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The anticancer effects of Shinan (Shinan-South Korea) sea salts on azoxymethane (AOM)/dextran sodium sulfate (DSS) with high fat diet (HFD)-induced colon cancer and obesity in C57BL/6N mice were studied. We prepared three types of sea salt: generally manufactured sea salt (GS), generally manufactured after filtering seawater (FS), and manufactured with only new seawater (NS). Sea salt intake increased colon length and reduced colon length/weight ratio, tumor number, and progression of colon cancer in colon tissue. The differently prepared sea salts also ameliorated liver injury. In addition, the mineral composition of each salt was different. Moreover, the sea salts enhanced activation of natural killer cell (CD56) expression in colon and spleen tissues. However, the mineral compositions of sea salts were not simply associated with anticancer effects in AOM/DSS+HFD induced mice. Thus, the anticolorectal cancer effects of sea salts may be mediated by different factors, which remain to be identified.

To evaluate the correlation between the maximum standardised uptake value (SUVmax) from bone single-photon-emission computed tomography/computed tomography (SPECT/CT) and other imaging parameters for medial compartment osteoarthritis (OA) of the knee.Patients (n=26; male:female=2:24; age, 55.3±5.8 years) underwent quantitative knee SPECT/CT using technetium-99m (Tc-99m) hydroxymethylene diphosphonate (HDP) before surgical operation for medial OA of the knee. SUVmax was calculated using dedicated quantitative software. Visual grades of tracer uptake on bone SPECT/CT and Kellgren-Lawrence (KL) OA scores on plain radiographs were assessed using a five-point scale. Magnetic resonance imaging (MRI) scores (n=22) and patient symptom scores were also assessed.The operated knees (n=34) had a greater SUVmax than the non-operated knees (n=18) in the medial compartment (14.1±6.1 versus 5.3±4.4, p0.0001). In the medial compartment, the SUVmax was significantly correlated with SPECT/CT visual grades (rho=0.794, p0.0001), KL scores (rho=0.703, p0.0001), and MRI scores (rho=0.714-0.808, p≤0.0002); however, SUVmax and other imaging parameters were not correlated with patient symptom scores (p0.05).The SUVmax of quantitative bone SPECT/CT was highly correlated with traditional imaging parameters for medial compartment OA severity of the knee. Quantitative bone SPECT/CT is a promising imaging technique for the objective assessment of knee OA.

To overcome the muscle atrophy, such as cachexia and sarcopenia, we tried to find myogenic agents from medicinal plants. From myogenic extract of Coptis japonica, we purified six isoquinoline alkaloids and evaluated their effects on transactivation of myoD and MHC expression in C2C12 cells during differentiation process. Among obtained compounds, magnoflorine most efficiently enhanced the myoblast differentiation by activating the p38 MAP kinase and Akt pathway, and also increased the number of multinucleated and cylinder-shaped myotubes. These results propose that magnoflorine from Coptis japonica might be a promising lead compound for the development of anti-muscle atrophy drug.

// Jin-Ock Kim 1 , So-Ra Kim 1 , Key-Hwan Lim 1 , Jun-Hyun Kim 1 , Brijesh Ajjappala 1 , Hey-Jin Lee 1 , Jee-In Choi 2 , Kwang-Hyun Baek 1 1 Department of Biomedical Science, CHA University, Bundang CHA Hospital, Gyeonggi-Do 463-400, Republic of Korea 2 Department of Rehabilitation Medicine, CHA University, Bundang CHA Hospital, Gyeonggi-Do 463-400, Republic of Korea Correspondence to: Kwang-Hyun Baek, e-mail: baek@cha.ac.kr Keywords: 14-3-3, cell proliferation, deubiquitinating enzyme, ubiquitin-specific protease Received: May 27, 2015 Accepted: September 14, 2015 Published: September 25, 2015 ABSTRACT 14-3-3 is a family of highly conserved protein that is involved in a number of cellular processes. In this study, we identified that the high expression of 14-3-3γ in various cancer cell lines correlates with the invasiveness of the cancer cells. Overexpression of 14-3-3γ causes changes to the morphologic characteristics of cell transformation, and promotes cell migration and invasion. The cells overexpressed with 14-3-3γ have been shown to stimulate foci and tumor formation in SCID-NOD mice in concert with signaling components as reported with the 14-3-3β. In our previous study, we demonstrated that 14-3-3γ inhibits apoptotic cell death and mediates the promotion of cell proliferation in immune cell lines. Earlier, binding partners for 14-3-3γ were defined by screening. We found that USP37, one of deubiquitinating enzymes (DUBs), belongs to this binding partner group. Therefore, we investigated whether 14-3-3γ mediates proliferation in cancer cells, and 14-3-3γ by USP37 is responsible for promoting cell proliferation. Importantly, we found that USP37 regulates the stability of ubiquitin-conjugated 14-3-3γ through its catalytic activity. This result implies that the interactive behavior between USP37 and 14-3-3γ could be involved in the regulation of 14-3-3γ degradation. When all these findings are considered together, USP37 is shown to be a specific DUB that prevents 14-3-3γ degradation, which may contribute to malignant transformation via MAPK signaling pathway, possibly providing a new target for therapeutic objectives of cancer.

