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Competing Interests: Declaration of competing interest The authors certify that all of their affiliations and/or financial involvement with any organization, or any financial conflicts with the subject matter discussed in the manuscript are disclosed in the Acknowledgement section of the manuscript.

Background: We previously reported the in vitro and in vivo antitumor effects of trametinib, a MEK inhibitor, on neuroblastoma with MAPK pathway mutations. As we observed eventual resistance to trametinib in our previous study, we evaluated the combination therapy of CA3, a YAP inhibitor, with trametinib, based on a recent report suggesting the potential involvement of YAP in the mechanism underlying the resistance to trametinib in neuroblastoma., Methods: SK-N-AS cells (a neuroblastoma cell line harboring RAS mutation) were treated with CA3 in vitro and subjected to a viability assay, immunocytochemistry and flow cytometry. Next, we analyzed the in vitro combination effect of CA3 and trametinib using the CompuSyn software program. Finally, we administered CA3, trametinib or both to SK-N-AS xenograft mice for 10 weeks to analyze the combination effect., Results: CA3 inhibited cell proliferation by both cell cycle arrest and apoptosis in vitro. Combination of CA3 and trametinib induced a significant synergistic effect in vitro (Combination Index <1). Regarding the in vivo experiment, combination therapy suppressed tumor growth, and 100% of mice in the combination therapy group survived, whereas the survival rates were 0% in the CA3 group and 33% in the trametinib group. However, despite this promising survival rate in the combination group, the tumors gradually grew after seven weeks with MAPK reactivation., Conclusion: Our results indicated that CA3 and trametinib exerted synergistic antitumor effects on neuroblastoma in vitro and in vivo, and CA3 may be a viable option for concomitant drug therapy with trametinib, since it suppressed the resistance to trametinib. However, this combination effect was not sufficient to achieve complete remission. Therefore, we need to adjust the protocol to obtain a better outcome by determining the mechanism underlying regrowth in the future., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Sequestosome 1 (p62) is a multifunctional adapter protein involved in various physiological functions, such as selective autophagy and oxidative stress response. Hence, aberrant expression and defective regulation of p62 are thought to lead to the onset of various diseases, including cancer. The expression of p62 has been shown to be increased in breast cancer tissues, and is correlated with a poor prognosis. However, the role of p62 in the breast cancer pathophysiology is still unclear. Here, we aimed to analyze the effect of changes in p62 expression on breast cancer cell lines. DNA microarray analysis revealed that the expression of progesterone receptor (PR), which is one of the indices for the classification of breast cancer subtypes, was markedly suppressed by forced expression of p62. The protein expression of PR was also decreased by forced expression of p62, but increased by knockdown of p62. Moreover, we found that p62 knockdown induced the protein expression of argonaute 2 (AGO2). Luciferase reporter assay results showed that the gene expression of PR was promoted by AGO2. Furthermore, results revealed that overexpression of AGO2 partially rescued the decrease in PR expression induced by forced expression of p62. Collectively, our findings indicated that p62 accumulation suppressed the expression of AGO2, which in turn decreased the expression of PR, suggesting that p62 may serve as a marker of aggressive breast cancer and poor prognosis. Moreover, the p62-AGO2-PR axis was identified as a crucial signaling cascade in breast cancer progression., Competing Interests: Declaration of competing interest The authors declare that they have no competing interests., (Copyright © 2020 Elsevier Inc. All rights reserved.)

