Interfacial and emulsifying properties of lentil protein isolate.

The dynamic interfacial tension (DIFT) at oil-water interface, diffusion coefficients, surface hydrophobicity, zeta potential and emulsifying properties, including emulsion activity index (EAI), emulsion stability index (ESI) and droplet size of lentil protein isolate (LPI), were measured at different pH and LPI concentration, in order to elucidate its emulsifying behaviour. Sodium caseinate (NaCas), whey protein isolate (WPI), bovine serum albumin (BSA) and lysozyme (Lys) were used as benchmark proteins and their emulsifying property was compared with that of LPI. The speed of diffusion-controlled migration of these proteins to the oil/water interface, was in the following order: NaCas>LPI>WPI>BSA>Lys, while their surface hydrophobicity was in the following order: BSA>LPI>NaCas>WPI>Lys. The EAI of emulsions stabilised by the above proteins ranged from 90.3 to 123.3 m(2)/g and it was 93.3 ± 0.2 m(2)/g in LPI-stabilised emulsion. However, the stability of LPI-stabilised emulsions was slightly lower compared to that of WPI and NaCas-stabilised emulsions at the same protein concentration at pH 7.0. The ESI of LPI emulsions improved substantially with decrease in droplet size when protein concentration was increased (20-30 mg/ml). Reduction of disulphide bonds enhanced both the EAI and ESI compared to untreated samples. Heat treatment of LPI dispersions resulted in poor emulsion stability due to molecular aggregation. The stability of LPI-stabilised emulsions was found to decrease in the presence of NaCl. This study showed that LPI can be as effective emulsifiers of oil-in-water emulsions as are WPI and NaCas at ≥20 mg/ml concentrations both at low and neutral pH. The emulsifying property of LPI can be improved by reducing the intra and inter-disulphide bond by using appropriate reducing agents.

Immunostimulatory principles from Chlorella pyrenoidosa--part 1: isolation and biological assessment in vitro.

Our proprietary preparation obtained by extraction of Chlorella pyrenoidosa cells, ONC-107 (Respondin), was recently found to selectively boost antibody response to the influenza vaccine in a human clinical trial. Respondin is a potent stimulator of mouse B cell proliferation and an activator of macrophages. Bioactivity-guided resolution concluded that Respondin is composed of a mixture of immunostimulatory principles of different chemical nature. A combination of size exclusion, anion exchange and hydrophobic interaction chromatography revealed that the bulk of the immunostimulatory activity resides in polysaccharide/protein complexes with molecular masses larger than 100 kDa that are composed primarily of galactose, rhamnose and arabinose.

An aqueous Chlorella extract inhibits IL-5 production by mast cells in vitro and reduces ovalbumin-induced eosinophil infiltration in the airway in mice in vivo.

An aqueous extract of the edible microalga (CP) (1), has recently been tested for its immunomodulatory effects in a human clinical trial. Here, the CP extract was dialyzed and fractionated using Sephadex G 100 chromatography. The effects of a dialyzed aqueous CP extract, fraction 2 , on mast cell mediator release in vitro and ovalbumin-induced allergic airway inflammation in vivo were examined. In vitro, treatment of mouse bone marrow-derived mast cells with 2 for 18 h significantly inhibited antigen (trinitrophenyl-BSA)-induced IL-5 production. In vivo, treatment of mice with 2 during ovalbumin sensitization and stimulation process significantly reduced eosinophil and neutrophil infiltration in the airways. Moreover, fractions obtained by size exclusion chromatography of 2 inhibited IgE-dependent cytokine GM-CSF production from human cord blood-derived mast cells. Taken together, these results suggest that 2 is composed of biopolymers with anti-allergic potential.

Alpha7-nicotinic acetylcholine receptors mediate an Abeta(1-42)-induced increase in the level of acetylcholinesterase in primary cortical neurones.