SDS3 is a key component of the histone deacetylase (HDAC)-dependent Sin3A co-repressor complex, serving to maintain its HDAC activity. Here, we report both exogenous and endogenous functional interaction between deubiquitinating enzyme USP17 and human SDS3 by MALDI-TOF-MS, co-immunoprecipitation assay, and GST pull-down assay. In this study, we demonstrated that SDS3 readily undergoes endogenous polyubiquitination, which is associated specifically with Lys-63-branched polyubiquitin chains and not with Lys-48-branched polyubiquitin chains. Further, we also demonstrated that USP17 specifically deubiquitinates Lys-63-linked ubiquitin chains from SDS3 and regulates its biological functions. The deubiquitinating activity of USP17 on SDS3 negatively regulates SDS3-associated HDAC activity. The constitutive expression of USP17 and its substrate SDS3 was involved in the inhibition of anchorage-independent tumor growth and blocks cell proliferation, leading to apoptosis in cervical carcinoma cells. Furthermore, we showed that USP17 and SDS3 mutually interact with each other to regulate cancer cell viability. These data support the possibility that SDS3, being a substrate of USP17, may play an important role in developing a novel therapeutic means to inhibit specific HDAC activities in cancer.

Translational studies have explored the therapeutic effects of stem cells, raising hopes for the treatment of numerous diseases. Here, we evaluated the therapeutic effect of chorionic plate-derived mesenchymal stem cells (CP-MSCs) isolated from human placenta and transplanted into rats with carbon tetrachloride (CCl(4))-injured livers. CP-MSCs were analyzed for hepatocyte-specific gene expression, indocyanine green (ICG) uptake, glycogen storage, and urea production following hepatogenic differentiation. PKH26-labeled CP-MSCs were directly transplanted into the livers of rats that had been exposed to CCl(4) (1.6 g/kg, twice per week for 9 weeks). Blood and liver tissue were analyzed at 1, 2, and 3 weeks post-transplantation. The expression of type I collagen (Col I) and matrix metalloproteinases (MMPs) was analyzed in rat T-HSC/Cl-6 hepatic stellate cells co-cultured with CP-MSCs following exposure to TGF-β. The expression levels of α-smooth muscle actin (α-SMA) and Col I were lower in transplanted (TP) rats than in non-transplanted (Non-TP) animals (P < 0.05), whereas the expression levels of albumin and MMP-9 were increased. TP rats exhibited significantly higher uptake/excretion of ICG than non-TP rats (P < 0.005). In addition, collagen synthesis in T-HSC/Cl-6 cells exposed to TGF-β was decreased by co-culture with CP-MSCs, which triggered the activation of MMP-2 and MMP-9. These results contribute to our understanding of the potential pathophysiological roles of CP-MSCs, including anti-fibrotic effects in liver disease, and provide a foundation for the development of new cell therapy-based strategies for the treatment of difficult-to-treat liver diseases.

