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.

Rhodium cationic complexes using dithioethers as chiral ligands. Application in styrene hydroformylation

Addition of the dithioethers (−)-DIOSR2 (R=Me, iPr) (2,3-O-isopropylidene-1,4-dimethyl (and diisopropyl) thioether-L-threitol) to a dichloromethane solution of [Rh(COD)2]ClO4 (COD=1,5-cyclooctadiene) yielded the mononuclear complexes [Rh(COD)(DIOSR2)]ClO4. X-ray diffraction methods showed that the [Rh(COD)(DIOSiPr2)]ClO4 complex had an square-planar coordination geometry at the rhodium atom with the iPr groups in anti position. Cyclooctadiene complexes react with carbon monoxide to form dinuclear tetracarbonylated complexes [(CO)2Rh(μ-DIOSR2)2(CO)2](ClO4)2. [Rh(COD)(DIOSR2)]ClO4 are active catalyst precursors in styrene hydroformylation at 30 atm and 65°C which give conversions of up to 99% with a regioselectivity in 2-phenylpropanal as high as 74%. In all cases enantioselectivities are low.

Lipid binding interactions of antimicrobial plant seed defence proteins: puroindoline-a and β-purothionin

The indolines and thionins are basic, amphiphilic and cysteine-rich proteins found in cereals; puroindoline-a (Pin-a) and β-purothionin (β-Pth) are members of these families in wheat (Triticum aestivum). Pin-a and β-Pth have been suggested to play a significant role in seed defence against microbial pathogens, making the interaction of these proteins with model bacterial membranes an area of potential interest. We have examined the binding of these proteins to lipid monolayers composed of 1,2-dipalmitoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (DPPG) using a combination of neutron reflectometry, Brewster angle microscopy, and infrared spectroscopy. Results showed that both Pin-a and β-Pth interact strongly with condensed phase DPPG monolayers, but the degree of penetration was different. β-Pth was shown to penetrate the lipid acyl chain region of the monolayer and remove lipids from the air/liquid interface during the adsorption process, suggesting this protein may be able to both form membrane spanning ion channels and remove membrane phospholipids in its lytic activity. Conversely, Pin-a was shown to interact mainly with the head-group region of the condensed phase DPPG monolayer and form a 33 Å thick layer below the lipid film. The differences between the interfacial structures formed by these two proteins may be related to the differing composition of the Pin-a and β-Pth hydrophobic regions.

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.

Production of angiotensin-I-converting enzyme (ACE) inhibitory activity in milk fermented with probiotic strains: Effects of calcium, pH and peptides on the ACE-inhibitory activity

Recently, probiotic fermented milk products have raised interest regarding their potential anti-hypertensive activity mainly due to the production of angiotensin-I-converting enzyme (ACE) inhibitory peptides. Ionic calcium released upon milk acidification during fermentation is also known to exert hypotensive activity. Thus, the main aim of this study was to screen probiotic strains for their ability to induce ACE-inhibitory activity upon fermentation of milk. The relationship of ACE-inhibitory activity percentage (ACEi%) with cell growth, pH, degree of hydrolysis and the concentration of ionic calcium released during the fermentation was also investigated. Compared with other lactic acid bacteria, Lactobacillus casei YIT 9029 and Bifidobacterium bifidum MF 20/5 were able to induce strong ACE-inhibitory activity. Furthermore, it was found that the ionic calcium released during milk fermentation could contribute to the ACE-inhibitory activity. These findings will contribute to the development of new probiotic dairy products with anti-hypertensive activity.

Recovery of gallic acid with colloidal gas aphrons generated from a cationic surfactant

In a previous study we have demonstrated that gallic acid (GA) in its anionic form can be recovered from aqueous solutions using colloidal gas aphrons (CGA) generated from the cationic surfactant cetyltrimethylammonium bromide (CTAB). The aim of the present work is to get a better understanding of the separation mechanism in order to determine the optimum operating conditions to maximise the recovery of GA while preserving its antioxidant properties. Zeta potential measurements were carried out to characterise the surface charge of GA, CTAB and their mixtures at three different pH conditions (both in buffers and in aqueous solutions). GA interacted strongly with CTAB at pH higher than its pKa 3.14 where it is ionised and negatively charged. However, at pH higher than 7 GA becomes oxidised and loses its antioxidant power. GA recovery was mainly affected by pH, ionic strength, surfactant/GA molar ratio, mixing conditions and contact time. Scale-up of the separation using a flotation column resulted in both higher recovery and reproducibility. Preliminary experiments with grape marc extracts confirmed the potential application of this separation for the recovery of polyphenols from complex feedstocks

Synthesis and incorporation into cyclic peptides of tolan amino acids and their hydrogenated congeners: construction of an array of A–B-loop mimetics of the Cε3 domain of human IgE

The disruption of the human immunolobulin E–high affinity receptor I (IgE–FcεRI) protein–protein interaction (PPI) is a validated strategy for the development of anti asthma therapeutics. Here, we describe the synthesis of an array of conformationally constrained cyclic peptides based on an epitope of the A–B loop within the Cε3 domain of IgE. The peptides contain various tolan (i.e., 1,2-biarylethyne) amino acids and their fully and partially hydrogenated congeners as conformational constraints. Modest antagonist activity (IC50 660 μM) is displayed by the peptide containing a 2,2′-tolan, which is the one predicted by molecular modeling to best mimic the conformation of the native A–B loop epitope in IgE.

