Involvement of transglutaminase in ex vivo maturation of cornified envelopes in the stratum corneum

The cornified envelope (CE) is a thin insoluble structure enveloping corneocytes, and is essential for the barrier function of the stratum corneum (SC). Our previous studies revealed that immature CEs are detected in the outermost layer of SC of barrier-impaired epidermis including the face and in various inflammatory disorders, using a non-invasive method to evaluate CE maturity. However, factors attributable for immaturity of CEs are still unclear. The aim of the present study is to clarify whether immature CEs in the SC have the potential to mature. SC samples, in which immature CEs abundantly exist, were collected from the cheek of healthy volunteers by tape-stripping, and were incubated ex vivo under the humidified air at 37 degrees C. Then, CE maturity was evaluated by staining with a combination of anti-involucrin and Nile red to detect involucrin antigenicity in the immature CEs and hydrophobicity in the mature CEs, respectively. Ex vivo incubation of the SC resulted in the conversion of immature CEs into mature CEs in terms of loss of involucrin antigenicity and acquisition of hydrophobicity. Application of buffer solutions of various pH onto the SC prior to incubation revealed that maturation of CEs was proceeded at range of pH 5-7, corresponding to intrinsic pH range within the SC. Chelating agents, ethylenediamine-N, N, N', N',-tetraacetic acid (EDTA) and ethyleneglycol bis(beta-aminoethylether)-N,N,N',N'-tetraacetic acid (EGTA), and thiol alkylating agents, N-ethylmaleimide and iodoacetamide, inhibited the maturation. Labelled cadaverine as an exogenous substrate for transglutaminase (TGase) could be incorporated into CEs during maturation. Extractable involucrin-like protein detected in the SC samples before incubation concomitantly disappeared with CE maturation, suggesting incorporation of endogenous substrates into the CEs. These results obviously demonstrate that maturation of CEs was mediated by TGase activity in the SC, and that immature CEs found in the outermost face SC have potential to mature by cross-linking of endogenous CE precursors present in the SC. Reduction of environmental humidity during ex vivo incubation of the SC resulted in marked suppression of maturation of CEs, and application of a moisturizer, glycerine, onto the SC replenished the suppression of maturation, suggesting that water content in the SC may affect the TGase reaction in the SC. Therefore, various factors, including a decrease in the water content in the SC, may account for impaired maturation of CEs in the face SC.     

Role of ceramides 2 and 5 in the structure of the stratum corneum lipid barrier

We utilized Fourier transform infrared (FTIR) spectroscopy to investigate headgroup and chain interactions in model SC lipid barriers containing equimolar amounts of deuterated hexadecanoic acid, cholesterol, and ceramide 2 (non-hydroxy sphingosine) or ceramide 5 (alpha-hydroxy sphingosine). In the ceramide 2 model the thermotropic response of the CD _ 2 and CH _ 2 stretching modes indicates that hexadecanoic acid begins to disorder at 42 degrees C while ceramide 2 remains ordered until 52 degrees C. Additionally, splitting of the CD _ 2 bending and CH _ 2 rocking modes provides evidence for separate orthorhombic hexadecanoic acid and ceramide domains. The ceramide amide I mode (1650 cm ; -1) is split into two components indicating strong intermolecular hydrogen bonding between headgroups. In the ceramide 5 model, the CH _ 2 and CD _ 2 stretching frequencies again reveal highly conformationally ordered ceramide 5 and hexadecanoic acid chains. Splitting of both the CD _ 2 bending and CH _ 2 rocking modes is observed. However, the CH _ 2 rocking frequencies indicate distorted packing of the ceramide. The collapse of these highly ordered phases, and the onset of conformational disorder, occurs at 50 degrees C for both ceramide 5 and hexadecanoic acid. The amide I and II frequencies of ceramide 5 indicate strong H-bonding, although neither mode is split. Our results demonstrate that model SC lipid systems have quite different physical properties depending on whether they contain ceramide 2 or 5. From this we infer that ceramide 2 and 5 make distinct contributions to the structural biophysics of the SC lipid barrier. Our observation of ordered lipid domains is also consistent with the recently proposed domain mosaic model of the skin barrier.     

