S-sulfonation of transthyretin is an important trigger step in the formation of transthyretin-related amyloid fibril

Senile systemic amyloidosis and familial amyloid polyneuropathy are caused by oxidative deposition of conformationally altered transthyretin (TTR). We identified oxidative modification of the 10th cysteine of TTR through S-sulfonation in vitro. Based on mass spectrometric analysis, we determined the spectrophotometric, western blotting, and fluororescent microscopic properties of TTR incubated with and without cysteine-S-sulfonate in acidic (pH 4) and alkaline (pH 8) conditions at 37°. The absorption of the aggregated TTR molecules increased more with incubation time and the concentration of cysteine-S-sulfonate at pH 4 than at pH 8. The Congo red binding to the S-sulfonated TTR at pH 4 was saturated with an apparent Bmax of 2.01 mol per mole of the S-sulfonated TTR and apparent K_D of 7.75 × 10⁻⁶ M. On the other hand, the Bmax of cysteinyl TTR was 1.38, and its K_D was 3.52 × 10⁻⁶ M while the Bmax of reduced TTR was 0.86, and its K_D was 2.86 × 10⁻⁶ M. Moreover, we detected positive amyloid fibril staining using Thioflavin T and Congo red with the S-sulfonated TTR but not with untreated or reduced TTR by microscopic fluororescent analysis. After modification of TTR in vitro, oligomers resisted reduction and denaturation was irreversibly induced, and which contributed differences in the Western blotting patterns obtained with four anti-TTR antibodies. In conclusion, this study showed that the formation of S-sulfonation of TTR through oxidative modifications of the thiol residue on the 10th cysteine of TTR is an important trigger step in the formation of transthyretin-related amyloid fibril.     

Neuroprotective effects of grape seed extract on neuronal injury by inhibiting DNA damage in the gerbil hippocampus after transient forebrain ischemia

Grape seed extract (GSE) possess cardioprotective abilities by functioning as in vivo antioxidants and by virtue of their ability to directly scavenge ROS including hydroxyl and peroxyl radicals. In the present study, we investigated the neuroprotective effects of grape seed extract (GSE) in the gerbil hippocampus after 5 min transient forebrain ischemia. Neuronal cell density in GSE-treated ischemic animals was significantly increased as compared with vehicle-treated ischemic animals 4 days after ischemic insult. In the GSE-treated groups, about 60% of pyramidal cells of the sham-operated group were stained with cresyl violet 4 days after ischemic insult. In this study, we found that GSE had neuroprotective effects on neuronal injury by inhibiting DNA damage in the CA1 region after ischemia. In vehicle-treated groups, 8-hydroxy-2'-deoxyguanosine (8-OHdG) immunoreactivity was significantly changed time-dependently, whereas the immunoreactivity in the GSE-treated group was similar to the sham-operated group. In addition, we confirmed that astrocytes and microglia did not show significant activation in the CA1 region 4 days after ischemia-reperfusion, because many CA1 pyramidal cells were not damaged. Therefore, these results suggest that GSE can protect ischemic neuronal damage by inhibiting DNA damage after transient forebrain ischemia.     

Synergic effect of grape seed extract with amphotericin B against disseminated candidiasis due to Candida albicans

Amphotericin B (Amp B) is considered as a drug of choice for treatment of fungal infections, but it causes severe side effects such as renal damage. To lessen the severity, it is often combined with the azole, but data reporting resistance of Candida albicans to the azole have been recently increasing. Thus, finding a new product that can reduce Amp B dose by combination seems to be important. In the present study, we investigated a synergic effect of grape seed extract (GSE) combined with Amp B against the fungus. Our results showed that the GSE alone inhibited growth of C. albicans yeast cells, and that in a murine model of disseminated candidiasis mice groups given GSE before intravenous inoculation with the yeast cells survived longer than diluent-received (control) mice groups (P<0.05). This GSE antifungal effect was dose-dependent. Upon combination of GSE plus Amp B, the combination therapy strikingly retarded the yeast growth as determined by the broth susceptibility method. Against the disseminated disease, mice given diluent (negative control), Amp B (0.5mg/kg of body weight), or GSE (2mg/kg of body weight) had mean survival times (MSTs) of approximately 11.4, 14.4, and 17.6 days, respectively. However, mice treated with the combination of the doses of Amp B and GSE had a MST value of 38.4 days, surviving an average of 24 days longer than Amp B alone-treated mice groups. This MST value from the combination-received mice group was greater than the MST value from the mice group given four times the Amp B dose (2mg/kg of body weight). All these data indicate that the combination therapy can reduce more than 75% of Amp B dose, implying that GSE has a synergic effect with Amp B.     

Proteomics analysis of the actions of grape seed extract in rat brain: technological and biological implications for the study of the actions of psychoactive compounds

Grape seed extract (GSE) is a commonly available dietary supplement taken for the anti-oxidant activity that's attributed to its proanthocyanidin (oligomers of monomeric polyphenols) content. Similar polyphenol-enriched preparations from blueberries and soy have shown protection against ovariectomy-induced or age-related cognitive deficits, suggesting that the molecular changes induced by these polyphenol preparations correlated with behavioral benefit. We hypothesized that ingestion of polyphenol-enriched preparations such as GSE would be manifested as protein changes that would be consistent with neuroprotection. Proteomics technology, namely 2D gel electrophoresis and mass spectrometry, identified quantitative changes in specific proteins induced in adult rat brain following ingestion of a powdered preparation of GSE. As recently reported [Deshane, J., Chaves, L., Sarikonda, K.V., Isbell, S., Wilson, L., Kirk, M., Grubbs, C., Barnes, S., Meleth, S. and Kim, H., 2004. Proteomics analysis of rat brain protein modulations by grape seed extract. Journal of Agricultural and Food Chemistry 52, 7872-7883.], the direction of change for the majority of the affected proteins was opposite to the direction the proteins were changed in either Alzheimer disease or in transgenic mouse models of dementia. A conservative conclusion is that GSE has neuroprotective activity, by affecting specific proteins in particular ways. In this chapter, elements of proteomics-type analysis are discussed that demonstrate the power of the technology to enable discovery of proteins involved in the response of the brain to a stimulus whether it be a dietary supplement, or a psychoactive drug. The fact that GSE affects proteins implicated in cognitive disorders suggests moreover that GSE may have impact on the actions of psychoactive drugs by maintaining an overall viability of the nervous system.     