Aim To compare the clinical effectiveness of percutaneous stent placement between T and criss-cross configuration techniques in patients with advanced malignant hilar biliary obstruction. Materials and methods Between January 2009 and December 2014, 59 patients who underwent percutaneous stent placement for malignant hilar obstruction were included in this retrospective study. T-configured stent placement (T group) was performed in 33 patients and criss-cross configured stent placement (criss-cross group) in 26 patients. Technical and clinical success, complications, patient survival, and stent patency were compared between the two groups. Results Stent placement was technically successful in all patients of the two groups. Clinical success was achieved in 30 (90.9%) patients of T group and 25 (96.9%) of criss-cross group (p=0.62). Two patients in the T group (6.1%) required additional stent placement for internal drainage of undrained sector. There were no major complications. Median survival was not statistically different between the two groups (128 days in the T group versus 183 days in the criss-cross group; p=0.33). Stent occlusion occurred in 15 patients in the T group and seven patients in the criss-cross group. The stent patency of the criss-cross group (median 330 days) was longer than that of the T group (median 132 days; p=0.0007). Conclusions Early clinical effectiveness is comparable between the two techniques; however, additional intervention is occasionally required for drainage of an undrained sector after T-configured stent placement. Stent placement with criss-cross configuration provides longer stent patency than T-configuration technique.

The aim of this research is to obtain a numerical material model for an amorphous glassy polymer, polycarbonate (PC), which can be used in finite element analysis (FEA) of the micro thermal imprint process near the glass transition temperature (Tg). Through understanding of the deformation behavior of the PC specimens was acquired by performing tensile stress relaxation tests. The viscoelastic material model based on generalized Maxwell model was introduced for the material near Tg to establish the FE model based on the commercial FEA code ABAQUS/Standard with a suitable set of parameters obtained for this material model from the test data. Further validation of the model and parameters was performed by comparing the analysis of FE model results to the experimental data. As a result, the feasibility of the established viscoelastic model for PC near Tg was confirmed and this material model can be used in FE analysis for the prediction and improvement of the micro thermal imprint process for pattern replication.

Mouse Usp42, a novel ubiquitin specific protease gene, was isolated from mouse embryonic stem cells. It consists of 1,324 amino acids with a predicted molecular weight of 146 kDa and contains the conserved Cys, Asp (I), His and Asn/Asp (II) domains defined as one of characteristics for deubiquitinating enzymes. RT-PCR analysis showed that the Usp42 transcript is expressed in NIH3T3 cells, B- and T-lymphocytes, and L1210 cells. Northern blot analysis revealed that Usp42 is expressed mainly in brain, lung, thymus and testis, and at mouse E10.5 the most during embryonic development. Usp42 expression rises from 2 weeks after birth to round-spermatid stage and decreases from condensing-spermatid stage during spermatogenesis. Deubiquitinating enzyme assays demonstrated that Usp42 can cleave ubiquitin from ubiquitinated substrates in vitro and in vivo. Taken all together, it is suggested that Usp42 is one of typical deubiquitinating enzymes, which may play an important role in mouse embryogenesis and spermatogenesis.

Deubiquitinating enzymes regulate a number of cellular mechanisms including pre-implantation, growth and differentiation, oncogenesis, cell cycle progression, transcriptional activation, and signal transduction. In this study, we have identified a novel human deubiquitinating enzyme gene, USP22, and its mouse homologue, Usp22. They encode 525 amino acids (approximate MW: 60kDa) and contains Cys, Asp (I), His and Asp/Asn (II), the highly conserved domains of the UBP family of deubiquitinating enzymes. The biochemical assay revealed that they have deubiquitinating enzyme activity. Northern blot analysis for USP22 showed moderate expression in various organs including human heart and skeletal muscle, and weak expression in lung and liver. However, Usp22 is expressed strongly in brain and weakly in other organs. We investigated the expression level of Usp22 mRNA and the localization during implantation and early pregnancy by in situ hybridization. Interestingly, Northern blot analysis showed the strong expression of Usp22 between embryonic days E10.5 and E12.5. Whole mount in situ hybridization staining revealed that Usp22 was expressed in the midbrain, forebrain, hindbrain and dorsal root ganglia of embryos at E12.5. Embryos at E12.5 showed the pronounced expression of Usp22 during the early embryonic development, although its expression was not detectable in the gut, liver and heart.

