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Background Ultraviolet (UV) irradiation to skin induces biological responses to protect and heal the wounded tissue. Cutaneous blood vessels play an important role in maintaining skin homeostasis, by inducing angiogenesis and vasodilation. However, the vascular dynamics in vivo, such as morphological changes over time and their depth dependency, are not fully understood. Methods Ten Asian males were enrolled in this study and received UV (UVA + UVB) irradiation at two minimal erythema dose (MED) to the inner upper arm. Changes in epidermal thickness and vascular structures associated with UV irradiation were evaluated over time for 28 days by optical coherence tomography angiography (OCTA). This technique enables non-invasive visualization of three-dimensional vascular networks in human skin based on OCT assessment of skin structures with near-infrared light. Results Notable dilation of vascular structures and increases in epidermal thickness were observed after UV irradiation. Vessel density was markedly increased from the papillary dermis to the upper reticular dermis at a depth of 200 µm. These increases in vascular density showed significant persistence even at 28 days after UV irradiation. Conclusion We visualized the vascular structural changes caused by UV irradiation and revealed that the effects of a single UV irradiation at 2 MED persisted for up to 28 days after exposure. The OCTA technique allows not only the in situ assessment of micro-vasculature in human skin but also its monitoring of vascular dynamics over time.

形態: カラー図版あり, Physical characteristics: Original contains color illustrations, Accepted: 2018-05-17, 資料番号: PA1910049000

It has been suggested that skin color changes not only with advancing age but also with the times. The purpose of this study was to investigate changes in Japanese women's facial skin color over 25 years, as well as the changes in skin pigmentation that affect skin color. First, skin color changes in terms of Munsell color values were investigated. A total of 3181 Japanese women residing in the greater Tokyo area were enrolled, and datasets were collected using spectrophotometers, designated as the 1991, 2001, 2005, and 2015 data. The mean Munsell hue, value, and chroma were calculated for each measurement year. Next, the concentrations of melanin and hemoglobin were calculated from spectral data, to investigate changes in skin pigmentations. Finally, Monte Carlo simulations were performed to verify that the suggested changes in skin pigmentations brought about skin color changes. As a result, skin color significantly changed toward high lightness, low saturation, and high yellowness from the 1991 data to the 2001 data. From the 2005 to 2015 data, the skin color distribution shifted toward lower saturation and increased redness. In addition, the concentration of hemoglobin decreased significantly from the 1991 data to the 2001 data, while the melanin concentration decreased significantly from the 2005 data to the 2015 data.

We have demonstrated tomographic imaging of in vivo human skin with an optical interferometric imaging technique using a monochromatic light source. The axial resolution of this method is determined by the center wavelength and the NA of the objective and is irrelevant to the bandwidth of the light source in contrast to optical coherence tomography. Our imaging system is constructed with low-priced and small-sized compact disk optical pickup components, a laser diode, a high NA objective, and a voice coil actuator. In spite of its low cost and small size, our imaging system can visualize the structure of human skin as clearly as a commercial reflectance confocal microscope.

Modification of internal configuration of a bubble column, airlift and stirred tank reactor (10-200 L) was made for root cultures of Bupleurum falcatum L. Agitation with an impeller covered with partition mesh was ineffective for a 10-L modified reactor, because it caused intensive foaming and subsequent overflow of the culture medium even at a low rotation speed of 50 rpm and a low aeration rate of 0.1 vvm (volume per volume of medium). In contrast, efficient aeration through a ceramic sparger placed at the bottom of a 20-L bubble column reactor yielded approximately 25 g/L of dry roots and 500 mg/L of saikosaponin-a and saikosaponin-d over 42 days. On a 200-L scale, however, the roots became flocculated under the upper perforated plate initially positioned near the middle of the reactor, forming a firm disk of roots and a large empty space between the disk and the medium. Thus, the roots had poor contact with the medium, which severely suppressed their growth. To avoid this flocculation, a bottom perforated plate and draft tube were installed as a partitioning device separating the culturing area (outside the draft tube) from the aeration area (inside the draft tube). The draft tube was made of a stainless steel mesh rather than a solid material, and the tube greatly increased the root yield in the 20-L reactor. This configuration was successfully applied at the 200-L scale, yielding 500-600 mg/L of saikosaponin-a and saikosaponin-d over 56 days.