The beta-amyloid protein (Abeta) is the major protein component of amyloid plaques found in the Alzheimer brain. Although there is a loss of acetylcholinesterase (AChE) from both cholinergic and non-cholinergic neurones in the brain of Alzheimer patients, the level of AChE is increased around amyloid plaques. Previous studies using P19 cells in culture and transgenic mice which overexpress human Abeta have suggested that this increase may be due to a direct action of Abeta on AChE expression in cells adjacent to amyloid plaques. The aim of the present study was to examine the mechanism by which Abeta increases levels of AChE in primary cortical neurones. Abeta1-42 was more potent than Abeta1-40 in its ability to increase AChE in primary cortical neurones. The increase in AChE was unrelated to the toxic effects of the Abeta peptides. The effect of Abeta1-42 on AChE was blocked by inhibitors of alpha7 nicotinic acetylcholine receptors (alpha7 nAChRs) as well as by inhibitors of L- or N-type voltage-dependent calcium channels (VDCCs), whereas agonists of alpha7 nAChRs (choline, nicotine) increased the level of AChE. The results demonstrate that the effect of Abeta1-42 on AChE is due to an agonist effect of Abeta1-42 on the alpha7 nAChR.

Improved preparation of amyloid-beta peptides using DBU as Nalpha-Fmoc deprotection reagent.

Previous studies have shown the amyloid peptides, Abeta 1-40/42, to be exceptionally difficult to assemble by Fmoc-solid phase peptide synthesis due to the high hydrophobicity of the C-terminal segment and resulting on-resin aggregation. We found that the use of the stronger and more efficient base, DBU, at a concentration of 2% in DMF for Nalpha-Fmoc deprotection allowed substantially improved continuous flow solid phase assembly of the model peptide Abeta 29-40/42 fragments. This suggested that, at least for these sequences, incomplete deprotection was a greater problem than incomplete amino acid acylation. This base was then used during the synthesis of both Abeta 1-40 and Abeta 1-42, up to and including Ser8, from which point 20% piperidine in DMF was utilized so as to avoid potential aspartimide formation at Asp7. By this means, the deprotection efficiency through the difficult C-terminal portion of the sequence was much improved and resulted in increased availability of terminal amino groups for acylation. This simple strategy that obviates the need for special conditions significantly improved crude peptide quality and allowed considerable facilitation of subsequent purification.

Copper and zinc binding modulates the aggregation and neurotoxic properties of the prion peptide PrP106-126.

The abnormal form of the prion protein (PrP) is believed to be responsible for the transmissible spongiform encephalopathies. A peptide encompassing residues 106-126 of human PrP (PrP106-126) is neurotoxic in vitro due its adoption of an amyloidogenic fibril structure. The Alzheimer's disease amyloid beta peptide (Abeta) also undergoes fibrillogenesis to become neurotoxic. Abeta aggregation and toxicity is highly sensitive to copper, zinc, or iron ions. We show that PrP106-126 aggregation, as assessed by turbidometry, is abolished in Chelex-100-treated buffer. ICP-MS analysis showed that the Chelex-100 treatment had reduced Cu(2+) and Zn(2+) levels approximately 3-fold. Restoring Cu(2+) and Zn(2+) to their original levels restored aggregation. Circular dichroism showed that the Chelex-100 treatment reduced the aggregated beta-sheet content of the peptide. Electron paramagnetic resonance spectroscopy identified a 2N1S1O coordination to the Cu(2+) atom, suggesting histidine 111 and methionine 109 or 112 are involved. Nuclear magnetic resonance confirmed Cu(2+) and Zn(2+) binding to His-111 and weaker binding to Met-112. An N-terminally acetylated PrP106-126 peptide did not bind Cu(2+), implicating the free amino group in metal binding. Mutagenesis of either His-111, Met-109, or Met-112 abolished PrP106-126 neurotoxicity and its ability to form fibrils. Therefore, Cu(2+) and/or Zn(2+) binding is critical for PrP106-126 aggregation and neurotoxicity.

The amyloid-beta peptide and its role in Alzheimer's disease.