Measurement of total water and bound water contents in human stratum corneum by in vitro proton nuclear magnetic resonance spectroscopy

The reliability of in vitro proton nuclear magnetic resonance (NMR) spectroscopy to specifically measure the water content of rehydrated human stratum corneum samples was assessed by comparison with the previously validated thermal desorption-mass spectrometry technique. The interest of proton NMR is that it can determine, in the same sample, both the amount of total water by recording the spectra at ambient temperature, and that of bound water (non-freezing water) by recording the spectra below 0 degrees C. Provided that enough samples of stratum corneum are analysed to average out the observed and well-known sample-to-sample variation, proton NMR may be of value in the evaluation of hygroscopic properties of raw materials for moisturizer formulations.     

Trends in stratum corneum research and the management of dry skin conditions

The structure, composition, formation and function of the stratum corneum have been the subject of intense research over the last few decades. As has become apparent, stratum corneum barrier function is not only dependent on one single component but also on its total architecture. Recent developments in understanding lipid composition have led to a new ceramide nomenclature system, a new proposal for a molecular model of the interactions between ceramides, cholesterol and fatty acids, and the demonstration of the presence of crystalline orthorhombic and gel hexagonal lipid phases in the stratum corneum. Linoleate-containing ceramide one, now known as CER EOS, have been shown to be essential for the formation of the 13 nm long periodicity phase (LPP) observed by electron microscopy and X-ray diffraction studies, whereas long-chain fatty acids are important for the formation of the crystalline lipid phases essential for barrier function. The role of the corneocyte envelope, its constituent proteins and its transglutaminase-mediated maturation processes have been shown to be essential for good skin condition. Several proteases may have a role in corneodesmolysis, particularly serine and cathepsin-like enzymes. Novel filaggrin polymorphisms have been identified that may be involved in the expression of a dry skin phenotype. Disturbances in lipid packing states, reduction in ceramide levels (particularly the phytosphingosine-containing ceramides), reductions in the levels of long-chain fatty acids and loss of the LPP largely account for the perturbations in lipid structure that occur in dry skin. The reduced corneodesmolysis that occurs in this xerotic skin disorder is now well accepted and is caused by reductions in the levels and activities of stratum corneum proteases together with elevated levels of corneodesmosomal glycoproteins in the superficial layers of the stratum corneum. Additionally, increased levels of fragile corneocytes are associated with reduced transglutaminase activity and corneocyte envelope cross-linking events. However, in comparison with the advances in our understanding of the textural changes that occur in dry skin, the somatosensory changes are poorly understood and the itching associated with dry skin is still an under-researched area. The unique biosensor role of the stratum corneum essential for a competent natural moisturizing barrier may also have a role to play in the action of anti-ageing technologies by controlling the expression and secretion of epidermal cytokines and growth factors. Technologies to treat the surface textural skin problems, enhance the differentiation process, particularly lipid biosynthesis, and to control the somatosensory problems in dry skin have received much attention in the last decade. This paper will review the state of the art of stratum corneum biology and the trends in the management of dry skin.     

Infrared spectroscopic studies of sodium dodecyl sulphate permeation and interaction with stratum corneum lipids in skin

The barrier function of skin is primarily provided by the lamellar lipid matrix of the stratum corneum (SC), which has been shown in previous infrared (IR) and related studies to consist predominantly of ordered lipids packed in orthorhombic and hexagonal domains. In the current work, we investigate the effects of the anionic surfactant, sodium dodecyl sulphate (SDS), on SC lipid packing and phase behaviour, using FT-IR spectroscopy. The use of acyl chain perdeuterated SDS allows unequivocal spectroscopic detection of both endogenous lipid and exogenous material in intact tissue. IR spectra were acquired as a function of temperature from isolated human SC exposed to SDS for various incubation periods at 34°C. SDS is found to enter the SC and is observed to be in a more ordered state in the SC than in solution, indicating that the SDS interacts with the ordered SC lipids. The results reveal that SDS reduces the amount of orthorhombic phase in the SC and increases the amount of hexagonally packed lipid at physiologically relevant temperatures. In addition, a small decrease in the lipid T(m) (acyl chain melting temperature) is observed. Furthermore, these SDS-induced changes were found to be strongly dependent on the time of exposure.     

