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.

Molecular characterisation of the gut microflora of healthy and inflammatory bowel disease cats using fluorescence in situ hybridisation with special reference to Desulfovibrio spp

Inflammatory bowel disease (IBD) is a common cause of chronic large bowel diarrhoea in cats. Although the aetiology of IBD is unknown, an immune-mediated response to a luminal antigen is thought to be involved. As knowledge concerning the colonic microflora of cats is limited and requires further investigation, the purpose of this study was to determine the presence of specific bacterial groups in normal and IBD cats, and the potential role they play in the health of the host. Total bacterial populations, Bacteroides spp., Bifidobacterium spp., Clostridium histolyticum subgp., Lactobacillus-Enterococcus subgp. and Desulfovibrio spp. were enumerated in 34 healthy cats and 11 IBD cats using fluorescence in situ hybridisation. The study is one of the first to show the presence of Desulfovibrio in cats. Total bacteria, Bifidobacterium spp. and Bacteroides spp. counts were all significantly higher in healthy cats when compared with IBD cats, whereas Desulfovibrio spp. (producers of toxic sulphides) numbers were found to be significantly higher in colitic cats. The information obtained from this study suggests that modulation of bacterial flora by increasing bifidobacteria and decreasing Desulfovibrio spp. may be beneficial to cats with IBD. Dietary intervention may be an important aspect of their treatment.

Fibrillisation of hydrophobically modified amyloid peptide fragments in an organic solvent

The self-assembly of a hydrophobically modified fragment of the amyloid beta(A beta) peptide has been studied in methanol. The peptide FFKLVFF is based on A beta(16-20) extended at the N terminus by two phenylalanine residues. The formation of amyloid-type fibrils is confirmed by Congo Red staining, thioflavin T fluorescence and circular dichroism experiments. FTIR points to the formation of beta-sheet structures in solution and in dried films and suggests that aggregation occurs at low concentration and is not strongly affected by further increase in concentration, i.e. the peptide is a strong fibril-former in methanol. UV fluorescence experiments on unstained peptide and CD point to the importance of aromatic interactions between phenylalanine groups in driving aggregation into beta-sheets. The CD spectrum differs from that usually observed for beta-sheet assemblies formed by larger peptides or proteins and this is discussed for solutions in methanol and also trifluoroethanol. The fibril structure is imaged by transmission electron microscopy and scanning electron microscopy on dried samples and is confirmed by small-angle X-ray scattering experiments in solution.

Effect of abomasal glucose infusion on plasma concentrations of gut peptides in periparturient dairy cows

Six Holstein cows fitted with ruminal cannulas and permanent indwelling catheters in the portal vein, hepatic vein, mesenteric vein, and an artery were used to study the effects of abomasal glucose infusion on splanchnic plasma concentrations of gut peptides. The experimental design was a randomized block design with repeated measurements. Cows were assigned to one of 2 treatments: control or infusion of 1,500 g of glucose/d into the abomasum from the day of parturition to 29 d in milk. Cows were sampled 12 ± 6 d prepartum and at 4, 15, and 29 d in milk. Concentrations of glucose-dependent insulinotropic polypeptide, glucagon-like peptide 1(7–36) amide, and oxyntomodulin were measured in pooled samples within cow and sampling day, whereas active ghrelin was measured in samples obtained 30 min before and after feeding at 0800 h. Postpartum, dry matter intake increased at a lower rate with infusion compared with the control. Arterial, portal venous, and hepatic venous plasma concentrations of the measured gut peptides were unaffected by abomasal glucose infusion. The arterial, portal venous, and hepatic venous plasma concentrations of glucose-dependent insulinotropic polypeptide and glucagon-like peptide 1(7–36) amide increased linearly from 12 d prepartum to 29 d postpartum. Plasma concentrations of oxyntomodulin were unaffected by day relative to parturition. Arterial and portal venous plasma concentrations of ghrelin were lower postfeeding compared with prefeeding concentrations. Arterial plasma concentrations of ghrelin were greatest prepartum and lowest at 4 d postpartum, giving a quadratic pattern of change over the transition period. Positive portal venous-arterial and hepatic venous–arterial concentration differences were observed for glucagon-like peptide 1(7–36) amide. A negative portal venous–arterial concentration difference was observed for ghrelin pre-feeding. The remaining portal venous–arterial and hepatic venous–arterial concentration differences of gut peptides did not differ from zero. In conclusion, increased postruminal glucose supply to postpartum transition dairy cows reduced feed intake relative to control cows, but did not affect arterial, portal venous, or hepatic venous plasma concentrations of gut peptide hormones. Instead, gut peptide plasma concentrations increased as lactation progressed. Thus, the lower feed intake of postpartum dairy cows receiving abomasal glucose infusion was not attributable to changes in gut peptide concentrations.