Amyloid formation plays a central role in the cause and progression of Alzheimer's disease. The major component of this amyloid is the amyloid-beta (A beta) peptide, which is currently the subject of intense study. This review discusses some recent studies in the area of A beta synthesis, purification and structural analysis. Also discussed are proposed mechanisms for A beta-induced neurotoxicity and some recent advances in the development of A beta-related therapeutic strategies.

The Val-210-Ile pathogenic Creutzfeldt-Jakob disease mutation increases both the helical and aggregation propensities of a sequence corresponding to helix-3 of PrP(C).

A peptide corresponding to the third helical region within the PrP(C) protein, from residues 198 to 218 (helix-3), was synthesised with and without the familial 210-Val to Ile Creutzfeldt-Jakob disease mutation. The NMR structure of PrP(C) predicts no global variation in stability for this mutation, indicating that local sequence rather than global structural factors are involved in the pathological effects of this mutation. 1H NMR analysis of peptides with and without this mutation indicated that it had no significant effect on local helical structure. Temperature denaturation studies monitored by CD showed that the mutation increased the helical content within this region (helical propensity), but did not stabilise the helix toward denaturation (helical stability). Aggregation data indicated that, in addition to increasing helical propensity, this mutation increased the aggregation propensity of this sequence. CD and NMR data indicate that helical interactions, stabilised by the Val-210-Ile mutation, may precede the formation of beta-sheet aggregates in this peptide sequence. Therefore, this pathological mutation probably does not facilitate PrP(C) to PrP(Sc) conversion by directly destabilising the helical structure of PrP(C), but may preferentially stabilise PrP(Sc) by facilitating beta-sheet formation within this sequence region of PrP. In addition, helical interactions between helix-3 in two or more PrP(C) molecules may promote conversion to PrP(Sc).

Sublethal concentrations of prion peptide PrP106-126 or the amyloid beta peptide of Alzheimer's disease activates expression of proapoptotic markers in primary cortical neurons.

Neurodegenerative disorders such as prion diseases and Alzheimer's disease (AD) are characterized by neuronal dysfunction and accumulation of amyloidogenic protein. In vitro studies have demonstrated that these amyloidogenic proteins can induce cellular oxidative stress and therefore may contribute to the neuronal dysfunction observed in these illnesses. Although the neurotoxic pathways are not fully elucidated, recent studies in AD have demonstrated up-regulation of caspases in neurons treated with amyloid beta (Abeta) peptide, suggesting involvement of apoptotic processes. To examine the role of proapoptotic pathways in prion diseases we treated primary mouse cortical neurons with the toxic prion protein peptide PrP106-126 and measured caspase activation and annexin V binding. We found that PrP106-126 induced a rapid and marked elevation in caspase 3, 6, and 8-like activity in neuronal cultures. Increased annexin V binding was observed predominantly on cortical cell neurites in peptide-treated cultures. Interestingly, these effects were induced by sublethal (5-50 microM) or lethal (100-200 microM) concentrations of PrP106-126. Sublethal concentrations of PrP106-126 maintained elevated caspase activation for at least 10 days with no loss of cell viability. Abeta1-40 also up-regulated caspase 3 activity and annexin V binding at both sublethal (5 microM) and lethal (25 microM) concentrations. There were no changes to proapoptotic marker expression in cultures treated with scrambled PrP106-126 (200 microM) or Abeta1-28 (25 microM) peptides. These studies demonstrate that amyloidogenic peptides can induce prolonged activation of proapoptotic marker expression in cultured neurons even at sublethal concentrations. These effects could contribute to chronic neuronal dysfunction and increase susceptibility to additional metabolic insults in neurodegenerative disorders. If so, targeting of therapeutic strategies against neuronal caspase activation early in the disease course could be beneficial in AD and prion diseases.

Alzheimer's disease amyloid-beta binds copper and zinc to generate an allosterically ordered membrane-penetrating structure containing superoxide dismutase-like subunits.