Increased carbonyl protein levels in the stratum corneum of the face during winter

The stratum corneum (SC) is the interface between the body and the environment, and is continuously exposed to oxidative stress that results in carbonyl modification of proteins. We previously developed a simple and non-invasive method to assess the stratum corneum carbonyl protein (SCCP) levels. In this study, we used this method to examine the seasonal changes in the SCCP levels and the relationship between the SCCP level and the physiological condition of the SC. SC was collected from the face of healthy Japanese volunteers by adhesive tape stripping and its carbonyl groups were determined by reaction with fluorescein-5-thiosemicarbazide. The average fluorescence intensity of the SC was calculated as the SCCP level. The SCCP level in the cheek was higher in winter than summer. The SCCP level was negatively correlated with the water content in the SC measured by the conductance and capacitance, and also negatively correlated with the extensibility of the skin measured by a Cutometer, suggesting that the mechanical properties of the skin can be affected by oxidative modification of the SC. These data suggest the involvement of oxidative modification of SC proteins in the generation of rough skin during winter.     

Utilization of fourier transform infrared spectroscopy for measurement of organic phosphorus and bound calcium in cheddar cheese

The methods available for measuring organic P and bound Ca in cheese are either cumbersome or involve dilution of the cheese. Dilution of the cheese can lead to erroneous results, particularly in the case of bound Ca. Hence, the objective of this study was to evaluate the feasibility of Fourier transform infrared (FTIR) spectroscopy for direct measurement of organic P and bound Ca in Cheddar cheese. Two hundred sixteen samples of cheese were analyzed for protein-bound organic P, bound Ca using a water-extraction based method, and buffering curves. Additionally, the infrared spectra of the cheeses were collected between 4,000 and 650 cm⁻¹, at a resolution of 4 cm⁻¹, and 256 scans per sample. The spectral shifts in the infrared region from 1,050 to 900 cm⁻¹, in addition to the measured concentrations of organic P, bound Ca, and buffering peak area at pH 5.1, were used to develop calibration models using partial least squares (PLS) regression analysis. The spectral region of 956 to 946 cm⁻¹ correlated with the measured concentrations of organic P and the overall PLS model had a correlation (R²) of 0.76 between the predicted and measured concentrations. The spectral region at ∼980 cm⁻¹ was correlated with the measured concentrations of bound Ca, and the overall PLS model had a correlation (R²) of 0.70 between the predicted and measured concentrations. A similar spectral region at ∼980 cm⁻¹ was also correlated with the measured buffering peak areas and the overall PLS model had a correlation (R²) of 0.64 between the predicted and measured peak areas. A linear regression analysis between the bound Ca and buffering peak area demonstrated that bound Ca was correlated (R² = 0.73) with buffering peak area. This study demonstrates that FTIR can be used to measure organic P in cheeses. It also has the potential to be used for measuring bound Ca in undiluted cheeses, and for prediction of the buffering capacity of cheese.     

A new technique for the evaluation of cosmetics effect on mechanical properties of stratum corneum and epidermis in vitro

The mechanical properties of stratum corneum and epidermis have been measured in vitro and a dispersity of approximately 30% to 50% between samples from the same donor has been found. To overcome such a limitation, a technique was developed in order to compare the mechanical properties of the same skin sample before and after treatment. The chosen parameter (initial slope of the stress-strain curve) appeared to be influenced by the topical application of products. A different time response was found with stratum corneum and epidermis reflecting their structural differences.
Une nouvelle technique d'evaluation de l'effet des produits cosmétique sur les propriétés mécaniques du stratum corneum et de l'épiderme humains (etude in vitro )     

傅立叶变换红外光谱法快速鉴别海参质量

利用傅立叶变换红外光谱(FTIR)快速鉴别了两种不同品质的海参,结果表明,优质海参与劣质海参的红外光谱图有明显的区别,利用谱图峰的波数位置及谱峰强度可鉴别海参质量的优劣。此方法具有简便、快速、用样品量少等特点。     

Increased mass levels of certain serine proteases in the stratum corneum in acute eczematous atopic skin