Longitudinal investigation of the faecal microbiota of healthy full-term infants using fluorescence in situ hybridization and denaturing gradient gel electrophoresis.

From birth onwards, the gastrointestinal (GI) tract of infants progressively acquires a complex range of micro-organisms. It is thought that by 2 years of age the GI microbial population has stabilized. Within the developmental period of the infant GI microbiota, weaning is considered to be most critical, as the infant switches from a milk-based diet (breast and/or formula) to a variety of food components. Longitudinal analysis of the biological succession of the infant GI/faecal microbiota is lacking. In this study, faecal samples were obtained regularly from 14 infants from 1 month to 18 months of age. Seven of the infants (including a set of twins) were exclusively breast-fed and seven were exclusively formula-fed prior to weaning, with 175 and 154 faecal samples, respectively, obtained from each group. Diversity and dynamics of the infant faecal microbiota were analysed by using fluorescence in situ hybridization and denaturing gradient gel electrophoresis. Overall, the data demonstrated large inter- and intra-individual differences in the faecal microbiological profiles during the study period. However, the infant faecal microbiota merged with time towards a climax community within and between feeding groups. Data from the twins showed the highest degree of similarity both quantitatively and qualitatively. Inter-individual variation was evident within the infant faecal microbiota and its development, even within exclusively formula-fed infants receiving the same diet. These data can be of help to future clinical trials (e.g. targeted weaning products) to organize protocols and obtain a more accurate outline of the changes and dynamics of the infant GI microbiota.

Evaluation of the time resolved fluorescence immunoassay (TRFIA) for the detection of varicella zoster virus (VZV) antibodies following vaccination of healthcare workers

Determination of varicella zoster virus (VZV) immunity in healthcare workers without a history of chickenpox is important for identifying those in need of vOka vaccination. Post immunisation, healthcare workers in the UK who work with high risk patients are tested for seroconversion. To assess the performance of the time-resolved fluorescence immunoassay (TRFIA) for the detection of antibody in vaccinated as well as unvaccinated individuals, a cut-off was first calculated. VZV-IgG specific avidity and titres six weeks after the first dose of vaccine were used to identify subjects with pre-existing immunity among a cohort of 110 healthcare workers. Those with high avidity (≥60%) were considered to have previous immunity to VZV and those with low or equivocal avidity (<60%) were considered naive. The former had antibody levels ≥400mIU/mL and latter had levels <400mIU/mL. Comparison of the baseline values of the naive and immune groups allowed the estimation of a TRFIA cut-off value of >130mIU/mL which best discriminated between the two groups and this was confirmed by ROC analysis. Using this value, the sensitivity and specificity of TRFIA cut-off were 90% (95% CI 79-96), and 78% (95% CI 61-90) respectively in this population. A subset of samples tested by the gold standard Fluorescence Antibody to Membrane Antigen (FAMA) test showed 84% (54/64) agreement with TRFIA.

Hydrogen-bond assisted stabilization of the less favored conformation of a tridentate Schiff base ligand in dinuclear nickel(II) complex: an experimental and theoretical study

The Schiff base ligand, HL (2-[1-(3-methylamino-propylimino)-ethyl]-phenol), the 1:1 condensation product of 2-hydroxy acetophenone and N-methyl-1,3-diaminopropane, has been synthesized and characterized by X-ray crystallography as the perchlorate salt [H2L]ClO4 (1). The structure consists of discrete [H2L](+) cations and perchlorate anions. Two dinuclear Ni-II complexes, [Ni2L2(NO2)(2)] (2), [Ni2L2(NO3)(2)] (3) have been synthesized using this ligand and characterized by single crystal X-ray analyses. Complexes 2 and 3 are centrosymmetric dimers in which the Ni-II ions are in distorted fac- and mer-octahedral environments, respectively, bridged by two mu(2)-phenolate ions of deprotonated ligand, L. The plane of the phenyl rings and the Ni2O2 basal plane are nearly coplanar in 2 but almost perpendicular in 3. We have studied and explained this different behavior using high level DFT calculations (RI-BP86/def2-TZVP level of theory). The conformation observed in 3, which is energetically less favorable, is stabilized via intermolecular non-covalent interactions. Under the excitation of ultraviolet light, characteristic fluorescence of compound 1 was observed; by comparison fluorescence intensity decreases in case of compound 3 and completely quenched in compound 2.