Isolation of induced pluripotent stem cells (iPSCs) from fully differentiated somatic cells has revolutionized existing concepts of cell differentiation and stem cells. Importantly, iPSCs generated from somatic cells of patients can be used to model different types of human diseases. They may also serve as autologous cell sources that can be used in transplantation therapy. In this study, we investigated the neuronal properties of an iPSC line that is derived from human neonatal foreskin fibroblasts (FS-1). We initially examined the morphology and marker expression of FS-1 cells at undifferentiated stage. We then spontaneously differentiated FS-1 cells in suspension culture and examined the expression of markers representing three germ layers. We finally differentiated FS-1 cells into neuronal lineages by co-culturing them with PA6 stromal cells, and found that, under the conditions we used, they have a tendency to differentiate into more forebrain-type neurons, suggesting that FS-1 iPSC-derived neural cells will be useful to be used in cell therapy of stroke or Huntington's disease, among others. Taken together, FS-1 cells derived from human neonatal fibroblasts exhibit very similar properties with human ES cells, and can provide useful sources for cell therapy and various other applications.

Background A plethora of biological metabolisms are regulated by the mechanisms of ubiquitination, wherein this process is balanced with the action of deubiquitination system. Dub-2 is an IL-2-inducible, immediate-early gene that encodes a deubiquitinating enzyme with growth regulatory activity. DUB-2 presumably removes ubiquitin from ubiquitin-conjugated target proteins regulating ubiquitin-mediated proteolysis, but its specific target proteins are unknown yet. Methodology/Principal Findings To elucidate the functional role of Dub-2, we generated genetically modified mice by introducing neo cassette into the second exon of Dub-2 and then homologous recombination was done to completely abrogate the activity of DUB-2 proteins. We generated Dub-2+/− heterozygous mice showing a normal phenotype and are fertile, whereas new born mouse of Dub-2−/− homozygous alleles could not survive. In addition, Dub-2−/− embryo could not be seen between E6.5 and E12.5 stages. Furthermore, the number of embryos showing normal embryonic development for further stages is decreased in heterozygotes. Even embryonic stem cells from inner cell mass of Dub-2−/− embryos could not be established. Conclusions Our study suggests that the targeted disruption of Dub-2 may cause embryonic lethality during early gestation, possibly due to the failure of cell proliferation during hatching process.

In general, the formation of embryoid bodies (EBs) is a commonly known method for initial induction of human embryonic stem cells (hESCs) into their derivatives in vitro. Despite the ability of EBs to mimic developmental processing, the specification and classifications of EBs are not yet well known. Because EBs show various differentiation potentials depending on the size and morphology of the aggregated cells, specification is difficult to attain. Here, we sought to classify the differentiation potentials of EBs by morphologies to enable one to control the differentiation of specific lineages from hESCs with high efficiency. To induce the differentiation of EB formation, we established floating cultures of undifferentiated hESCs in Petri dishes with hESC medium lacking basic fibroblast growth factor. Cells first aggregated into balls; ∼10 days after suspension culture, some different types of EB morphology were present, which we classified as cystic-, bright cavity-, and dark cavity-type EBs. Next, we analyzed the characteristics of each type of EB for its capacity to differentiate into the 3 germ layers via multiplex polymerase chain reaction (PCR), real-time PCR, and immunocytochemistry. Our results indicated that most cells within the cystic EBs were composed of endoderm lineage populations, and both of the cavity EB types were well organized with 3 germ-layer cells. However, the differentiation capacity of the bright cavity EBs was faster than that of the dark cavity EBs. Thus, the bright cavity EBs in this study, which showed equal differentiation tendencies compared with other types of EBs, may serve as the standard for in vitro engineering of EBs. These results indicate that the classification of EB morphologies allows the estimation of the differentiation status of the EBs and may allow the delineation of subsets of conditions necessary for EBs to differentiate into specific cell types.