Acute eczematous atopic dermatitis (AD) is associated with increases in stratum corneum (SC) serine protease activity. The purpose of this study was to examine whether the increased SC protease activities in acute eczematous atopic dermatitis were associated with increased mass levels of SC proteases. Six subjects with healthy skin and six patients with AD each with non-lesional skin or lesional acute eczematous skin had the mass levels of their extractable SC kallikreins (KLK), plasmin and urokinase quantified using Luminex multiplex bead-based assays from SC tape strippings. The mass levels of KLK5 and KLK14 together with urokinase were not elevated in the SC in atopic skin. However, the mass levels of KLK7 and KLK11 together with plasmin were greatly elevated compared with the extracts from the non-lesional and the healthy skin and correlated with the corresponding enzymatic activities.     

An analytical technique for measuring protein carbonyl in the stratum corneum using surface plasmon resonance

Protein carbonyl groups in the stratum corneum may be used as a biomarker for skin photo-stress. To evaluate the relationship between the protein carbonyl to total protein (TP) ratio (carbonylation ratio) and skin photo-stress, the authors established a methodology by which protein carbonyl can be easily and highly sensitively analysed using an optical technique based on surface plasmon resonance (SPR). To collect the stratum corneum, tape stripping was employed. Firstly, the protein carbonyl was reacted with 2,4-dinitrobenzenesulfonic acid dihydrate, and the quantity of dinitrophenylated (DNP)-protein carbonyl was determined using an anti-dinitrophenyl (anti-DNP) antibody. The mass of DNP-protein carbonyl was measured using SPR. A truncated sampling-reporting cycle of <5 min allowed speedy reporting of DNP-protein carbonyl levels. A significant difference was observed in the protein carbonyl/TP ratio (carbonylation ratio) between a sun-protected area (mid-ventral arm) and a sun-exposed area (upper cheek, P < 0.05). Additionally, the carbonylation ratio of the sun-exposed area showed a higher value than that of the sun-protected area. It was suggested that the carbonylation ratio might be a useful index of skin photo-stress.     

Comparison of fourier transform mid infrared spectroscopy and near infrared reflectance spectroscopy with differential scanning calorimetry for the study of the staling of bread

The staling of bread has previously been studied by differential scanning calorimetry and by simple rheological measurements. In this collaborative study, two spectroscopic techniques (Fourier transform mid infrared spectroscopy and near infrared reflectance spectroscopy) have been used in addition to differential scanning calorimetry to follow the progress of bread staling. Using each technique, changes in measured properties were apparent which, when fitted by first order exponential equations, gave calculated rate constants of similar magnitude. It is postulated that each technique gives independent information about the crystallisation process in the amylopectin fraction of the bread crumb.     

Selection for milk coagulation properties predicted by Fourier transform infrared spectroscopy in the Italian Holstein-Friesian breed

Milk coagulation is based on a series of physicochemical changes at the casein micelle level, resulting in formation of a gel. Milk coagulation properties (MCP) are relevant for cheese quality and yield, important factors for the dairy industry. They are also evaluated in herd bulk milk to reward or penalize producers of Protected Designation of Origin cheeses. The economic importance of improving MCP justifies the need to account for this trait in the selection process. A pilot study was carried out to determine the feasibility of including MCP in the selection schemes of the Italian Holstein. The MCP were predicted in 1,055 individual milk samples collected in 16 herds (66 ± 24 cows per herd) located in Brescia province (northeastern Italy) by means of Fourier transform infrared (FTIR) spectroscopy. The coefficient of determination of prediction models indicated moderate predictions for milk rennet coagulation time (RCT = 0.65) and curd firmness (a₃₀ = 0.68), and poor predictions for curd-firming time (k₂₀ = 0.49), whereas the range error ratio (8.9, 6.9, and 9.5 for RCT, k₂₀, and a₃₀, respectively) indicated good practical utility of the predictive models for all parameters. Milk proteins were genotyped and casein haplotypes (α_S1-, β-, α_S2-, and κ-casein) were reconstructed. Data from 51 half-sib families (19.9 ± 16.4 daughters per sire) were analyzed by an animal model to estimate (1) the genetic parameters of predicted RCT, k₂₀, and a₃₀; (2) the breeding values for these predicted clotting variables; and (3) the effect of milk protein genotypes and casein haplotypes on predicted MCP (pMCP). This is the first study to estimate both genetic parameters and breeding values of pMCP, together with the effects of milk protein genotypes and casein haplotypes, that also considered k₂₀, probably the most important parameter for the dairy industry (because it indicates the time for the beginning of curd-cutting). Heritability of predicted RCT (0.26) and k₂₀ (0.31) were close to the average heritability described in literature, whereas the heritability of a₃₀ was higher (0.52 vs. 0.27). The effects of milk proteins were statistically significant and similar to those obtained on measured MCP. In particular, haplotypes including uncommon variants showed positive (B-I-A-B) or negative (B-A¹-A-E) effects. Based on these findings, FTIR spectroscopy-pMCP is proposed as a potential selection criterion for the Italian Holstein.     