Hydrogelation of self-assembling RGD-based peptides

The self-assembly of tripeptides based on the RGD cell adhesion motif is investigated. Two tripeptides containing the Fmoc [N-(fluorenyl)-9-methoxycarbonyl] aromatic unit were synthesized, Fmoc-RGD and a control peptide containing a scrambled sequence, Fmoc-GRD. The Fmoc is used to control selfassembly via aromatic stacking interactions. The self-assembly and hydrogelation properties of the two Fmoc-tripeptides are compared. Both form well defined amyloid fibrils (as shown by cryo-TEM and SAXS) with b-sheet features in their circular dichroism and FTIR spectra. Both peptides form selfsupporting hydrogels, the dynamic shear modulus of which was measured. Preliminary cell culture experiments reveal that Fmoc-RGD can be used as a support for bovine fibroblasts, but not Fmoc- GRD, consistent with the incorporation of the cell adhesion motif in the former peptide.

Self-assembly of amphiphilic peptides

The self-assembly of amphiphilic peptides is reviewed. The review covers surfactant-like peptides with amphiphilicity arising from the sequence of natural amino acids, and also peptide amphiphiles (PAs) in which lipid chains are attached to hydrophilic peptide sequences containing charged residues. The influence of the secondary structure on the self-assembled structure and vice versa is discussed. For surfactant-like peptides structures including fibrils, nanotubes, micelles and vesicles have been reported. A particularly common motif for PAs is beta-sheet based fibrils, although other structures have been observed. In these structures, the peptide epitope is presented at the surface of the nanostructure, providing remarkable bioactivity. Recent discoveries of potential, and actual, applications of these materials in biomedicine and bionanotechnology are discussed.

Inhibition of synaptic transmission and G protein modulation by synthetic CaV2.2 Ca2+ channel peptides

Abstract: Modulation of presynaptic voltage-dependent Ca+ channels is a major means of controlling neurotransmitter release. The CaV 2.2 Ca2+ channel subunit contains several inhibitory interaction sites for Gβγ subunits, including the amino terminal (NT) and I–II loop. The NT and I–II loop have also been proposed to undergo a G protein-gated inhibitory interaction, whilst the NT itself has also been proposed to suppress CaV 2 channel activity. Here, we investigate the effects of an amino terminal (CaV 2.2[45–55]) ‘NT peptide’ and a I–II loop alpha interaction domain (CaV 2.2[377–393]) ‘AID peptide’ on synaptic transmission, Ca2+ channel activity and G protein modulation in superior cervical ganglion neurones (SCGNs). Presynaptic injection of NT or AID peptide into SCGN synapses inhibited synaptic transmission and also attenuated noradrenaline-induced G protein modulation. In isolated SCGNs, NT and AID peptides reduced whole-cell Ca2+ current amplitude, modified voltage dependence of Ca2+ channel activation and attenuated noradrenaline-induced G protein modulation. Co-application of NT and AID peptide negated inhibitory actions. Together, these data favour direct peptide interaction with presynaptic Ca2+ channels, with effects on current amplitude and gating representing likely mechanisms responsible for inhibition of synaptic transmission. Mutations to residues reported as determinants of Ca2+ channel function within the NT peptide negated inhibitory effects on synaptic transmission, Ca2+ current amplitude and gating and G protein modulation. A mutation within the proposed QXXER motif for G protein modulation did not abolish inhibitory effects of the AID peptide. This study suggests that the CaV 2.2 amino terminal and I–II loop contribute molecular determinants for Ca2+ channel function; the data favour a direct interaction of peptides with Ca2+ channels to inhibit synaptic transmission and attenuate G protein modulation. Non-technical summary: Nerve cells (neurones) in the body communicate with each other by releasing chemicals (neurotransmitters) which act on proteins called receptors. An important group of receptors (called G protein coupled receptors, GPCRs) regulate the release of neurotransmitters by an action on the ion channels that let calcium into the cell. Here, we show for the first time that small peptides based on specific regions of calcium ion channels involved in GPCR signalling can themselves inhibit nerve cell communication. We show that these peptides act directly on calcium channels to make them more difficult to open and thus reduce calcium influx into native neurones. These peptides also reduce GPCR-mediated signalling. This work is important in increasing our knowledge about modulation of the calcium ion channel protein; such knowledge may help in the development of drugs to prevent signalling in pathways such as those involved in pain perception.

Amyloid peptides incorporating a core sequence from the amyloid beta peptide and gamma amino acids: relating bioactivity to self-assembly

A series of heptapeptides comprising the core sequence Ab(16–20), KLVFF, of the amyloid b peptide coupled with paired N-terminal c-amino acids are investigated in terms of cytotoxicity reduction and binding to the full Ab peptide, both pointing to inhibition of fibrillisation for selected compounds. This is related to the self-assembly capacity of the heptapeptides.

