The structural basis of TGF-beta, bone morphogenetic protein, and activin ligand binding

The transforming growth factor-beta (TGF-beta) superfamily is a large group of structurally related growth factors that play prominent roles in a variety of cellular processes. The importance and prevalence of TGF-beta signaling are also reflected by the complex network of check points that exist along the signaling pathway, including a number of extracellular antagonists and membrane-level signaling modulators. Recently, a number of important TGF-beta crystal structures have emerged and given us an unprecedented clarity on several aspects of the signal transduction process. This review will highlight these latest advances and present our current understanding on the mechanisms of specificity and regulation on TGF-beta signaling outside the cell.     

Eucalyptus increases ceramide levels in keratinocytes and improves stratum corneum function

The objectives of this study were to identify a plant extract that would improve stratum corneum functions and to elucidate the mechanism(s) involved. Based on the information that stratum corneum functions depend on the level of ceramide in the stratum corneum, we identified a Eucalyptus extract that was able to increase the level of ceramide in human keratinocytes in culture and in human stratum corneum and that improves the stratum corneum water holding and barrier functions. Addition of the Eucalyptus extract to human keratinocytes in culture increased the level of ceramide in a dose-dependent manner and also increased the biosynthesis of ceramide, glucosylceramide and sphingomyelin. Topical application of the Eucalyptus extract on the dry skin of human subjects induced by acetone and diethylether treatment resulted in a significant increase in ceramide level in the stratum corneum, a significant improvement in its water-holding function and an improvement in its barrier function. The addition of macrocarpal A, one of the main components of the Eucalyptus extract, to human keratinocytes in culture increased the level of ceramide and the mRNA expression of serine palmitoyltransferase, acid sphingomyelinase, neutral sphingomyelinase, glucosylceramide synthase and glucocerebrosidase in a dose-dependent manner. Our results indicate that the increased content of ceramides in the stratum corneum may underlie the therapeutic effect of the Eucalyptus extract. Our results also indicate the possibility that macrocarpal A is the key component that stimulates the synthesis of ceramide in the stratum corneum.     

Cell cycle analysis of Chinese hamster ovary cells stimulated by phosphatidic acid in serum-free culture

When recombinant Chinese hamster ovary cells were treated with pertussis toxin or genistein, not only lysophosphatidic acid (LPA) but also phosphatidic acid (PA) failed to stimulate progression through the cell cycle in serum-free culture, suggesting that PA and LPA induce cell growth through the same signal transduction pathway. Cell cycle analysis also indicates that cell growth promoted by PA results in enhanced protein production.     

Proliferation and stratification of keratinocyte on cultured amniotic epithelial cells for tissue engineering

Human amniotic epithelial cells (HAECs) are formed from amnioblasts, separated from the epiblast at about the 8th day after fertilization. Recent studies suggest that HAECs can produce various biologically active substances. In this study, the effects of cultured HAECs on keratinocytes were investigated. First of all, the effect of the medium conditioned by cultured HAECs on the proliferation of keratinocytes was examined. The conditioned medium significantly enhanced the proliferation (P<0.05). Next, the effect of co-culture with HAECs was also examined. The keratinocytes formed a stratified epithelium on day 7 after the start of co-culture. The cultured epithelium formed by the co-culture was five to six layers thick, could be detached by dispase treatment, and had sufficient strength as a sheet. These results suggest that HAECs will be a novel supplemental material for the tissue engineering of skin.     

Elevation of ceramide in Acetobacter malorum S24 by low pH stress and high temperature stress

Acetic acid bacteria have unique and highly pure membrane lipid components, such as 2-hydroxypalmitoyl-sphinganine (dihydroceramide) and can grow and produce acetic acid at around pH 3.0, suggesting that ceramide in cell membranes may be involved in the tolerance to acidic pH. Acetobacter malorum S24 was selected for the production of ceramide and grown in YPG medium containing 0.8% ethanol. Ceramide biosynthesis was induced at pH 4 and below, suggesting that ceramide biosynthesis is induced by low pH stress. Elevation of ceramide was also induced by high temperature stress (40–70 °C). After the strain was cultured in an optimal growth medium, the cells were collected and treated at pH 3 and 40 °C for 4 days, resulting in a 30-fold elevation of both the yield and content of ceramide.     

