Article

The physical and chemical disruption of human hair after bleaching – studies by transmission electron microscopy and redox proteomics

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Abstract

Objective To understand the structural and chemical effects of cosmetic peroxide bleaching on human hair. Methods Human hair was progressively bleached using alkaline peroxide‐persulfate treatment. Proteins lost through leaching were examined using amino acid analysis and mass spectrometric sequencing. Fibre damage was assessed using transmission electron microscopy, amino acid analysis, and redox proteomics. Results Protein loss through leaching increased with bleaching severity. Leached proteins were not limited to the cuticle, but also included cortical intermediate filaments and matrix keratin‐associated proteins. The leached proteins were progressively oxidised as bleaching severity increased. Bleached fibres demonstrated substantial damage to the cuticle layers and to the cortex. Extensive melanin granule degradation was present after the mildest bleach treatment. Protein oxidation in bleached fibres was principally in cortical intermediate filaments – the most abundant hair proteins – and targeted the sulfur‐containing amino acids, particularly the conversion of cystine disulfide bonds to cysteic acid. Conclusion Peroxide chemical treatments quickly access the cortex, causing untargeted oxidative damage across the fibre in addition to the desired loss of melanin. Peroxide ingress is likely facilitated by the considerable structural degradation caused to the cuticle layers of hair fibres. The consequences of the peroxide action within the cuticle and cortex are oxidation of the proteins, and subsequent protein loss from the fibre that correlates to bleaching severity. This article is protected by copyright. All rights reserved.

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... The effect of RH on the water content of virgin human hair is in full agreement with the literature [1]. Furthermore, the evaluation of water loss from both sets of data confirms previous observations showing that bleaching leads to liquid retention [1,15,[21][22][23][24][25]. On further heating, i.e. above 180 • C, and independently of measurement approach, it was observed that the bleached hair showed a lower mass loss when compared to the virgin sample. ...
... On further heating, i.e. above 180 • C, and independently of measurement approach, it was observed that the bleached hair showed a lower mass loss when compared to the virgin sample. These results substantiate previous findings, where it was considered that in the process of bleaching the hair loses mass (proteins and lipids) [1,[21][22][23]. In addition, the oxidative treatment causes hydrolysis of the amide groups of the amino acids plus formation of cysteic acid residues, resulting in an increase of the acidic groups in the fibers followed by a decrease of their isoelectric and isoionic points [1,23,26]. ...
... Such increase of the denaturation peak maximum temperature (T D ) has been previously observed in samples where the oxidative treatment was reapplied [22]. These effects can be related to the loss of crystalline material (α-keratin IFs) promoting the weakening of the fiber, and consequent alteration of their properties [3,21,22]. ...
Article
Hair analysis plays an important role in forensic toxicology and biomonitoring tests. However, cosmetic treatments cause changes to the hair. Thus, a better understanding of the hair’s structure and the factors that influence its composition is critical. It is known that oxidative treatments modify the hair chemical, structural and mechanical properties. These treatments also cause degradation of the melanin as well as of the structures present in the hair cuticle and cortex. Considering that the literature is unanimous regarding the increase in hydrophilicity and porosity promoted in human hair by bleaching, in this work we investigated how this oxidative damage is triggered. By combining several techniques, inelastic and quasi-elastic neutron scattering, differential scanning calorimetry, thermal gravimetric analysis and X-ray diffraction, we were able to connect the chemical and structural changes to a subtle dynamic modification of the proton mobility in the hair fibers. In addition, alterations in the thermal behavior evidenced a small denaturation of α-keratin intermediate filaments and a slight decrease in the amount of confined water in the hair fibers. Moreover, data obtained by neutron spectroscopy indicated that bleaching attacks the thioester groups of the proteins causing larger proton mobility of the hydrogenous components (water, protein and/or lipids).
... In recent studies by Joo et al. and Grosvenor et al., the influence of hair bleaching on amino acids and lipids and proteins was investigated, respectively. 18,19 The authors could show that oxidative hair treatment has a significant impact on the studied compounds. However, these studies were conducted for a small selection of endogenous compounds and aimed to analyze the impact of hair bleaching on hair damage. ...
... These bleaching conditions do not only lead to a visible brightening of the hair but also the hair structure carries away a severe damage as can already be seen by microscopic observation. 19,32 This might also facilitate the leakage and the extraction from the hair matrix. Nevertheless, it is to be expected that individuals adulterating their hair on purpose with the goal of a negative hair testing result would accept and encourage a considerably high hair damage. ...
Article
Full-text available
Hair analysis has become an integral part in forensic toxicological laboratories for e.g. assessment of drug or alcohol abstinence. However, hair samples can be manipulated by cosmetic treatments, altering drug concentrations which eventually leads to false negative hair test results. Especially oxidative bleaching of hair samples under alkaline conditions significantly affects incorporated drug concentrations. To date, current techniques to detect cosmetic hair adulterations bear limitations such as the implementation of cut-off values or the requirement of specialized instrumentations. As a new approach, untargeted hair metabolomics analysis was applied to detect altered, endogenous biomolecules that could be used as biomarkers for oxidative cosmetic hair treatments. For this, genuine hair samples were treated in vitro with 9 % hydrogen peroxide (H2O2) during 30 minutes. Untreated and treated hair samples were analyzed using liquid-chromatography high-resolution time-of-flight mass spectrometry. In total, 69 metabolites could be identified as significantly altered after hair bleaching. The majority of metabolites decreased after bleaching, yet totally degraded metabolites were most promising as suitable biomarkers. The formation of biomarker ratios of metabolites decreasing and increasing in concentrations improved the discrimination of untreated and treated hair samples. With the results of this study, the high variety of identified biomarkers now offers the possibility to include single biomarkers or biomarker selections into routine screening methods for improved data interpretation of hair test results.
... Although biochemical analyses of human hair fibers oxidative damage have been mainly focused on the redox analysis of cystine (the oxidized dimer form of the amino acid cysteine) and in particular cysteic acid formation, it counts only for 9% of hair fiber amino acid composition [3,7]. The simplicity of the method for assessing cysteic acid in hair fibers could explain its popularity; however an important lack of sensibility is known for this method [8]. ...
Article
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(1) Background: Environmental factors, such as airborne pollutants and solar UV, induce oxidative damage to proteins and lipids on hair fibers, leading to decreased hair strength and shine, increased fiber porosity, brittleness, dryness, and stiffness. Traditional methods used for hair damage/protection/reparation assessment show limitations in sensitivity or specificity for evidencing the benefits to be gained from the protection/reparation of hair fibers against environmental stressors. (2) Methods: Ex vivo experimental models of hair fibers exposed to urban pollutants and UV irradiation were developed. Targeted proteomics approaches for the quantification of oxidatively damaged (carbonylated) proteins on hair fibers were optimized. (3) Results: A significant dose-dependent increase in carbonylation both in the cuticle and cortex proteins was observed upon exposure of hair fibers to particulate matter and UV-A radiation, at daily stress equivalent doses. Increased protein carbonylation on keratins and keratin-associated proteins led to loss of hair fiber structural integrity. The oxidative modification of proteins induced by urban pollution exposure led to hair cuticle structural damage revealed by an increased permeability. However, protein carbonylation was prevented in the presence of antioxidant compounds. (4) Conclusions: Protein carbonylation is an early event in hair fiber damage which can be used as a reliable biomarker for the efficacy of hair care interventions against environmental stressors.
... This would account for the relatively small increase of protein leak in BH compared to VH. Wolfram et al. (1970) suggested that the reaction of hydrogen peroxide on hair keratin was mostly limited to cystine amino acid based on the fact that the amount of cysteic acid in the protein extracted from bleached hair quadrupled that of the protein extracted from untreated hair. High alkaline condition of bleaching which swells and opens up the cuticle layer would allow bleaching agent to pass through the cuticle, and as a result protein of cell membrane complex and also the matrix protein of cortex, which are more susceptible than the α-helix protein, could be oxidized into smaller molecules which could leak by cosmetic treatments such as shampooing, permanent wave, etc. (Grosvenor et al., 2018;Rele & Mohile, 2003). The effect of bleaching reported in this research was based on the usage of a specific brand of the bleaching agent chosen for this study. ...
Article
Effect of hair conditioner formulated with Argan oil or Camellia oil was investigated on the protection of hair damaged by bleaching. Six different rinse-off type hair conditioners were made with the basic ingredients of hair conditioner and one of the following conditioning agent; Argan oil (AO), Camellia oil (CO), Palmitic acid (PA), Stearic acid (SA), Oleic acid (OA), and Linoleic acid (LA). L*, a*, b* color values and tensile strength, elongation were measured, and the amount of protein leak was examined using the Bradford Protein Assay. Statistical significance was tested using the SPSS statistical software. Although both AO and CO were effective in protecting the tensile properties of bleached hair, significant effects were observed with AO in enhancing the tensile strength and retaining the color of bleached hair. This might be due possibly to the difference in the composition of four major fatty acids in Argan oil and Camellia oil.
... The conventional bleaching process to remove the natural yellowish impurities from cotton -usually undertaken at 98 • C for 60 min in alkaline, is incompatible with yak hair. Because the protein structure displays higher degradation propensity than cellulose under such harsh condition, which leads to the mechanical loss of fibre [10]. As yet, much less emphasize has been placed on the decolouration of yak hair than cotton and wool. ...
Article
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Promoting processing efficiency and taking advantage of agricultural by-products are two promising ways to achieve sustainable textile industry. This study presents a customized efficient ultrasonic-assisted bleaching strategy for yak hair – a widely existing but underexploited secondary dark shade fibre from yak. A melanin-targeted Fenton oxidation process is established which involves three phases, i.e., (I) incorporation of Fe²⁺ ions with melanin, (II) catalytic oxidative bleaching using hydrogen peroxide (H2O2), and (III) reductive cleansing. The bleaching efficacy, dyeing performance and tensile property of yak hair treated with and without ultrasound were explored and compared. Further, the ultrasonic bleaching mechanism in terms of the catalytic effect of Fe²⁺ ions, the promotion of H2O2 decomposition, removal of melanin granule from yak hair, were demonstrated. Finally, the main effects and interactions of parameters in phase II, and optimal condition were obtained through mathematical modelling based on a central composite design (CCD). Results reveal that ultrasonic bleaching dramatically enhances the whiteness index (WI) of yak hair from 11 to 45 which is 44.6% higher than those bleached without ultrasound, and also promotes the uptake of acid dyes. There is only 15% tensile strength loss and 14% elongation increment of yak hair after ultrasonic bleaching, rising from a slight damage of cuticle layer and cleavage of disulfide bonds, respectively. In the study of bleaching mechanism, Fe²⁺ ion is confirmed to improve the H2O2 decomposition rate by 20.9% which further runs up to 35.9% after introducing ultrasound. Ultrasound increases the concentration of hydroxyl radicals (HO) by 94% which are the main oxidative species participating in bleaching confirmed by HO scavenging experiment. The porous structure was observed on the cross section of yak hair stemming from the removal of melanin granules contributed by the cleaning action of ultrasound. A theoretical highest WI of 52.4 can be achieved under an optimal condition based on the CCD study. In general, the proposed melanin-targeted bleaching strategy for yak hair that integrates ultrasonic technology and Fenton reaction, is beneficial to the development of sustainable textile industry from material and processing perspectives.
