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Hairless (HR) has been shown to regulate hair follicle (HF) morphogenesis and hair cycling. The Hr mutant hair loss mouse referred to as 'hairpoor' ( Hr Hp) displays overexpression of the HR protein through translational derepression. In this study, we found that 64 miRNAs were differentially expressed between the skin of Hr Hp/ Hr Hp and wild type mice at P7 using miRNA-microarray analysis and miR-31 displayed the most reduced expression in Hr Hp/ Hr Hp skin. In vivo observation and investigation using an in vitro reporter expression system revealed that miR-31 and pri-miR-31 were consistently down-regulated in the HR over-expressed condition. In addition, we found that the transforming growth factor β 2 ( Tgf-β2), a known catagen inducer, is the putative target of miR-31. Furthermore, Tgf-β2 level was also increased in HR over-expressed keratinocyte and Hr Hp/ Hr Hp mice. These study results suggest that HR controls Tgf-β2 expression via regulation of miR-31, thus causing abnormal hair cycle in Hr Hp/ Hr Hp mice. J. Cell. Physiol. 230: 2075-2085, 2015. © 2015 Wiley Periodicals, Inc. [ABSTRACT FROM AUTHOR]

In this study, a micro-pull-off testing machine is developed for reliable measurement of the adhesive force on micro- and nano-scale contact areas. The measuring scheme and working principle of the equipment are based on a reliable measurement technique that uses a commercial atomic force microscope (AFM). Adhesive-force-only measuring equipment is manufactured to overcome the inherent limitations of commercial AFMs, and an adhesive force map with a 1-μm interval is obtained around a micro-machined pattern of radius 2.25 μm. Use of the proposed technique and equipment could reduce deviations of the measured bond strength of an adhesive protein compared with those we have previously reported. Moreover, the proposed technique and equipment could be further developed to measure the binding force of even a single molecule if a nano-scale pattern is fabricated. [ABSTRACT FROM AUTHOR]

The ubiquitin fusion degradation (UFD) pathway is a proteolytic system conserved in yeast and mammals in which an uncleavable ubiquitin moiety linked to the N terminus of a protein functions as a degradation signal of the fusion protein. Although key components of the UFD pathway in yeast have been identified, the E3 enzyme of the human UFD pathway has not been studied. In this work, we show that TRIP12 is the E3 enzyme of the human UFD pathway. Thus, TRIP12 catalyzes in vitro ubiquitination of UFD substrates in conjunction with E1, E2, and E4 enzymes. Knockdown of TRIP12 stabilizes not only artificial UFD substrates but a physiological substrate UBB[sup+1. Moreover, TRIP12 knockdown reduces UBB[sup+1]-induced cell death in human neuroblastoma cells. Surprisingly, complementation of TRIP12 knockdown cells with the TRIP12HECT domain mostly restores efficient degradation of UFD substrates, indicating that the TRIP12 HECT domain can act as the E3 enzyme for the UFD pathway in human cells. The TRIP12 HECT domain directs ubiquitination of UFD substrates in vitro and can be specifically cross-linked to the ubiquitin moiety of the substrates in vivo, suggesting that the TRIP12 HECT domain possesses a noncovalent ubiquitin-binding site. In addition, we demonstrate that UbΔGG, a mutant ubiquitin that cannot be conjugated to other proteins, is a substrate of the TRIP12HECT domain both in vivo and in vitro, indicating that the C-terminal extension fused to the uncleavable ubiquitin is not required for substrate recognition in the UFD pathway. These results provide new insights into the mechanism of the mammalian UFD pathway and the functional non equivalence of different HECT domains. [ABSTRACT FROM AUTHOR]

Abstract: The NEDD8 pathway plays an essential role in various physiological processes, such as cell cycle progression and signal transduction. The conjugation of NEDD8 to target proteins is initiated by the NEDD8-activating enzyme composed of APP-BP1 and Uba3. In the present study, we show that APP-BP1 is degraded by ubiquitin-dependent proteolysis. To study biological functions of TRIP12, a HECT domain-containing E3 ubiquitin ligase, we used the yeast two-hybrid system and identified APP-BP1 as its binding partner. Immunoprecipitation analysis showed that TRIP12 specifically interacts with the APP-BP1 monomer but not with the APP-BP1/Uba3 heterodimer. Overexpression of TRIP12 enhanced the degradation of APP-BP1, whereas knockdown of TRIP12 stabilized it. In vitro ubiquitination assays revealed that TRIP12 functions as an E3 enzyme of APP-BP1 and additionally requires an E4 activity for polyubiquitination of APP-BP1. Moreover, neddylation of endogenous CUL1 was increased in TRIP12 knockdown cells, while complementation of the knockdown cells with TRIP12 lowered neddylated CUL1. Our data suggest that that TRIP12 promotes degradation of APP-BP1 by catalyzing its ubiquitination, which in turn modulates the neddylation pathway. [Copyright &y& Elsevier]

A new method is proposed and developed to measure adhesive forces by use of the force-distance curve of a micro cantilever with an extremely small amount of testing material such as adhesive proteins. The contact area should be well-controlled at a reasonable value. Even though the area is desired to be as small as possible, a contact region of several micrometers by several micrometers is adopted in order to avoid obtaining meaningless measured values and uncertainty in the contact areas. An AFM cantilever is used after having been modified with a micro glass bead to enlarge the contact area for adhesion. A glass plate with micro-scale circular patterns is fabricated from a glass wafer by micro-machining processes in order to control precisely the contact area in adhesion tests. In the proposed method the adhesive materials are directly applied to the bead attached at the AFM cantilever before it is applied on the top area of the truncated cone on the fabricated glass plate. The developed method is applied to measure the adhesive forces of Cell-Tak® (which is a commercial extracted mussel adhesive) and recombinant Mgfp-5 (which is a recombinant mussel adhesive protein) and the statistical credibility of the measured adhesive force data is enormously improved as a result. [ABSTRACT FROM AUTHOR]

