SummaryHyoscyamus albus hairy roots with/without an exogenous gene (11 clones) were established by inoculation of Agrobacterium rhizogenes. All clones cultured under iron deficient condition secreted riboflavin from root tips into the culture medium and the productivity depended on the number and size of root tips among the clones, although the addition of sucrose was essential for riboflavin production. A decline of pH was observed before riboflavin production and root development using either a root tip or propagated roots: propagated roots were employed for further work due to their lesser variation.Additions of proton-pump inhibitors, N,N'-dicyclohexylcarbodiimide (DCCD) at 100 and 10 μM and erythrosine B at 100 μM, suppressed the pH decline at 100 and 10 μM accompanied by inhibition of riboflavin secretion and root growth; at 10 μM of erythrosine B, pH decline occurred with a moderate delay, but both growth and riboflavin efflux were inhibited. Neither inhibition of the pH decline nor riboflavin production was observed at 1 μM. To examine the necessity of acidification and riboflavin secretion by the roots themselves, artificial pH reduction of culture medium with organic acids and the addition of exogenous riboflavin with/without pH reduction were performed. When hairy roots were cultured in iron-deficient medium acidified with citric acid (pH 4.0) or malic acid (pH 3.7), pH increased rapidly to around 5 overnight, following which riboflavin production and root growth occurred. Addition of riboflavin did not affect riboflavin secretion by the roots, but acidification with citric acid (pH 4.0) helped achiever greater riboflavin production and earlier pH elevation. These results indicate that riboflavin efflux does not directly connected to active pH reduction, and more significantly active riboflavin secretion occurs by internal requirement in H. albus hairy roots under iron deficiency.

Neurotoxicity caused by peptide and protein aggregates is associated with the onset of neurodegenerative diseases. Accumulation of the amyloid β protein (Aβ) induced by neuronal ganglioside-enriched nanodomains (nanoclusters) in the presynaptic neuronal membrane, resulting in toxic oligomeric and fibrous forms, is implicated in the onset of Alzheimer's disease (AD). In the current study, we found that the ganglioside clusterb i n d i n g p e p t i d e ( G C B P ) , a p e n t a d e c a p e p t i d e VWRLLAPPFSNRLLP that binds to ganglioside-enriched nanoclusters, inhibits the formation of Aβ assemblies with an IC 50 of 12 pM and also removes Aβ fibrils deposited on the lipid membrane. Thus, in addition to inhibiting Aβ assembly formation, GCBP effectively clears toxic Aβ assemblies as well, thereby suppressing neuronal cellular damage and death induced by such assemblies. These results indicate that ganglioside cluster-binding molecules may act as novel Aβ-targeting drugs with a unique mechanism of action that may be utilized to ameliorate AD.

The specific features of the lateral distribution of gangliosides play key roles in cell−cell communications and the onset of various diseases related to the plasma membrane. We herein demonstrated that an artificial peptide identified from a phage-displayed library is available as a molecular probe for specific ganglioside nanoclustering sites in caveolae/membrane rafts on the cell surface. Atomic force microscopy studies indicated that the peptide specifically binds to the highly enriched monosialoganglioside GM1 nanodomains of reconstituted lipid bilayers composed of GM1, sphingomyelin, cholesterol, and unsaturated phospholipids. The ganglioside-containing area recognized by the peptide on the surface of PC12 cells was part of the area recognized by the cholera toxin B subunit, which has high affinity for GM1. Furthermore, the peptide bound to the cell surface after a treatment with methyl-β-cyclodextrin (MβCD), which disrupts membrane rafts by removing cholesterol. The present results indicate that there are heterogeneous ganglioside clusters with different ganglioside densities in caveolae/membrane rafts, and the peptidyl probe selectively recognizes the high-density ganglioside nanodomain that resists the MβCD treatment. This peptidyl probe will be useful for obtaining information on the lipid organization of the cell membrane and will help clarify the mechanisms by which the lateral distribution of gangliosides affects biological functions and the onset of diseases.