The food additive fast green FCF inhibits α-synuclein aggregation, disassembles mature fibrils and protects against amyloid-induced neurotoxicity.

α-Synuclein (α-syn) aggregates into cytotoxic amyloid fibrils, which are recognized as the defining neuropathological feature of Parkinson's disease (PD). Therefore, inhibiting α-syn fibrillogenesis and disrupting the preformed fibrils are both considered attractive strategies to cure PD. We discovered that a safe food additive, fast green FCF, is capable of inhibiting α-synuclein fibrillogenesis and reducing the related cytotoxicity. Thioflavin T fluorescence assays demonstrated that fast green FCF could inhibit the fibrillogenesis α-synuclein. In the presence of 100 µM fast green FCF, amorphous aggregates were formed and observed by atomic force microscopy. Toxicity assays in cell cultures revealed that fast green FCF significantly reduced the cytotoxicity of α-syn. Molecular dynamics simulations revealed the potential mechanism of the interactions between fast green FCF and α-synuclein. Fast green FCF greatly disrupted the α-synuclein pentamer and reduced the ß-sheet content by reducing both nonpolar and polar interactions. Furthermore, two binding sites were identified, named region I (Y39-K45) and region II (H50-Q62). Our data reveal that electrostatic interactions, hydrogen bonds, and π-π interactions synergistically contribute to the binding of fast green FCF to the α-synuclein pentamer. These results indicate that fast green FCF is a candidate prototype for the development of drugs against the aggregation of amyloid fibrils in PD.

Inhibition of glycosaminoglycan-mediated amyloid formation by islet amyloid polypeptide and proIAPP processing intermediates.

Islet amyloid polypeptide (IAPP; also known as amylin) is responsible for islet amyloid formation in type 2 diabetes, and IAPP-induced toxicity is believed to contribute to the loss of ß-cell mass associated with the late stages of type 2 diabetes. Islet amyloid formation may also play a role in graft failure after transplantation. IAPP is produced as a prohormone, pro-islet amyloid polypeptide (proIAPP), and processed in the secretory granules of the pancreatic ß-cells. Partially processed forms of proIAPP are found in amyloid deposits; most notable is a 48-residue intermediate, proIAPP(1-48), which includes the N-terminal pro-extension, but which has been properly processed at the C-terminus. Incomplete processing may play a role in islet amyloid formation by promoting interactions with sulfated proteoglycans of the extracellular matrix, which, in turn, promote amyloid formation. We show that acid fuchsin (3-(1-(4-amino-3-methyl-5-sulphonatophenyl)-1-(4-amino-3-sulphonatophenyl)methylene)cyclohexa-1,4-dienesulphonic acid), a simple sulfonated triphenyl methyl derivative, is a potent inhibitor of amyloid formation by the proIAPP(1-48) intermediate. The more complicated triphenyl methane derivative fast green FCF {ethyl-[4-[[4-[ethyl-[(3-sulfophenyl)methyl]amino]phenyl]-(4-hydroxy-2-sulfophenyl)methylidene]-1-cyclohexa-2,5-dienylidene]-[(3-sulfophenyl)methyl]azanium} also inhibits amyloid formation by IAPP and the proIAPP processing intermediate. Both compounds inhibit amyloid formation by mixtures of the proIAPP intermediate and the model glycosaminoglycan heparan sulfate. Acid fuchsin also inhibits glycosaminoglycan-mediated amyloid formation by mature IAPP. The ability to inhibit amyloid formation is not simply due to the compounds being sulfonated, since the sulfonated inhibitor of amyloid-ß, tramiprosate, is not an inhibitor of amyloid formation by proIAPP(1-48).

Staining properties of deepithelialized human amniotic membrane.

PURPOSE: The surgical use of human amniotic membrane (HAM) can be challenging because of its transparency, thin structure, and adhesive quality. Staining of HAM has been proposed to facilitate intraoperative visualization. We evaluated the in vitro staining properties of deepithelialized HAM using 5 vital dyes. METHODS: Deepithelialized HAM was stained with indocyanine green (1.0%, 0.5%, 0.1%), fluorescein sodium (1.0%, 0.25%, 0.1%), lissamine green (0.5%, 0.1%), rose bengal (1.0%, 0.1%), and trypan blue (0.5%, 0.1%). The staining of each dyed sample was evaluated using a x10 operating microscope by a single observer. Each membrane was then rinsed, resuspended (3 mL of balanced salt solution), and then reevaluated for staining at 30-minute intervals for a total of 4 hours. All remaining stained samples were then placed in 5 mL balanced salt solution and then reevaluated for staining at 24 hours. RESULTS: All concentrations of the 5 dyes stained the membrane initially. After 120 minutes, fluorescein sodium 0.1% and lissamine green 0.5% and 0.1% no longer stained the membrane. Fluorescein sodium 0.5% no longer stained at 210 minutes, and fluorescein sodium 1.0% no longer stained at 24 hours. All concentrations of indocyanine green, rose bengal, and trypan blue stained positively at 24 hours. CONCLUSIONS: All 5 dyes stain deepithelialized HAM initially. Tested concentrations of fluorescein sodium and lissamine green may be superior to other tested dyes for intraoperative use because these dyes stain the membrane and then fade. Tested concentrations of rose bengal, trypan blue, and ICG may not be ideal for clinical use because they demonstrate persistent staining at 24 hours.

Articular cartilage fibrillation and permeability to Light Green SF dye. A method for the detection of pre-microscopic disease?

We describe a method which may be useful for the selection of samples for the study of early fibrillation in human articular cartilage. Blocks of cartilage and bone were cut post-mortem from the medial tibial condyles of 29 male and 31 female subjects and the grade of fibrillation was assessed from sections. Contiguous, unfixed blocks of cartilage from the same surface were immersed in a solution of the dye Light Green SF. Sections of these blocks were cut and the rate of penetration of the dye measured at 30 equidistant points across the condylar surface. The relationship between the grade of fibrillation and the rate of dye diffusion was then determined. We demonstrated a significant correlation between the two variables. This technique may make it possible to detect a pre-fibrillary state in apparently normal specimens.