A symposium entitled “Women in Physiology” was held on 30 July 2009 during IUPS in Kyoto. Seven female researchers from five countries presented their views. This article includes the summary of each speaker’s talk. Figures are not included here because they have been uploaded to the web site of Women Physiologists of Japan (WPJ) ( http://square.umin.ac.jp/wpj/ ) with the permission of each speaker. Please visit this internet site, download the files freely, and use them for presentations.

Squamous cell carcinoma antigen 1 (SCCA1), which belongs to serine proteinase inhibitor (serpin) superfamily, inhibits papain-like cysteine proteinase. Recently, it has been reported that SCCA1 acts not only as a proteinase inhibitor but also as an inhibitor of UV-induced apoptosis via suppression of the activity of c-Jun NH(2)-terminal kinase (JNK1). The present study determined the crystal structure of SCCA1, suggesting that the reactive center loop (RCL) of SCCA1, a recognition site of proteinase, is very flexible and located away form the main-body of SCCA1. We show that the inhibitory effect of SCCA1 on the kinase activity of JNK1 is lost when the RCL was truncated. Furthermore, we found that a mutant protein created by replacing one amino acid in RCL maintain the suppressive activity to JNK1, whereas the inhibitory effect to proteinase is obviously decreased.

Background Facial skin pigmentation is one of the most prominent visible features of skin aging and often affects perception of health and beauty. To date, facial pigmentation has been evaluated using various image analysis methods developed for the cosmetic and esthetic fields. However, existing methods cannot provide precise information on pigmented spots, such as variations in size, color shade, and distribution pattern. The purpose of this study is the development of image evaluation methods to analyze individual pigmented spots and acquire detailed information on their age-related changes. Methods To characterize the individual pigmented spots within a cheek image, we established a simple object-counting algorithm. First, we captured cheek images using an original imaging system equipped with an illumination unit and a high-resolution digital camera. The acquired images were converted into melanin concentration images using compensation formulae. Next, the melanin images were converted into binary images. The binary images were then subjected to noise reduction. Finally, we calculated parameters such as the melanin concentration, quantity, and size of individual pigmented spots using a connected-components labeling algorithm, which assigns a unique label to each separate group of connected pixels. Results The cheek image analysis was evaluated on 643 female Japanese subjects. We confirmed that the proposed method was sufficiently sensitive to measure the melanin concentration, and the numbers and sizes of individual pigmented spots through manual evaluation of the cheek images. The image analysis results for the 643 Japanese women indicated clear relationships between age and the changes in the pigmented spots. Conclusion We developed a new quantitative evaluation method for individual pigmented spots in facial skin. This method facilitates the analysis of the characteristics of various pigmented facial spots and is directly applicable to the fields of dermatology, pharmacology, and esthetic cosmetology.

Chemical peeling with salicylic acid in polyethylene glycol vehicle (SA-PEG), which specifically acts on the stratum corneum, suppresses the development of skin tumors in UVB-irradiated hairless mice. To elucidate the mechanism through which chemical peeling with SA-PEG suppresses skin tumor development, the effects of chemical peeling on photodamaged keratinocytes and cornified envelopes (CEs) were evaluated in vivo. Among UVB-irradiated hairless mice, the structural atypia and expression of p53 protein in keratinocytes induced by UVB irradiation were intensely suppressed in the SA-PEG-treated mice 28 days after the start of weekly SA-PEG treatments when compared to that in the control UVB-irradiated mice. Incomplete expression of filaggrin and loricrin in keratinocytes from the control mice was also improved in keratinocytes from the SA-PEG-treated mice. In photo-exposed human facial skin, immature CEs were replaced with mature CEs 4 weeks after treatment with SA-PEG. Restoration of photodamaged stratum corneum by treatment with SA-PEG, which may affect remodeling of the structural environment of the keratinocytes, involved the normalization of keratinocyte differentiation and suppression of skin tumor development. These results suggest that the stratum corneum plays a protective role against carcinogenesis, and provide a novel strategy for the prevention of photo-induced skin tumors.