Lysophospholipids improve skin moisturization by modulating of calcium-dependent cell differentiation pathway

Recent studies have demonstrated that lysophospholipids (LPL) play critical roles in several biological signal transduction pathways to maintain the homoeostasis of cells, tissues and organs. Among them, lysophosphatidic acid (LPA) has been identified as a lipid mediator that induces morphological improvement in the epidermis in mice. In this study, we examined the effects of LPL (soybean-derived phospholipids modified with phospholipase A2 and C) compared with LPA. We initially examined the effects of LPA on normal human epidermal keratinocytes (NHEK) focusing on the expression of profilaggrin and serine palmitoyltransferase (SPT) mRNAs. LPA enhanced the expression of profilaggrin and SPT mRNAs via the modulation of Ca(2+) influx. Based on those results, the influence of LPL on NHEK was examined and was expanded to analyse the expression of two tight junction-related proteins, occludin and claudin-1. LPL had similar effects to increase profilaggrin and SPT mRNA expression and also stimulated the expression of occludin and claudin-1 at the mRNA and protein levels. In accordance with these results, LPL elicited significant improvements in surface water content in human skin. These findings indicate that LPL has the potential to strengthen the skin moisturizing capability by up-regulating the expression of mRNAs encoding components important to skin barrier function and skin hydration.     

No. 3Vol. 42, No. 2, pp. 94–101, 2008Effect of Birch (Betula platyphylla Sukatchev var. japonica Hara) sap on cultured human epidermal keratinocyte differentiation

The beauty of ideal skin texture is closely associated with dermal moisture factors. The key factors of skin moisture are NMF (natural moisturizing factor) and skin normal barrier function. The former keeps dermal surface moisture, and the later protects from excess water loss. So we have searched for the ingredient that improves these factors. Birch sap has been widely used as an effective drink for anti-fatigue and anti-stress. However, the effect of birch sap on skin as a cosmetic agent has not been known entirely. In this study, we investigated the effects of birch ( Betula platyphylla Sukatchev var. japonica Hara) sap on human skin. Birch sap induced epidermal keratinocyte differentiation properties in vitro . We assessed two epidermal differentiation agents. Filaggrin is a precursor protein of NMF, and involucrin is one of the precursor proteins of the cornified cell envelope (CE), which is related to normal barrier function. We have evaluated the production of these proteins where birch sap was applied to human normal keratinocytes. Birch sap not only increased mRNA expression of filaggrin and involucrin, but also accelerated these proteins production. Otherwise, birch sap did not have any influence for IL-6 production, which is related to inflammatory and aberrant keratinocyte proliferation. The results of induced differentiation properties on birch sap-treated keratinocytes are very similar to the differentiation induced by calcium in vitro . This similarity suggested that birch sap has a differentiation inducible property on in vitro cultured keratinocytes. Our study suggested that birch sap is able to control both moisturizing- and barrier-related factor production. From these effects, birch sap provides appropriate epidermal functions and skin homeostasis, and revealed itself as a very useful ingredient in the cosmetic field.     

A Zn(II)-glycine complex suppresses UVB-induced melanin production by stimulating metallothionein expression