... Therefore, major targets of bleaching damage in the cortex are the disulfide bonds of the amorphous phase proteins. Measurements including tensile strength, fatigue testing, vibrational spectroscopy, liquid retention, DSC, thermogravimetric analysis (TGA), scanning electron microscopy, and amino acid analysis, have been extensively used by researchers to survey chemical and structural damage to hair fibers as a function of alkaline bleaching treatments (2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19). In one of the earliest studies, Edman and Marti provided empirical evidence that hydrogen peroxide degrades disulfide bonds and diminishes the work required to stretch fibers (5). ...
Article
Full-text available
In this study, we sought to determine a practical correlation between disulfide bond oxidation and the thermal response of chemically bleached hair fibers. Bleaching processes, and the alkaline environment under which they are applied, cause scission of native covalent cystine cross-links in virgin hair fibers to form cysteine-sulfenic, cysteine-sulfinic, and cysteine-sulfonic (cysteic) acids in the cuticle, cortex, and, to a lesser extent, in the medulla. To further our understanding of hair bleaching kinetics, results from Fourier transform infrared (FTIR) chemical imaging, FTIR-attenuated total reflectance (FTIR-ATR), and Raman spectroscopic measurements were correlated with results from high pressure differential scanning calorimetry (HPDSC), dry differential scanning calorimetry (DSC), dynamic vapor sorption (DVS), and modulated thermogravimetric analysis (MTGA). Spectroscopic results were used to calculate an equivalent damage factor (EDF), which was used to index bleaching damage to the cuticular and cortical compartments of the hair fiber. Spectrofluorescence and colorimetry measurements were performed on bleached whole fiber hair tresses. Fluorescence measurements provided a means to monitor changes in the tryptophan and kynurenine levels, and colorimetry measurements were conducted to quantify the overall color change (ΔE) of hair at various bleaching intervals. FTIR imaging showed that cysteic acid levels in the fibers increased with increasing bleaching time and that the spatial distribution of cysteic acid builds from the outer cortex to the inner cortex, which further validates that bleaching is a diffusion-controlled process. FTIR- ATR studies with whole fiber hair tresses and 3-μm cross-sections showed that the cuticular cysteic acid concentration changes abruptly, whereas conversion of cortical cystine to cysteic acid is diffusion limited. Raman spectroscopy perfectly complemented FTIR-ATR and FTIR imaging, in which case Raman was used to directly follow changes in cystine (509 cm−1) as a function of bleaching time, whereas FTIR spectroscopy monitored increases in cysteic acid concentration (1040 cm−1). The cortical EDF values for Raman and FTIR spectroscopic techniques correlated linearly (R2=0.93–0.99), whereas the association between whole tress and cortical EDF results was poor (R2=0.61–0.73). For the series of bleached fibers, changes in the denaturation temperature (TD) from HPDSC analyses obeyed Fick’s laws of diffusion (R2 = 0.99), where the diffusion constant was estimated to be 1.1×10−8 cm2min−1. Using the peak in TD, the model-free Ozawa method was applied to approximate changes in the activation energy of intermediate filament denaturation as a function of increasing bleaching time. After 90 min of bleaching, the HPDSC activation energies plateaued at 180±8 kJ/mol against increasing cysteic acid concentration. Dry DSC results showed that conversion of cystine to cysteic acid increased the cortical mobility temperature, advocating that ionic and hydrogen-bonded networks stabilized components of the dry cortex during excessive heating. The MTGA pyrolysis onset temperatures ranged from 237°C to 248°C for virgin and 240 min bleached hair tresses, respectively, where the onsets positively and linearly correlated with increases in cysteic acid concentration (R2=0.95); however, the activation energy for pyrolysis of dry fibers showed a curvilinear correlation with Raman EDF, with a peak activation energy (554±9 kJ/mol) corresponding to 60–90 min bleaching times. To establish connections between water management properties and cystine oxidation, linear trends in denaturation temperature against the normalized Raman cystine band at 509 cm−1 demonstrated that decreased cross-link density is directly connected to greater steady-state moisture regains (R2=0.94). For hair tresses, low EDF correlated with high tryptophan levels; however, with increased bleaching, tryptophan and cystine levels rapidly decreased. As expected, longer bleaching times produced increased differences in color, as indexed by ΔE.
... For the damaged surfaces, the outer F-layer is partially removed, which occurs during a single bleach treatment. 11 More severe damage, such as that caused by repeated bleaching, 81 can lead to cuticle removal. This can change the microscale topography 82 and porosity 83 of the hair surface, which cannot be accounted for in the current CG-MD simulation framework. ...
Article
Full-text available
We present a coarse-grained molecular model of the surface of human hair, which consists of a supported lipid monolayer, in the MARTINI framework. Using coarse-grained molecular dynamics (MD) simulations, we...
... For the damaged surfaces, the outer F-layer is partially removed, which occurs during a single bleach treatment. 11 More severe damage, such as that caused by repeated bleaching, 71 can lead to cuticle removal. This can change the microscale topography 72 and porosity 73 of the hair surface, which cannot be accounted for in the current CG-MD simulation framework. ...
Preprint
Full-text available
We present a coarse-grained molecular model of the surface of human hair, which consists of a lipid monolayer, in the MARTINI framework. Using molecular dynamics simulations, we identify a lipid grafting distance that yields a monolayer thickness consistent with atomistic simulations and experimental measurements of hair surfaces. Coarse-grained models for fully-functionalised, partially damaged, and fully damaged hair surfaces are created by randomly replacing neutral thioesters with anionic sulfonate groups. This mimics the progressive removal of fatty acids from the hair surface by bleaching. We study the structure of the lipid monolayer at different degrees of damage using molecular dynamics simulations in vacuum as well as in polar (water) and non-polar (n-hexadecane) solvents. We also compare the wetting behaviour of water and n-hexadecane on the hair surfaces through contact angle measurements conducted using molecular dynamics simulations and experiments. Our model captures the experimentally-observed transition of the hair surface from hydrophobic (and oleophilic) to hydrophilic (and oleophobic) as the level of bleaching damage increases. By using surfaces with different damage ratios, we obtain contact angles from the simulations that are in good agreement with experiments for both solvents on virgin and bleached human hairs. In both the molecular dynamics simulations and further experiments using biomimetic surfaces, the cosine of the water contact angle increases linearly with the sulfonate group surface coverage. We expect that the proposed systems will be useful for future molecular dynamics simulations of the adsorption and tribological behaviour of hair surfaces.
... Oxidation of cysteine to cysteic acid by OsO 4 has a measured half time of 10 min in actin filaments. Oxidative modifications to keratins and KAPs not only affect structure in mature hair shafts, 54 but also cause widespread peptide backbone cleavage and affect the secondary structure of both the soluble and membrane proteins. 55 Moreover, additives such as potassium dichromate that mitigate OsO 4 's slow penetration rate and protein extraction, also yield lower contrast, resulting in osmium dioxide deposits during dehydration 52 and detrimentally affects sectioning. ...
Article
Transmission electron microscopy (TEM) has greatly advanced our knowledge of hair growth and follicle morphogenesis, but complex preparations such as fixation, dehydration and embedding compromise ultrastructure. While recent developments with cryofixation have been shown to preserve the ultrastructure of biological materials close to native state, they do have limitations. This review will focus on each stage of the TEM sample preparation process and their effects on the structural integrity of follicles.
... Disulphide bonds, which are responsible for maintaining the hair integrity, when broken, increase the hydrophilicity of the hair fibres, leading to higher rates of swelling. It also causes increased material diffusion [7,9]. Cationic compounds, the main components of conditioner formulations, can penetrate the cortex and protect the hair fibres from fatigue stress. ...
Article
Consumers are attracted to the latest fashion trends and different looks. This drives the search for novel hair treatments. Some chemicals present in hair treatment products can penetrate the hair shaft. These materials can either nourish or injure the hair cortex. Different techniques have been used to investigate the mechanism of molecule penetration and the conditions under which penetration occurs. This article reviews the techniques applied for this purpose. Various microscopy techniques are used to capture clear and colorful images to determine the diffusion pathways and the exact location of the molecules under study. However, the laborious sample preparation often leads to sample destruction since cross‐sectioning is often required. While various other techniques have been successfully used for investigating the penetration methods most of these require different amounts of work to be put in for sample preparation and instrumentation. Several spectroscopic techniques have been used to study the penetration of the molecules because of the high levels of accuracy and the quick response time of these techniques. Moreover, the samples are not damaged during the investigation.
... In addition to changing the pigmentary profile of hair, bleaching has a number of side effects that result in damage to its For example, it is well known that bleaching treatments oxidize disulphide residues resulting in the formation of cysteic acid and also damage other protein structures in hair [40]. In addition, the fibre becomes extremely porous and contains cracks, crevices and other asperities throughout its structure [41]. ...
Article
Full-text available
Objective: The tactile sensation of hair is an important consumer-perceivable attribute. There are limited instrumental options to measure the haptic properties of hair. In this study, we introduce a novel technique using the acoustic emissions produced when skin comes in contact with dry hair in a stroking motion. Methods: Using a free-field microphone with a frequency response of 8-12,500 Hz, we recorded acoustic emission data of the interaction of skin with hair. Data were captured with Electroacoustics Toolbox software and analysed with Matlab. Acoustic emission profiles were generated allowing us to monitor the acoustic response at distinct frequencies. Results: Various experiments were conducted to develop this novel technique as a suitable measure to monitor the surface properties of hair. Increasing the normal force and velocity of the interaction led to an increase in acoustic emissions. We also examined the acoustic profile of hair that underwent chemical treatment. For example, bleached hair produced a much higher magnitude acoustic response than the corresponding virgin hair. On the other hand, hair conditioner systems mitigated the acoustic response. Finally, investigations of textured hair revealed that the three-dimensional structure of the hair fibre assembly and its ability to return to its original state when perturbed produce the most dominant acoustic response for this type of hair. Conclusion: We introduce a cutting-edge method to reproducibly evaluate the surface properties of hair. Different types of hair geometry produce unique acoustic profiles as do hair types that experience harsh damaging treatments. This is also a very practical and efficient way to evaluate the degree of protection or conditioning of the fibre.