Background: Seasonal changes affect the condition of skin and may trigger various cutaneous disorders. Objective: To clarify the effects of the environmental humidity on the skin pathology, we studied the effects of the humidity on a water-holding function of the stratum corneum. Methods: We evaluated the skin surface conductance, amino acid in the stratum corneum, and immunoreactivity of filaggrin of the epidermis of hairless mice kept in different environmental humidity. Results: Skin surface conductance in the stratum corneum of hairless mice 3–7 days after transfer from a humid environment (>80% relative humidity) to a dry (

角層では, 乾燥した外部環境に対処するために, 遊離アミノ酸を主成分とする天然保湿因子 (NMF) が水分を保持している。ドライスキンでは角層水分量とともに角層アミノ酸量が低下することが報告されているが, その機構については不明な点が多い。角層アミノ酸はタンパク質「フィラグリン」が分解して生ずるが, われわれは培養ヒトケラチノサイトを気相に曝すことにより, プロフィラグリンmRNA量, タンパク量が減少することを見出した。この系で薬剤をスクリーニングしたところ, メキシコ産植物Palo Azulの抽出液がプロフィラグリンmRNA量減少を効果的に抑制した。また, この抽出液を配合した化粧水は, SDSで誘導したヒトの実験的ドライスキンに対して角層水分量, 角層アミノ酸量低下を抑制する効果も有していた。これらの結果は, 気相に曝された培養ヒトケラチノサイトがヒトのドライスキンに類似したモデル, ならびに薬剤のスクリーニング系になり得ることを示唆している。そして, 乾燥そのものがフィラグリン量の低下を引き起こすという, 乾燥による角層アミノ酸低下の一因を示唆している。

The epidermal permeability barrier of premature infants matures rapidly following birth. Previous studies suggest that air exposure could contribute to this acceleration, because: (i) development of a structurally and functionally mature barrier accelerates when fetal rat skin explants are incubated at an air-medium interface, and (ii) occlusion with a water-impermeable membrane prevents this acceleration. To investigate further the effects of air exposure on epidermal barrier ontogenesis, we compared the activities of several key enzymes of lipid metabolism and gene expression of protein markers of epidermal differentiation in fetal rat skin explants grown immersed versus air exposed. The rate-limiting enzymes of cholesterol (HMG CoA reductase) and ceramide (serine palmitoyl transferase) synthesis were not affected. In contrast, the normal developmental increases in activities of glucosylceramide synthase and cholesterol sulfotransferase, responsible for the synthesis of glucosylceramides and cholesterol sulfate, respectively, were accelerated further by air exposure. Additionally, two enzymes required for the final stages of barrier maturation and essential for normal stratum corneum function, beta-glucocerebrosidase, which converts glucosylceramide to ceramide, and steroid sulfatase, which desulfates cholesterol sulfate, also increased with air exposure. Furthermore, filaggrin and loricrin mRNA levels, and filaggrin, loricrin, and involucrin protein levels all increased with air exposure. Finally, occlusion with a water-impermeable membrane prevented both the air-exposure-induced increase in lipid enzyme activity, and the expression of loricrin, filaggrin, and involucrin. Thus, air exposure stimulates selected lipid metabolic enzymes and the gene expression of key structural proteins in fetal epidermis, providing a biochemical basis for air-induced acceleration of permeability barrier maturation in premature infants.

Lipids in the stratum corneum (SC) are organized into lamellar membrane unit structures that provide the permeability barrier. Cholesterol sulfate, a SC membrane lipid, is synthesized by cholesterol sulfotransferase (CSTase) in the lower epidermis and hydrolyzed to cholesterol by steroid sulfatase (SSase) in the SC. To determine whether these enzymes are induced during barrier ontogenesis, we examined their activity in epidermis of fetal rats before (gestational day 17), during (day 19), and after (day 21) barrier formation. CSTase activity increased ∼10-fold between day 17 and day 19 then declined between day 19 and day 21. In contrast, SSase activity reached its peak activity on day 21, increasing >5-fold. Fetal rat skin explants develop a SC and barrier over the same dine course in vitro as in utero . Likewise, CSTase and SSase activities during in vitro ontogenesis precisely mirrored those obtained in utero . Moreover, hormones that accelerate barrier ontogenesis ( e.g. glucocorticoids, thyroid hormone, and estrogen) accelerated the increase in CSTase and SSase activities during in vitro ontogenesis. mRNA levels of SSase increased in parallel with enzymatic activity, suggesting that these developmental changes are regulated at the genomic level. Finally, addition of exogenous cholesterol sulfate to explants in vitro did not accelerate either SC development or barrier formation. These studies suggest that induction of the cholesterol sulfate cycle enzymes during SC ontogenesis is a component of the fetal epidermal differentiation program and that the synthetic and degradative enzymes of this pathway are differentially regulated.