Purpose: This study elucidates the mechanism of the physiological effect of cannabidiol (CBD) by assessing its impact on lipopolysaccharide (LPS)-induced inflammation in RWPE-1 cells and prostatitis-induced by 17β-estradiol and dihydrotestosterone in a rat model, focusing on its therapeutic potential for chronic prostatitis/chronic pelvic pain syndrome (CP/CPPS). Materials and Methods: RWPE-1 cells were stratified in vitro into three groups: (1) controls, (2) cells with LPS-induced inflammation, and (3) cells with LPS-induced inflammation and treated with CBD. Enzyme-linked immunosorbent assays and western blots were performed on cellular components and supernatants after administration of CBD. Five groups of six Sprague– Dawley male rats were assigned: (1) control, (2) CP/CPPS, (3) CP/CPPS and treated with 50 mg/kg CBD, (4) CP/CPPS and treated with 100 mg/kg CBD, and (5) CP/CPPS and treated with 150 mg/kg CBD. Prostatitis was induced through administration of 17β-estradiol and dihydrotestosterone. After four weeks of CBD treatment, a pain index was evaluated, and prostate tissue was collected for subsequent histologic examination and western blot analysis. Results: CBD demonstrated efficacy in vivo for CP/CPPS and in vitro for inflammation. It inhibited the toll-like receptor 4 (TLR4)/nuclear factor-kappa B (NF-κB) pathway by activating the CB2 receptor, reducing expression of interleukin-6, tumor necrosis factor-alpha, and cyclooxygenase-2 (COX2) (p<0.01). CBD exhibited analgesic effects by activating and desensitizing the TRPV1 receptor. Conclusions: CBD inhibits the TLR4/NF-κB pathway by activating the CB2 receptor, desensitizes the TRPV1 receptor, and decreases the release of COX2. This results in relief of inflammation and pain in patients with CP/CPPS, indicating CBD as a potential treatment for CP/CPPS. [ABSTRACT FROM AUTHOR]

The E3 ubiquitin ligase membrane-associated ring-CH–type finger 2 (MARCH2) is known to be involved in intracellular vesicular trafficking, but its role in the early secretory pathway between the endoplasmic reticulum (ER) and Golgi compartments is largely unknown. Human ER–Golgi intermediate compartment protein 2 (ERGIC2) and ERGIC3 are orthologs of Erv41 and Erv46 in yeast, proteins that form a heteromeric complex, cycle between the ER and Golgi, and function as cargo receptors in both anterograde and retrograde protein trafficking. Here, we report that MARCH2 directs ubiquitination and subsequent degradation of ERGIC3 and that MARCH2 depletion increases endogenous ERGIC3 levels. We provide evidence that the lysine residues at positions 6 and 8 of ERGIC3 are the major sites of MARCH2-mediated ubiquitination. Of note, MARCH2 did not significantly decrease the levels of an ERGIC3 variant with lysine-to-arginine substitutions at residues 6 and 8. Wealso show that ERGIC3 binds to itself or to ERGIC2, whereas ERGIC2 is unable to interact with itself. Our results indicate that α1-antitrypsin and haptoglobin are likely to be cargo proteins of ERGIC3. We further observed that α1-antitrypsin and haptoglobin specifically bind to ERGIC3 and that ERGIC3 depletion decreases their secretion. Moreover, MARCH2 reduced secretion of α1-antitrypsin and haptoglobin, and coexpression of the ubiquitination-resistant ERGIC3 variant largely restored their secretion, suggesting that MARCH2-mediated ERGIC3 ubiquitination is the major cause of the decrease in trafficking of ERGIC3-binding secretory proteins. Our findings provide detailed insights into the regulation of the early secretory pathway by MARCH2 and into ERGIC3 function. [ABSTRACT FROM AUTHOR]

Dystroglycan is a ubiquitous membrane protein that functions as a mechanical connection between the extracellular matrix and cytoskeleton. In skeletal muscle, dystroglycan plays an indispensable role in regulating muscle regeneration; a malfunction in dystroglycan is associated with muscular dystrophy. The regulation of dystroglycan stability is poorly understood. Here, we report that WWP1, a member of NEDD4 E3 ubiquitin ligase family, promotes ubiquitination and subsequent degradation of β-dystroglycan. Our results indicate that dystrophin and utrophin protect β-dystroglycan from WWP1-mediated degradation by competing with WWP1 for the shared binding site at the cytosolic tail of β-dystroglycan. In addition, we show that a missense mutation (arginine 440 to glutamine) in WWP1—which is known to cause muscular dystrophy in chickens—increases the ubiquitin ligase-mediated ubiquitination of both β-dystroglycan and WWP1. The R440Q missense mutation in WWP1 decreases HECT domain-mediated intramolecular interactions to relieve autoinhibition of the enzyme. Our results provide new insight into the regulation of β-dystroglycan degradation by WWP1 and other Nedd4 family members and improves our understanding of dystroglycan-related disorders. [ABSTRACT FROM AUTHOR]

