mutual attenuation or potentiation of biological responses in the cardiovascular system and elsewhere. The chemical basis of this interaction is elusive. Moreover, polysulfides recently emerged as potential mediators of H2S/sulfide signaling, but their biosynthesis and relationship to NO remain enigmatic. We sought to characterize the nature, chemical biology, and bioactivity of key reaction products formed in the NO/sulfide system. At physiological pH, we find that NO and sulfide form a network of cascading chemical reactions that generate radical intermediates as well as anionic and uncharged solutes, with accumulation of three major products: nitrosopersulfide (SSNO−), polysulfides, and dinitrososulfite N-nitrosohydroxylamine-N-sulfonate (SULFI/NO), each with a distinct chemical biology and in vitro and in vivo bioactivity. SSNO− is resistant to thiols and cyanolysis, efficiently donates both sulfane sulfur and NO, and potently lowers blood pressure. Polysulfides are both intermediates and products of SSNO− synthesis/decomposition, and they also decrease blood pressure and enhance arterial compliance. SULFI/NO is a weak combined NO/nitroxyl donor that releases mainly N2O on decomposition; although it affects blood pressure only mildly, it markedly increases cardiac contractility, and formation of its precursor sulfite likely contributes to NO scavenging. Our results unveil an unexpectedly rich network of coupled chemical reactions between NO and H2S/sulfide, suggesting that the bioactivity of either transmitter is governed by concomitant formation of polysulfides and anionic S/N-hybrid species. This conceptual framework would seem to offer ample opportunities for the modulation of fundamental biological processes governed by redox switching and sulfur trafficking.

Production of bioactive diterpenoids in the Euphorbiaceae depends on evolutionarily conserved gene clusters

The Euphorbiaceae produce a diverse range of diterpenoids, many of which have pharmacological activities. These diterpenoids include ingenol mebutate, which is licensed for the treatment of a precancerous skin condition (actinic keratosis), and phorbol derivatives such as resiniferatoxin and prostratin, which are undergoing investigation for the treatment of severe pain and HIV, respectively. Despite the interest in these diterpenoids, their biosynthesis is poorly understood at present, with the only characterized step being the conversion of geranylgeranyl pyrophosphate into casbene. Here, we report a physical cluster of diterpenoid biosynthetic genes from castor (Ricinus communis), including casbene synthases and cytochrome P450s from the CYP726A subfamily. CYP726A14, CYP726A17, and CYP726A18 were able to catalyze 5-oxidation of casbene, a conserved oxidation step in the biosynthesis of this family of medicinally important diterpenoids. CYP726A16 catalyzed 7,8-epoxidation of 5-keto-casbene and CYP726A15 catalyzed 5-oxidation of neocembrene. Evidence of similar gene clustering was also found in two other Euphorbiaceae, including Euphorbia peplus, the source organism of ingenol mebutate. These results demonstrate conservation of gene clusters at the higher taxonomic level of the plant family and that this phenomenon could prove useful in further elucidating diterpenoid biosynthetic pathways.