来るべき高齢化社会において, 化粧品や医薬部外品は重要な役割を果たすと考えられているが, 我々は皮膚老化を防御する戦略として, 乾燥防御, 紫外線防御に加えて酸化防御を提唱してきた。本報告では, 特に酸化防御の重要性について論じた。今までに著者らは, CL-HPLCシステムを用いた皮表における脂質過酸化反応の検討から, ヒト皮膚においては一重項酸素がキーとなっている活性酸素種であることを報告してきたが, 本報告において, 皮表脂質とヒポタウリンの一重項酸素との反応速度定数を測定して明らかにした. さらに光酸化条件下において, ヒト皮表において第一の酸化ターゲットであるスクアレンの酸化防御効果をヒポタウリンが発現することを明らかにした。一方ヒポタウリンは, 哺乳類において含硫アミノ酸の代謝産物の一つであることが知られており, 皮膚, 眼球, 肝臓, 脳, 心臓, 血清中等でその存在が認められている。またその酸化ストレスへの生体防御機構としての存在意義も議論されている。皮膚において生体の酸化防御機構を再構築, 強化する有用性を化粧品における新たなコンセプトとして提案した。

The assay conditions for protein phosphatase 2B (PP2B) in crude extracts from mouse lymphoid tissues and lymphocytes were extensively investigated. Under the conditions elucidated, the PP2B activity was measured in autoimmune-prone MRL/MpJ-lpr/lpr mice (MRL/lpr mice) and two control strains, MRL/MpJ- +/+ mice (MRL/+/+ mice) and C3H/HeJ mice. In the control mice, PP2B activity was distinctly higher in spleen and thymus than brain and liver. PP2B activity was further elevated in spleen of MRL/lpr mice than in the controls. Furthermore, we observed a specific increase in PP2B activity in T lymphocytes from MRL/lpr mice as compared with in those from control mice. On the other hand, no alteration was observed in PP2B activity in B lymphocytes. These results suggest the involvement of PP2B in the abnormal signal transduction and proliferation of T lymphocytes in MRL/lpr mice.

Restricted expression of caspase-14 in differentiating keratinocytes suggests the involvement of caspase-14 in terminal differentiation. We purified active caspase-14 from human cornified cells with sequential chromatographic procedures. Specific activity increased 764-fold with a yield of 9.1%. Purified caspase-14 revealed the highest activity on WEHD-methylcoumaryl-amide (MCA), although YVAD-MCA, another caspase-1 substrate, was poorly hydrolyzed. The purified protein was a heterodimer with 17 and 11 kDa subunits. N-terminal and C-terminal analyses demonstrated that the large subunit consisted of Ser(6)-Asp(146) and N-terminal of small subunit was identified as Lys(153). We successfully developed an antiserum (anti-h14D146) directed against the Asp(146) cleavage site, which reacted only with active caspase-14 but not with procaspase-14. Furthermore we confirmed that anti-h14D146 did not show any reactivity to the active forms of other caspases. Immunohistochemical analysis demonstrated that anti-h14D146 staining was mostly restricted to the cornified layer and co-localized with some of the TUNEL positive-granular cells in the normal human epidermis. UV radiation study demonstrated that caspase-3 was activated and co-localized with TUNEL-positive cells in the middle layer of human epidermis. In contrast, we could not detect caspase-14 activation in response to UV. Our study revealed tightly regulated action of caspase-14, in which only the terminal differentiation of keratinocytes controls its activation process.

