We report for the
very first time the discovery of amyloid-like
self-assemblies formed by the nonaromatic single amino acids cysteine
(Cys) and methionine (Met) under neutral aqueous conditions. The structure
formation was assessed and characterized by various microscopic and
spectroscopic techniques such as optical microscopy, phase contrast
microscopy, scanning electron microscopy, and transmission electron
microscopy. The mechanism of self-assembly and the role of hydrogen
bonding and thiol interactions of Cys and Met were assessed by Fourier
transform infrared spectroscopy, thermogravimetric analysis, X-ray
diffraction, and solid state NMR along with various control experiments.
In addition, molecular dynamics simulations were carried out to gain
insight into assembly initiation. Further, Thioflavin T and Congo
red binding assays with Cys and Met structures indicated that these
single amino acid assemblies may have amyloid-like characteristics.
To understand the biological significance of the Cys and Met structures,
cytotoxicity assays of the assemblies were performed on human neuroblastoma
IMR-32 cells and monkey kidney cells (COS-7). The results revealed
that both Cys and Met fibers were cytotoxic. The cell viability assay
further supported the hypothesis that aggregation of single amino
acid may contribute to the etiology of metabolic disorders like cystinuria
and hypermethioninemia. The results presented in this study are striking,
and to the best of our knowledge this is the first report which demonstrates
that nonaromatic amino acids like Cys and Met can undergo spontaneous
self-assembly to form amyloidogenic aggregates. The results presented
are also consistent with the established generic amyloid hypothesis
and support a new paradigm for the study of the etiology of single
amino acid initiated metabolic disorders in amyloid related diseases.
The specific binding of two model drugs for photodynamic therapy, namely chlorin p6 and purpurin 18 in the vicinity of Sudlow's Site I of HSA has been investigated by monitoring the intrinsic fluorescence of single tryptophanyl residue and by competitive binding with warfarin. The distance from the tryptophanyl residue has been ascertained by FRET from Trp to the chlorins and has been found to indicate a binding to Sudlow's Site I. The principal driving force for the interaction is found to be the hydrophobic effect. The main mechanism of protein fluorescence quenching was static. Time-resolved fluorescence results of competitive binding with warfarin are found to confirm that they bind to the warfarin binding site.
While the frequency of patch test reactivity to many cosmetic allergens has decreased over the last 20 years, we have previously shown that in our clinic, the patch test reactivity to p-phenylenediamine (PPD) has remained stubbornly high between 2.5% and 4.2% in the years when patch testing was performed with 1% PPD. Further retrospective analysis of the PPD patch test frequency over the last 6 years shows an increasing rate of PPD patch test frequency, showing an upward linear trend. This increasing trend cannot be fully explained by any increase in patch testing of Southern Asian patients or of sensitization caused by PPD exposure from 'temporary henna tattoos'. An alternative explanation may be the increasing use of permanent hair dyes.
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