Ian F. Musgrave

(-)-Epigallocatechin-3-gallate (EGCG) maintains κ-casein in its pre-fibrillar state without redirecting its aggregation pathway

The polyphenol (-)-epigallocatechin-3-gallate (EGCG) has recently attracted much research interest in the field of protein-misfolding diseases because of its potent anti-amyloid activity against amyloid-beta, alpha-synuclein and huntingtin, the amyloid-fibril-forming proteins involved in Alzheimers, Parkinsons and Huntingtons diseases, respectively. EGCG redirects the aggregation of these polypeptides to a disordered off-folding pathway that results in the formation of non-toxic amorphous aggregates. Whether this anti-fibril activity is specific to these disease-related target proteins or is more generic remains to be established. In addition, the mechanism by which EGCG exerts its effects, as with all anti-amyloidogenic polyphenols, remains unclear. To address these aspects, we have investigated the ability of EGCG to inhibit amyloidogenesis of the generic model fibril-forming protein RCMkappa-CN (reduced and carboxymethylated kappa-casein) and thereby protect pheochromocytoma-12 cells from RCMkappa-CN amyloid-induced toxicity. We found that EGCG potently inhibits in vitro fibril formation by RCMkappa-CN [the IC(50) for 50 microM RCMkappa-CN is 13+/-1 microM]. Biophysical studies reveal that EGCG prevents RCMkappa-CN fibril formation by stabilising RCMkappa-CN in its native-like state rather than by redirecting its aggregation to the disordered, amorphous aggregation pathway. Thus, while it appears that EGCG is a generic inhibitor of amyloid-fibril formation, the mechanism by which it achieves this inhibition is specific to the target fibril-forming polypeptide. It is proposed that EGCG is directed to the amyloidogenic sheet-turn-sheet motif of monomeric RCMkappa-CN with high affinity by strong non-specific hydrophobic associations. Additional non-covalent pi-pi stacking interactions between the polyphenolic and aromatic residues common to the amyloidogenic sequence are also implicated.

Gallic acid is the major component of grape seed extract that inhibits amyloid fibril formation

Many protein misfolding diseases, for example, Alzheimers, Parkinsons and Huntingtons, are characterised by the accumulation of protein aggregates in an amyloid fibrillar form. Natural products which inhibit fibril formation are a promising avenue to explore as therapeutics for the treatment of these diseases. In this study we have shown, using in vitro thioflavin T assays and transmission electron microscopy, that grape seed extract inhibits fibril formation of kappa-casein (κ-CN), a milk protein which forms amyloid fibrils spontaneously under physiological conditions. Among the components of grape seed extract, gallic acid was the most active component at inhibiting κ-CN fibril formation, by stabilizing κ-CN to prevent its aggregation. Concomitantly, gallic acid significantly reduced the toxicity of κ-CN to pheochromocytoma12 cells. Furthermore, gallic acid effectively inhibited fibril formation by the amyloid-beta peptide, the putative causative agent in Alzheimers disease. It is concluded that the gallate moiety has the fibril-inhibitory activity.

Metabolism of selenite in human lung cancer cells: X-ray absorption and fluorescence studies

Selenite is an inorganic form of selenium that has a cytotoxic effect against several human cancer cell lines: one or more selenite metabolites are considered to be responsible for its toxicity. X-ray absorption spectroscopy was used to monitor Se speciation in A549 human lung cancer cells incubated with selenite over 72 h. As anticipated, selenodiglutathione and elemental Se both comprised a large proportion of Se in the cells between 4 and 72 h after treatment, which is in accordance with the reductive metabolism of selenite in the presence of glutathione and glutathione reductase/NADPH system. Selenocystine was also present in the cells but was only detected as a significant component between 24 and 48 h concomitant with a decrease in the proportion of selenocysteine and the viability of the cells. The change in speciation from the selenol, selenocysteine, to the diselenide, selenocystine, is indicative of a change in the redox status of the cells to a more oxidizing environment, likely brought about by metabolites of selenite. X-ray fluorescence microscopy of single cells treated with selenite for 24 h revealed a punctate distribution of Se in the cytoplasm. The accumulation of Se was associated with a greater than 2-fold increase in Cu, which was colocalized with Se. Selenium K-edge extended X-ray absorption fine structure (EXAFS) spectroscopy revealed Se-Se and Se-S bonding, but not Se-Cu bonding, despite the spatial association of Se and Cu. Microprobe X-ray absorption near-edge structure spectroscopy (μ-XANES) showed that the highly localized Se species was mostly elemental Se.