Signaling through many transmembrane receptors is terminated by their sorting to the intraluminal vesicles (ILVs) of multivescular bodies (MVBs) and subsequent lysosomal degradation. ILV formation requires the endosomal sorting complex required for transport (ESCRT) machinery. CC2D1A and CC2D1B interact with the CHMP4 family of proteins, the major subunit of the ESCRT-III complex, however, their roles in receptor degradation and signaling are poorly defined. Here, we report that CC2D1A binds to CHMP4B polymers formed on endosomes to regulate the endosomal sorting pathway. We show that depletion of CC2D1A and B accelerates degradation of EGFR and elicits rapid termination of its downstream signaling through ERK1 and 2. Depletion of CC2D1A and B promotes sorting of EGFR to ILV leading to its rapid lysosomal degradation. In addition, we show that knockdown of CC2D1A and B has similar effects on degradation and downstream signaling of another membrane receptor, TLR4. Thus, these findings suggest that CC2D1A and B may have broad effects on transmembrane receptors by preventing premature ILV sorting and termination of signaling. [ABSTRACT FROM AUTHOR]

Background MicroRNAs (miRNAs) are small non-coding RNA molecules that mediate the biological cellular processes via regulation of target genes through translational repression or mRNA degradation. Among various miRNAs, miRNA-199a (miR-199a) has been known to be involved in cancer development and progression, protection of cardiomyocyte, and skeletal formation. Objective Although miR-199a-5p was studied in various cell types, the role of miR-199a-5p and its target genes in skin keratinocyte have not been documented. In this study, we identified target genes of miR-199a-5p in skin keratinocyte. Methods In order to identify the target of miR-199a-5p in keratinocyte, microarray analysis was performed. The relative expression of candidate target genes was investigated using quantitative RT-PCR and western blot analysis. To determine whether their expression was directly regulated by miR-199a-5p, luciferase reporter assay was performed. In order to investigate expression of target genes in cutaneous squamous cell carcinoma, immunohistochemistry was performed. Results We identified new target genes, Bcam , Fzd6 , and Wnt7a , as well as previously known targets, Ddr1 and Podxl. We found that their expressions were directly regulated by miR-199a-5p in the skin keratinocyte using in vitro study and observed that expression of miR-199a-5p was inversely correlated with those of BCAM, FZD6 and DDR1 in the cSCC. In addition, overexpression of miR-199a-5p resulted in inhibition of the migratory capability of the skin keratinocyte. Conclusion These results suggested that miR-199a-5p plays a role in pathogenesis of cSCC via inhibition of invasiveness through regulation of BCAM, FZD6 and DDR1 expression. [ABSTRACT FROM AUTHOR]

Background: Hair follicle stem cells (HFSC) play an essential role in the maintenance of hair homeostasis; during the hair cycle, HFSC remain quiescent for most of its duration. The hairpoor mouse (+ /HrHp), an animal model of Marie-Unna hypotrichosis (MUHH), overexpresses hairless in the bulge, inner root sheath, and outer root sheath of HF and shows the same phenotype as in MUHH patients manifesting sparse hair with progression to alopecia with age. The aim of this study was to gain an understanding of the hair cycle and the status of HFSC during the hair cycle of the hairpoor mouse in order to delineate the pathogenesis of MUHH. Methods: H&E staining was performed in order to define the state of the hair follicle. FACS analysis and immunostaining were performed at the 1st and 2nd telogen stages for observation of the HFSC. A label retaining assay was performed to determine the quiescent state of hair follicles. qRT-PCR was performed to determine expression of factors involved in niche signaling and Wnt signaling. Results: We observed a drastic decrease in the number of hair follicles after the 1st telogen, followed by an intensified disturbance in the hair cycle with shorter anagen as well as 2nd telogen in the hairpoor mouse. A dramatic reduction in the number of CD34 expressing bulges as well as cells was observed at the telogen of the HFs, with prominent high proliferation of bulge cells, suggesting the loss of HFSC quiescence in the hairpoor mouse. The increased cell proliferation in HF was reiterated following the synchronization of the hair cycle, leading to acceleration of HF cycling. Reduced expression of Fgf18 and Bmp6, the factors involved in HFSC quiescence, was observed in the HFSC niche of the hairpoor mouse. In addition, disturbed expression of Wnt signaling molecules including Wnt7b, Wnt10b, and Sfrp1 was observed, which induced the telogen-to-anagen transition of HFs in the hairpoor mouse. Conclusions: These results indicate that the quiescent state of HFSC is not properly maintained in the hairpoor mouse, consequently leading HFs to the completely disarrayed hair cycle. These findings may provide an understanding of an underlying mechanism for development of alopecia with age in MUHH patients. [ABSTRACT FROM AUTHOR]

The 26 S proteasome is a large multi-subunit protein complex that degrades ubiquitinated proteins in eukaryotic cells. Proteasome assembly is a complex process that involves formation of six- and seven-membered ring structures from homologous subunits. Here we report that the assembly of hexameric Rpt ring of the 19 S regulatory particle (RP) requires nucleotide binding but not ATP hydrolysis. Disruption of nucleotide binding to an Rpt subunit by mutation in the Walker A motif inhibits the assembly of the Rpt ring without affecting heterodimer formation with its partner Rpt subunit. Coexpression of the base assembly chaperones S5b and PAAF1with mutant Rpt1 and Rpt6, respectively, relieves assembly inhibition of mutant Rpts by facilitating their interaction with adjacent Rpt dimers. The mutation in the Walker B motif which impairs ATP hydrolysis does not affect Rpt ring formation. Incorporation of a Walker B mutant Rpt subunit abrogates the ATPase activity of the 19 S RP, suggesting that failure of the mutant Rpt to undergo the conformational transition from an ATP-bound to an ADP-bound state impairs conformational changes in the other five wild-type Rpts in the Rpt ring. In addition, we demonstrate that the C-terminal tails of Rpt subunits possessing core particle (CP)-binding affinities facilitate the cellular assembly of the 19 SRP, implying that the 20 SCP may function as a template for base assembly in human cells. Taken together, these results suggest that the ATP bound conformational state of an Rpt subunit with the exposed C-terminal tail is competent for cellular proteasome assembly. [ABSTRACT FROM AUTHOR]

