Takafumi Inoue’s research while affiliated with Japan Synchrotron Radiation Research Institute and other places

This article deals with the structure of the cell membrane complex (CMC) in the human hair cuticle. The microbeam X-ray provided a pattern of small-angle scattering from the CMC in the cuticle with no sample preparations, including slicing and pre-staining of hair. The thickness of the beta-and delta-layers, substructure in CMC, was estimated by analysis of the scattering pattern. We used hair samples extracted with several solvents, and found that solvent extraction changed the thickness of the beta-and delta-layers in a manner dependent on the type of solvent. Extraction of hair with solvent was also shown to have effects on the extent of dyeing. There was a high correlation between the extent of dyeing and the thickness of the delta-layer, i.e., a thin layer tended to show a high amount of dyeing, whereas there was no significant correlation between the thickness of the beta-layer and the extent of dyeing.

Hair treatment chemicals induce sudden and severe hair damage. In this study, we examined cuticles from untreated, permed, and bleached hair that were mechanically discriminated by shaking in water. Both perming and bleaching treatments are prone to easily delaminate cuticles. Confocal microscopy revealed that the cuticles of permed hair were delaminated with larger pieces than untreated ones. On the other hand, the cuticles of bleached hair tend to fragment into small peptides. At the minimum concentration of thioglycolate required to elute S100A3 protein from the endocuticle into the reductive permanent waving lotion, enlarged delaminated cuticle fragments were observed. Although S100A3 is retained in bleached hair, S100A3 is irreversibly oxidized upon bleaching treatment. It is likely that the oxidative cleavage of disulfide bonds between cuticle-constituting proteins, including S100A3, results in the fragile property of cuticles. Here we present a more comprehensive model of hair damage based on a diverse mechanism of cuticle delamination.

Because of small fluctuations, it is difficult to evaluate hair damage caused by bleaching using previously utilized hair damage indexes. Application of commercial bleaching products elevates partially extractable labile hair protein amounts in the range of 0.4-1.2 mg/g of hair. Within this range, the level of labile protein fluctuates greatly, depending on the extent of bleaching. In the current study, it was found that the effects of alkaline constituents and various peptides contained in bleaching lotions on hair damage could be evaluated by measuring labile protein amounts without employing harsher bleaching conditions.

S100A3, a unique protein among all members of the calcium-binding S100 family, is specifically expressed at the inner endocuticle of human hair fibers. Upon hair damage, S100A3 is released from hair fibers and possibly destabilizes the hair tissue architecture. This study describes the purification and characterization of native S100A3 isolated from human hair fibers. We extracted native S100A3 from cuticles and purified the protein by anion-exchange chromatography. The results of 2D gel electrophoresis showed that cuticle S100A3 has a slightly lower isoelectric point compared to the recombinant protein. Tandem mass spectrometry of the peptides resulting from endoproteinase digest of cuticle S100A3 revealed that the N-terminal methionine is replaced with an acetyl group. This is the first report on biochemical characteristics of S100A3 in hair cuticle.

We previously found that certain hair proteins were soluble by means of a partial extraction method. In this study, we demonstrate that the amount of soluble proteins internally formed in permed and bleached hair, labile proteins, is a useful index for hair damage assessment. Compared to tensile property changes, this index rose in widely dynamic ranges as the time of either permanent waving or bleaching treatments increased. The amount of labile proteins was much larger than that of proteins eluted into perming and bleaching lotions. However, the labile proteins showed electrophoretic profiles similar to those of the eluted proteins. These results suggest that a portion of the stable proteins in normal hair was transformed into labile proteins upon permanent waving and bleaching treatments. Consequently, permed and bleached hair tends to release the resultant labile proteins.

Partial protein extracts were prepared from hair, nail, and stratum corneum in the absence of urea and interfacial surfactant. Tricine-sodium dodecyl sulfate polyacrylamide gel electrophoreses of these extracts showed low-molecular weight protein-rich patterns apparently different from those of whole protein extracts, which mainly consist of keratin bands. Several protein bands characterized each keratinized tissue or its derived species. In addition, we identified a major band of approximately 7 kDa as ubiquitin, a ubiquitously distributed protein that mediates non-lysosomal protein degradation, through direct amino acid sequence analysis of the electro-blotted protein band. The partial extraction is useful for investigation of soluble proteins retained in the keratinized tissues.

Protein is the most abundant component (65-95%) of hair, and has been targeted as an important subject for hair science research. Chemical treatments, such as permanent waving or bleaching, are widely known to be a major cause of hair damage. However, the constituent proteins in the effluent from these treatments have not been characterized in detail. In this study, we performed Tricine-SDS-polyacrylamide gel electrophoresis for detailed analyses of the proteins eluted under various chemical treatment conditions. Effluents from permanent waving or bleaching showed similar electrophoretic profiles, and both types contained a major protein band of approximately 7kDa. Through immunoblot analyses, this protein band was identified as ubiquitin, a ubiquitously distributed protein that mediates non-lysosomal protein degradation in eukaryotic cells. Comparative analyses of the ubiquitin-signal-intensities revealed that natural hair extracts derived from distal parts contained a lower ubiquitin content than those from proximal parts. These results suggested that ubiquitin was released during the course of natural occurring hair damage. Therefore, we speculated that the major components lost from hair were likely to be soluble proteins that were neither keratin intermediate filament proteins nor keratin associated proteins.

In hair fiber, a cysteine-rich calcium-binding S100A3 protein is segregated in the inner part of the cuticle and postulated to play an important role in the attachment to the adjacent cuticular scale. In this study, elution of S100A3 from hair fiber was examined under various conditions by means of immunoblot analyses. The exposure of hair fiber to permanent waving lotions resulted in recoveries of substantial amounts of S100A3 by elution. Ultraviolet-light radiation and perming also increased the elution of S100A3 even without reductant. The distal part of hair fiber eluted less S100A3, as compared to the proximal section, under reducing conditions. These results suggest that S100A3 is eluted preferentially by daily washing and rinsing, especially from damaged hair. Given the presence of soluble S100A3 in the inner part of cuticle, we propose a new mechanism of hair damage in which the elution of S100A3 plays a major role.

We have characterized the subcellular distribution of S100A3, a cysteine-rich calcium binding protein, in human scalp hair shaft. This was accomplished using rapid-freezing immunocytochemistry, a technique that combines rapid-freezing, freeze-substitution fixation without chemical fixatives, and subsequent electron microscopic detection of immunocytochemical labeling. This technique preserves both the antigenicity and the ultrastructural integrity of fully keratinized tissues, which are highly unmanageable when prepared for immunoelectron microscopy. In the hair shaft, S100A3 was primarily identified in the endocuticle and was also present in the intermacrofibrillar matrix surrounding macrofibril bundles of intermediate filament keratins in cortex cells. Double immunolabeling of S100A3 and hair keratins revealed the in situ spatial relationship between them. In the endocuticle, S100A3 was present on the inner portion of the endocuticle adjacent to the cell membrane complex, whereas hair keratins were present on the outer portion. These results provide the first ultrastructural evidence that an S100 protein is localized in specific subcompartments in human hair cells. (J Histochem Cytochem 47:525-532, 1999)