Cdo activates Akt via indirect interaction with APPL1 during myoblast differentiation, and this complex likely mediates some of the promyogenic effect of cell–cell interaction. The promyogenic function of Cdo involves a coordinated activation of p38MAPK and Akt via interaction with scaffold proteins, JLP and Bnip-2 for p38MAPK and APPL1 for Akt., Cell–cell interactions between muscle precursors are required for myogenic differentiation; however, underlying mechanisms are largely unknown. Promyogenic cell surface protein Cdo functions as a component of multiprotein complexes containing other cell adhesion molecules, Boc, Neogenin and N-cadherin, and mediates some of signals triggered by cell–cell interactions between muscle precursors. Cdo activates p38MAPK via interaction with two scaffold proteins JLP and Bnip-2 to promote myogenesis. p38MAPK and Akt signaling are required for myogenic differentiation and activation of both signaling pathways is crucial for efficient myogenic differentiation. We report here that APPL1, an interacting partner of Akt, forms complexes with Cdo and Boc in differentiating myoblasts. Both Cdo and APPL1 are required for efficient Akt activation during myoblast differentiation. The defective differentiation of Cdo-depleted cells is fully rescued by overexpression of a constitutively active form of Akt, whereas overexpression of APPL1 fails to do so. Taken together, Cdo activates Akt through association with APPL1 during myoblast differentiation, and this complex likely mediates some of the promyogenic effect of cell–cell interaction. The promyogenic function of Cdo involves a coordinated activation of p38MAPK and Akt via association with scaffold proteins, JLP and Bnip-2 for p38MAPK and APPL1 for Akt.

In regulation of the developmental process, the balance between cellular proliferation and cell death is critical. Placental development tightly controls this mechanism, and increased apoptosis of placental trophoblasts can cause a variety of gynecological diseases. Members of the immortalization-upregulated protein (IMUP) family are nuclear proteins implicated in SV40-mediated immortalization and cellular proliferation; however, the mechanisms by which their expression is regulated in placental development are still unknown. We compared IMUP-2 expression in normal and pre-eclamptic placental tissues and evaluated the function of IMUP-2 in HTR-8/SVneo trophoblast cells under hypoxic conditions. IMUP-2 was expressed in syncytiotrophoblasts and syncytial knots of the placental villi. IMUP-2 expression was significantly higher in preterm pre-eclampsia patients than in patients who went to term (P < 0.001); however, we observed no differences in IMUP-2 expression between normal term patients with and without pre-eclampsia. Hypoxic conditions increased apoptosis of HTR8/SVneo trophoblast cells and induced IMUP-2 expression. Also, apoptosis of HTR-8/SVneo trophoblast cells was increased after IMUP-2 gene transfection. These results suggest that IMUP-2 expression is specifically elevated in preterm pre-eclampsia and under hypoxic conditions, and that IMUP-2 induces apoptosis of the trophoblast. Therefore, IMUP-2 might have functional involvement in placental development and gynecological diseases such as pre-eclampsia.

Ubiquitination and deubiquitination have a critical role in protein homoeostasis in the cell. Here, we have characterized a novel USP44 (ubiquitin-specific protease 44), which has a ZnF-UBP (zinc-finger ubiquitin-specific protease) domain and conserved cysteine, histidine and asparagine/aspartic acid residues characteristic of deubiquitinating enzymes. The biochemical assay revealed that USP44 can cleave ubiquitin from ubiquitinated substrates both in vitro and in vivo. Further, USP44 undergoes both lysine 48- and lysine 63-linked polyubiquitination. In situ hybridization using mouse tissues showed a basal detection level in all organs tested, with strong detection in lung, pancreas, skin, liver, stomach and intestine. RT-PCR (reverse-transcription PCR) analysis showed high levels of detection of USP44 mRNA in testis, spleen, lung, stomach and ovary. Furthermore, we raised a polyclonal antibody against USP44 and checked its endogenous protein expression in different cell lines. A localization study of USP44 showed its predominant expression in the nucleus.