In many neurodegenerative diseases, mitochondria are actively involved in the onset and/or progression of diseases because the energy depletion of the neuronal cells directly leads to the dysfunction and degeneration of cells. In the case of prion diseases, mitochondrial involvement has been reported recently and evidence that prion protein (PrP) is localized in mitochondria is increasing. Despite these findings, the precise molecular mechanism by which PrP targets mitochondria remains unclear. PrP is a secretory protein and does not have a pre-sequence that targets the mitochondria, therefore, we thought that there was a covert signal in the amino acid sequence of PrP. To find the sequence, we constructed various GFP-fused PrP-truncations and colocalization with mitochondria was verified by live-cell imaging. Consequently, we found that 18 amino acids, PrP (122-139), are indispensable for the mitochondrial targeting of PrP. In addition, fluorescent microscopy observation revealed that PrP-localized mitochondria were accumulated at the perinuclear region in neuronal cells such as mouse neuroblastoma Neuro2a (N2a) and prion persistent infection N2a strain (ScN2a), anterograde movement of the mitochondria toward the cell membrane was completely inhibited because of the stacking of PrP on the outer membrane. The cristae formation of perinuclear accumulated mitochondria was disappeared indicating the reduced mitochondrial activity. Surprisingly, PrP-dependent mitochondrial perinuclear accumulation was specifically occurred on neuronal cells, whereas in epithelial HeLa cells and fibroblast COS-7 cells, no perinuclear accumulation observed even after the mitochondrial targeting of PrP., Competing Interests: Declaration of competing interest The authors declare no competing financial interests., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Osteoclast lysosomes secrete lytic enzymes into bone resorption lacunae, and sort the lysosomal proton pumping vacuolar-type ATPase (V-ATPase) to the plasma membrane to form the acidic environment required for bone digestion. The a3 isoform of V-ATPase is essential for outward trafficking of the secretory lysosomes and interacts physically with Rab7, a small GTPase that regulates trafficking of late endosomes and lysosomes, to recruit it to lysosomes. However, it is unclear whether organelle acidification by V-ATPase is required for the lysosome trafficking. Here, we showed that incubation of osteoclasts with the V-ATPase inhibitor bafilomycin A1 abolished the osteoclast-characteristic peripheral localization of secretory lysosomes, Rab7, and α-tubulin. Although bafilomycin A1 had little or no effect on Rab7 activation and its interaction with a3, treatment with the inhibitor significantly reduced the lysosomal localization of Rab7. Even constitutively active Rab7 did not localize to lysosomes in the presence of the inhibitor. These results suggest that organelle acidification by V-ATPase is required for localization of activated Rab7 to lysosomes., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Previous studies showed that gold nanoparticles (AuNPs) are useful radiosensitizers which optimize radiation therapy under low-dose radiation. However, the mechanisms of AuNP radiosensitization, including the amount and localization of the AuNPs interacting with cancer cells, has not yet been quantified. To answer these questions, we prepared AuNPs conjugated with anti-human epidermal growth factor receptor type 2 (HER2) antibody via polyethylene glycol (PEG) chains (AuNP-PEG-HER2ab). AuNP-PEG-HER2ab specifically bound to the HER2-expressing cancer cells and entered the cells via endocytosis. Whether endocytosis of AuNP-PEG-HER2ab occurred had no effect on radiosensitization efficacy by AuNP-PEG-HER2ab in vitro. The radiosensitization efficacy in vitro depended on dose of AuNP-PEG-HER2ab or dose of X-ray. Moreover, AuNP-PEG-HER2ab administrated into tumor-bearing mice was localized to both the periphery of the tumor tissue and near the nuclei in cancer cells in tumor deep tissue. The localization of AuNP-PEG-HER2ab in tumor tissues was important factors for in vivo powerful radiosensitization efficacy., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Rubisco, an enzyme for photosynthetic carbon dioxide fixation, is a major green leaf protein and known as the most abundant protein on the Earth. We found that Rubisco digested mimicking gastrointestinal enzymatic conditions exhibited anxiolytic-like effects after oral administration in mice. Based on a comprehensive peptide analysis of the digest using nanoLC-Orbitrap-MS and the structure-activity relationship of known anxiolytic-like peptides, we identified SYLPPLTT, SYLPPLT and YHIEPV [termed Rubisco anxiolytic-like peptide (rALP)-1, rALP-1(1-7) and rALP-2, respectively], which exhibited potent anxiolytic-like effects after oral administration. The anxiolytic-like effects of rALP-1/rALP-1(1-7) were blocked by a serotonin 5-HT 1A receptor antagonist, whereas rALP-2-induced effects were inhibited by a δ-opioid receptor antagonist. In conclusion, novel Rubisco-derived anxiolytic-like peptides, rALP-1/rALP-1(1-7) and rALP-2, act via independent neural pathways., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Porphyromonas gingivalis is a well-known Gram-negative bacterium that causes periodontal disease. The bacterium metabolizes amino acids and peptides to obtain energy. An ion gradient across its plasma membrane is thought to be essential for nutrient import. However, it is unclear whether an ion-pumping ATPase responsible for the gradient is required for bacterial growth. Here, we report the inhibitory effect of protonophores and inhibitors of a proton-pumping ATPase on the growth of P. gingivalis. Among the compounds examined, curcumin and citreoviridin appreciably reduced the bacterial growth. Furthermore, these compounds inhibited the ATPase activity in the bacterial membrane, where the A-type proton-pumping ATPase (A-ATPase) is located. This study suggests that curcumin and citreoviridin inhibit the bacterial growth by inhibiting the A-ATPase in the P. gingivalis membrane., (Copyright © 2018 Elsevier Inc. All rights reserved.)