... Such damage is manifested in changes to the surface properties of hair, as monitored by surface techniques such as combing force and surface tension measurements, as well as damage to the internal components of the fi ber, which can be followed by tensile strength and differential scanning calorimetry measurements of hair (54,55). At the chemical level, one of the hallmarks of hair bleaching is the breakdown of disulfi de bonds, resulting in the formation of cysteic acid residues (56,57). In addition, the lipid structures in hair are also vulnerable to the bleaching process (58). ...
Article
Full-text available
The internal ultrafine structure of human hair was explored with atomic force microscopy (AFM). Cross sections of hair were prepared by a proprietary technique that provided a smooth surface for effective imaging in contact-mode AFM. Investigations of virgin hair revealed structural details of cortical and cuticle cells consistent with previous transmission electron microscopy (TEM) studies, in addition to the identification of a boundary region surrounding macrofibrils of the cortex. The effects of bleaching and solvent extraction on the internal structure of hair were also investigated. In the cuticle cell, bleaching causes the most damage to the endocuticle and cell membrane complex, evident by erosion of these components. Similarly, bleaching results in crevices, cracks, and asperities in the cortex of hair. In addition, the cortical cell membrane complex appears compromised along with either lipid or protein structures at the outer boundaries of macrofibrils. In delipidated hair, most structural components of the fiber appear intact with the exception of an overall swollen nature of the various morphological components.
... It is during this process that the greatest damage occurs, for example, hydrogen peroxide at high percentage concentrations can burn holes in the cuticle, damaging it beyond repair. 5 This damage caused by bleaching is well known and documented, [6][7][8][9] with many researchers concluding 2 the cause is a mixture of mechanical stresses and disruption to the chemical composition of the hair. ...
Thesis
In this thesis, the advanced spectroscopic techniques of atomic force microscopy - infrared (AFM-IR) spectroscopy and sum-frequency generation (SFG) spectroscopy are explored in detail for their innate ability to unravel the complexities of biological interfaces. The benefits of both techniques are demonstrated through applications to a variety of different interfaces, ranging from whole cells and aggregates (red blood cells and hair fibres) to artificial mimics (hydrogels) and thin molecular films (phospholipid monolayers). Specifically, the lateral chemical resolution and surface sensitivity of AFM-IR are used to chemically characterise the constituent components of biologically relevant systems below the diffraction limit whilst correlating these measurements to the nanoscale physical properties of the surface, such as topography, friction, and adhesion, that are accessible through traditional AFM. Similarly, the inherent surface specificity and sub-monolayer sensitivity of SFG is utilised, along with its ability to elucidate chemical structure and conformation, to probe oxidation in cell membranes, both in whole cells and monolayer mimics, thereby yielding a much deeper understanding of the oxidation mechanism due to reactive oxygen species (ROS) in physiological systems. Furthermore, some limitations and challenges associated with the use of these techniques in biologically relevant systems are identified, characterised and discussed, along with potential ways to circumvent them or minimise their impact in such investigations. Specifically, the inability of AFM-IR to probe thick substrates under aqueous conditions is discussed due to the relevance to studying many biological systems under physiological conditions. A novel method of sample preparation is proposed to maintain pseudo-aqueous conditions for the substrate whilst avoiding the limitations associated with AFM-IR measurement. Additionally, the common application of SFG to study surfactant monolayers at the air-water interface, particularly phospholipids due to them modelling cell membranes, is critically assessed for some inherent issues. The first example of which is associated with the inherent fluidity of the monolayer that makes it highly susceptible to local heating from the necessarily large incident fields. The effects of this local heating are thoroughly investigated and modelled, leading to conclusions about how to minimise this disruption in future investigations. Finally, SFG simulations are then used to quantitatively assess the validity of a common assumption associated with SFG investigations of phospholipids, namely that both alkyl chains yield the same spectral contributions despite having different orientational distributions.
Article
Objective Human hair is regularly subjected to chemical and physical insults, such as heat, UV-irradiation and alkaline hair care products. These insults result in molecular modifications at the hair protein level that underpin mechanical and sensory property changes in the fibers. These changes can manifest itself in reduced hair quality and performance attributes observable to the consumer. In this work, changes in protein modification as a result of heat and alkaline treatments are determined. Methods Redox proteomic profiling using high-resolution mass spectrometry was applied to map and evaluate amino acid residue modifications in human hair exposed to a combination of thermal treatments and alkali exposure with the aim to understand the underlying chemical processes. Results Our results show that an increase in redox-related modifications is associated with exposure to higher levels of hydrothermal and alkaline insult. Post-translational modification (PTM) profiling at the protein primary structural level delivered some further insights into the site-specificity of these modifications, with a clear increase in the number of cysteic acid modifications noticed in samples subjected to more extreme insults. Conclusion Pinpointing modification sides within proteins and the hair shaft proteome can be used as a basis for employing mitigation or repair strategies of hair protein damage caused by environmental or hair treatment-related insults.
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As a natural biomaterial with outstanding properties, keratin fibres hold promising potential in many application fields. The reduction of disulphides in keratin to the corresponding thiols plays a central role e.g. in modification and functionalisation of wool, in hair cosmetics, as well as in advanced applications using keratin as biomaterial. To achieve controlled conditions, the need for a quantitative determination of thiols on the solid wool fibre is evident. This work presents an advanced method of heterogeneous potentiometric back titration method for thiol detection in intact keratin fibres. First, keratin is reductively treated with NaBH4, then the thiol content in keratin is determined using K3[Fe(CN)6] and K2CrO4 as reagents and potentiometric endpoint detection. Thiol concentration in the range between 6.83 and 26.17 (±2.13) μmol/g can be detected with the heterogeneous back titration. Thus, the method is simple in execution and reliable in performance and of significant potential, both as scientific technique to quantify thiols in insoluble keratin material, as well as for process and quality control in industry.
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The structure and bleaching/dyeing behaviour of hair sampled from people of various ages were investigated to provide guidance for wig‐making. Hair surface and cross‐sectional morphology were investigated by scanning electron microscopy, whereas the number density and size of melanin granules were evaluated by transmission electron microscopy. The suitability of hair for dyeing was determined by the trade‐off between the fracture strength and lightness of bleached samples. Hair collected from 26‐year‐olds showed the highest number density and the lowest size of melanin granules, thus featuring the lowest mechanical strength reduction and the highest lightness increase after bleaching. The optimal bleaching conditions for such hair were identified as bleaching time = 80 minutes, hydrogen peroxide (H2O2) concentration = 60 mL/L, and bleaching temperature = 75°C. Considering its resilience to damage and favourable dyeing behaviour, hair sampled from 26‐year‐olds was concluded to be most suitable for wig‐making. Thus, the present research provides guidance for the selection of raw materials for wig‐making.
Article
The purpose of this study was to measure and visualize the protective effect of a patented combination of the active ingredients succinic acid, lysine and arginine on human hair fibers during oxidative bleaching treatment. Human hair was bleached using alkaline hydrogen peroxide and alkaline hydrogen peroxide persulfate treatment, respectively. The loss of elasticity of human hair fiber was examined using tensile strength measurements. The structure of cross-sections of human hair fiber resulting from the bleaching treatments was directly observed using transmission electron microscopy. Hair fiber damage was analyzed at the molecular level using Fourier transform near-infrared spectroscopy. In addition, the effect of the addition of a patented mixture containing succinic acid, lysine and arginine during the bleaching treatment was evaluated using these methods. The bleaching treatment resulted in the oxidation of cystine with the associated cysteic acid formation, with oxidation of cystine increasing as bleaching progressed. A decrease in tensile strength in the wet condition was proved. Bleaching led to substantial ultrastructural damage not only to the cuticle but also to the cortex. Addition of the patented mixture containing succinic acid, lysine and arginine during the bleaching treatment improved mechanical properties and inhibited oxidation of the cystine. However, the lightening effect was not impaired. The results suggested that the addition of a combination of succinic acid, lysine and arginine to a hair bleaching formulation is a potential technology to prevent the undesirable side effects of oxidative bleaching on human hair. Furthermore, these results indicate the involvement of these ingredients in a milder bleaching process.
Article
Objectives To evaluate improved protein extraction and two‐dimensional electrophoresis (2DE) separation methods with Japanese reference human hair (JRH). To determine if fibre curvature is related to protein composition in curly and straight Japanese women’s human hair (JHH) samples. To identify proteins from JRH 2DE maps and expression differences between curly and straight JHH. Methods Hair keratin and keratin‐associated proteins (KAPs) were extracted intact with dithiothreitol or tris(2‐carboxyethyl) phosphine from JRH or from curved or straight JHH. Extracted proteins were isoelectric‐focused on first‐dimension pH gradient gel strips then separated by molecular weight on laboratory‐made, second‐dimension, large format gels. The software compared protein abundance between duplicate 2DE gels of curved and straight JHH. Thirty‐eight proteins from a JRH 2DE gel were enzyme cleaved for MALDI‐TOF‐MS analysis to determine peptide composition, and where possible, de novo sequencing gave peptide sequence data. An in‐house human hair protein database incorporating ninety‐eight annotated protein sequences assisted MS analysis. Results 2DE gels of tris(2‐carboxyethyl) phosphine extracted JRH improved keratin and KAP resolution and number compared to those of dithiothreitol extracted JRH and published commercially‐made second‐dimension gels. Silver‐stained 2DE gels of the straight or curved JHH sets were remarkably similar. Over‐staining to reveal basic proteins caused poor resolution of the major acidic protein classes. Software comparisons of fifty‐nine resolved proteins revealed two were significantly different in abundance between curved and straight hairs but in insufficient amounts for MS analysis. MS identified twelve proteins from a JRH CBBG‐stained 2DE gel: six Type II keratins, three Type I keratins, and three high sulphur proteins. A further eight were potential conformational isoforms and isoelectric variants of the identified proteins bringing the total to twenty identified or partially identified proteins. Conclusion Root‐end human hair extraction with tris(2‐carboxyethyl) phosphine improves protein resolution and visualises more proteins on large format 2DE gels. The two minor protein differences between duplicate straight or curved JHH 2DE gels were unlikely to change fibre structure from straight to curved hair. MS results confirmed that multiple isoforms exist of various hair proteins. Low sequence coverage prevented distinction between members in rows of homologous protein spots of similar molecular weight.