Osmotic stress causes profound perturbations of cell functions. Although the adaptive responses required for cell survival upon osmotic stress are being unraveled, little is known about the effects of osmotic stress on ubiquitin-dependent proteolysis. We now report that hyperosmotic stress inhibits proteasome activity by activating p38 MAPK. Osmotic stress increased the level of polyubiquitinated proteins in the cell. The selective p38 inhibitor SB202190 decreased osmotic stress-associated accumulation of polyubiquitinated proteins, indicating that p38 MAPK plays an inhibitory role in the ubiquitin proteasome system. Activated p38 MAPK stabilized various substrates of the proteasome and increased polyubiquitinated proteins. Proteasome preparations purified from cells expressing activated p38 MAPK had substantially lower peptidase activities than control proteasome samples. Proteasome phosphorylation sites dependent on p38 were identified by measuring changes in the extent of proteasome phosphorylation in response to p38 MAPK activation. The residue Thr-273 of Rpn2 is the major phosphorylation site affected by p38 MAPK. The mutation T273A in Rpn2 blocked the proteasome inhibition that is mediated by p38 MAPK. These results suggest that p38 MAPK negatively regulates the proteasome activity by phosphorylating Thr-273 of Rpn2. [ABSTRACT FROM AUTHOR]

Abstract: ER-associated protein degradation (ERAD) is a protein quality control system of ER, which eliminates misfolded proteins by proteasome-dependent degradation and ensures export of only properly folded proteins from ER. Herp, an ER membrane protein upregulated by ER stress, is implicated in regulation of ERAD. In the present study, we show that Herp interacts with members of the ubiquilin family, which function as a shuttle factor to deliver ubiquitinated substrates to the proteasome for degradation. Knockdown of ubiquilin expression by small interfering RNA stabilized the ERAD substrate CD3δ, whereas it did not alter or increased degradation of non-ERAD substrates tested. CD3δ was stabilized by overexpressed Herp mutants which were capable of binding to ubiquilins but were impaired in ER membrane targeting by deletion of the transmembrane domain. Our data suggest that Herp binding to ubiquilin proteins plays an important role in the ERAD pathway and that ubiquilins are specifically involved in degradation of only a subset of ubiquitinated targets, including Herp-dependent ERAD substrates. [Copyright &y& Elsevier]

T cells play an important role in the initiation and the progression of rheumatoid arthritis (RA) and depletion of potentially pathogenic T cells was suggested as an important therapeutic protocol. We determined if rosmarinic acid (RosA), known as a secondary metabolite from herbal plants, had apoptotic activity toward T cells from RA patients and further verified target T-cell subsets. CD3+CD25+activated T-cell subsets from most of the RA patients displayed significantly higher apoptosis rates than did the PBMCs and total CD3+T cells. Furthermore, activated and effector CD4+T cells, including CD4+CD25+and CD4+CD45RO+T cells, had a tendency of being more susceptible to RosA-induced apoptosis than that of resting and na?ve T-cell subsets. RosA induced the release of cytochromecfrom mitochondria and the blockage of mitochondrial depolarization inhibited apoptosis. Taken together, these results suggest that RosA induces apoptosis of activated T-cell subsets from RA patients via a mitochondrial pathway. [ABSTRACT FROM AUTHOR]

Human Fas associated factor 1 protein (hFAF1) is involved in the positive regulation of Fas signaling even though it can not initiate the signal for itself. By chromosomal assignment using somatic cell hybrids (CASH), the hFAF1 gene was located on human chromosome I between markers D1S443 and DIS197. The hFAF1 gene was mapped to human chromosome band 1p32 by FISH utilizing a genomic PAC clone containing the gene. In genomic Southern analysis using hFAF1 cDNA as a probe, several bands appeared in three different restriction enzyme digestions. The single band appearance in FISH analysis compared to several bands in Southern blots implies that the hFAF1 gene would he rather big or that an additional hFAF1 gene isotype(s) might be present in close vicinity. [ABSTRACT FROM AUTHOR]

Background: Tuberculosis is a major health concern in not only humans, but also in non-human primates. In this paper, we report recent cases of Mycobacterium tuberculosis in cynomolgus monkeys from Cambodia used in transplantation research in a Korean facility and describe a program instituted to prevent and control subsequent infections. Case presentation: All monkeys were antibody negative for tuberculosis during quarantine; however, suspected tuberculosis gross lesions were observed in two cynomolgus monkeys who underwent allograft kidney transplantation. Lung tissue from one monkey was found to be weakly positive by PCR for detection of M. tuberculosis. After PCR confirmation of tuberculosis, we decided to sacrifice the remaining animals and instituted a program for preventing subsequent infections. During necropsy of the remaining monkeys, two additional suspected tuberculosis cases were observed. A total of four monkeys with nodular lesions in the respiratory tract, suspected to be tuberculosis, demonstrated no clinical signs. Acid-fast bacilli were identified on slides from the lung or liver in all four monkeys. Two of four monkeys tested PCR positive. We decided that new monkeys entering from Cambodia should undergo a single gastric aspiration PCR and tuberculin skin testing (TST) every 2 weeks until four consecutive negatives to detect latent tuberculosis are obtained before starting experiments. Monkeys should then undergo a chest X-ray monthly and TST every 6 months. Conclusions: Detection of latent tuberculosis by an effective preventive screening program before starting experiments is an essential process to reduce the risk of reactivation of tuberculosis, especially in studies using immunosuppressive drugs. It also serves to protect the health of captive non-human primates, their caretakers and researchers. [ABSTRACT FROM AUTHOR]