This study aimed to evaluate the effects of combination therapy with a dipeptidyl peptidase-4 inhibitor, alogliptin, and a peroxisome proliferator-activated receptor-γ agonist, pioglitazone, in a preclinical model of nonalcoholic steatohepatitis using low-density lipoprotein receptor-knockout mice fed a modified choline-deficient l-amino acid-defined diet. Monotherapy with either alogliptin (10-200 mg/kg) or pioglitazone (6-20 mg/kg) significantly decreased hepatic triglyceride content and fibrosis. The concomitant treatment of alogliptin (30 mg/kg), pioglitazone (20 mg/kg) also decreased hepatic triglyceride and hepatic collagen-I mRNA at greater extent compared to monotherapy. Hepatic expression of CD11b mRNA and monocyte chemoattractant protein-1 were also reduced by the concomitant treatment. These results suggest that via an anti-inflammatory potential in addition to anti-metabolic effects, the combination therapy of alogliptin and pioglitazone may provide therapeutic benefits to type 2 diabetes patients with nonalcoholic steatohepatitis, which will be proven in controlled clinical trials., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Brain edema is a severe complication that accompanies ischemic stroke. Increasing evidence shows that inflammatory cytokines impair tight junctions of the blood-brain barrier, suggesting the involvement of microglia in brain edema. In this study, we examined the role of microglia in the progression of ischemic brain edema using mice with permanent middle cerebral artery occlusion. The intensity of T2-weighted imaging (T2WI) in the cerebral cortex and the striatum was elevated 3 h after occlusion and spread to peripheral regions of the ischemic hemisphere. Merged images of 2,3,5-triphenyl tetrazolium chloride staining and T2WI revealed the exact vasogenic edema region, which spread from the ischemic core to outside the ischemic region. Microglia were strongly activated in the ischemic region 3 h after occlusion and, notably, activated microglia were observed in the non-ischemic region 24 h after occlusion. Pretreatment with minocycline, an inhibitor of microglial activation clearly suppressed not only vasogenic edema but also infarct formation. We demonstrated in this study that vasogenic edema spreads from the ischemic core to the peripheral region, which can be elicited, at least in part, by microglial activation induced by ischemia., (Copyright © 2018 Elsevier Inc. All rights reserved.)

To establish prognostic biomarkers and to identify potential novel therapeutic targets, we performed integrative immunomonitoring of blood and tumor in patients with resectable pancreatic cancer. Flow cytometry (FC) was employed for phenotyping immune cells, multiplex bead assays for plasma cytokine and chemokine determination, and RNA-Seq for the analysis of gene expression in the tumor. Nineteen pancreatic cancer patients were stratified into those with longer or shorter than median recurrence-free survival after surgery (median, 426 days). There were no significant differences between the two groups for clinical parameters including age, sex, surgical procedure, stage, or postoperative adjuvant therapy. However, we found that the percentages of NK cells as assessed by FC in peripheral blood mononuclear cells were higher in patients with late recurrence (P = .037). RNA-Seq data indicated no differences in the amount of immune cells or stromal cells between the two groups, although NK cells in the tumor did tend to be higher in patients with late recurrence (P = .058). Type I and II IFN signatures were enriched in late-recurring tumors (FDR q-value <0.001), while genes related to KRAS signaling and the epithelial mesenchymal transition (EMT) were enriched in early recurrence. We conclude that tumor-intrinsic properties of metastasis and recurrence influence prognosis, whereas NK cells that might contribute to prevent metastasis are associated with longer recurrence-free survival. Therefore, enhancement of NK cell activity and inhibition of the EMT and KRAS signaling might represent appropriate therapeutic targets following surgical resection of pancreatic cancer., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Human herpesvirus 6B (HHV-6B) causes exanthema subitum in infants and is known to be mildly pathogenic. However, HHV-6B infection can induce febrile seizures in a high percentage of patients, and in rare cases, result in encephalitis. We detected higher levels of interleukin (IL)-1β and basic fibroblast growth factor (bFGF) in the cerebrospinal fluid (CFS) of patients with HHV-6B encephalitis when compared to those in patients with non-HHV-6B-induced febrile seizures. In vitro, IL-1β and bFGF enhanced HHV-6B gene expression in infected U373 astrocytes during the initial and maintenance phases of infection, respectively. These findings indicated that IL-1β and bFGF contribute to HHV-6B growth and the onset of encephalitis., (Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.)