Oxidative stress caused by ultraviolet (UV) radiation generates reactive oxygen species (ROS) in the skin, induces the secretion of melanocyte growth and activating factors from keratinocytes, which results in the formation of cutaneous hyper-pigmentation. Thus, increasing the anti-oxidative ability of skin cells is expected to be a good strategy for skin-lightening cosmetics. Metallothionein (MT) is one of the stress-induced proteins and is known to exhibit a strong anti-oxidative property. We previously reported that a zinc(II) complex with glycine (Zn(II)(Gly)(2)) effectively induces MT expression in cultured human keratinocytes. To determine its potential as a new skin lightening active, we examined whether Zn(II)(Gly)(2) regulates the release of melanocyte-activating factors from UVB-irradiated keratinocytes and affects melanin production in a reconstructed human epidermal equivalent. Conditioned medium from UVB-irradiated keratinocytes accelerated melanocyte proliferation to 110%, and that increase could be prevented by pre-treatment with Zn(II)(Gly)(2). In addition, Zn(II)(Gly)(2) significantly reduced both the production of prostaglandin E(2) and proopiomelanocortin expression in UVB-irradiated keratinocytes. Zn(II)(Gly)(2) also decreased melanin production in a reconstructed human epidermal equivalent. These results indicate that MT-induction in the epidermis effectively up-regulates tolerance against oxidative stress and inhibits the secretion of melanocyte growth and activating factors from keratinocytes. Thus, Zn(II)(Gly)(2) is a good candidate as a new skin-lightening active.     

A novel anti‐ageing mechanism for retinol: induction of dermal elastin synthesis and elastin fibre formation

Dermal elastic fibres are extracellular matrix protein complexes produced by fibroblasts and involved in skin elasticity. Elastin fibres decrease with age as a result of reduced synthesis and increased degradation, resulting in skin sagging and reduced skin elasticity. In this study, we show that retinol (ROL), known to enhance dermal collagen production, is also enhancing elastin fibre formation. ROL induced elastin gene expression and elastin fibre formation in cultured human dermal fibroblasts. Topical treatment of cultured human skin explants with a low dose (0.04%) of ROL increased mRNA and protein levels of tropoelastin and of fibrillin‐1, an elastin accessory protein, as documented by QPCR and immunohistochemistry staining. Luna staining confirmed the increased elastin fibre network in the ROL‐treated skin explants, as compared with untreated controls. These data demonstrate that ROL exerts its anti‐ageing benefits not only via enhanced epidermal proliferation and increased collagen production, but also through an increase in elastin production and assembly.     

A Simplified Method for the Isolation and Estimation of Cell Wall Bound Glycogen in Saccharomyces cerevisiae

Glycogen is an important storage reserve in yeast. In Saccharomyces cerevisiae glycogen is present in two pools, an intracellular soluble pool and a cell wall bound, insoluble extra‐cellular pool. The present method uses a 20% KOH treatment to separate the two pools, which are then estimated using amyloglucosidase. The amount of soluble glycogen was found to be 6.5 mg/g of wet weight of yeast while that of cell wall bound glycogen was found to be almost three times that of the soluble, viz., 18 mg/g of wet weight of yeast. The data is compared with two earlier commonly used methods of yeast carbohydrate fractionation, which reported glycogen in totality. Reviewing these methods in the light of finding two pools of glycogen revealed that both the methods can be demonstrated to yield soluble glycogen in the range of 6–9 mg/g of yeast and 18–21 mg/g of wet weight of yeast of cell wall bound glycogen.     

果蔬诱导抗病性中的信号转导途径

果蔬诱导抗病性在果蔬采后病害防治中是一种新的生物防治技术,其中,研究果蔬诱导抗病的信号转导途径对认识果蔬诱导抗病的分子机制具有重要意义。本文从激发子激发的最初防卫反应信号、细胞外的信号传导和细胞内的信号传导三方面进行阐述。     

蜂胶复方产品对心肌肥厚改善机制的转录组学研究

为了利用转录组学研究蜂胶复方产品改善心肌肥厚的机制,以自发性高血压大鼠(SHR)为研究对象,连续给药35d后剖取心脏、测定左心室指数,对药效最佳组和模型组的心肌进行转录组测序,筛选出差异基因(DGEs),并进行GO富集和KEGG代谢通路分析。结果表明:蜂胶复方产品组与模型对照组有1 297个DEGs(91个DEGs上调、1 206个DEGs下调)。KEGG代谢通路结果显示,有410个DEGs可富集到182个代谢通路中,DEGs富集较多的前3个通路与胰岛素信号转导密切相关,分别是PI3K-Akt信号通路、胰岛素信号通路、MAPK信号通路,提示该蜂胶复方产品通过调控与胰岛素信号转导相关的信号通路从而改善SHR的心肌肥厚。     