Superior effect of isostearyl isostearate on improvement in stratum corneum water permeability barrier function as examined by the plastic occlusion stress test

Dry skin is a major dermatological problem and consumer research indicates that although current moisturizers are effective they are not completely meeting consumer expectation. Several technological approaches have been taken but influencing stratum corneum (SC) lipid phase behaviour as a novel water permeability barrier‐enhancing and moisturizing mechanism has only been started to be investigated recently. Both the long periodicity SC lipid lamellar phase and the orthorhombic lipid packing state have been proposed to define optimal SC water permeability barrier properties. Several lipophillic moisturizers have been tested for their ability to modify SC lipid lateral packing namely glyceryl monoisostearate (GMIS), isopropyl isostearate (IPIS) and isostearyl isostearate (ISIS) of which IPIS and ISIS are reported to induce the orthorhombic phase. Despite the improvements in the lateral packing of SC lipids, these ingredients have been shown not to improve transepidermal water loss. However, using a novel skin surface water loss method we have observed for the first time significant improvements in SC water permeability barrier function for ISIS compared with IPIS, GMIS and petrolatum. However, using synthetic membranes and measuring water vapour transport rates we showed that the isostearyl esters were not occlusive like petrolatum. As the effects of ISIS were not because of what would be considered as true occlusion, we propose that the differences in the SC water permeability barrier properties from use of ISIS to the other ingredients tested are because of its reported effects on SC lipid phase behaviour. Further studies probably using spectroscopic approaches, however, will be needed to specifically test this hypothesis in vivo.     

织物傅里叶变换衰减全反射红外光谱库的建立及应用

针对常规方法鉴别纯纺织物及预测混纺织物组分含量工序多、耗时长且污染环境的问题,采用傅里叶变换红外光谱仪,结合衰减全反射(ATR)附件测试各纯纺及二组分混纺织物的傅里叶变换衰减全反射红外光谱(ATR-IR)图,并从已测的753个样品中筛选出正反经纬组分一致的纯纺及混纺织物样品205个,建立了涤纶/棉、涤纶/羊毛、涤纶/锦纶、蚕丝/棉和涤纶/粘胶等纯纺及混纺织物的ATR-IR谱库。并利用自建谱库的检索功能,对20个未知纤维织物样品进行快速无损鉴别及含量预测。研究表明:对于纯纺织物,识别准确率为100%;对于混纺织物,当误差≤3% 时,通过T检验,其定量预测结果与国标法测定值无显著差异,方便了样品的快速检验与含量预测。     

食醋总酸近红外快速检测方法的研究

利用傅立叶变换近红外光谱技术结合偏最小二乘法（PLS）对食醋的总酸进行快速检测分析。随机采集106个不同酸度食醋的实验数据、扫描近红外光谱图,选择最优的光谱预处理方法优化、建立模型。利用模型对15个未参与建模的食醋样品总酸的含量进行预测,外部验证模型的准确性。结果表明,食醋总酸实测值与对应采集的近红外漫反射光谱相关联建立的快速检测模型,其近红外食醋总酸模型的交叉验证决定系数（R2）为0.972 3,交叉验证均方差（RMSECV）为0.062 1。经外部验证后,该模型食醋总酸预测值和实测值的绝对偏差平均值为0.035,最大相对误差为1.383%,两者间相关性系数为0.995。该方法可以快速、高效、简便地用于食醋总酸的快速检测。