Recently, hydropersulfide (RSSH) was found to exist in mammalian tissues and fluids. Cysteine hydropersulfide can be found in free cysteine residues as well as in proteins, and it has potent antioxidative activity. Human serum albumin (HSA) is the most abundant protein in mammalian serum. HSA possesses a free thiol group in Cys-34 that could be a site for hydropersulfide formation. HSA hydropersulfide of high purity as a positive control was prepared by treatment of HSA with Na 2 S. The presence of HSA hydropersulfide was confirmed by spectroscopy and ESI-TOFMS analysis where molecular weights of HSA hydropersulfide by increments of approximately 32 Da (Sulfur atom) were detected. The fluorescent probe results showed that Alexa Fluor 680 conjugated maleimide (Red-Mal) was a suitable assay and bromotrimethylammoniumbimane bromide appeared to be a selective reagent for hydropersulfide. The effect of oxidative stress related disease on the existence of albumin hydropersulfides was examined in rat 5/6 nephrectomy model of chronic kidney disease (CKD). Interestingly, the level of hydropersulfides in rat 5/6 nephrectomy model serum was decreased by a uremic toxin that increases oxidative stress in rat 5/6 nephrectomy model. Furthermore, we demonstrated that the levels of HSA hydropersulfide in human subjects were reduced in CKD but restored by hemodialysis using Red-Mal assay. We conclude that HSA hydropersulfide could potentially play an important role in biological anti-oxidative defense, and it is a promising diagnostic and therapeutic marker of oxidative diseases., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Fatigue reduces productivity and is a risk factor for lifestyle diseases and mental disorders. Everyone experiences physiological fatigue and recovers with rest. Pathological fatigue, however, greatly reduces quality of life and requires therapeutic interventions. It is therefore necessary to distinguish between the two but there has been no biomarker for this. We report on the measurement of salivary human herpesvirus (HHV-) 6 and HHV-7 as biomarkers for quantifying physiological fatigue. They increased with military training and work and rapidly decreased with rest. Our results suggested that macrophage activation and differentiation were necessary for virus reactivation. However, HHV-6 and HHV-7 did not increase in obstructive sleep apnea syndrome (OSAS), chronic fatigue syndrome (CFS) and major depressive disorder (MDD), which are thought to cause pathological fatigue. Thus, HHV-6 and HHV-7 would be useful biomarkers for distinguishing between physiological and pathological fatigue. Our findings suggest a fundamentally new approach to evaluating fatigue and preventing fatigue-related diseases., (Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.)

Hepcidin is a main regulator of iron metabolism, of which abnormal expression affects intestinal absorption and reticuloendothelial sequestration of iron by interacting with ferroportin. It is also noted that abnormal iron accumulation is one of the key factors to facilitate promotion and progression of cancer including hepatoma. By RT-PCR/agarose gel electrophoresis of hepcidin mRNA in a hepatocellular carcinoma cell line HLF, a smaller mRNA band was shown in addition to the wild-type hepcidin mRNA. From sequencing analysis, this additional band was a selective splicing variant of hepcidin mRNA lacking exon 2 of HAMP gene, producing the transcript that encodes truncated peptide lacking 20 amino acids at the middle of preprohepcidin. In the present study, we used the digital PCR, because such a small amount of variant mRNA was difficult to quantitate by the conventional RT-PCR amplification. Among seven hepatoma-derived cell lines, six cell lines have significant copy numbers of this variant mRNA, but not in one cell line. In the transient transfection analysis of variant-type hepcidin cDNA, truncated preprohepcidin has a different character comparing with native preprohepcidin: its product is insensitive to digestion, and secreted into the medium as a whole preprohepcidin form without maturation. Loss or reduction of function of HAMP gene by aberrantly splicing may be a suitable phenomenon to obtain the proliferating advantage of hepatoma cells., (Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.)

