Citrus sudachi Peel Extract Suppresses Cell Proliferation and Promotes the Differentiation of Keratinocytes through Inhibition of the EGFR–ERK Signaling Pathway

Citrus sudachi is a well-known fruit in Tokushima Prefecture, Japan, and its peels are rich in phytochemicals, including phenolic compounds. Although it is expected that the extract of the C. sudachi peel elicits various beneficial physiological activities, the effect on the skin has not been investigated. In this study, we report that the aqueous extract from the peel of C. sudachi suppresses cell proliferation of the immortalized human keratinocyte cell line, HaCaT, and primary normal human epidermal keratinocytes. The extract of C. sudachi peel suppressed epidermal growth factor (EGF)-induced EGF receptor activation and tumor necrosis factor (TNF)-α-induced extracellular regulated kinase (ERK) 1/2 activation, which suggests that the extract exerts its inhibitory effect through inhibition of both the EGF receptor (EGFR) and its downstream molecules. Additionally, the extract of C. sudachi peel potentiated calcium-induced keratinocyte differentiation. These results suggest that the extract of C. sudachi peel may have beneficial effects against skin diseases that are characterized by hyperproliferation of epidermal keratinocytes, such as those seen in psoriasis and in cutaneous squamous cell carcinoma

Cys34-cysteinylated human serum albumin is a sensitive plasma marker in oxidative stress-related chronic diseases

The degree of oxidized cysteine (Cys) 34 in human serum albumin (HSA), as determined by high performance liquid chromatography (HPLC), is correlated with oxidative stress related pathological conditions. In order to further characterize the oxidation of Cys34-HSA at the molecular level and to develop a suitable analytical method for a rapid and sensitive clinical laboratory analysis, the use of electrospray ionization time-of-flight mass spectrometer (ESI-TOFMS) was evaluated. A marked increase in the cysteinylation of Cys34 occurs in chronic liver and kidney diseases and diabetes mellitus. A significant positive correlation was observed between the Cys-Cys34-HSA fraction of plasma samples obtained from 229 patients, as determined by ESI-TOFMS, and the degree of oxidized Cys34-HSA determined by HPLC. The Cys-Cys34-HSA fraction was significantly increased with the progression of liver cirrhosis, and was reduced by branched chain amino acids (BCAA) treatment. The changes in the Cys-Cys34-HSA fraction were significantly correlated with the alternations of the plasma levels of advanced oxidized protein products, an oxidative stress marker for proteins. The binding ability of endogenous substances (bilirubin and tryptophan) and drugs (warfarin and diazepam) to HSA purified from chronic liver disease patients were significantly suppressed but significantly improved by BCAA supplementation. Interestingly, the changes in this physiological function of HSA in chronic liver disease were correlated with the Cys-Cys34-HSA fraction. In conclusion, ESI-TOFMS is a suitable high throughput method for the rapid and sensitive quantification of Cys-Cys34-HSA in a large number of samples for evaluating oxidative stress related chronic disease progression or in response to a treatment

Auxin-dependent interaction of IAA8 with the TIR1 auxin receptor.

<p>(A) Yeast (EGY48::pJK103) cells were co-transformed with bait plasmid, which contained a LexA DNA-binding domain (BD)-TIR1 fusion (BD-TIR1) in pEG202, and prey plasmid, which contained activation domain (AD)-Aux/IAA fusions (AD-Aux/IAA) in pJG4-5. Auxin-dependent interactions in each transformant were assayed by plating on SD(Gal)/-Ura/-His/-Trp/-Leu medium with 100 μM IAA (+IAA). Auxin-independent interactions were examined using the same medium without IAA (−IAA). (B) The BiFC assay was used to detect protein-protein interactions between Aux/IAA repressors and TIR1. <i>nYFP-Aux/IAA</i> fusions or <i>nYFP-GUS</i> and <i>cYFP-TIR1</i> were transiently co-expressed in <i>Arabidopsis</i> mesophyll protoplasts with a nucleus marker plasmid, NLS-tdTomato. To monitor the auxin-dependent interaction, 10 μM IAA was added before microscopic observation (+IAA). BiFC, NLS and BF (top) represent YFP, tdTomato fluorescence, and bright field images, respectively. n: nucleus. Bars  = 10 μm.</p

Subcellular localization of IAA7, IAA8, and IAA17.

<p>GFP fusions of IAA7, IAA8, and IAA17 were transiently expressed in <i>Arabidopsis</i> mesophyll protoplasts. NLS-tdTomato was co-introduced both as a nuclear marker and as a control for transformation. GFP, NLS, and BF (top) represent GFP and tdTomato fluorescence and bright field images, respectively. n: nucleus, c: cytosol. Bars  = 10 μm.</p

Detection of the interaction between IAA8 and ARFs by yeast two-hybrid and BiFC assays.

<p>(A) Yeast (EGY48::pJK103) cells were co-transformed with BD-IAA8 bait plasmid and AD-ARFCTD prey plasmid as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043414#pone-0043414-g004" target="_blank">Figure 4</a>. The strength of each interaction was evaluated by measuring β-gal activity using the o-nitrophenyl β-D-galactopyranoside (ONPG) method. Vector indicates empty vector (negative control experiment). (B) A BiFC assay was carried out to monitor the protein-protein interaction between IAA8 and ARFs. <i>ARF-nYFP</i> fusions or <i>nYFP-GUS</i> and <i>IAA8-cYFP</i> were transiently co-expressed in <i>Arabidopsis</i> mesophyll protoplasts with a nuclear marker plasmid, NLS-tdTomato. BiFC, NLS, and BF (top) represent YFP, tdTomato fluorescence, and bright field images, respectively. n: nucleus. Bars  = 10 μm.</p

<i>IAA8</i> overexpression suppresses the auxin-inducible expression of early auxin-responsive genes in roots.

<p>Wild-type (Col-0) and XVE::IAA8 (line #8, #11) roots treated first with 10 μM β-estradiol for 24 h and then with 1 μM IAA for 0 to 60 min were subjected to semi-quantitative RT-PCR analysis of the auxin marker genes, <i>IAA1</i>, <i>IAA19</i> and <i>GH3.3</i> together with endogenous <i>IAA8</i> gene expression (end) and of both endogenous and exogenous (end + exo) <i>IAA8</i> gene expression. EF1α was used as an internal control. All semi-quantitative RT-PCR figures are representative of three biological replicates.</p