αB-Crystallin inhibits the cell toxicity associated with amyloid fibril formation by κ-casein and the amyloid-β peptide

Amyloid fibril formation is associated with diseases such as Alzheimer’s, Parkinson’s, and prion diseases. Inhibition of amyloid fibril formation by molecular chaperone proteins, such as the small heat-shock protein αB-crystallin, may play a protective role in preventing the toxicity associated with this form of protein misfolding. Reduced and carboxymethylated κ-casein (RCMκ-CN), a protein derived from milk, readily and reproducibly forms fibrils at physiological temperature and pH. We investigated the toxicity of fibril formation by RCMκ-CN using neuronal model PC12 cells and determined whether the inhibition of fibril formation altered its cell toxicity. To resolve ambiguities in the literature, we also investigated whether fibril formation by amyloid-β1–40 (Aβ1–40), the peptide associated with Alzheimer’s disease, was inhibited by αB-crystallin and if this affected the toxicity of Aβ. To this end, either RCMκ-CN or Aβ1–40 was incubated at neutral pH to induce fibril formation before treating PC12 cells and assessing cell viability. Incubated (fibrillar) RCMκ-CN was more toxic to PC12 cells than native RCMκ-CN with the highest level of toxicity being associated with mature fibrils and protofibrils. Furthermore, the toxicity of RCMκ-CN was attenuated when its fibril formation was inhibited, either through the chaperone action of αB-crystallin or when it interacted with its natural binding partners in milk, αS- and β-casein. Likewise, incubating Aβ1–40 with αB-crystallin inhibited both Aβ1–40 fibril formation and the associated cell toxicity. Importantly, by inhibiting fibril formation, αB-crystallin prevents the cell toxicity associated with protein misfolding.

Uptake, Distribution, and Speciation of Selenoamino Acids by Human Cancer Cells: X-ray Absorption and Fluorescence Methods

Selenium compounds exhibit chemopreventative properties at supranutritional doses, but the efficacy of selenium supplementation in cancer prevention is dependent on the chemical speciation of the selenium supplement and its metabolites. The uptake, speciation, and distribution of the common selenoamino acid supplements, selenomethionine (SeMet) and Se-methylselenocysteine (MeSeCys), in A549 human lung cancer cells were investigated using X-ray absorption and fluorescence spectroscopies. X-ray absorption spectroscopy of bulk cell pellets treated with the selenoamino acids for 24 h showed that while selenium was found exclusively in carbon-bound forms in SeMet-treated cells, a diselenide component was identified in MeSeCys-treated cells in addition to the carbon-bound selenium species. X-ray fluorescence microscopy of single cells showed that selenium accumulated with sulfur in the perinuclear region of SeMet-treated cells after 24 h, but microprobe selenium X-ray absorption near-edge spectroscopy in this region indicated that selenium was carbon-bound rather than sulfur-bound. X-ray absorption and X-ray fluorescence studies both showed that the selenium content of MeSeCys-treated cells was much lower than that of SeMet-treated cells. Selenium was distributed homogeneously throughout the MeSeCys-treated cells.

Dietary polyphenol-derived protection against neurotoxic β-amyloid protein: from molecular to clinical.

Polyphenolic compounds derived mainly from plant products have demonstrated neuroprotective properties in a number of experimental settings. Such protective effects have often been ascribed to antioxidant capacity, but specific augmentation of other cellular defences and direct interactions with neurotoxic proteins have also been demonstrated. With an emphasis on neurodegenerative conditions, such as Alzheimers disease, we highlight recent findings on the neuroprotection ascribed to bioactive polyphenols capable of directly interfering with the Alzheimers disease hallmark toxic β-amyloid protein (Aβ), thereby inhibiting fibril and aggregate formation. This includes compounds such as the green tea polyphenol (-)-epigallocatechin-3-gallate (EGCG) and the phytoalexin resveratrol. Targeted studies on the biomolecular interactions between dietary polyphenolics and Aβ have not only improved our understanding of the pathogenic role of β-amyloid, but also offer fundamentally novel treatment options for Alzheimers disease and potentially other amyloidoses.

Alteration of the Fc gamma RIIa dimer interface affects receptor signaling but not ligand binding.