Ceramide, a crucial functional lipid, and its metabolic regulation by acid ceramidase

Over two decades ago, ceramide, a precursor of most complex sphingolipids, was recognized as a second messenger that plays important roles in signal transduction and cell regulation. Acid ceramidase (N-acylsphingosine deacylase, EC 3.5.1.23; AC) is a lysosomal hydrolase that catalyzes the breakdown of ceramide into sphingosine (Sph) and free fatty acid, thereby controlling the concentration of ceramide. Sph can be further phosphorylated by Sph kinase to generate sphingosine 1-phosphate (S1P), which is considered as a bioactive lipid that antagonizes ceramide-induced apoptosis. Importantly, AC can also synthesize ceramide from Sph and free fatty acids, suggesting that the enzyme may determine cell fate, namely, death or survival, by controlling the balance between the intracellular levels of ceramide and S1P. Recent studies have also shown that aberrant AC activity is a relevant feature of human disease. This review will discuss the roles of ceramide, the current findings regarding AC, and its important roles in mammalian development and human diseases.     

基于转录组的氯吡脲对草莓果实成熟的调控机制研究

目的 通过对对照组和白熟期外源施用氯吡脲处理组的成熟草莓果实进行转录组学分析,探讨外源喷施氯吡脲影响草莓果实成熟的调控机制。方法 利用高通量测序技术RNA-Seq,结合生物信息学方法,进行差异基因的筛选和其通路的功能富集分析。结果 草莓白熟期喷施氯吡脲,成熟果实中有1176个基因表达上调,2031个基因表达下调。差异基因的富集分析发现,植物-病原体相互作用、植物激素信号传导、淀粉和蔗糖代谢、内质网蛋白加工以及次级代谢物的生物合成等通路为差异基因主要富集的代谢途径。结论 植物激素信号转导、植物-病原体相互作用和次级代谢物的生物合成等通路受氯吡脲影响最为显著,其中植物激素信号转导通路是通过不同激素的共同调控来影响草莓的生长成熟。研究结果为氯吡脲影响草莓果实成熟调控基因的挖掘和深入研究奠定了基础。。     

Ultrastructural assessments of the melanosome distribution patterns and pigmentation features in human epidermal cells after UV irradiation and kojic acid treatment

It is well-known that skin pigmentation depends, among others, on number, aggregation and distribution of melanosomes in the epidermis. Here we describe a correlative microscopy-based ultrastructural approach that investigates the spatial distribution and pigmentation features of the melanosomes within melanocytes and keratinocytes. Data obtained from control skin, ultraviolet (UV)-stimulated tissue and kojic acid-treated UV-irradiated explants are compared. We introduce original parameters for the evaluation of the aggregation and pigmentation features of the melanosomes: the aggregation and pigmentation indexes. The aggregation index evaluates the presence of clustered melanosomes when the pigmentation index expresses the electron-density level of the pigment granules. The present study demonstrates that the last parameters clearly express histological effects induced by UVB irradiation. Results indicate that UV light did not change the number of melanosomes within either melanocytes or keratinocytes, but it definitely modified the distribution patterns of the pigment granules in both cell types. It also enhanced the pigmentation state of the epidermal cells. Moreover, statistical analysis concerning keratinocytes discloses a significant decrease in the mean pigmentation index when explants exposed to UV light were treated with kojic acid. Obviously, the present numerical findings point out the relevance of the introduced parameters to characterize the pigmentation state of skin.     