Amyloid beta peptide (Abeta) is the major constituent of extracellular plaques and perivascular amyloid deposits, the pathognomonic neuropathological lesions of Alzheimer's disease. Cu(2+) and Zn(2+) bind Abeta, inducing aggregation and giving rise to reactive oxygen species. These reactions may play a deleterious role in the disease state, because high concentrations of iron, copper, and zinc have been located in amyloid in diseased brains. Here we show that coordination of metal ions to Abeta is the same in both aqueous solution and lipid environments, with His(6), His(13), and His(14) all involved. At Cu(2+)/peptide molar ratios >0.3, Abeta coordinated a second Cu(2+) atom in a highly cooperative manner. This effect was abolished if the histidine residues were methylated at N(epsilon)2, indicating the presence of bridging histidine residues, as found in the active site of superoxide dismutase. Addition of Cu(2+) or Zn(2+) to Abeta in a negatively charged lipid environment caused a conformational change from beta-sheet to alpha-helix, accompanied by peptide oligomerization and membrane penetration. These results suggest that metal binding to Abeta generated an allosterically ordered membrane-penetrating oligomer linked by superoxide dismutase-like bridging histidine residues.

Homocysteine potentiates copper- and amyloid beta peptide-mediated toxicity in primary neuronal cultures: possible risk factors in the Alzheimer's-type neurodegenerative pathways.

Oxidative stress may have an important role in the progression of neurodegenerative disorders such as Alzheimer's disease (AD) and prion diseases. Oxidative damage could result from interactions between highly reactive transition metals such as copper (Cu) and endogenous reducing and/or oxidizing molecules in the brain. One such molecule, homocysteine, a thiol-containing amino acid, has previously been shown to modulate Cu toxicity in HeLa and endothelial cells in vitro. Due to a possible link between hyperhomocysteinemia and AD, we examined whether interaction between homocysteine and Cu could potentiate Cu neurotoxicity. Primary mouse neuronal cultures were treated with homocysteine and either Cu (II), Fe (II or III) or Zn (II). Homocysteine was shown to selectively potentiate toxicity from low micromolar concentrations of Cu. The toxicity of homocysteine/Cu coincubation was dependent on the ability of homocysteine to reduce Cu (II) as reflected by the inhibition of toxicity with the Cu (I)-specific chelator, bathocuproine disulphonate. This was supported by data showing that homocysteine reduced Cu (II) more effectively than cysteine or methionine but did not reduce Fe (III) to Fe (II). Homocysteine also generated high levels of hydrogen peroxide in the presence of Cu (II) and promoted Abeta/Cu-mediated hydrogen peroxide production and neurotoxicity. The potentiation of metal toxicity did not involve excitotoxicity as ionotropic glutamate receptor antagonists had no effect on neurotoxicity. Homocysteine alone also had no effect on neuronal glutathione levels. These studies suggest that increased copper and/or homocysteine levels in the elderly could promote significant oxidant damage to neurons and may represent additional risk factor pathways which conspire to produce AD or related neurodegenerative conditions.

Amyloidogenicity and neurotoxicity of peptides corresponding to the helical regions of PrP(C).

An alpha-helical to beta-sheet conformational change in the prion protein, PrP(C), is believed to be causative in transmissible spongiform encephalopathies. Recent nuclear magnetic resonance structures of PrP(C) have identified three helical regions in the normal full-length protein. We have synthesised peptides corresponding to these helical regions (PrP144-154, helical region one; PrP178-193, helical region two; and PrP198-218, helical region three). Circular dichroism results show that the peptide corresponding to helical region one is unstructured, while peptides corresponding to the second and third helical regions have a high propensity to form beta-sheet structure in a pH-dependent manner in aqueous solutions. Peptides corresponding to the second helical region, PrP180-193 and PrP178-193, are the only ones that form amyloid by electron microscopy and congo red birefringence. PrP178-193 and the amyloidogenic Alzheimer's disease Abeta25-25 peptide were found to promote Cu (II)-induced lipid peroxidation and cytotoxicity in primary neuronal cultures, while PrP144-154, PrP198-218 and the nonamyloidogenic Abeta1-28 had no effect on Cu (II) toxicity. There was no increase in toxicity induced by PrP178-193 in cultures treated with Fe (II) or hydrogen peroxide, indicating a preferential modulatory effect on Cu (II) toxicity by PrP178-193. The data suggest that the PrP178-193 peptide has both structural and bioactive properties in common with Abeta25-35 and that the second putative helical region of PrP could be involved in modulation of Cu (II)-mediated toxicity in neurons during prion disease.