Article
Hair analysis has become a valuable tool in forensic toxicology to assess drug or alcohol abstinence. Yet, hair adulteration by cosmetic products presents a major challenge for forensic hair analysis. Oxidative treatments, e.g. bleaching, may lead to analyte loss and thereby to false negative results. Currently, the eumelanin degradation product 1H‐pyrrole‐2,3,5‐tricarboxylic acid (PTCA) serves as a marker for oxidative hair treatment, but requires the definition of cut‐off values. To investigate further eumelanin degradation products as markers for oxidative hair treatment, hair samples with and without in vitro bleaching (hydrogen peroxide (H2O2) concentrations 1.9 % up to 12 %; incubation times 15 min, 30 min, 60 min) were analyzed by liquid chromatography coupled to high‐resolution time of flight mass spectrometry (HPLC‐HRMS). The distribution of eumelanin degradation products along the hair shaft was investigated for routine applicability after segmentation of cosmetically untreated hair samples and authentically treated hair samples. The signals of the eumelanin degradation products PTCA, 1H‐pyrrole‐2,3,4‐tricarboxylic acid (isoPTCA) and 1H‐pyrrole‐2,3,4,5‐tetracarboxylic acid (PTeCA) were found to be significantly elevated after in vitro bleaching already with low H2O2 concentrations and after short incubation times. In contrast to PTCA and isoPTCA, PTeCA was not detectable in cosmetically untreated segments up to 12 cm from hair root and was only formed through the oxidation process. The results of the study show that the detection of PTeCA within the proximal 3 to 6 cm segment can be applied to reliably detect hair adulteration attempts through hair bleaching.
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Significance Mechanical properties of tissues often emerge from fibrous protein networks spanning multiple cell lengths. For the first time, to our knowledge, atomic force microscopy was used to measure the mechanical properties of the human hair follicle. We find a considerable stiffening along the first millimeter that we link to changes in the keratin network architecture and composition. In early keratinization stages, the thickening, densification, and increasing orientation of fibers are responsible for the mechanical stiffening, whereas in later stages, intermolecular cross-linking becomes predominant. Our results corroborate the known biological and structural events during hair keratinization and underline the link between the mechanical properties of the hair follicle and its multiscale tridimensional organization.
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Keratin 15 (K15) is type I keratin protein co-expressed with the K5/K14 pair present in the basal keratinocytes of all stratified epithelia. Although it is a minor component of the cytoskeleton with a variable expression pattern, nonetheless its expression has been reported as a stem cell marker in the bulge of hair follicles. Conversely, suprabasal expression of K15 has also been reported in both normal and diseased tissues, which is inconsistent with its role as a stem cell marker. Our recently published work has given evidence of the molecular pathways that seem to control the expression of K15 in undifferentiated and differentiated cells. In this article, we have critically reviewed the published work to establish the reliability of K15 as an epidermal stem cell marker.
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Dynamic phosphorylation is one mechanism that regulates the more than 20 keratin type I and II intermediate filament proteins in epithelial cells. The major type II keratin in “simple type” glandular epithelia is keratin 8 (K8). We used biochemical and mutational approaches to localize two major in vivo phosphorylation sites of human K8 to the head (Ser-23) and tail (Ser-431) domains. Since Ser-23 of K8 is highly conserved among all type II keratins, we also examined if the corresponding Ser-59 in stratified epithelial keratin 6e is phosphorylated. Mutation of K6e Ser-59 abolished its phosphorylation in 32PO4-labeled baby hamster kidney cell transfectants. With regard to K8 phosphorylation at Ser-431, it increases dramatically upon stimulation of cells with epidermal growth factor (EGF) or after mitotic arrest and is the major K8 phosphorylated residue after incubating K8 immunoprecipitates with mitogen-activated protein or cdc2 kinases. A monoclonal antibody that specifically recognizes phosphoserine 431-K8 manifests increased reactivity with K8 and recognizes reorganized K8/18 filaments after EGF stimulation. Our results suggest that in vivo serine phosphorylation of K8 and K6e within the conserved head domain motif is likely to reflect a conserved phosphorylation site of most if not all type II keratins. Furthermore, K8 Ser-431 phosphorylation occurs after EGF stimulation and during mitotic arrest and is likely to be mediated by mitogen-activated protein and cdc2 kinases, respectively.
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Monilethrix is a congenital hair shaft disorder with associated fragility. Many of the changes seen in monilethrix hair on light microscopy and scanning electron microscopy are also seen in hair weathering and cosmetic damage to hair. We used monilethrix as a model to investigate the relationship between hair protein structure and hair strength and resistance to cosmetic insult. We applied proteomic techniques to identify novel peptide damage markers for chemical oxidative damage to hair. The findings suggest that specific sites in the protein structure of hair are targeted during oxidative damage from bleaching, a unique insight into how chemical damage compromises the structural integrity of the hair shaft at the molecular level. Applying proteomics to the study of congenital and acquired hair shaft disorders can deliver new insights into hair damage and novel strategies to strengthen hair.
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Sunlight on human hair causes photo-degradation. This results in bleaching due to melanin oxidation through free radicals, and induces keratin impairment. Protein degradation, tryptophan degradation, lipidic peroxidation and electron paramagnetic resonance can be used to evaluate proteic and lipidic photodecomposition and free radical formation in hair fibres subjected to antioxidant action and different UV intensities. All these methodologies have been optimised to determine protein, lipid and melanin degradation in hair subjected to different UV intensities.
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Protein and peptide oxidation is a key feature in the progression of a variety of disease states and in the poor performance of protein-based products. The present work demonstrates a mass spectrometry-based approach to profiling degradation at the amino acid residue level. Synthetic peptides containing the photosensitive residues, tryptophan and tyrosine, were used as models for protein-bound residue photodegradation. Electrospray ionisation tandem mass spectrometry (ESI-MS/MS) was utilised to characterise and provide relative quantitative information on the formation of photoproducts localised to specific residues, including the characterisation of low abundance photomodifications not previously reported, including W + 4O modification, hydroxy-bis-tryptophandione and topaquinone. Other photoproducts observed were consistent with the formation of tyrosine-derived dihydroxyphenylalanine (dopa), trihydroxyphenylalanine, dopa-quinone and nitrotyrosine, and tryptophan-derived hydroxytryptophan, dihydroxytryptophan/N-formylkynurenine, kynurenine, hydroxyformylkynurenine, tryptophandiones, tetrahydro-beta-carboline and nitrotryptophan. This approach combined product identification and abundance tracking to generate a photodegradation profile of the model system. The profile of products formed yields information on formative mechanisms. Profiling of product formation offers new routes to identify damage markers for use in tracking and controlling oxidative damage to polypeptides.
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We have investigated the expression of 52 of the 54 keratins in beard hair medulla. We found that not only 12 hair keratins but, unexpectedly, also 12 epithelial keratins are potentially expressed in medulla cells. The latter comprise keratins also present in outer- and inner-root sheaths and in the companion layer. Keratins K5, K14, K17, K25, K27, K28, and K75 define a "pre-medulla," composed of cells apposed to the upper dermal papilla. Besides K6, K16, K7, K19, and K80, all pre-medullary epithelial keratins continue to be expressed in the medulla proper, along with the 12 hair keratins. Besides this unique feature of cellular keratin co-expression, the keratin pattern itself is highly variable in individual medulla cells. Remarkably, both epithelial and hair keratins behave highly promiscuously with regard to heterodimer- and IF formation, which also includes keratin chain interactions in IF bundles. We also identified cortex cells within the medullary column. These exhibit all the properties of genuine cortex cells, including a particular type of keratin heterogeneity of their compact IF bundles. In both keratin expression profile and keratin number, medulla cells are distinct from all other cells of the hair follicle or from any other epithelium.
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Keratin 6 (K6) expression in the epidermis has two components: constitutive expression in the innermost layer of the outer root sheath (ORS) of hair follicles and inducible expression in the interfollicular epidermis in response to stressful stimuli such as wounding. Mice express two K6 isoforms, MK6a and MK6b. To gain insight into the functional significance of these isoforms, we generated MK6a-deficient mice through mouse embryonic stem cell technology. Upon wounding, MK6a was induced in the outer ORS and the interfollicular epidermis including the basal cell layer of MK6a+/+ mice, whereas MK6b induction in MK6a−/− mice was restricted to the suprabasal layers of the epidermis. After superficial wounding of the epidermis by tape stripping, MK6a−/− mice showed a delay in reepithelialization from the hair follicle. However, the healing of full-thickness skin wounds was not impaired in MK6a−/−animals. Migration and proliferation of MK6a−/−keratinocytes were not impaired in vitro. Furthermore, the migrating and the proliferating keratinocytes of full-thickness wounds in MK6a−/− animals expressed neither MK6a nor MK6b. These data indicate that MK6a does not play a major role in keratinocyte proliferation or migration but point to a role in the activation of follicular keratinocytes after wounding. This study represents the first report of a keratin null mutation that results in a wound healing defect.
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The human type II hair keratin subfamily consists of six individual members and can be divided into two groups. The group A members hHb1, hHb3, and hHb6 are structurally related, whereas group C members hHb2, hHb4, and hHb5 are rather distinct. Specific antisera against the individual hair keratins were used to establish the two-dimensional catalog of human type II hair keratins. In this catalog, hHb5 showed up as a series of isoelectric variants, well separated from a lower, more acidic, and complex protein streak containing isoelectric variants of hair keratins hHb1, hHb2, hHb3, and hHb6. Both in situ hybridization and immunohistochemistry on anagen hair follicles showed that hHb5 and hHb2 defined early stages of hair differentiation in the matrix (hHb5) and cuticle (hHb5 and hHb2), respectively. Although cuticular differentiation proceeded without the expression of further type II hair keratins, cortex cells simultaneously expressed hHb1, hHb3, and hHb6 at an advanced stage of differentiation. In contrast, hHb4, which is undetectable in hair follicle extracts and sections, could be identified as the largest and most alkaline member of this subfamily in cytoskeletal extracts of dorsal tongue. This hair keratin was localized in the posterior compartment of the tongue filiform papillae. Comparative analysis of type II with the previously published type I hair keratin expression profiles suggested specific, but more likely, random keratin-pairing principles during trichocyte differentiation. Finally, by combining the previously published type I hair keratin catalog with the type II hair keratin catalog and integrating both into the existing catalog of human epithelial keratins, we present a two-dimensional compilation of the presently known human keratins.