The formation of a novel supramolecular structure by amyloid of poly-L-glutamic acid

Polyglutamic acid (PE) has been shown to form amyloid fibrils in vitro under pH value of 4.0. However, under the pH of 2.0, a further self-association process resulting in a novel supramolecular structure was observed. These supramolecular assemblies had diameters ranging from 1 to 20 μm and lengths up to several hundred microns, which were significantly larger than those of typical “amyloid fibrils”. The existence of amyloid-like structure within these assemblies was confirmed with Fourier transform infrared spectroscopy and Thioflavin T fluorescence assay. The aggregation process of PE was studied by direct observation of electronic microscopy. The supramolecular assemblies appeared to be formed in a hierarchical process in which the preformed amyloid-like subunits self-assembled into higher-order assemblies in a well-organized pattern.     

Oleic acid inhibits amyloid formation of the intermediate of alpha-lactalbumin at moderately acidic pH

The effects of oleic acid on amyloid formation of Ca2+-depleted bovine alpha-lactalbumin (apo-BLA) at low pH and the biological impact of the effects were investigated by using thioflavin T, Congo red, far-UV circular dichroism, atomic force microscopy, transmission electron microscopy, and other biophysical methods. The results from the phase diagram method of fluorescence show that two intermediates exist in the conformational transition of apo-BLA induced by low pH. One intermediate populated at pH 3.0 is characterized as a molten globule state and the other accumulates with stable secondary structure and exposed hydrophobic surface at pH 4.0-4.5. Amyloid formation of apo-BLA takes place upon decreasing the pH to 4.5 and is accelerated remarkably as the pH is decreased further. However, amyloid fibrils of apo-BLA are not observed in the pH range of 5.0-7.0 on a time-scale of 30 days. The lag time of fibrillation at pH 4.0 is greatly elongated by the presence of oleic acid, accompanied by a remarkable decline of the maximum thioflavin T intensity. Furthermore, amyloid formation of apo-BLA at pH 4.5 is inhibited completely by oleic acid, and insoluble aggregates are observed. In contrast, the effects of oleic acid on amyloid formation are not remarkable at pH 3.0 or at pH 2.0. Our data demonstrate that oleic acid specifically induces the intermediate of apo-BLA at pH 4.0-4.5 to form insoluble amorphous aggregates, which is responsible for the inhibition of amyloid formation of the protein by oleic acid in this range of pH values.     

Unusual sequence and characteristics of a chick-pea seed protein which is regulated by abscisic acid and is similar to late-embryogenesis-abundant proteins

The cDNA clone GAB-9 was selected from a cDNA gene library constructed from the mRNA of embryonic axes of chick-pea (Cicer arietinum L.) seeds imbibed for 12 h in the presence of abscisic acid. The sequence of this cDNA has an open reading frame of 546 nucleotides that code for 182 amino acids. The polypeptide encoded by the corresponding mRNA is of approx. 20.5 kDa, is basic, and has a broad hydrophobic central region flanked by two hydrophilic regions. The unusual characteristics of this protein, which is similar to late-embryogenesis-abundant proteins, and its possible function are discussed.Abbreviations ABA abscisic acid - HSP heat-shock proteins - LEA late embryogenesis abundant The nucleotide sequence data reported appear in the EMBL under the accession number X79680.We thank Dr J.L. Revuelta for helpful discussion. This work was supported by grants from Direction General de Investigation Científica y Técnica, Spain (PB90-0536) and Junta de Castilla y León (SA-33/11/92).     

The effect of ethylene, octanoic acid and a plant-derived smoke extract on the germination of light-sensitive lettuce seeds

The effects of an aqueous plant-derived smoke extract, octanoic acid and ethylene on germination of light-sensitive Grand Rapids lettuce seeds were investigated. The smoke extract brought about a concentration dependent increase in germination and a complete inhibition of germination at high concentrations. Octanoic acid could not induce germination. Ethylene at concentrations over 5 L L^–1 increased lettuce seed germination, but not to the same degree as smoke. Aqueous smoke in combination with ethylene showed a synergistic effect on germination at suboptimal smoke concentrations. At high smoke concentrations the effect of ethylene was almost completely inhibited. Octanoic acid in combination with ethylene brought about a higher level of germination than with ethylene alone, but only at the highest concentration of octanoic acid tested (1 mM). Standardized hexane and dichloromethane-partitioned smoke extracts and octanoic acid were subjected to TLC separation. The R _f -fractions in the smoke lanes showing activity in the lettuce seed bioassay did not correspond to the R _f -value of octanoic acid. As aqueous smoke can withstand autoclaving and can be separated by TLC and HPLC without loosing activity it is unlikely that the activity of aqueous smoke is linked to ethylene. It thus appears that the active compound in smoke is neither octanoic acid nor ethylene.Abbreviations TLC thin layer chromatography - HPLC High performance liquid chromatography