A beta peptides and calcium influence secretion of the amyloid protein precursor from chick sympathetic neurons in culture.

The major constituent of amyloid plaques in the Alzheimer disease (AD) brain is the amyloid protein (A beta). A beta has been shown to be neurotoxic to cells, but the exact mechanism of its effects are still not known. Most studies have focussed on A beta neurotoxicity, but little is known about the effect of A beta peptides on cellular protein metabolism and secretion. To examine the effect of A beta peptides on APP secretion, chick sympathetic neurons were metabolically labeled with [(35)S]methionine and the amounts of radiolabeled APP and A beta quantitated. Several A beta peptides (A beta(25-35), [pyroglu(3)]A beta(3-40), and [pyroglu(11)]A beta(11-40)) inhibited secretion of [(35)S]APP and increased cell-associated [(35)S]APP. There was also a 2-2.5-fold increase in secretion of several other proteins when cells were incubated with A beta(25-35). However, the amount of A beta secreted into the medium was decreased. Treatment of cells with the calcium ionophore A23187 caused a 1.5-fold increase in secreted [(35)S]APP and a decrease in cell-associated [(35)S]APP. Although L-type voltage-dependent calcium channels (VDCC) have been implicated in A beta toxicity, the effect of L-type VDCC on APP secretion has not previously been examined. The L-type VDCC antagonists nifedipine and diltiazem both increased [(35)S]APP secretion into the medium but did not influence the effect of A beta on [(35)S]APP secretion. These studies suggest that A beta interferes with the secretory pathway of APP. Insofar as secreted APP has been proposed to have a neuroprotective function, the accumulation of A beta in the AD brain could decrease secreted APP and thereby indirectly increase A beta toxicity.

Peptide thioester preparation by Fmoc solid phase peptide synthesis for use in native chemical ligation.

Established methodology for the preparation of peptide thioesters requires the use of t-butoxycarbonyl chemistry owing to the lability of thioester linkers to the nucleophilic reagents used in Fmoc solid phase peptide synthesis. Both the greater ease of use and the broad applicability of the method has led to the development of an Fmoc-based methodology for direct peptide thioester synthesis. It was found that successful preparation of a peptide thioester could be achieved when the non-nucleophilic base, 1,8-diazabicyclo[5.4.0]undec-7-ene, together with 1-hydroxybenzotriazole in dimethylformamide, were used as the N(alpha)-Fmoc deprotection reagent. Native chemical ligation of the resulting thioester product to an N-terminal cysteine-containing peptide was successfully performed in aqueous solution to produce a fragment peptide of human alpha-synuclein. The formation of aspartimide (cyclic imide) in a base-sensitive hexapeptide fragment of scorpion toxin II was found to be significant under the deprotection conditions used. However, this could be controlled by the judicious protection of sensitive residues using the 2-hydroxy-4-methoxybenzyl group.

Substrate-bound beta-amyloid peptides inhibit cell adhesion and neurite outgrowth in primary neuronal cultures.

Accumulation of the beta-amyloid protein (Abeta) in the brain is an important step in the pathogenesis of Alzheimer's disease. However, the mechanism of Abeta toxicity remains unclear. Abeta can bind to the extracellular matrix, a structure that regulates adhesive events such as neurite outgrowth and synaptogenesis. The binding of Abeta to the extracellular matrix suggests that Abeta may disrupt cell-substrate interactions. Therefore, the effect of substrate-bound Abeta on the growth of isolated chick sympathetic and mouse cortical neurons was examined. Abeta1-40 and Abeta1-42 had dose-dependent effects on cell morphology. When tissue culture plates were coated with 0.1-10 ng/well Abeta, neurite outgrowth increased. Higher amounts of Abeta peptides (> or =3 microg/well) inhibited outgrowth. The inhibitory effect was related to aggregation of the peptide, as preincubation of Abeta1-40 for 24 h at 37 degrees C (a process known to increase amyloid fibril formation) was necessary for inhibition of neurite outgrowth. Abeta29-42, but not Abeta1-28, also inhibited neurite outgrowth at high concentrations, demonstrating that the inhibitory domain is located within the hydrophobic C-terminal region. Abeta1-40, Abeta1-42, and Abeta29-42 also inhibited cell-substrate adhesion, indicating that the effect on neurite outgrowth may have been due to inhibition of cell adhesion. The results suggest that accumulation of Abeta may disrupt cell-adhesion mechanisms in vivo.