The establishment of new technology for genetic modification in human embryonic stem (ES) cell lines has raised great hopes for achieving new ground in basic and clinical research. Recently, lentiviral vector technology has been shown to be highly effective and therefore could emerge as a popular tool for human ES cell genetic modification. The objectives of this study were to evaluate the efficiency of promoters in lentiviral gene delivery systems in mammalian ES cells, including mouse, monkey, and human, and to construct efficient and optimized conditions for lentivirus-mediated transfection systems. Mammalian ES cells were transfected with self-inactivating (SIN) human immunodeficiency virus type-1 (HIV-1)-based lentiviral vectors containing the human polypeptide chain elongation factor-1alpha (EF-1alpha) promoter or cytomegalovirus (CMV) promoter and analyzed by fluorescence-activated cell sorting (FACS) analysis for the expression of the enhanced green fluorescent protein (eGFP) reporter gene. The efficiency of the EF-1alpha promoter was higher than that of the CMV promoter in all ES cells tested. The EF-1alpha promoter efficiently drove gene expression (14.74%) compared with CMV promoter (3.69%) in human ES cells. We generated a stable eGFP+ human ES cell line (CHA3-EGFP human ES cells) that continuously expressed high levels of EGFP ( approximately 95%) from the EF-1alpha promoter and was maintained for up to 60 weeks with undifferentiated proliferation. The established CHA3-EGFP human ES cell lines were characterized as being negative for nondifferentiation markers and teratoma formation. These results imply that genetic modification by lentiviral vectors with specific promoters in ES cells constitute a powerful tool for guided differentiation as well as gene therapy.

p53 tumor suppressor protein is stabilized by the herpes-virus-associated ubiquitin-specific protease (HAUSP), a deubiquitinating enzyme. We previously isolated a mouse orthologue of HAUSP, mHAUSP, encoding 1103 amino acids with a molecular weight of approximately 135 kDa containing highly conserved Cys, Asp (I), His, and Asn/ Asp (II) domains. In this study, we investigated the temporal and spatial expression of mHAUSP during the early mouse embryonic development. Northern blot analysis revealed that the expression of mHAUSP was detected throughout the process of embryonic development with the maximal expression between E10.5 and E13.5. In situ hybridization study showed the global expression of mHA USP in various organs of embryos, including mesencephalon, spinal cord, lung and genital eminence. In addition, we carried out biochemical analysis for 6 conserved amino acids (Cys224, Gln231, Asp296, His457, His465, and Asp482) in Cys box, QQD box, and His box in order to investigate their structural and functional roles of these amino acid residues. The conserved Gln231 was not essential for the catalytic activity of mHAUSP. However, other conserved amino acids were required for deubiquitinating enzyme activity of mHAUSP. Moreover, we observed that the overexpression of mHAUSP induces cell death in HeLa cells.

Platelet-derived growth factor (PDGF) has multiple functions including inhibition of apoptosis and promotion of cell proliferation. In this study, we show that Na(+)/H(+) exchanger regulatory factor 2 (NHERF2) binds to the carboxyl-terminal PDZ domain-binding motif of the PDGF receptor through a PDZ domain-mediated interaction, and evaluate the consequence on PDGF-induced proliferation. Stable transfection with NHERF2 increased the PDGF-induced phosphorylation of ERK and Akt in Rat1 embryonic fibroblasts. The phosphorylation of Akt was blocked by pretreatment with LY294002, a PI-3-kinase inhibitor, in both Rat1/NHERF2 and Rat1/vector cells. In Rat1/vector cells, PDGF-induced phosphorylation of ERK was completely inhibited by pretreatment with PD98059, a MEK inhibitor. In contrast, the NHERF2-dependent increase of ERK phosphorylation was not affected by pretreatment with PD98059 in Rat1/NHERF2 cells. Thus, the NHERF2-dependent increase of ERK phosphorylation occurs in a MEK-independent fashion. Pretreatment with PP2, a specific inhibitor of Src family tyrosine kinase, completely blocked the NHERF2-dependent increase of the phosphorylation of ERK and Akt, suggesting that NHERF2 up-regulates Erk phosphorylation through a Src family kinase-dependent pathway. Consistent with these results, the PDGF-induced thymidine incorporation was increased in Rat1/NHERF2 cells, and the NHERF2-dependent increase of thymidine incorporation was prevented by treatment with LY294002 and PP2 but not with PD98059. These results suggest that NHERF2 stimulates PDGF-induced proliferation by increasing PI-3-kinase/Akt, MEKindependent ERK, and Src family kinase-mediated signaling pathways.