The mammalian maintenance methyltransferase DNMT1 [DNA (cytosine-5-)-methyltransferase 1] mediates the inheritance of the DNA methylation pattern during replication. Previous studies have shown that depletion of DNMT1 causes a severe growth defect and apoptosis in differentiated cells. However, the detailed mechanisms behind this phenomenon remain poorly understood. Here we show that conditional ablation of Dnmt1 in murine embryonic fibroblasts (MEFs) resulted in an aberrant DNA replication program showing an accumulation of late-S phase replication and causing severely defective growth. Furthermore, we found that the catalytic activity and replication focus targeting sequence of DNMT1 are required for a proper DNA replication program. Taken together, our findings suggest that the maintenance of DNA methylation by DNMT1 plays a critical role in proper regulation of DNA replication in mammalian cells., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Osteosarcoma is a common metastatic bone cancer that predominantly develops in children and adolescents. Metastatic osteosarcoma remains associated with a poor prognosis; therefore, more effective anti-metastatic drugs are needed. Differentiation-inducing factor-1 (DIF-1), -2, and -3 are novel lead anti-tumor agents that were originally isolated from the cellular slime mold Dictyostelium discoideum. Here we investigated the effects of a panel of DIF derivatives on lysophosphatidic acid (LPA)-induced migration of mouse osteosarcoma LM8 cells by using a Boyden chamber assay. Some DIF derivatives such as Br-DIF-1, DIF-3(+2), and Bu-DIF-3 (5-20 μM) dose-dependently suppressed LPA-induced cell migration with associated IC50 values of 5.5, 4.6, and 4.2 μM, respectively. On the other hand, the IC50 values of Br-DIF-1, DIF-3(+2), and Bu-DIF-3 versus cell proliferation were 18.5, 7.2, and 2.0 μM, respectively, in LM8 cells, and >20, 14.8, and 4.3 μM, respectively, in mouse 3T3-L1 fibroblasts (non-transformed). Together, our results demonstrate that Br-DIF-1 in particular may be a valuable tool for the analysis of cancer cell migration, and that DIF derivatives such as DIF-3(+2) and Bu-DIF-3 are promising lead anti-tumor agents for the development of therapies that suppress osteosarcoma cell proliferation, migration, and metastasis., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Epigenetic modifications such as DNA methylation and histone H3 lysine 27 methylation (H3K27me) are repressive marks that silence gene expression. The M phase phosphoprotein (MPP8) associates with proteins involved in both DNA methylation and histone modifications, and therefore, is a potential candidate to mediate crosstalk between repressive epigenetic pathways. Here, by performing immunohistochemical analyses we demonstrate that MPP8 is expressed in the rodent testis, especially in spermatocytes, suggesting a role in spermatogenesis. Interestingly, we found that MPP8 physically interacts with PRC1 (Polycomb Repressive Complex 1) components which are known to possess essential function in testis development by modulating monoubiquitination of Histone H2A (uH2A) and trimethylation of Histone H3 Lysine 27 (H3K27me3) residues. Knockdown analysis of MPP8 in HeLa cells resulted in derepression of a set of genes that are normally expressed in spermatogonia, spermatids and mature sperm, thereby indicating a role for this molecule in silencing testis-related genes in somatic cells. In addition, depletion of MPP8 in murine ES cells specifically induced expression of genes involved in mesoderm differentiation, such as Cdx2 and Brachyury even in the presence of LIF, which implicated that MPP8 might be required to repress differentiation associated genes during early development. Taken together, our results indicate that MPP8 could have a role for silencing genes that are associated with differentiation of the testis and the mesoderm by interacting with epigenetic repressors modules such as the PRC1 complex., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Galectins are a group of animal lectins characterized by their specificity for β-galactosides. Galectin-2 (Gal-2) is predominantly expressed in the gastrointestinal tract. A proteomic analysis identified Gal-2 as a protein that was S-nitrosylated when mouse gastric mucosal lysates were reacted with S-nitrosoglutathione, a physiologically relevant S-nitrosylating agent. In the present study, recombinant mouse (m)Gal-2 was S-nitrosylated using nitrosocysteine (CysNO), which had no effect on the sugar-binding specificity and dimerization capacity of the protein. On the other hand, mGal-2 oxidation by H2O2 resulted in the loss of sugar-binding ability, while S-nitrosylation prevented H2O2-inducted inactivation, presumably by protecting the Cys residue(s) in the protein. These results suggest that S-nitrosylation by nitric oxides protect Gal-2 from oxidative stress in the gastrointestinal tract., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Introduction: Liver fibrosis is characterized by excessive accumulation of extracellular matrix. In a mouse model of liver fibrosis, systemic injection of bone marrow mesenchymal stem cells (BM-MSCs) was considered to rescue the diseased phenotype. The aim of this study was to assess the effectiveness of human adipose tissue-derived multi-lineage progenitor cells (hADMPCs) in improving liver fibrosis., Methods and Results: hADMPCs were isolated from subcutaneous adipose tissues of healthy volunteers and expanded. Six week-old male nude mice were treated with carbon tetra-chloride (CCl4) by intraperitoneal injection twice a week for 6 weeks, followed by a tail vein injection of hADMPCs or placebo control. After 6 more weeks of CCl4 injection (12 weeks in all), nude mice with hADMPCs transplants exhibited a significant reduction in liver fibrosis, as evidenced by Sirius Red staining, compared with nude mice treated with CCl4 for 12 weeks without hADMPCs transplants. Moreover, serum glutamic pyruvate transaminase and total bilirubin levels in hADMPCs-treated nude mice were lower levels than those in placebo controls. Production of fibrinolytic enzyme MMPs from hADMPCs were examined by ELISA and compared to that from BM-MSCs. MMP-2 levels in the culture media were not significantly different, whereas those of MMP-3 and -9 of hADMPCs were higher than those by BM-MSCs., Conclusion: These results showed the mode of action and proof of concept of systemic injection of hADMPCs, which is a promising therapeutic intervention for the treatment of patients with liver fibrosis., (Copyright © 2014 Elsevier Inc. All rights reserved.)