Purpose: MicroRNAs (miRNAs) are noncoding RNAs that play roles as tumor suppressors or oncogenes by regulating the expression of target genes via binding to seed-match sequences. Polymorphisms in the miRNA-binding site of a target gene can alter miRNA binding and potentially affect the risk of cancer. The objective of this study was to identify singlenucleotide polymorphisms (SNPs) in miRNA-binding sites and assess their involvement in the risk of colorectal cancer (CRC). Materials and methods: SNPs in the 3' untranslated regions of genes were selected and assessed for their effects on CRC risk in Korean population using participants in Korean Cancer Prevention Study-II. A detailed study was carried out with the SNP rs7930 in the 3' untranslated region of the translocase of outer mitochondrial membrane 20 (TOMM20) gene. A case-control study (1,545 controls and 620 CRC cases) was conducted to analyze the relationship between polymorphism at rs7930 and the risk of CRC. An interacting miRNA was predicted using webbased software programs, and its interaction with rs7930 in CRC cell lines was investigated by using a luciferase assay. Results: Individuals carrying the rs7930 AG genotype (G allele) had a 1.721-fold increased risk for CRC in comparison with those with the AA genotype (A allele). The miRNA miR-4273-5p was found to specifically interact with the A allele of rs7930 and to suppress the expression of the target gene (TOMM20) in CRC cell lines. Conclusion: rs7930 is an independent genetic risk factor for CRC susceptibility. Our study suggests a mechanism of how this SNP contributes to CRC carcinogenesis. [ABSTRACT FROM AUTHOR]

Srpr is a gene encoding α subunit of the signal recognition particle receptor which is involved in the targeting and translocation of nascent secretory and membrane proteins to the endoplasmic reticulum. Previous studies showed aberrant expression of Srpr in several cell types with abnormal growth rate. Although Srpr is expressed in various tissues including skin, the role of Srpr in keratinocytes and regulation of its expression by miRNAs have not been studied. In this study, we investigated the role of SRPR and regulation of its expression by miRNA in skin keratinocytes. We found that SRPR was highly expressed in epidermal keratinocytes and regulated keratinocyte proliferation by affecting cell cycle progression. We also demonstrated that miR-330-5p directly inhibits Srpr expression. These data suggest that miR-330-5p-mediated regulation of the SRPR level is needed for the regulation of proliferation of epidermal keratinocytes. [ABSTRACT FROM AUTHOR]

The microRNAs (miRNAs) are a class of small non-coding RNA molecules that play important roles in cellular processes by regulating gene expression at the post-transcriptional level. In skin biology, several miRNAs have been shown to be associated with differentiation, migration and apoptosis of keratinocytes, and regulation of the hair cycle. Although the biological role of miR-330-5p has been reported in several cancers and cells, the function and molecular mechanism of miR-330-5p in skin keratinocytes have not been identified. In this study, we found that miR-330- 5p inhibited the proliferation and migration of mouse keratinocytes. Among the candidate target genes of miR-330-5p searched using microarray analysis, we found that the expression of Pdia3 was directly regulated by miR-330-5p in the mouse keratinocyte. Moreover, inhibition of Pdia3 expression caused decreased proliferation and migration ability of mouse keratinocytes. Additionally, expressions of miR-330-5p and Pdia3 displayed an inverse correlation with respect to the hair cycle stage. These results indicated that regulation of Pdia3 expression by miR-330-5p is important in maintaining the hair cycle through regulation of the proliferation and migration capability of keratinocytes. [ABSTRACT FROM AUTHOR]

Background: Adiponectin levels have been shown to be associated with colorectal cancer (CRC). Furthermore, a newly identified adiponectin receptor, T-cadherin, has been associated with plasma adiponectin levels. Therefore, we investigated the potential for a genetic association between T-cadherin and CRC risk. Result: We conducted a case-control study using the Korean Cancer Prevention study-II cohort, which is composed of 325 CRC patients and 977 normal individuals. Study results revealed that rs3865188 in the 5' flanking region of the T-cadherin gene (CDH13) was significantly associated with CRC (p = 0.0474). The odds ratio (OR) for the TT genotype as compared to the TA + AA genotype was 1.577 (p = 0.0144). In addition, the interaction between CDH13 and the adiponectin gene (APN) for CRC risk was investigated using a logistic regression analysis. Among six APN single nucleotide polymorphisms (rs182052, rs17366568, rs2241767, rs3821799, rs3774261, and rs6773957), an interaction with the rs3865188 was found for four (rs2241767, rs3821799, rs3774261, and rs6773957). The group with combined genotypes of TT for rs3865188 and GG for rs377426 displayed the highest risk for CRC development as compared to those with the other genotype combinations. The OR for the TT/GG genotype as compared to the AA/AA genotype was 4.108 (p = 0.004). Furthermore, the plasma adiponectin level showed a correlation with the gene-gene interaction, and the group with the highest risk for CRC had the lowest adiponectin level (median, 4.8 μg/mL for the TT/GG genotype vs.7.835 μg/mL for the AA/AA genotype, p = 0.0017). Conclusions: The present study identified a new genetic factor for CRC risk and an interaction between CDH13 and APN in CRC risk. These genetic factors may be useful for predicting CRC risk. [ABSTRACT FROM AUTHOR]