Invited review: Sphingolipid biology in the dairy cow: The emerging role of ceramide

The physiological control of lactation through coordinated adaptations is of fundamental importance for mammalian neonatal life. The putative actions of reduced insulin sensitivity and responsiveness and enhanced adipose tissue lipolysis spare glucose for the mammary synthesis of milk. However, severe insulin antagonism and body fat mobilization may jeopardize hepatic health and lactation in dairy cattle. Interestingly, lipolysis- and dietary-derived fatty acids may impair insulin sensitivity in cows. The mechanisms are undefined yet have major implications for the development of postpartum fatty liver disease. In nonruminants, the sphingolipid ceramide is a potent mediator of saturated fat-induced insulin resistance that defines in part the mechanisms of type 2 diabetes mellitus and nonalcoholic fatty liver disease. In ruminants including the lactating dairy cow, the functions of ceramide had remained virtually undescribed. Through a series of hypothesis-centered studies, ceramide has emerged as a potential antagonist of insulin-stimulated glucose utilization by adipose and skeletal muscle tissues in dairy cattle. Importantly, bovine data suggest that the ability of ceramide to inhibit insulin action likely depends on the lipolysis-dependent hepatic synthesis and secretion of ceramide during early lactation. Although these mechanisms appear to fade as lactation advances beyond peak milk production, early evidence suggests that palmitic acid feeding is a means to augment ceramide supply. Herein, we review a body of work that focuses on sphingolipid biology and the role of ceramide in the dairy cow within the framework of hepatic and fatty acid metabolism, insulin function, and lactation. The potential involvement of ceramide within the endocrine control of lactation is also considered.     

Inhibiting or enhancing effect of sulfuric acid-treated wheat starch on antibody production induced by two types of adjuvant

Glycogen has been reported to have immune-regulating activity. We examined in this study the immune-regulating activity of wheat starch of various molecular weights, because both starch and glycogen are made from glucose components linked by α-1,4 and α-1,6 glycoside bonds. Wheat starch was treated by sulfuric acid to prepare starch samples with differing molecular weight. The acid-treated starch inhibited cytokine production from murine splenocytes when the splenocytes were incubated with the antigen and a starch sample. The activity depended on the treatment time by sulfuric acid. Mice were then i.p. immunized with some antigens and the starch mixed with two types of adjuvant. The starch also inhibited the in vivo antibody production when administered with an alum adjuvant. In contrast, the starch enhanced the antibody response when administered with complete Fround adjuvant, indicating that the starch regulated immune responses depending on the molecular weight and surrounding circumstances.     

新橙皮苷对3T3-L1前脂肪细胞分化的影响及其作用机制

为探讨新橙皮苷对脂肪细胞脂肪形成的影响及其作用机制,采用MTS法检测新橙皮苷对3T3-L1前脂肪细胞的细胞活力的影响,确定新橙皮苷的作用浓度后,油红O染色法和分光光度法测定3T3-L1脂肪细胞的分化及胞内甘油三酯的含量,RT-PCR测定脂肪形成相关基因CCAAT增强子结合蛋白α（CCAAT/enhancer binding proteinα,C/EBPα）和过氧化物酶体增殖激活受体γ（Peroxisome proliferators-activated receptorγ,PPARγ）mRNA表达,Western蛋白印迹法检测蛋白激酶B（Protein kinase B,PKB/Akt）、糖原合成酶激酶3β（Glycogen synthase kinase3β,GSK3β）和糖原合成酶（Glycogen synthase,GS）的磷酸化水平;利用GSK3β选择性的抑制剂LiCl作用3T3-L1脂肪细胞后,检测新橙皮苷对3T3-L1细胞分化、胞内甘油三酯含量、C/EBPα、PPARγ及aP2蛋白表达的影响。结果表明,新橙皮苷能极显著抑制脂肪细胞分化和胞内甘油三酯的形成（P<0.01）,激活Akt信号通路,促进p-Akt和p-GSK3β水平增加,极显著抑制C/EBPα、PPARγ、aP2 mRNA和蛋白表达（P<0.01）。新橙皮苷的这些影响部分地被GSK3β抑制剂LiCl所抑制（P<0.01）。新橙皮苷能够通过激活Akt/GSK3β信号通路抑制脂肪细胞分化。