Background: Parakeratosis, the persistent presence of nuclei in the stratum corneum (SC) is associated with serious disruption of skin barrier function. Squamous cell carcinoma antigen 1 (SCCA1) is strongly up-regulated in inflamed and parakeratotic skin. Objective: To find a biochemical marker for the SC barrier disruption, especially the disruption associated with parakeratosis. Methods: An ELISA assay system was established to quantify SCCA1 in the extract of tape-stripped cornified cells. Transepidermal water loss (TEWL) and other skin parameters were measured and compared with the amount of SCCA1. Localization of SCCA1 was investigated immunohistochemically in various skin diseases with parakeratosis. Nuclei and SCCA1 on the skin surface were detected by staining of corniocytes collected on an adhesive-coated slide glass. Results: SCCA1 showed strong up-regulation in lesional skin with psoriasis (466-fold), hayfever skin caused by Japanese ceder pollen (232-fold) and sun-exposed skin of healthy individuals (90-fold) compared to their normal sun-protected skin. The increased levels of SCCA1 were well correlated with increased values of TEWL and the number of parakeratotic cells in the SC. Furthermore, subjects with high levels of SCCA1 in the epidermis were more susceptible to barrier disruption by external stimuli, and this was accompanied with a further increase of SCCA1. We confirmed that localization of SCCA1 was limited to parakeratotic areas by using the skin surface staining technique. Immunohistochemical study also demonstrated that SCCA1 was always present at high levels in parakeratotic epidermis. Conclusion: All of our findings indicate that SCCA1 plays an important role in the induction of epidermal barrier disruption. SCCA1 may be a critical determinant of barrier function in the epidermis.

S100A8 and S100A9 are known to be up-regulated in hyperproliferative and psoriatic epidermis, but their function in epidermal keratinocytes remains largely unknown. Here we show that (1) S100A8 and S100A9 are secreted by cultured normal human keratinocytes (NHK) in a cytokine-dependent manner, (2) when applied to NHK, recombinant S100A8/A9 (a 1:1 mixture of S100A8 and S100A9) induced expression of a number of cytokine genes such as IL-8/CXCL8, CXCL1, CXCL2, CXCL3, CCL20, IL-6, and TNFα that are known to be up-regulated in psoriatic epidermis, (3) the S100A8/A9-induced cytokines in turn enhanced production and secretion of S100A8 and S100A9 by NHK, and (4) S100A8 and S100A8/A9 stimulated the growth of NHK at a concentration as low as 1 ng/ml. These results indicate the presence of a positive feedback loop for growth stimulation involving S100A8/A9 and cytokines in human epidermal keratinocytes, implicating the relevance of the positive feedback loop to the etiology of hyperproliferative skin diseases, including psoriasis. J. Cell. Biochem. 104: 453–464, 2008. © 2007 Wiley-Liss, Inc.

Summary Background c-Jun N-terminal kinase (JNK) plays a critical role in UV-induced apoptotic cell death. Although three isoforms are known in mammals, physiological roles of each isoform are still obscure. Furthermore, our recent findings show that serpin squamous cell carcinoma antigen (SCCA1) binds to JNK. Objective To determine which isoform is responsible for the UV signal transduction in human epidermis and whether SCCA1 is capable to regulate kinase activity of a specific isoform. Methods Immunohistochemical localization of each JNK isoform was investigated after UV irradiation in vivo and in vitro. Effect of recombinant SCCA1 on JNK kinase activity was also analyzed. Results Immunostaining for JNK1, 2 and 3 demonstrated marked elevation of JNK1 in spinous to granular cells of UV-irradiated skin, whereas they were expressed weakly in upper epidermis of the sun-protected, buttock skin. In cultured keratinocytes, only JNK1 is translocated into nucleus after UV irradiation. JNK2, which localized in the cytoplasm, or JNK3, which was confined in nucleus, remained in the same compartment after UV irradiation. We confirmed that only JNK1 mRNA was up-regulated after UV irradiation in cultured keratinocytes. In addition, recombinant SCCA1 suppressed kinase activity of JNK1 but did not affect JNK2 or JNK3 kinase activity. Conclusion JNK1 is associated with UV signal transduction in human epidermis and SCCA1 is a suppressor of this process.