Exposure to several persistent organic pollutants (POPs) has been linked to alteration of lipid metabolism. Adipokines, such as leptin and adiponectin, are hormones that play roles in lipid metabolism, and have been suggested as markers of health effects that may lead to obesity. To date, only serum adipokines have been associated with POPs exposure. In the present study, for the first time, the associations between leptin and adiponectin in breast milk, and several POPs in serum were investigated among 82 lactating Korean women between 25 and 46 years of age. Breast milk adipokines are important because adipokine intake of infant through breastfeeding may influence the growth of infants. The median concentrations of leptin and adiponectin in skimmed milk of the Korean lactating women were 17.9 ng/L and 16.5 μg/L, respectively. Leptin concentrations in breast milk were negatively associated with ∑hexachlorohexane (HCH), oxychlordane, ∑chlordane, or 2,2′,4,4′,5,5′-hexachlorobiphenyl (CB 153) levels in maternal serum. Linear relationships between adiponectin and POPs concentrations were not observed, however, nonmonotonic relationship which showed generally positive associations was suggested for p,p′-dichlorodiphenyltrichloroethane (DDT) and ∑chlordane in quartile analysis. Our observations show that POPs at the current level of exposure may be associated with the alteration of lipid metabolism among lactating women. Implication of adipokine transfer to breastfed infants is of concern and deserves further investigation. [ABSTRACT FROM AUTHOR]

The Hairless (Hr), a transcription factor, is expressed in the suprabasal cell layer of the interfollicular epidermis and the lower portion of the hair follicle epithelium, where its expression is dependent on the hair cycle. Recently, we reported a new Hr mutant mouse, HrHp, in which the hairless protein (HR) was overexpressed. In this study, we documented abnormal formation of inner root sheath (IRS), suppressed expression of Dlx3, and IRS keratins in the HrHp/HrHp skin. We also found that HR down-regulated Dlx3 mRNA expression through suppression of Dlx3 promoter activity. In addition, we showed that Dlx3 regulated the expression of IRS keratins. Our results demonstrate that regulation of Dlx3 by HR affects the IRS keratin expression, thus modulating the formation of IRS of hair follicle. [ABSTRACT FROM AUTHOR]

Abstract: Ethnopharmacological relevance: Dioscorea japonica Thunb. has been traditionally used to treat polyuria and diabetes in Korea. Aim of the study: We previously report the effects of Dioscorea japonica Thunb. extract on glucose control, NGF induction, and neuroprotection in a rodent diabetic model. Since the most potent fraction, DA-9801, was identified from a mixture of Dioscorea japonica Thunb. (DJ) and Dioscorea nipponica Makino (DN) following bioactivity-guided fractionation, here, we investigated the potential mechanism of the extract activity against diabetic peripheral neuropathy (DPN). Materials and methods: A 1:3 mixture of DJ and DN was extracted with ethanol (DA-9801) and further fractionated into an ethylacetate-soluble fraction (DA-9801E). Effects of these extracts on neurite outgrowth were measured in PC-12 cells and DRG neurons. Effects on cell viability and TrkA phosphorylation were evaluated in PC-12 cells. NGF induction effect was determined in primary Schwann cells as well as IMS32 cells (immortalized Schwann cells). Results: No cytotoxicity was observed in PC-12 cells at the concentration below 500μg/ml of either DA-9801 or DA-9801E. DA-9801 and DA-9801E at 100μg/ml and 10μg/ml, respectively, showed a significant effect on neurite outgrowth in PC-12 cells and DRG neurons in the presence of or absence a low concentration of NGF (2ng/ml). The Trk-A phosphorylation effect of DA9801 was confirmed in PC-12 cells. An NGF induction effect of these extracts was not detected in either IMS-32 cells, or primary Schwann cells. Conclusions: The NGF agonistic activity of DA-9801 and DA-9801E was demonstrated, which may contribute to their neuroprotective effect against DPN. Studies of the detailed mechanism of these extracts as well as identification of the active components are warranted for the development of an anti-DPN drug from DJ and DN. [Copyright &y& Elsevier]

Abstract: Ginseng (the root of Panax ginseng C.A. Meyer, family Araliaceae) possesses various biological activities, including anti-inflammatory and anti-tumor actions. However, their topical effect on atopic dermatitis (AD) has not been studied yet. The aim of this study was to examine the therapeutic effects of topical Korean red ginseng saponin fraction (KRGS) and its genuine constituents on AD-like skin lesions in an AD mouse model. The therapeutic effect of topical KRGS and ginsenosides in 2-chloro-1,3,5-trinitrobenzene (TNCB)-treated NC/Nga mice was assessed by measuring the skin severity scores, ear thickness, histological changes of lesional skin including mast cell count, tissue tumor necrosis factor (TNF)-α, interleukin (IL)-4, and interferon (IFN)-γ mRNA expression, and total serum IgE. Topical administration of 0.1% KRGS, 0.1% Rh2 and 0.1% Rh2+0.1% Rg3 significantly reduced the clinical skin severity scores, ear thickness and mast cell infiltration in the TNCB-induced AD-like skin lesions. Topical application of KRGS and its constituents significantly reduced TNCB-induced increase in ear TNF-α and IL-4 mRNA expression, but not IFN-γ mRNA expression. There was little change of serum total IgE level by topical KRGS and its constituents. In this study, topical KRGS and ginsenosides Rh2 and Rg3 were found to exert an anti-inflammatory effect in vivo and proved to be beneficial in an animal model of AD. Our results suggest that KRGS and its ginsenosides Rh2 and Rg3 have potential as a topical agent for the treatment of AD. [ABSTRACT FROM AUTHOR]