On-line high-performance liquid chromatography/mass spectrometric investigation of amyloid-beta peptide variants found in Alzheimer's disease.

Abeta peptides are the major components of amyloid deposits in Alzheimer's disease. The presence of N-terminally truncated Abeta variants in amyloid may be a critical factor in Alzheimer's disease pathogenesis. These Abeta variants are less soluble and more amyloidogenic than full-length Abeta, making their separation, purification and identification difficult. High-performance liquid chromatography (HPLC) at elevated temperatures, coupled to electrospray ionization (ES) mass spectrometry (MS), enables rapid separation and identification of N-terminally truncated Abeta variants. This methodology provides a potential tool for exploring the importance of these Abeta variants in both the pathogenesis and diagnosis of Alzheimer's disease.

The hydrophobic core sequence modulates the neurotoxic and secondary structure properties of the prion peptide 106-126.

The neurodegeneration seen in spongiform encephalopathies is believed to be mediated by protease-resistant forms of the prion protein (PrP). A peptide encompassing residues 106-126 of human PrP has been shown to be neurotoxic in vitro. The neurotoxicity of PrP106-126 appears to be dependent upon its adoption of an aggregated fibril structure. To examine the role of the hydrophobic core, AGAAAAGA, on PrP106-126 toxicity, we performed structure-activity analyses by substituting two or more hydrophobic residues for the hydrophilic serine residue to decrease its hydrophobicity. A peptide with a deleted alanine was also synthesized. We found all the peptides except the deletion mutant were no longer toxic on mouse cerebellar neuronal cultures. Circular dichroism analysis showed that the nontoxic PrP peptides had a marked decrease in beta-sheet structure. In addition, the mutants had alterations in aggregability as measured by turbidity, Congo red binding, and fibril staining using electron microscopy. These data show that the hydrophobic core sequence is important for PrP106-126 toxicity probably by influencing its assembly into a neurotoxic structure. The hydrophobic sequence may similarly affect aggregation and toxicity observed in prion diseases.

The A beta 3-pyroglutamyl and 11-pyroglutamyl peptides found in senile plaque have greater beta-sheet forming and aggregation propensities in vitro than full-length A beta.

A beta isolated from neuritic plaque and vascular walls of the brains of patients with Alzheimer's disease has been shown to contain significant quantities of A beta peptides which begin at residue 3Glu or 11Glu in the form of pyroglutamyl residues (A beta 3pE and A beta 11pE). To investigate the effects of these N-terminal modifications on the biophysical properties of A beta, peptides A beta 1-40, A beta 3pE-40, A beta 11pE-40, A beta 1-28, A beta 3pE-28, and A beta 11pE-28 were synthesized. Using circular dichroism spectroscopy, we determined that the pyroglutamyl-containing peptides form beta-sheet structure more readily than the corresponding full-length A beta peptides, both in aqueous solutions and in 10% sodium dodecyl sulfate micelles. Trifluoroethanol spectra indicated that the relative beta-sheet to alpha-helical stability is higher for the pyroglutamyl-containing peptides. Sedimentation experiments show that the pyroglutamyl-containing peptides have greater aggregation propensities than the corresponding full-length peptides. Comparison between the A beta 40 and the A beta 28 series indicated that the greater beta-sheet forming and aggregation propensities of the pyroglutamyl peptides are not simply due to an increase in hydrophobicity.