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The murine genome is known to have two keratin 6 (K6) genes, mouse K6 (MK6)a and MK6b. These genes display a complex expression pattern with constitutive expression in the epithelia of oral mucosa, hair follicles, and nail beds. We generated mice deficient for both genes through embryonic stem cell technology. The majority of MK6a/b-/- mice die of starvation within the first two weeks of life. This is due to a localized disintegration of the dorsal tongue epithelium, which results in the build up of a plaque of cell debris that severely impairs feeding. However, approximately 25% of MK6a/b-/- mice survive to adulthood. Remarkably, the surviving MK6a/b-/- mice have normal hair and nails. To our surprise, we discovered MK6 staining both in the hair follicle and the nail bed of MK6a/b-/- mice, indicating the presence of a third MK6 gene. We cloned this previously unknown murine keratin gene and found it to be highly homologous to human K6hf, which is expressed in hair follicles. We therefore termed this gene MK6 hair follicle (MK6hf). The presence of MK6hf in the MK6a/b-/- follicles and nails offers an explanation for the absence of hair and nail defects in MK6a/b-/- animals.
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Epiplakin, a giant epithelial protein of >700 kDa, belongs to the plakin family of cytolinker proteins. It represents an atypical family member, however, as it consists entirely of plakin repeat domains but lacks any of the other domains commonly shared by plakins. Hence, its putative function as a cytolinker protein remains to be shown. To investigate epiplakin's biological role, we generated epiplakin-deficient mice by gene targeting in embryonic stem cells. Epiplakin-deficient mice were viable and fertile, without developing any discernible phenotype. Ultrastructurally, their epidermis revealed no differences compared to wild-type littermates, and cornified envelopes isolated from skin showed no alterations in shape or stability. Furthermore, neither embryonal formation nor later function of the epithelial barrier was affected. In primary cultures of epiplakin-deficient keratinocytes, the organization of actin filaments, microtubules, and keratin networks was found to be normal. Similarly, no alterations in keratin network organization were observed in simple epithelia of small intestine and liver or in primary hepatocytes. We conclude that, despite epiplakin's abundant and highly specific expression in stratified and simple epithelia, its absence in mice does not lead to severe skin dysfunctions, nor has it detectable consequences for keratin filament organization and cytoarchitecture of cells.
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Protein oxidative degradation is implicated in a wide range of deleterious effects. For human hair, this oxidative damage can lead to significant observable changes in fibre physical and visual properties. A redox proteomic approach was applied to map molecular modification in human hair proteins and correlate this modification with the abundance of copper (II) ions, the levels of UV exposure and the general level of hair pigmentation. An increase in oxidative modification was observed with increasing copper (II) ion levels, regardless of the pigmentation level. Significantly, increased protein oxidative modification was also observed to occur in both lightly and darkly pigmented hair tresses even in the absence of irradiation, albeit at lower relative levels. Modification levels increased with increased copper (II) ion concentration. This new finding indicates that the level of copper (II) ions in human hair plays a key role in mediating protein oxidation, with or without exposure to UV light. Overall, these results strongly suggest that minimization of the level of copper (II) ions in human hair will mitigate and/or slow protein oxidative modification and therefore lower overall hair damage. This article is protected by copyright. All rights reserved.
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This paper describes a convenient method for mounting fibres for processing and embedding. Prior to processing, the fibres are mounted across windows in acetate frames. These frames protect the fibres from impact damage during processing, keep the fibres straight, and make alignment of the fibres for sectioning simple and accurate. The frames are produced using purpose-built devices which are also described in this paper.
Chapter
Human hair consists of proteins, lipids, water, trace elements and pigments. The composition of the first four of these components is the focus of this Chapter. About two decades ago the emphasis on the proteins of hair was on its amino acid constituents which provided important information on the relative amounts of different functional groups in different types of hair and in different regions of the fiber. However, as a result of advances in the characterization and classification of the different proteins and genes of keratins and keratin associated proteins the focus today is on the proteins themselves. Several important new contributions to the composition of the surface layers of hair and the proteins of the cell membrane complex have been and are continuing and therefore are summarized in this Chapter. The current state of changes in the amino acids, proteins and lipids of hair by morphological region (including KAP and keratin proteins and where they reside), chemical and sunlight damage, diet, puberty and menopause, and other factors have been and are being made and are summarized here. An expanded section on metals in hair, where in the fiber these metals reside and the functional groups that they bind to and their effects on hair chemistry, toxicity and disorders are included.
Article
The major structural proteins of epithelia, the keratins, and the keratin filament-associated protein, filaggrin, were analyzed in more than 50 samples of human embryonic and fetal skin by one-dimensional SDS PAGE and immunoblotting with monoclonal and polyclonal antibodies. Companion samples were examined by immunohistochemistry and electron microscopy. Based on structural characteristics of the epidermis, four periods of human epidermal development were identified. The first is the embryonic period (before 9 wk estimated gestational age), and the others are within the fetal period: stratification (9-14 wk), follicular keratinization (14-24 wk), and interfollicular keratinization (beginning at approximately 24 wk). Keratin proteins of both the acidic (AE1-reactive, type I) and the basic (AE3-reactive, type II) subfamilies were present throughout development. Keratin intermediate filaments were recognized in the tissue by electron microscopy and immunohistochemical staining. Keratins of 50 and 58 kD were present in the epidermis at all ages studied (8 wk to birth), and those of 56.5 and 67 kD were expressed at the time of stratification and increased in abundance as development proceeded. 40- and 52-kD keratins were present early in development but disappeared with keratinization. Immunohistochemical staining suggested the presence of keratins of 50 and 58 kD in basal cells, 56.5 and 67 kD in intermediate cells, and 40 and 52 kD in the periderm as well as in the basal cells between the time of stratification and birth. Filaggrin was first detected biochemically at approximately 15 wk and was localized immunohistochemically in the keratinizing cells that surround hair follicles. It was identified 8-10 wk later in the granular and cornified cell layers of keratinized interfollicular epidermis. These results demonstrate the following. An intimate relationship exists between expression of structural proteins and morphologic changes during development of the epidermis. The order of expression of individual keratins is consistent with the known expression of keratins in simple vs. stratified vs. keratinized epithelia. Expression of keratins typical of stratified epithelia (50 and 58 kD) precedes stratification, and expression of keratins typical of keratinization (56.5 and 67 kD) precedes keratinization, which suggests that their expression marks the tissue commitment to those processes. Because only keratins that have been demonstrated in various adult tissues are expressed during fetal development, we conclude that there are no "fetal" keratins per se.(ABSTRACT TRUNCATED AT 400 WORDS)
Article
Damage to hair from UV exposure has been well reported in the literature and is known to be a highly complex process involving initiation via absorption of UV light followed by formation and propagation of reactive oxygen species (ROS). The objective of this work was to understand these mechanisms, explain the role of copper in accelerating the formation of ROS and identify strategies to reduce the hair damage caused by these reactive species. The location of copper in hair was measured by Transmission electron microscopy (TEM) x-ray energy dispersive spectroscopy (XEDS) and levels measured by ICP-OES. Protein changes were measured as total protein loss via the Lowry assay and MALDI ToF was used to identify the biomarker protein fragments. TBARS assay was used to measure lipid peroxide formation. Sensory methods and dry combing friction were used to measure hair damage due to copper and UV exposure and to demonstrate the efficacy of EDDS and histidine chelants to reduce this damage RESULTS: In this work a biomarker protein fragment formed during UV exposure is identified using mass spectrometry. This fragment originates from the calcium binding protein S100A3. Also shown is the accelerated formation of this peptide fragment in hair containing low levels of copper absorbed from hair during washing with tap water containing copper ions. Transmission electron microscopy (TEM) x-ray energy dispersive spectroscopy (XEDS) studies indicate copper is located in the sulfur-poor endo-cuticle region; a region where the S100A3 protein is concentrated. A mechanism for formation of this peptide fragment is proposed in addition to the possible role of lipids in UV oxidation. A shampoo and conditioner containing chelants (EDDS in shampoo and histidine in conditioner) is shown to reduce copper uptake from tap water, reduce protein loss and formation of S100A3 protein fragment. In addition, the long term consequences of UV oxidation and additional damage induced by copper are illustrated in a four month wear study where hair was treated with a consumer relevant protocol of hair coloring treatments, UV exposure and regular shampoo and conditioning. The role of copper in accelerating UV damage to hair has been demonstrated as well as the ability of chelants such as EDDS and histidine in shampoo and conditioner products to reduce this damage. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.
Article
In this study, we investigated the effects of oxidative permanent hair coloring agents on the dyeability and the damage of human hair. p-phenylenediamine and toluene-2.5-diamine sulfate were used as a hair coloring agent precursers. The degree of dyeability was checked by the change of CIELAB value according to dyeing time. And the damage of hair was evaluated by the tensile strength and morphological change of hair in SEM. When the hair was dyed, the CIELAB value was decreased with dyeing time regardless of the type of precursers. But when the hair was dyed after nutritional treatment, the CIELAB value showed lower level. This means that the nutritional treatment covers the scale of hair and protects the hair from the chemicals.
Article
Yellowing is an undesirable phenomenon that is common in people with white and grey hair. Because white hair has no melanin, the pigment responsible for hair colour, the effects of photodegradation are more visible in this type of hair. The origin of yellowing and its relation to photodegradation processes are not properly established, and many questions remain open in this field. In this work, the photodegradation of grey hair was investigated as a function of the wavelength of incident radiation, and its ultrastructure was determined, always comparing the results obtained for the white and black fibres present in grey hair with the results of white wool.
Article
There is increasing awareness of the importance of reductive and oxidative (redox) protein damage in protein-based materials including, hair, wool, nails, and skin. Light-induced damage to protein-based materials is of particular concern because of its impact on age-related degradation and product life spans. Consequently, cosmetic applications frequently target hair and skin restoration, where the integrity of the constituent filamentous proteins is essential to a healthy appearance. The keratins constitute an important subset of the structural proteins within skin, hair, and wool. We will introduce a means to assess damage to this important group of proteins at the molecular level, utilizing proteomic techniques to track the formation or degradation of sensitive peptides within intermediate filament proteins. The degradation of three molecular markers of redox damage, the peptides SFGYR, LASDDFR, and DVEEWYIR, along with the formation of their oxidized products, is demonstrated after exposure to ultraviolet A, ultraviolet B, and blue light. The method is shown to be suitable for evaluating the protective effect of treatments, as lower levels of oxidative markers were observed after the application of a protective fiber treatment. Molecular-level redox tracking will allow more targeted design and evaluation of protection and repair treatments for protein systems.