Fluorescence lifetime imaging microscopy (FLIM) to demonstrate the nuclear binding of flavanols and (--epigallocatechin gallate

The use of light microscopy and DMACA staining strongly suggested that plant and animal cell nuclei act as sinks for flavanols [1, 2]. Detailed uv-vis spectroscopic titration experiments indicated that histone proteins are the likely binding sites in the nucleus [2]. Here we report the development of a multi-photon excitation microscopy technique combined with fluorescent lifetime measurements of flavanols. Using this technique, (+) catechin, (-) epicatechin and (-) epigallocatechin gallate (EGCG) showed strikingly different excited state lifetimes in solution. Interaction of histone proteins with flavanols was indicated by the appearance of a significant τ2-component of 1.7 to 4.0ns. Tryptophan interference could be circumvented in the in vivo fluorescence lifetime imaging microscopy (FLIM) experiments with 2-photon excitation at 630nm. This enabled visualisation and semi-quantitative measurements that demonstrated unequivocally the absorption of (+)catechin, (-)epicatechin and EGCG by nuclei of onion cells. 3D FLIM revealed for the first time that externally added EGCG penetrated the whole nucleus in onion cells. The relative proportions of EGCG in cytoplasm: nucleus: nucleoli were ca. 1:10:100. FLIM experiments may therefore facilitate probing the health effects of EGCG, which is the major constituent of green tea.

Evaluation of the environmental specificity of fluorescence in situ hybridization (FISH) using fluorescence-activated cell sorting (FACS) of probe (PSE1284)-positive cells extracted from rhizosphere soil

We explicitly tested for the first time the ‘environmental specificity’ of traditional 16S rRNAtargeted fluorescence in situ hybridization (FISH) through comparison of the bacterial diversity actually targeted in the environment with the diversity that should be exactly targeted (i.e. without mismatches) according to in silico analysis. To do this, we exploited advances in modern Flow Cytometry that enabled improved detection and therefore sorting of sub-micron-sized particles and used probe PSE1284 (designed to target Pseudomonads) applied to Lolium perenne rhizosphere soil as our test system. The 6-carboxyfluorescein (6-FAM)-PSE1284-hybridised population, defined as displaying enhanced green fluorescence in Flow Cytometry, represented 3.51±1.28% of the total detected population when corrected using a nonsense (NON-EUB338) probe control. Analysis of 16S rRNA gene libraries constructed from Fluorescence Activated Cell Sorted (FACS) -recovered fluorescent populations (n=3), revealed that 98.5% (Pseudomonas spp. comprised 68.7% and Burkholderia spp. 29.8%) of the total sorted population was specifically targeted as evidenced by the homology of the 16S rRNA sequences to the probe sequence. In silico evaluation of probe PSE1284 with the use of RDP-10 probeMatch justified the existence of Burkholderia spp. among the sorted cells. The lack of novelty in Pseudomonas spp. sequences uncovered was notable, probably reflecting the well-studied nature of this functionally important genus. To judge the diversity recorded within the FACS-sorted population, rarefaction and DGGE analysis were used to evaluate, respectively, the proportion of Pseudomonas diversity uncovered by the sequencing effort and the representativeness of the Nycodenz® method for the extraction of bacterial cells from soil.

Two-photon excitation with pico-second fluorescence lifetime imaging to detect nuclear association of flavanols

Two-photon excitation enabled for the first time the observation and measurement of excited state fluorescence lifetimes from three flavanols in solution, which were ∼1.0 ns for catechin and epicatechin, but <45 ps for epigallocatechin gallate (EGCG). The shorter lifetime for EGCG is in line with a lower fluorescence quantum yield of 0.003 compared to catechin (0.015) and epicatechin (0.018). In vivo experiments with onion cells demonstrated that tryptophan and quercetin, which tend to be major contributors of background fluorescence in plant cells, have sufficiently low cross sections for two-photon excitation at 630 nm and therefore do not interfere with detection of externally added or endogenous flavanols in Allium cepa or Taxus baccata cells. Applying two-photon excitation to flavanols enabled 3-D fluorescence lifetime imaging microscopy and showed that added EGCG penetrated the whole nucleus of onion cells. Interestingly, EGCG and catechin showed different lifetime behaviour when bound to the nucleus: EGCG lifetime increased from <45 to 200 ps, whilst catechin lifetime decreased from 1.0 ns to 500 ps. Semi-quantitative measurements revealed that the relative ratios of EGCG concentrations in nucleoli associated vesicles: nucleus: cytoplasm were ca. 100:10:1. Solution experiments with catechin, epicatechin and histone proteins provided preliminary evidence, via the appearance of a second lifetime (τ2 = 1.9–3.1 ns), that both flavanols may be interacting with histone proteins. We conclude that there is significant nuclear absorption of flavanols. This advanced imaging using two-photon excitation and biophysical techniques described here will prove valuable for probing the intracellular trafficking and functions of flavanols, such as EGCG, which is the major flavanol of green tea.