Fibrillisation of ring-closed amyloid peptides

Ring-closing olefin metathesis reactions are used to create intramolecularly ring closed peptides or inter-molecularly ring-closed peptide dimers based on a designed amyloid peptide sequence. The uncrosslinked peptide self-assembles into high aspect ratio nanotubes, however ring-closing leads to the formation of fibrillar and twisted/helical ribbon structures.

Control of strand registry by attachment of PEG chains to amyloid peptides influences nanostructure

The self-assembly in aqueous solution of PEG-peptide conjugates comprising a model amyloid peptide sequence FFKLVFF that contains the Ab(16–20) KLVFF motif is investigated. X-ray diffraction reveals different packing motifs dependent on PEG chain length. This is correlated to remarkable differences in self-assembled nanostructures. The control of strand registry points to a subtle interplay between aromatic stacking, electrostatic and amphiphilic interactions.

Production of angiotensin converting enzyme inhibitory peptides from β-lactoglobulin and casein derived peptides: an integrative approach

Angiotensin I-converting enzyme (ACE) inhibition is one of the mechanisms by which reduction in blood pressure is exerted. Whey proteins are a rich source of ACE inhibitory peptides and have shown a blood pressure reduction effect i.e. antihypertensive activity. The aim of this work was to develop a simplified process using a combination of adsorption and microfiltration steps for the production of hydrolysates from whey with high ACE inhibitory activity and potency; the latter was measured as the IC50, which is the peptide concentration required to reduce ACE activity by half. This process integrates the selective separation of β-lactoglobulin and casein derived peptides (CDP) from rennet whey and their hydrolysis, which results in partially pure, less complex hydrolysates with high bioactive potency. Hydrolysis was carried out with protease N ‘Amano’ in a thermostatically controlled membrane reactor operated in a batch mode. By applying the integrative approach it was possible to produce from the same feedstock two different hydrolysates that exhibited high ACE inhibition. One hydrolysate was mainly composed of casein-derived peptides with IC50= 285 μg/mL. In this hydrolysate we identified the well known potent ACE-I and anti-hypertensive tri-peptide Ile-Pro-Pro (IPP) and another novel octa-peptide Gln-Asp-Lys-Thr-Glu-Ile-Pro-Thr (QDKTEIPT). The second hydrolysate was mainly composed of β-lactoglobulin derived peptides with IC50=128 µg/mL. This hydrolysate contained a tetra-peptide (Ile-Ile-Ala-Glu) IIAE as one of the two major peptides. A further advantage to this process is that enzyme activity was substantially increased as enzyme product inhibition was reduced.

Spatial and temporal patterns in antimicrobial resistance of Salmonella Typhimurium in cattle in England and Wales

Salmonella is the second most commonly reported human foodborne pathogen in England and Wales, and antimicrobial-resistant strains of Salmonella are an increasing problem in both human and veterinary medicine. In this work we used a generalized linear spatial model to estimate the spatial and temporal patterns of antimicrobial resistance in Salmonella Typhimurium in England and Wales. Of the antimicrobials considered we found a common peak in the probability that an S. Typhimurium incident will show resistance to a given antimicrobial in late spring and in mid to late autumn; however, for one of the antimicrobials (streptomycin) there was a sharp drop, over the last 18 months of the period of investigation, in the probability of resistance. We also found a higher probability of resistance in North Wales which is consistent across the antimicrobials considered. This information contributes to our understanding of the epidemiology of antimicrobial resistance in Salmonella.

Identifying antimicrobial resistance genes of human clinical relevance within Salmonella isolated from food animals in Great Britain

To investigate the occurrence of antimicrobial resistance genes of human clinical relevance in Salmonella isolated from livestock in Great Britain. Two hundred and twenty-five Salmonella enterica isolates were characterized using an antimicrobial resistance gene chip and disc diffusion assays. Plasmid profiling, conjugation experiments and identification of Salmonella genomic island 1 (SGI1) were performed for selected isolates. Approximately 43% of Salmonella harboured single or multiple antimicrobial resistance genes with pig isolates showing the highest numbers where 96% of Salmonella Typhimurium harboured one or more resistance genes. Isolates harbouring multiple resistances divided into three groups. Group 1 isolates harboured ampicillin/streptomycin/sulphonamide/tetracycline resistance and similar phenotypes. This group contained isolates from pigs, cattle and poultry that were from several serovars including Typhimurium, 4,[5],12:i:-, Derby, Ohio and Indiana. All Group 2 isolates were from pigs and were Salmonella Typhimurium. They contained a non-sul-type class 1 integron and up to 13 transferrable resistances. All Group 3 isolates harboured a class 1 integron and were isolated from all animal species included in the study. Most isolates were Salmonella Typhimurium and harboured SGI1. Salmonella isolated from livestock was shown to harbour antimicrobial resistance genes although no or little resistance to third-generation cephalosporins or ciprofloxacin, respectively, was detected. The preponderance in pigs of multidrug-resistant Salmonella Typhimurium makes it important to introduce control measures such as improved biosecurity to ensure that they do not pass through the food chain and limit human therapeutic options.