The aggregation of cell surface FcRs by immune complexes induces a number of important Ab-dependent effector functions. However, despite numerous studies that examine receptor function, very little is known about the molecular organization of these receptors within the cell. In this study, protein complementation, mutagenesis, and ligand binding analyses demonstrate that human FcγRIIa is present as a noncovalent dimer form. Protein complementation studies found that FcγRIIa molecules are closely associated. Mutagenesis of the dimer interface, as identified by crystallographic analyses, did not affect ligand binding yet caused significant alteration to the magnitude and kinetics of receptor phosphorylation. The data suggest that the ligand binding and the dimer interface are distinct regions within the receptor, and noncovalent dimerization of FcγRIIa may be an essential feature of the FcγRIIa signaling cascade.

Combined DNA, toxicological and heavy metal analyses provides an auditing toolkit to improve pharmacovigilance of traditional Chinese medicine (TCM).

Globally, there has been an increase in the use of herbal remedies including traditional Chinese medicine (TCM). There is a perception that products are natural, safe and effectively regulated, however, regulatory agencies are hampered by a lack of a toolkit to audit ingredient lists, adulterants and constituent active compounds. Here, for the first time, a multidisciplinary approach to assessing the molecular content of 26 TCMs is described. Next generation DNA sequencing is combined with toxicological and heavy metal screening by separation techniques and mass spectrometry (MS) to provide a comprehensive audit. Genetic analysis revealed that 50% of samples contained DNA of undeclared plant or animal taxa, including an endangered species of Panthera (snow leopard). In 50% of the TCMs, an undeclared pharmaceutical agent was detected including warfarin, dexamethasone, diclofenac, cyproheptadine and paracetamol. Mass spectrometry revealed heavy metals including arsenic, lead and cadmium, one with a level of arsenic >10 times the acceptable limit. The study showed 92% of the TCMs examined were found to have some form of contamination and/or substitution. This study demonstrates that a combination of molecular methodologies can provide an effective means by which to audit complementary and alternative medicines.

An immunomodulator used to protect young in the pouch of the Tammar wallaby, Macropus eugenii

Eugenin [pGluGlnAspTyr(SO3)ValPheMetHisProPhe‐NH2] has been isolated from the pouches of female Tammar wallabies (Macropus eugenii) carrying young in the early lactation period. The sequence of eugenin has been determined using a combination of positive and negative ion electrospray mass spectrometry. This compound bears some structural resemblance to the mammalian neuropeptide cholecystokinin 8 [AspTyr(SO3)MetGlyTrpMetAspPhe‐NH2] and to the amphibian caerulein peptides [caerulein: pGluGlnAspTyr(SO3)ThrGlyTrpMetAspPhe‐NH2]. Eugenin has been synthesized by a route which causes only minor hydrolysis of the sulfate group when the peptide is removed from the resin support. Biological activity tests with eugenin indicate that it contracts smooth muscle at a concentration of 10−9 m, and enhances the proliferation of splenocytes at 10−7 m, probably via activation of CCK2 receptors. The activity of eugenin on splenocytes suggests that it is an immunomodulator peptide which plays a role in the protection of pouch young.

Açaí (Euterpe oleraceae Mart.) berry extract exerts neuroprotective effects against β-amyloid exposure in vitro

The native South American palm açaí berry (Euterpe oleraceae Mart.) has high polyphenolic and antioxidant levels. This study examined whether açaí berry extract afforded protection against β-amyloid (Aβ)-mediated loss of cell viability and oxidative stress associated with anti-fibrillar effects. PC12 cells were exposed to either Aβ1-42, Aβ25-35 or tert butyl hydroperoxide (t-BHP), alone or in the presence of açaí extract (0.5-50μg/ml). Thioflavin T (ThT) binding assay and transmission electron microscopy were used to determine effects of açaí extract on Aβ1-42 fibril morphology and compared to açaí phenolics gallic acid, cyanidin rutinoside and cyanidin glucoside. Exposure to Aβ1-42, Aβ25-35 or t-BHP decreased PC12 cell viability. Pretreatment with açaí extract significantly improved cell viability following Aβ1-42 exposure, however Aβ25-35 or t-BHP-mediated viability loss was unaltered. Açaí extract inhibited ThT fluorescence and disrupted Aβ1-42 fibril and aggregate morphology. In comparison with other phenolics, açaí was most effective at inhibiting Aβ1-42 aggregation. Inhibition of β-amyloid aggregation may underlie a neuroprotective effect of açaí.