Article
Human scalp hairs are comprised of a central cortex enveloped by plate-like cuticle cells. The elongate cortex cells of mature fibres are composed primarily of macrofibrils - bundles of hard-keratin intermediate filaments (IFs) chemically cross-linked within a globular protein matrix. In wool, three cell types (ortho-, meso- and paracortex) contain macrofibrils with distinctly different filament arrangements and matrix fractions, but in human hair macrofibril-cell type relationships are less clear. Here we show that hair macrofibrils all have a similar matrix fraction (∼0.4) and are typically composed of a double-twist architecture in which a central IF is surrounded by concentric rings of tangentially-angled IFs. The defining parameter is the incremental angle increase (IF-increment) between IFs of successive rings. Unlike the wool orthocortex, hair double-twist macrofibrils have considerable inter-macrofibril variation in IF increment (0.05-0.35 °/nm), and macrofibril size and IF increment are negatively correlated. Correspondingly, angular difference between central and outer-most IFs is up to 40° in small macrofibrils, but only 5-10° in large macrofibrils. Single cells were observed containing mixtures of macrofibrils with different diameters. These new observations advance our understanding of the nano-level and cell-level organization of human hair, with implications for interpretation of structure with respect the potential roles of cortex cell types in defining the mechanical properties of hair.
Article
Multiple type I and II hair keratins are expressed in hair-forming cells but the role of each protein in hair fiber formation remains obscure. In this study, recombinant proteins of human type I hair keratins (K35, K36 and K38) and type II hair keratins (K81 and K85) were prepared using bacterial expression systems. The heterotypic subunit interactions between the type I and II hair keratins were characterized using two-dimensional gel electrophoresis and surface plasmon resonance (SPR). Gel electrophoresis showed that the heterotypic complex-forming urea concentrations differ depending on the combination of keratins. K35-K85 and K36-K81 formed relatively stable heterotypic complexes. SPR revealed that soluble K35 bound to immobilized K85 with a higher affinity than to immobilized K81. The in vitro intermediate filament (IF) assembly of the hair keratins was explored by negative-staining electron microscopy. While K35-K81, K36-K81 and K35-K36-K81 formed IFs, K35-K85 afforded tight bundles of short IFs and large paracrystalline assemblies, and K36-K85 formed IF tangles. K85 promotes lateral association rather than elongation of short IFs. The in vitro assembly properties of hair keratins depended on the combination of type I and II hair keratins. Our data suggest the functional significance of K35-K85 and K36-K81 with distinct assembly properties in the formation of macrofibrils.
Article
Objective: The objective of this work was to identify whether low levels of redox metals such as copper will accelerate damage to hair on exposure to UV irradiation and whether this damage can be prevented. Methods: The methods used were proteomics to measure the protein damage via protein loss after different periods of exposure and mass spectroscopy methods to identify specific marker peptides that are specifically created by this type of damage. Results: In this work, we have developed new insights into the mechanism of UV damage using these proteomic methods. A marker fragment in the hair protein loss extract was identified (m/z = 1279) that is unique to UV exposure and increases with time of UV exposure. We have also identified for the first time in hair the role of exogenous copper in increasing UV damage both in terms of total protein degradation and also increased formation of the marker fragment and proposed a mechanism of action. It has been demonstrated that shampoo treatment containing a chelant such as N,N'-ethylenediamine disuccinic acid (EDDS) reduced copper accumulation in hair. Conclusion: This work provides evidence for the role of copper in UV-induced damage to hair and strategies to reduce copper levels in hair using a chelant such as EDDS.
Article
Protein modification and damage in human hair, resulting from environmental, cosmetic and grooming stresses, creates changes to visual and tactile characteristics and correlates to consumer perception of quality. This study outlines molecular-level evaluation of modification resulting from peroxide (bleaching) and alkaline straightening (relaxing) treatments. Redox proteomic profiling of virgin, bleached and relaxed hair tresses was performed, with comprehensive qualitative characterisation of modification and semi-quantitative evaluation of damage through adaptation of a new damage scoring system Modifications were mapped to specific locations in the hair proteome and a range of potential damage marker peptides identified. Virgin hair contained a baseline level of modification, consistent with environmental oxidative insult during hair growth. Hydrogen peroxide bleaching resulted in significantly increased levels of oxidative damage observable at the molecular level. This treatment also resulted in enhanced levels of dehydroalanine and dehydration products; modifications typically associated with alkali or thermal treatment and not previously been reported as a product of hair bleaching. Relaxation treatment with sodium hydroxide increased formation of dehydroalanine and dehydration products, and moderately enhanced levels of oxidation. Cysteine was the predominant modification site for both bleaching and alkali damage. This study validates the utility and power of redox proteomic-based approaches to characterising hair modification. This offers potential application to a wide range of damage types; as well as evaluation of new damage mitigation and repair technologies. This article is protected by copyright. All rights reserved.
Article
Since their first finding in wool 50 years ago, keratin-associated proteins (KAPs), which are classified into 3 groups; high sulfur (HS) KAPs, ultra high sulfur (UHS) KAPs, and high glycine-tyrosine (HGT) KAPs, have been the target of curiosity for scientists due to their characteristic amino acid sequences. While HS and UHS KAPs are known to function in disulfide bond crosslinking, the function of HGT KAPs remains unknown. To clarify the function as well as the binding partners of HGT KAPs, we prepared KAP8.1 and other KAP family proteins, the trichocyte intermediate filament proteins (IFP) K85 and K35, the head domain of K85, and the C subdomain of desmoplakin C-terminus (DPCT-C) and investigated the interactions between them in vitro. Western blot analysis and isothermal titration calorimetry (ITC) indicate that KAP8.1 binds to the head domain of K85, which is helically aligned around the axis of the intermediate filament (IF). From these results and transmission electron microscopy (TEM) observations of bundled filament complex in vitro, we propose that the helical arrangement of IFs found in the orthocortex, which is uniquely distributed on the convex fiber side of the hair, is regulated by KAP8.1. Structure-dependent binding of DPCT-C to trichocyte IFP was confirmed by Western blotting, ITC, and circular dichroism. Moreover, DPCT-C also binds to some HGT KAPs. It is probable that such bidirectional binding property of HGT KAPs contribute to the mechanical robustness of hair.
Article
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.
Article
Synopsis---The CUTICLE of human HAIR has been isolated in bulk by a new method involving vigorous agitation of fibres in water. The cuticle fractions have been shown to be of high morpho- logical purity using various techniques of ELECTRON MICROSCOPY. The significance of AMINO ACID ANALYSES is discussed.
Article
The medulla is the innermost structure of the mammalian hair fibre. Unlike most mammals, in man only androgen-dependent sexual hairs, i.e. beard, pubic and axillary hairs, are regularly medullated, while the frequency of medullation in scalp hairs varies among individuals and ethnicities(1,2) . Recently, we reported that out of the 54 human keratins,12 epithelial keratins [comprising members of the outer and inner root sheath (ORS, IRS), the companion layer (cl), and the almost ubiquitously expressed keratin K80(3) , as well as 12 hair cortex keratins] are present in the medulla of beard hairs(4) .
Article
The human type I hair keratin subfamily comprises nine individual members, which can be subdivided into three groups. Group A (hHa1, hHa3-I, hHa3-II, hHa4) and B (hHa7, hHa8) each contains structurally related hair keratins, whereas group C members hHa2, hHa5, and hHa6 represent structurally rather unrelated hair keratins. Antibodies produced against these individual hair keratins, first analyzed for specificity by one- dimensional Western blots of total hair keratins, were used to establish the two-dimensional catalog of the human type I hair keratin subfamily. The catalog comprises two different series of type I hair keratins: a strongly expressed, Coomassie-stainable series containing hair keratins hHa1, hHa3-I/II, hHa4, and hHa5, and a weakly expressed, immunodetectable series harboring hHa2, hHa6 hHa7, and hHa8. In situ hybridization and immunohistochemical expression studies on scalp follicles show that two hair keratins, hHa2 and hHa5, define the early stage of hair differentiation, i.e. hHa5 expression in hair matrix and hHa5/hHa2 coexpression in the early hair cuticle cells. Whereas cuticular differentiation proceeds without the expression of further type I hair keratins, matrix cells embark on the cortical pathway by sequentially expressing hHa1, hHa3-I/II, and hHa4, which are supplemented by hHa6 at an advanced stage of cortical differentiation, and hHa8, which is expressed heterogeneously in cortex cells. Thus, six type I hair keratins are involved in the terminal differentiation of anagen hairs. The expression of hHa7 is conspicuously different from that of the other hair keratins in that it does not occur in the large anagen follicles of terminal scalp hairs but only in central cortex cells of the rare and small follicle type that gives rise to vellus hairs.
Article
Human hair follicle keratin-associated proteins (KAPs) comprise a large multigene family of proteins thought to be responsible for the bundling of keratin intermediate filaments. Recently, four new KAP family members KAP24.1, KAP25.1, KAP26.1 and KAP27.1 were identified from the genome, but the expression of only one, KAP24.1, was investigated and shown in hair follicles. In the current study, the expression of the remaining members of the family were analysed. Reverse transcriptase-polymerase chain reaction analysis of samples from numerous human organs was used. Only KAP26.1 showed expression, which was limited to the hair follicle. By in situ hybridization and immunohistochemistry using a specific antiserum, KAP26.1 was localized to the differentiated portion of the hair cuticle. As well as KAP24.1 in hair follicles, expression of KAP26.1 was shown and is found in the differentiated part of the hair cuticle.
Article
The human keratin family comprises 54 members, 28 type I and 26 type II. Out of the 28 type I keratins, 17 are epithelial and 11 are hair keratins. Similarly, the 26 type II members comprise 20 epithelial and 6 hair keratins. As, however, 9 out of the 37 epithelial keratins are specifically expressed in the hair follicle, the total number of hair follicle-specific keratins (26) almost equals that of those expressed in the various forms of epithelia (28). Up to now, more than half of the latter have been found to be involved in inherited diseases, with mutated type I and type II members being roughly equally causal. In contrast, out of the 26 hair follicle-specific keratins only 5 have, at present, been associated with inherited hair disorders, while one keratin merely acts as a risk factor. In addition, all hair follicle-specific keratins involved in pathologies are type II keratins. Here we provide a detailed description of the respective hair diseases which are either due to mutations in hair keratins (monilethrix, ectodermal dysplasia of hair and nail type) or hair follicle-specific epithelial keratins (two mouse models, RCO3 and Ca(Rin) as well as pseudofolliculitis barbae).
Article
Keratin IF (KRT) and keratin-associated protein genes encode the majority of wool and hair proteins. We have identified cDNA sequences representing nine novel sheep KRT genes, increasing the known active genes from eight to 17, a number comparable to that in the human. However, the absence of KRT37 in the type I family and the discovery of type II KRT87 in sheep exemplify species-specific compositional differences in hair KRT genes. Phylogenetic analysis of hair KRT genes within type I and type II families in the sheep, cattle and human genomes revealed a high degree of consistency in their sequence conservation and grouping. However, there were differences in the fibre compartmentalisation and keratinisation zones for the expression of six ovine KRT genes compared with their human orthologs. Transcripts of three genes (KRT40, KRT82 and KRT84) were only present in the fibre cuticle. KRT32, KRT35 and KRT85 were expressed in both the cuticle and the fibre cortex. The remaining 11 genes (KRT31, KRT33A, KRT33B, KRT34, KRT36, KRT38-39, KRT81, KRT83 and KRT86-87) were expressed only in the cortex. Species-specific differences in the expressed keratin gene sets, their relative expression levels and compartmentalisation are discussed in the context of their underlying roles in wool and hair developmental programmes and the distinctive characteristics of the fibres produced.