Adiponectin is associated with obesity and insulin resistance. To date, there has been no genome-wide association study (GWAS) of adiponectin levels in Asians. Here we present a GWAS of a cohort of Korean volunteers. A total of 4,001 subjects were genotyped by using a genome-wide marker panel in a two-stage design (979 subjects initially and 3,022 in a second stage). Another 2,304 subjects were used for follow-up replication studies with selected markers. In the discovery phase, the top SNP associated with mean log adiponectin was rs3865188 in CDH13 on chromosome 16 (p = 1.69 × 10−15 in the initial sample, p = 6.58 × 10−39 in the second genome-wide sample, and p = 2.12 × 10−32 in the replication sample). The meta-analysis p value for rs3865188 in all 6,305 individuals was 2.82 × 10−83. The association of rs3865188 with high-molecular-weight adiponectin (p = 7.36 × 10−58) was even stronger in the third sample. A reporter assay that evaluated the effects of a CDH13 promoter SNP in complete linkage disequilibrium with rs3865188 revealed that the major allele increased expression 2.2-fold. This study clearly shows that genetic variants in CDH13 influence adiponectin levels in Korean adults. [ABSTRACT FROM AUTHOR]

Hairpoor mice ( Hr Hp) were derived through N-ethyl- N-nitrosourea (ENU) mutagenesis. These mice display sparse and short hair in the Hr Hp/ + heterozygous state and complete baldness in the Hr Hp/ Hr Hp homozygous state. This phenotype was irreversible and was inherited in an autosomal semidominant manner. Hair follicles (HFs) of Hr Hp/ + mice underwent normal cycling and appeared normal, although smaller than those of the wild-type mice. In contrast, HFs of Hr Hp/ Hr Hp mice became cyst-like structures by postnatal day (P) 21. The number and length of vibrissae decreased in a dose-dependent manner as the number of mutant alleles increased. A positional candidate gene approach was used to identify the gene responsible for the hairpoor phenotype. Genetic linkage analysis determined that the hairpoor locus is 2 cm from D14Mit34 on chromosome 14. Sequence analysis of the exons of the candidate gene hairless revealed a T-to-A transversion mutation at nucleotide position 403 (exon 2), presumably resulting in abolishment of an upstream open reading frame (uORF). In addition, we also found that the near-naked mouse ( Hr N), a spontaneously arising mutant, harbors a A402G transition in its genome. Both mutations were in the uATG codon of the second uORF in the 5′ UTR and corresponded to the mutations identified in Marie Unna Hereditary Hypotrichosis (MUHH) patients. In the present study we describe the phenotype, histological morphology, and molecular etiology of an animal model of MUHH, the hairpoor mouse. [ABSTRACT FROM AUTHOR]

Abstract??In this paper, we present a simple and cost-effective replication method of high-aspect-ratio polymer nanopillar array as a biomimetic gecko?s foot hair prototype. A UV nano embossing process was applied for the replication of polymer nanopillar arrays. Highly ordered straight nanoporous AAO (anodic aluminum oxide) templates were utilized as reusable master molds. Densely arranged high-aspect-ratio nanopillar arrays have been successfully fabricated by means of the UV nano embossing process with the AAO mold. Pull-off force measurements were carried out to characterize the adhesive force of the replicated nanopillar arrays on the polymer substrates based on the force?distance curves obtained from the atomic force microscope (AFM) with a modified AFM cantilever. The force measurement results showed that the larger diameter and the taller height of the nanopillars result in the larger adhesive force. [ABSTRACT FROM AUTHOR]

Abstract: Brugada syndrome (BS) is an inherited cardiac disorder associated with a high risk of sudden cardiac death and is caused by mutations in the SCN5A gene encoding the cardiac sodium channel α-subunit (Nav1.5). The aim of this study was to identify the genetic cause of familial BS and characterize the electrophysiological properties of a novel SCN5A mutation (W1191X). Four families and one patient with BS were screened for SCN5A mutations by PCR and direct sequencing. Wild-type (WT) and mutant Nav1.5 channels were expressed in tsA201 cells, and the sodium currents (I Na) were analyzed using the whole-cell patch-clamp technique. A novel mutation, W1191X, was identified in a family with BS. Expression of the WT or the mutant channel (Nav1.5/W1191X) co-transfected with the β1-subunit in tsA201 cells resulted in a loss of function of Nav1.5 channels. While voltage-clamp recordings of the WT channel showed a distinct acceleration of Nav1.5 activation and fast inactivation kinetics, the Nav1.5/W1191X mutant failed to generate any currents. Co-expression of the WT channel and the mutant channel resulted in a 50% reduction in I Na. No effect on activation and inactivation were observed with this heterozygous expression. The W1191X mutation is associated with BS and resulted in the loss of function of the cardiac sodium channel. [Copyright &y& Elsevier]

Abstract: A novel autosomal recessive mutant was produced using N-ethyl-N-nitrosourea mutagenesis. The characteristics of the mutant mice included progressive irreversible hair loss within a month of birth, wrinkled skin, and long curved nails. Linkage analysis revealed that the causative gene is linked to D14Mit193 on chromosome 14. Sequence analysis of the complete cDNA of the candidate gene, hairless (Hr), identified a homozygous G-to-T transition at nucleotide 3572, leading to the substitution of glycine by tryptophan, designated Gly960Trp. This missense mutation occurs in the vicinity of repression domain 3 of the hairless protein (HR). This allele was named Hr m1Enu . The relative amounts of Hr mRNA and HR protein determined by real-time PCR and Western blot analyses, respectively, were slightly elevated in the mutant mice. Quantitative real-time PCR analysis revealed the increased expression of Kc1 and Vdr in the mutant mice, whereas the expression of Nrs1 and Krtap16-6 was decreased. These results suggest that the Gly960Trp substitution in HR protein in Hr m1Enu mice may alter the function of HR as a transcriptional corepressor. [Copyright &y& Elsevier]