ATP synthase (F-ATPase) function depends upon catalytic and rotation cycles of the F1 sector. Previously, we found that F1 ATPase activity is inhibited by the dietary polyphenols, curcumin, quercetin, and piceatannol, but that the inhibitory kinetics of curcumin differs from that of the other two polyphenols (Sekiya et al., 2012, 2014). In the present study, we analyzed Escherichia coli F1 ATPase rotational catalysis to identify differences in the inhibitory mechanism of curcumin versus quercetin and piceatannol. These compounds did not affect the 120° rotation step for ATP binding and ADP release, though they significantly increased the catalytic dwell duration for ATP hydrolysis. Analysis of wild-type F1 and a mutant lacking part of the piceatannol binding site (γΔ277-286) indicates that curcumin binds to F1 differently from piceatannol and quercetin. The unique inhibitory mechanism of curcumin is also suggested from its effect on F1 mutants with defective β-γ subunit interactions (γMet23 to Lys) or β conformational changes (βSer174 to Phe). These results confirm that smooth interaction between each β subunit and entire γ subunit in F1 is pertinent for rotational catalysis., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Transcription factor GATA-6 plays essential roles in developmental processes and tissue specific functions through regulation of gene expression. GATA-6 mRNA utilizes two Met-codons in frame as translational initiation codons. Deletion of the nucleotide sequence encoding the PEST sequence (Glu(31)-Cys(46)) between the two initiation codons unusually reduced the protein molecular size on SDS-polyacrylamide gel-electrophoresis, and re-introduction of this sequence reversed this change. The long-type (L-type) GATA-6 containing this PEST sequence self-associated similarly to the short-type (S-type) GATA-6, as determined on co-immunoprecipitation of Myc-tagged GATA-6 with HA-tagged GATA-6. The L-type and S-type GATA-6 also interacted mutually. The L-type GATA-6 without the PEST sequence also self-associated and interacted with the S-type GATA-6. The transcriptional activation potential of L-type GATA-6 is higher than that of S-type GATA-6. When the PEST sequence (Glu(31)-Cys(46)) was inserted into the L-type GATA-6 without Arg(13)-Gly(101), the resultant recombinant protein showed significantly higher transcriptional activity, while the construct with an unrelated sequence exhibited lower activity. These results suggest that the Glu(31)-Cys(46) segment plays an important role in the transcriptional activation, although it does not participate in the self-association., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Intra-molecular rotation of FOF1 ATP synthase enables cooperative synthesis and hydrolysis of ATP. In this study, using a small gold bead probe, we observed fast rotation close to the real rate that would be exhibited without probes. Using this experimental system, we tested the rotation of FOF1 with the ε subunit connected to a globular protein [cytochrome b562 (ε-Cyt) or flavodoxin reductase (ε-FlavR)], which is apparently larger than the space between the central and the peripheral stalks. The enzymes containing ε-Cyt and ε-FlavR showed continual rotations with average rates of 185 and 148 rps, respectively, similar to the wild type (172 rps). However, the enzymes with ε-Cyt or ε-FlavR showed a reduced proton transport. These results indicate that the intra-molecular rotation is elastic but proton transport requires more strict subunit/subunit interaction., (Copyright © 2014 Elsevier Inc. All rights reserved.)