Article
Protein oxidative damage lies behind skin and hair degradation and the deterioration of protein-based products, such as wool and meat, in addition to a range of serious health problems. Effective strategies to ameliorate degenerative processes require detailed fundamental knowledge of the chemistry at the molecular level, including specific residue-level products and their relative abundance. This paper presents a new means of tracking damage-induced side-chain modification in peptides using a novel application for isobaric label quantification. Following exposure to heat and UVA and UVB irradiation, tryptophan and tyrosine damage products in synthetic peptides were characterized and tracked using ESI-MS/MS and iTRAQ labeling-based relative quantification. An in-depth degradation profile of these peptides was generated, enabling the formation of even low-abundance single-residue-level modifications to be sensitively monitored. The development of this novel approach to profiling and tracking residue-level protein damage offers significant potential for application in the development and validation of protein protection treatments.
Article
The bulge stem cell region is a structure important for the regeneration of the pilosebaceous unit. Measurements of the different compartments of a hair follicle may have implications in hair transplantation and hair regeneration studies. To measure the length of the different portions of the occipital scalp hair and to estimate at what depth they are located. Hair follicles from the occipital scalp were obtained from 29 individuals. Measurements were performed on digital pictures using a software imaging system. Antibody anticytokeratin (CK), 15 was used as a bulge stem cell marker. The mean length of a scalp hair follicle is 4.16 mm. The infundibulum measures 0.76 mm, the isthmus 0.89 mm, and the inferior portion 2.5 mm. The insertion of the arrector pili muscle is located 1.65 mm deep. CK15 immunoreactivity starts at a depth of 1 mm and extends down to 1.8 mm. The ideal depth for the trichophytic procedure is to cut the wound edge at a depth of less than 1 mm to avoid the bulge zone. The data provided can serve as an objective anatomical reference in hair regeneration studies using horizontally transected follicles.
Article
Keratin-associated proteins (KAPs) are important constituents of the wool cuticle, comprised of the endo-, exocuticle and a-layers, which contribute significantly to the fibre's molecular and mechanical characteristics. Relatively little is known about the distribution of specific KAPs across these layers, and correct protein identification of individual KAPs is difficult due to extensive homology and identity among individual KAPs. We here present evidence that, by specifically exploiting the high-cysteine content of KAPs in the wool cuticle, using 2-nitro-5-thiocyanobenzoic acid (NTCB) cleavage in combination with tryptic digestion, a larger number of KAPs can be identified than with standard trypsin-only digests. A total of 27 KAPs were identified, six of which could only be identified using NTCB. Furthermore, NTCB-mediated cleavage of cuticle proteins generated unique peptides critical for unambiguous identification of two KAPs, as well as significantly increasing the overall sequence coverage of most identified KAPs. Interestingly, some of the peptides found to be unique to particular KAPs could only be found in either the exo- or endocuticle. We conclude that for the analysis of high sulphur proteomes, specific targeting of cysteine residues using chemical agents such as NTCB can provide critical information for unambiguous protein identification.
Article
Background: The macrolide sirolimus (rapamycin) selectively blocks translation of mRNAs containing a terminal 5' oligopyrimidine (TOP) tract by altering the activity of mammalian target of rapamycin (mTOR) and inhibiting downstream mTOR pathway components involved in TOP mRNA translation. The skin disorder pachyonychia congenita (PC) is caused by mutations in the inducible keratins (K) including K6a, K6b, K16 and K17. Published sequence data suggest the 5' untranslated regions of K6a and K6b mRNAs contain 5' TOP motifs and therefore may be sensitive to rapamycin treatment. Objective: Determine if mTOR inhibitors (rapamycin, temsirolimus or everolimus) are viable drug candidates for treatment of PC and other disorders caused by inappropriate expression of K6a and K6b. Methods: 5' RACE analysis was used to map the transcriptional start sites for K5, K6a, K6b, K14, K16 and K17. The sensitivity of these keratins to mTOR inhibitors was determined by Western and qPCR analysis following treatment of a human HaCaT keratinocyte cell line with rapamycin, temsirolimus or everolimus. A small off-label study was undertaken using orally administered rapamycin in three PC patients and the effects were monitored by clinical examination, photography, a validated Dermatology Life Quality Index (DLQI) and a pain and activity diary. Results: Sequence comparison and 5' RACE analysis of the 5' untranslated regions of K6a and K6b revealed putative TOP regulatory elements. Treatment of a human HaCaT keratinocyte cell line with mTOR inhibitors (rapamycin, temsirolimus or everolimus) resulted in selective K6a repression. Furthermore, treatment of this HaCaT cell line with siRNAs targeting components of the mTOR pathway altered the levels of K6a expression. To test the ability of rapamycin to ameliorate PC symptoms, an off-label study was conducted. PC patient clinical responses to oral rapamycin showed a therapeutic response in callus character as well as subjective improvement. Of particular note, rapamycin greatly reduced the presence of painful cutaneous thromboses after reaching therapeutic serum levels. The well-known rapamycin side effects led to the early withdrawal of all of the patients from the study. Conclusion: Rapamycin selectively blocks K6a expression in human keratinocytes. The improvement of symptoms in PC patients following rapamycin treatment suggests rapamycin (or rapamycin analogs) may be a therapeutic option, particularly if topical formulations can be developed that avoid the side effects associated with systemic administration.
Article
Historically, the term 'keratin' stood for all of the proteins extracted from skin modifications, such as horns, claws and hooves. Subsequently, it was realized that this keratin is actually a mixture of keratins, keratin filament-associated proteins and other proteins, such as enzymes. Keratins were then defined as certain filament-forming proteins with specific physicochemical properties and extracted from the cornified layer of the epidermis, whereas those filament-forming proteins that were extracted from the living layers of the epidermis were grouped as 'prekeratins' or 'cytokeratins'. Currently, the term 'keratin' covers all intermediate filament-forming proteins with specific physicochemical properties and produced in any vertebrate epithelia. Similarly, the nomenclature of epithelia as cornified, keratinized or non-keratinized is based historically on the notion that only the epidermis of skin modifications such as horns, claws and hooves is cornified, that the non-modified epidermis is a keratinized stratified epithelium, and that all other stratified and non-stratified epithelia are non-keratinized epithelia. At this point in time, the concepts of keratins and of keratinized or cornified epithelia need clarification and revision concerning the structure and function of keratin and keratin filaments in various epithelia of different species, as well as of keratin genes and their modifications, in view of recent research, such as the sequencing of keratin proteins and their genes, cell culture, transfection of epithelial cells, immunohistochemistry and immunoblotting. Recently, new functions of keratins and keratin filaments in cell signaling and intracellular vesicle transport have been discovered. It is currently understood that all stratified epithelia are keratinized and that some of these keratinized stratified epithelia cornify by forming a Stratum corneum. The processes of keratinization and cornification in skin modifications are different especially with respect to the keratins that are produced. Future research in keratins will provide a better understanding of the processes of keratinization and cornification of stratified epithelia, including those of skin modifications, of the adaptability of epithelia in general, of skin diseases, and of the changes in structure and function of epithelia in the course of evolution. This review focuses on keratins and keratin filaments in mammalian tissue but keratins in the tissues of some other vertebrates are also considered.
Article
The major structural proteins of epithelia, the keratins, and the keratin filament-associated protein, filaggrin, were analyzed in more than 50 samples of human embryonic and fetal skin by one-dimensional SDS PAGE and immunoblotting with monoclonal and polyclonal antibodies. Companion samples were examined by immunohistochemistry and electron microscopy. Based on structural characteristics of the epidermis, four periods of human epidermal development were identified. The first is the embryonic period (before 9 wk estimated gestational age), and the others are within the fetal period: stratification (9-14 wk), follicular keratinization (14-24 wk), and interfollicular keratinization (beginning at approximately 24 wk). Keratin proteins of both the acidic (AE1-reactive, type I) and the basic (AE3-reactive, type II) subfamilies were present throughout development. Keratin intermediate filaments were recognized in the tissue by electron microscopy and immunohistochemical staining. Keratins of 50 and 58 kD were present in the epidermis at all ages studied (8 wk to birth), and those of 56.5 and 67 kD were expressed at the time of stratification and increased in abundance as development proceeded. 40- and 52-kD keratins were present early in development but disappeared with keratinization. Immunohistochemical staining suggested the presence of keratins of 50 and 58 kD in basal cells, 56.5 and 67 kD in intermediate cells, and 40 and 52 kD in the periderm as well as in the basal cells between the time of stratification and birth. Filaggrin was first detected biochemically at approximately 15 wk and was localized immunohistochemically in the keratinizing cells that surround hair follicles. It was identified 8-10 wk later in the granular and cornified cell layers of keratinized interfollicular epidermis. These results demonstrate the following. An intimate relationship exists between expression of structural proteins and morphologic changes during development of the epidermis. The order of expression of individual keratins is consistent with the known expression of keratins in simple vs. stratified vs. keratinized epithelia. Expression of keratins typical of stratified epithelia (50 and 58 kD) precedes stratification, and expression of keratins typical of keratinization (56.5 and 67 kD) precedes keratinization, which suggests that their expression marks the tissue commitment to those processes. Because only keratins that have been demonstrated in various adult tissues are expressed during fetal development, we conclude that there are no "fetal" keratins per se.(ABSTRACT TRUNCATED AT 400 WORDS)
Article
The cytokeratin family of intermediate filament (IF) proteins can be grouped into the epithelial polypeptides ("soft alpha-keratins"), of which at least 19 exist in the various human epithelia, and the hair-type cytokeratins ("hard alpha-keratins"), which are typical of trichocytes, i.e., the living hair-forming cells. We have recently shown [34] that the hair follicles from diverse mammalian species contain a set of eight major cytokeratin polypeptides, four each of the acidic (type I) and the basic (type II) subfamily, which are different from all known epithelial cytokeratins. In addition, we have identified two new minor trichocytic cytokeratin polypeptides, designated Hax (type I) and Hbx (type II). Antibodies against trichocytic cytokeratins that do not crossreact with any of the epithelial cytokeratins have enabled us to study the expression of both kinds of cytokeratin in the various cell types of human and bovine hair follicles. Using immunofluorescence microscopy, we have observed intense reactions of trichocytic cytokeratins only in cells contributing to the forming hairs, i.e., hair shaft, medulla and cuticle, whereas immunostaining of the peribulbar matrix cells was weaker, if at all detectable. In contrast, epithelial cytokeratins were localized in both the inner and outer root sheath epithelia but, surprisingly, also in certain portions of the trichocyte column, notably cells of the cuticle, certain medullary cells, and trichocytes of the basalmost peripapillary cell layers. Cells coexpressing trichocytic and epithelial cytokeratins have been identified by double-label immunofluorescence microscopy. Epithelial cytokeratins of the inner and outer root sheath epithelia include, most remarkably, "simple-epithelium-type" cytokeratins 8, 18, and 19; these occur in certain peribulbar regions, in distinct patterns, but with variable frequencies. The occurrence of simple epithelial cytokeratins in hair follicles has also been confirmed by high-sensitivity immunoblotting of follicular polypeptides separated by gel electrophoresis. Vimentin-positive cells were abundantly interspersed (in some follicles, but not in all) between the trichocytes of the peripapillary cone, most of them probably being melanocytes. The cell-type complexity of the hair follicle and the different patterns of cytoskeletal protein expression in the various hair follicle cells are discussed in relation to the development and growth of this organ.