Abstract: Cullin-RING ligases (CRLs) regulate diverse cellular functions such as cell cycle progression and cytokine signaling by ubiquitinating key regulatory proteins. The activity of CRLs is controlled by Nedd8 modification of the cullin subunits. Recent reports have suggested that CAND1, which specifically binds to unmodified CUL1 but not to neddylated one, is required for the in vivo function of SCFs, the CUL1-containing CRLs. We show here that CAND1 and COP9 signalosome (CSN), the major deneddylase of cullins, bind to unneddylated CUL1 in a mutually exclusive way. The suppression of CAND1 expression by small inhibitory RNA enhanced the interaction between CUL1 and CSN, suggesting that CAND1 inhibited the binding of CSN to CUL1. We found that the binding of CSN to CUL1 required the four helix bundle in CUL1 C-terminal domain, which was wrapped around by CAND1 in the CAND1-CUL1-Rbx1 complex. CAND1 greatly facilitated CSN-mediated deneddylation of CUL1 in vitro, which was dependent on its binding to CUL1. Our data suggest that enhancement of CSN-mediated deneddylation by CAND1 may contribute to its function as a positive regulator of SCFs in vivo. [Copyright &y& Elsevier]

The 26S proteasome, composed of the 20S core and the 19S regulatory complex, plays a central role in ubiquitin-dependent proteolysis by catalyzing degradation of polyubiquitinated proteins. In a search for proteins involved in regulation of the proteasome, we affinity purified the 19S regulatory complex from HeLa cells and identified a novel protein of 43 kDa in size as an associated protein. Immunoprecipitation analyses suggested that this protein specifically interacted with the proteasomal ATPases. Hence the protein was named proteasomal ATPase-associated factor 1 (PAAF1). Immunoaffinity purification of PAAF1 confirmed its interaction with the 19S regulatory complex and further showed that the 19S regulatory complex bound with PAAF1 was not stably associated with the 20S core. Overexpression of PAAF1 in HeLa cells decreased the level of the 20S core associated with the 19S complex in a dose-dependent fashion, suggesting that PAAF1 binding to proteasomal ATPases inhibited the assembly of the 26S proteasome. Proteasomal degradation assays using reporters based on green fluorescent protein revealed that overexpression of PAAF1 inhibited the proteasome activity in vivo. Furthermore, the suppression of PAAF1 expression that is mediated by small inhibitory RNA enhanced the proteasome activity. These results suggest that PAAF1 functions as a negative regulator of the proteasome by controlling the assembly/disassembly of the proteasome. [ABSTRACT FROM AUTHOR]

In a process seeking out a good model cell line for Epstein–Barr virus (EBV)-associated gastric cancer, we found that one previously established gastric adenocarcinoma cell line is infected with type 1 EBV. This SNU-719 cell line from a Korean patient expressed cytokeratin without CD19 or CD21 expression. In SNU-719, EBNA1 and LMP2A were expressed, while LMP1 and EBNA2 were not. None of the tested lytic EBV proteins were detected in this cell line unless stimulated with phorbol ester. EBV infection was also shown in the original carcinoma tissue of SNU-719 cell line. Our results support the possibility of a CD21-independent EBV infection of gastric epithelial cells in vivo. As the latent EBV gene expression pattern of SNU-719 closely resembles that of the EBV-associated gastric cancer, this naturally derived cell line may serve as a valuable model system to clarify the precise role of EBV in gastric carcinogenesis. [Copyright &y& Elsevier]

Purpose: Although mutations are associated with carcinogenesis, little is known about survival-specific genes in clear cell renal cell carcinoma (ccRCC). We developed a customized next-generation sequencing (NGS) gene panel with 156 genes. The purpose of this study was to investigate whether the survival-specific genes we found were present in Korean ccRCC patients, and their association with clinicopathological findings. Materials and Methods: DNA was extracted from the formalin-fixed, paraffin-embedded tissue of 22 ccRCC patients. NGS was performed using our survival-specific gene panel with an Illumina MiSeq. We analyzed NGS data and the correlations between mutations and clinicopathological findings and also compared them with data from the Cancer Genome Atlas-Kidney Renal Clear Cell Carcinoma (TCGA-KIRC) and Renal Cell Cancer-European Union (RECA-EU). Results: We found a total of 100 mutations in 37 of the 156 genes (23.7%) in 22 ccRCC patients. Of the 37 mutated genes, 11 were identified as clinicopathologically significant. Six were novel survival-specific genes (ADAMTS10, CARD6, NLRP2, OBSCN, SECISBP2L, and USP40), and five were top-ranked mutated genes (AKAP9, ARID1A, BAP1, KDM5C, and SETD2). Only CARD6 was validated as an overall survival-specific gene in this Korean study (p = 0.04, r = −0.441), TCGA-KIRC cohort (p = 0.0003), RECA-EU (p = 0.0005). The 10 remaining gene mutations were associated with clinicopathological findings; disease-free survival, mortality, nuclear grade, sarcomatoid component, N-stage, sex, and tumor size. Conclusions: We discovered 11 survival-specific genes in ccRCC using data from TCGA-KIRC, RECA-EU, and Korean patients. We are the first to find a correlation between CARD6 and overall survival in ccRCC. The 11 genes, including CARD6, NLRP2, OBSCN, and USP40, could be useful diagnostic, prognostic, and therapeutic markers in ccRCC. [ABSTRACT FROM AUTHOR]