The LIM-homeodomain transcription factor Islet2a establishes neuronal identity in the developing nervous system. Our previous study showed that Islet2a function is crucial for extending peripheral axons of sensory neurons in zebrafish embryo. Overexpressing a dominant-negative form of Islet2a significantly reduced peripheral axon extension in zebrafish sensory neurons, implicating Islet2a in the gene regulation required for neurite formation or proper axon growth in developing sensory neurons. Based on this, we conducted systematic screening to isolate genes regulated by Islet2a and affecting the development of axon growth in embryonic zebrafish sensory neurons. The 26 genes selected included some encoding factors involved in neuronal differentiation, axon growth, cellular signaling, and structural integrity of neurons, as well as genes whose functions are not fully determined. We chose four representative candidates as possible Islet2a downstream functional targets (simplet, tppp, tusc5 and tmem59l) and analyzed their respective mRNA expressions in dominant-negative Islet2a-expressing embryos. They are not reported the involvement of axonal extension or their functions in neural cells. Finally, knockdown of these genes suggested their direct actual involvement in the extension of peripheral axons in sensory neurons., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Skeletal muscle is a primary organ that uses blood glucose. Insulin- and 5'AMP-activated protein kinase (AMPK)-regulated intracellular signaling pathways are known as major mechanisms that regulate muscle glucose transport. It has been reported that macrophage migration inhibitory factor (MIF) is secreted from adipose tissue and heart, and affects these two pathways. In this study, we examined whether MIF is a myokine that is secreted from skeletal muscles and affects muscle glucose transport induced by these two pathways. We found that MIF is expressed in several different types of skeletal muscle. Its secretion was also confirmed in C2C12 myotubes, a skeletal muscle cell line. Next, the extensor digitorum longus (EDL) and soleus muscles were isolated from mice and treated with recombinant MIF in an in vitro muscle incubation system. MIF itself did not have any effect on glucose transport in both types of muscles. However, glucose transport induced by a submaximal dose of insulin was diminished by co-incubation with MIF in the soleus muscle. MIF also diminished glucose transport induced by a maximal dose of 5-aminoimidazole-4-carboxyamide ribonucleoside (AICAR), an AMPK activator, in the EDL muscle. These results suggest that MIF is a negative regulator of insulin- and AICAR-induced glucose transport in skeletal muscle. Since MIF secretion from C2C12 myotubes to the culture medium decreased during contraction evoked by electrical stimulations, MIF may be involved in the mechanisms underlying exercise-induced sensitization of glucose transport in skeletal muscle., (Copyright © 2014 Elsevier Inc. All rights reserved.)

We have shown previously that the Streptococcus mutans F-type H(+)-ATPase (F(O)F(1)) c subunit gene could complement Escherichia coli defective in the corresponding gene, particularly at acidic pH (Araki et al., (2013) [14]). In this study, the entire S. mutans F(O)F(1) was functionally assembled in the E. coli plasma membrane (SF(O)F(1)). Membrane SF(O)F(1) ATPase showed optimum activity at pH 7, essentially the same as that of the S. mutans, although the activity of E. coli F(O)F(1) (EF(O)F(1)) was optimum at pH≥9. The membranes showed detectable ATP-dependent H(+)-translocation at pH 5.5-6.5, but not at neutral conditions (pH≥7), consistent with the role of S. mutans F(O)F(1) to pump H(+) out of the acidic cytoplasm. A hybrid F(O)F(1), consisting of membrane-integrated F(O) and -peripheral F(1) sectors from S. mutans and E. coli (SF(O)EF(1)), respectively, essentially showed the same pH profile as that of EF(O)F(1) ATPase. However, ATP-driven H(+)-transport was similar to that by SF(O)F(1), with activity at acidic pH. Replacement of the conserved c subunit Glu53 in SF(O)F(1) abolished H(+)-transport at pH 6 or 7, suggesting its role in H(+) transport. Mutations in the SF(O)F(1) c subunit, Ser17Ala or Glu20Ile, changed the pH dependency of H(+)-transport, and the F(O) could transport H(+) at pH 7, as the membranes with EF(O)F(1). Ser17, Glu20, and their vicinity were suggested to be involved in H(+)-transport in S. mutans at acidic pH., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Autophagy is an intracellular degradative system that is believed to be involved in the aging process. The contribution of autophagy to age-related changes in the human skin is unclear. In this study, we examined the relationship between autophagy and skin aging. Transmission electron microscopy and immunofluorescence microscopy analyses of skin tissue and cultured dermal fibroblasts derived from women of different ages revealed an increase in the number of nascent double-membrane autophagosomes with age. Western blot analysis showed that the amount of LC3-II, a form associated with autophagic vacuolar membranes, was significantly increased in aged dermal fibroblasts compared with that in young dermal fibroblasts. Aged dermal fibroblasts were minimally affected by inhibition of autophagic activity. Although lipofuscin autofluorescence was elevated in aged dermal fibroblasts, the expression of Beclin-1 and Atg5-genes essential for autophagosome formation-was similar between young and aged dermal fibroblasts, suggesting that the increase of autophagosomes in aged dermal fibroblasts was due to impaired autophagic flux rather than an increase in autophagosome formation. Treatment of young dermal fibroblasts with lysosomal protease inhibitors, which mimic the condition of aged dermal fibroblasts with reduced autophagic activity, altered the fibroblast content of type I procollagen, hyaluronan and elastin, and caused a breakdown of collagen fibrils. Collectively, these findings suggest that the autophagy pathway is impaired in aged dermal fibroblasts, which leads to deterioration of dermal integrity and skin fragility., (Crown Copyright © 2013. Published by Elsevier Inc. All rights reserved.)