Article
Three different methods for hydrolysis and determination of amino acid composition of peptides and proteins were compared. We found, that the method of Matsubara and Sasaki (using 6N HCl and thioglycolic acid) gives comparatively low recoveries for tryptophan, while Liu and Chang's method, using p-toluenesulfonic acid and tryptamine, is more suitable. To eliminate the difficulties of the latter method, we used mercaptoethane-sulfonic acid, which, in the concentration used, results in total hydrolysis of peptide bonds within 22 hr and gives very high tryptophan recoveries. Both sulfonic acid methods were used for hydrolysis of the pentapeptide “pentagastrine” as well as of the proteins lysozyme, cytochrome c, and chymotrypsine. Their amino acid composition was determined using an automatic amino acid analyzer. Similarly to the p-toluenesulfonic acid method, the results of our method are totally reliable only for pure peptides and proteins, though the results obtained with our method using samples containing carbohydrates are better than those of all earlier methods.
Article
A cDNA library was constructed with poly(A)+ RNA from mouse tail epidermis which contained all hair follicles of tail skin. The library was subjected to sequential screening procedures aimed at selecting cDNA clones coding for acidic, type I hair keratins. Two clones, pktI-2 and pktI-3, encoded keratins that could be identified as murine type I hair keratins mHa2 and mHa3, respectively, by positive hybridization selection analysis. Sequence comparisons with the known murine type I hair keratins mHa1 (Bertolino et al., J. Invest. Dermatol. 91, 541-546, 1988) and mHa4 (Bertolino et al., J. Invest. Dermatol. 94, 297-303, 1990) revealed a structural heterogeneity within the type I hair keratin subfamily. Three keratins, mHa1, mHa3, and mHa4, are highly related, differing mainly in the penultimate part of their amino and carboxy termini. In contrast, mHa2 is structurally distinct from the three other keratins in both the alpha-helix and, in particular, the non-alpha-helical domains. These findings are confirmed by evolutionary investigations and flexibility calculations which indicate a more flexible nature of the mHa2 amino terminus when compared to the corresponding region of the three other keratins. In situ hybridization experiments with specific 3' fragments of mHa2 and mHa3 show that mHa3 is expressed in cortex cells, whereas mHa2 transcripts are strictly limited to the cuticle of the hair shaft. mHa3 mRNA expression can also be demonstrated in the central unit of the murine lingual filiform papillae, whereas the cuticular keratin mHa2 is not expressed in this body site. These data indicate that the structural heterogeneity within the type I hair keratin subfamily is functionally relevant in the morphogenesis of hard alpha-keratin-expressing tissues.
Article
Dynamic phosphorylation is one mechanism that regulates the more than 20 keratin type I and II intermediate filament proteins in epithelial cells. The major type II keratin in "simple type" glandular epithelia is keratin 8 (K8). We used biochemical and mutational approaches to localize two major in vivo phosphorylation sites of human K8 to the head (Ser-23) and tail (Ser-431) domains. Since Ser-23 of K8 is highly conserved among all type II keratins, we also examined if the corresponding Ser-59 in stratified epithelial keratin 6e is phosphorylated. Mutation of K6e Ser-59 abolished its phosphorylation in 32PO4-labeled baby hamster kidney cell transfectants. With regard to K8 phosphorylation at Ser-431, it increases dramatically upon stimulation of cells with epidermal growth factor (EGF) or after mitotic arrest and is the major K8 phosphorylated residue after incubating K8 immunoprecipitates with mitogen-activated protein or cdc2 kinases. A monoclonal antibody that specifically recognizes phosphoserine 431-K8 manifests increased reactivity with K8 and recognizes reorganized K8/18 filaments after EGF stimulation. Our results suggest that in vivo serine phosphorylation of K8 and K6e within the conserved head domain motif is likely to reflect a conserved phosphorylation site of most if not all type II keratins. Furthermore, K8 Ser-431 phosphorylation occurs after EGF stimulation and during mitotic arrest and is likely to be mediated by mitogen-activated protein and cdc2 kinases, respectively.
Article
The cytoplasm of animal cells is structured by a scaffolding composed of actin microfilaments, microtubules, and intermediate filaments. Intermediate filaments, so named because their 10-nanometer diameter is intermediate between that of microfilaments (6 nanometers) and microtubules (23 nanometers), assemble into an anastomosed network within the cytoplasm. In combination with a recently identified class of cross-linking proteins that mediate interactions between intermediate filaments and the other cytoskeletal networks, evidence is reviewed here that intermediate filaments provide a flexible intracellular scaffolding whose function is to structure cytoplasm and to resist stresses externally applied to the cell. Mutations that weaken this structural framework increase the risk of cell rupture and cause a variety of human disorders.
Article
To examine the effects of external modulation of epithelial-mesenchymal interaction on conjunctival epithelial cell differentiation characteristics. Keratin K4 and laminin beta2 chain protein localization was examined in an organotypic model which facilitates the comparison of differentiation characteristics of conjunctival epithelium interacting with conjunctival basement membrane or corneal basement membrane. In addition, keratin K4 and laminin beta2 chain localization was examined in primary cultures of conjunctival epithelial cells and fibroblasts. The synthesis and secretion of laminin beta2 chain by conjunctival fibroblasts in culture was determined by western blot analysis and immunoprecipitation. The ability of conjunctival epithelium to respond to exogenous laminin beta2 chain was assayed by culturing epithelial cells on a laminin matrix isolated from human placenta. In culture, conjunctival fibroblasts synthesize and secrete laminin beta2 chain but do not deposit this chain into an extracellular matrix substrate or basement membrane-like structure. The lack of extracellular deposition of this chain correlates to the gradual loss of keratin K4 protein in conjunctival epithelial cell culture. Conjunctival epithelium remains responsive to laminin beta2 chain in vitro because keratin K4 localization can be rescued in these cells by culture on a substrate of exogenous placental laminin. In vitro, alterations in native conjunctival epithelial-mesenchymal interactions results in aberrant basement membrane laminin isoform composition. This, in turn, leads to the loss of adult epithelial cell phenotype characteristics, suggesting that at least some aspects of conjunctival epithelial cell differentiation are regulated by the extracellular matrix.
Article
Desmosomes are important epidermal adhesion complexes that are characterized by a cell-specific expression of transmembrane cadherins and plaque-associated molecules. Desmosomes have so far, been implicated in three main disease types: autoimmune diseases that involve desmosome components (such as pemphigus vulgaris and pemphigus foliaceus), congenital diseases that affect intracellular calcium channels (such as Hailey-Hailey disease and Darier disease) and congenital diseases that directly affect desmosomal structural components. The identification of the first congenital defect affecting a desmosome component was in the gene for plakophilin I which caused an autosomal recessive skin fragility-ectodermal dysplasia syndrome with skin, hair and nail defects. Subsequently, either a haploinsufficiency of desmoplakin or a defect in desmoglein 1 was found to underlie the autosomal dominant condition Striate Palmoplantar Keratoderma. In addition, plakoglobin has been shown to be defective in Naxos disease, which results in a cardiomyopathy and growth of abnormal hair. These findings pave the way for the discovery of further cell cohesion-related diseases and will help to greatly increase our understanding of the specific function of desmosome and other epithelial junction components.
Article
In this study we have characterized a novel human type II keratin, hK6irs1, which is specifically expressed in the inner root sheath of the hair follicle. This keratin represents the ortholog of the recently described mouse inner root sheath keratin mK6irs. The two keratins were highly related and migrated at the same height as keratin 6 in two-dimensional gel electrophoresis. Both RNA in situ hybridization and indirect immunofluorescence studies of human hair follicles demonstrated hK6irs1 expression in the Henle and Huxley layers as well as in the cuticle of the inner root sheath. In all three layers, the expression of hK6irs1 mRNA and protein began simultaneously in adjacent cells of the lowermost bulb above the germinative cell pool. Higher up in the follicle, the detection limits for both hK6irs1 mRNA and protein precisely coincided with the asynchronous onset of abrupt terminal differentiation of the Henle layer, inner root sheath cuticle, and Huxley layer. Mainly above the level of terminal Henle cell differentiation, both indirect immunofluorescence and immunoelectron microscopy revealed the occurrence of distinct Huxley cells that developed pseudopodal hK6irs1-positive extensions passing through the fully keratinized Henle layer. These outwardly protruding foot processes abutted upon cells of the companion layer, with which they were connected by numerous desmosomes. These specialized Huxley cells have previously been termed "Flügelzellen", which means "winged cells", with reference to their characteristic foot processes. We provide evidence that, together with Henle cells, Flügelzellen ensure the maintenance of a continuous desmosomal anchorage of the companion layer along the entire inner root sheath. This tightly connected companion layer/inner root sheath unit provides an optimal molding and guidance of the growing hair shaft.
Article
Substantial progress has been made regarding the elucidation of differentiation processes of the human hair follicle. This review first describes the genomic organization of the human hair keratin gene family and the complex expression characteristics of hair keratins in the hair-forming compartment. Sections describe the role and fate of hair keratins in the diseased hair follicle, particularly hereditary disorders and hair follicle-derived tumors. Also included is a report on the actual state of knowledge concerning the regulation of hair keratin expression. In the second part of this review, essentially the same principles are applied to outline more recent and, thus, occasionally fewer data on specialized epithelial keratins expressed in various tissue constituents of the external sheaths and the companion layer of the follicle. A closing outlook highlights issues that need to be explored further to deepen our insight into the biology and genetics of the hair follicle.