A murine macrophage-derived line, RAW264.7, becomes multinuclear on stimulation with lipopolysaccharide (LPS), an outer membrane component of Gram-negative bacteria. These multinuclear cells internalized more polystyrene beads than mononuclear cells or osteoclasts (Nakanishi-Matsui, M., Yano, S., Matsumoto, N., and Futai, M., 2012). In this study, we analyzed the time courses of cell fusion in the presence of large beads. They were internalized into cells actively fusing to become multinuclear. However, the multinuclear cells once formed showed only low phagocytosis activity. These results suggest that formation of the multinuclear cells and bead internalization took place simultaneously. The formation of multinuclear cells was blocked by inhibitors for phosphoinositide 3-kinase, phospholipase C, calcineurin, and c-Jun N-terminal kinase. In addition, interleukin 6 and 10 also exhibited inhibitory effects. These signaling molecules and cytokines may play a crucial role in the LPS-induced multinuclear cell formation., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Our aims were to evaluate the involvement of heparin-binding EGF-like growth factor (HB-EGF) in liver fibrogenesis of humans and mice and to elucidate the effect of HB-EGF deficiency on cholestatic liver fibrosis using conditional HB-EGF knockout (KO) mice. We first demonstrated that gene expression of HB-EGF had a positive significant correlation with that of collagen in human fibrotic livers, and was increased in bile duct ligation (BDL)-induced fibrotic livers in mouse. We then generated conditional HB-EGF knockout (KO) mice using the interferon inducible Mx-1 promoter driven Cre recombinase transgene and wild type (WT) and KO mice were subjected to BDL. After BDL, KO mice exhibited enhanced liver fibrosis with increased expression of collagen, compared with WT mice. Finally, we used mouse hepatic stellate cells (HSCs) to examine the role of HB-EGF in the activation of these cells and showed that HB-EGF antagonized TGF-β-induced gene expression of collagen in mouse primary HSCs. Interestingly, HB-EGF did not prevent the TGF-β-induced nuclear accumulation of Smad3, but did lead to stabilization of the Smad transcriptional co-repressor TG-interacting factor. In conclusion, our data suggest a possible protective role of HB-EGF in cholestatic liver fibrosis., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Galactoseβ1-4Fucose (Galβ1-4Fuc) is a unique disaccharide exclusively found in N-glycans of protostomia, and is recognized by some galectins of Caenorhabditis elegans and Coprinopsis cinerea. In the present study, we investigated whether mammalian galectins also bind such a disaccharide. We examined sugar-binding ability of human galectin-1 (hGal-1) and found that hGal-1 preferentially binds Galβ1-4Fuc compared to Galβ1-4GlcNAc, which is its endogenous recognition unit. We also tested other human and mouse galectins, i.e., hGal-3, and -9 and mGal-1, 2, 3, 4, 8, and 9. All of them also showed substantial affinity to Galβ1-4Fuc disaccharide. Further, we assessed the inhibitory effect of Galβ1-4Fuc, Galβ1-4Glc, and Gal on the interaction between hGal-1 and its model ligand glycan, and found that Galβ1-4Fuc is the most effective. Although the biological significance of galectin-Galβ1-4Fuc interaction is obscure, it might be possible that Galβ1-4Fuc disaccharide is recognized as a non-self-glycan antigen. Furthermore, Galβ1-4Fuc could be a promising seed compound for the synthesis of novel galectin inhibitors., (Copyright © 2013 Elsevier Inc. All rights reserved.)

DNA methylation is a key epigenetic contributor to gene regulation in mammals. We have recently found that in the mouse liver, the promoter region of glycerol-3-phosphate acyltransferase 1, a rate-limiting enzyme of de novo lipogenesis, is regulated by DNA methylation, which is mediated by Dnmt3b, an enzyme required for the initiation of de novo methylation. In this study, using primary cultures of mouse hepatocytes with adenoviral overexpression of Dnmt3b, we characterized Dnmt3b-dependent DNA methylation on a genome-wide basis. A genome-wide DNA methylation analysis, called microarray-based integrated analysis of methylation by isoschizomers, identified 108 genes with Dnmt3b dependent DNA methylation. In DNA expression array analysis, expression of some genes with Dnmt3b-dependent DNA methylation was suppressed. Studies with primary mouse hepatocytes overexpressing Dnmt3b or Dnmt3a revealed that many genes with Dnmt3b-dependent methylation are not methylated by Dnmt3a, whereas those methylated by Dnmt3a are mostly methylated by Dnmt3b. Bioinformatic analysis showed that the CANAGCTG and CCGGWNCSC (N denotes A, T, G, or C; W denotes A or T; and S denotes C or G) sequences are enriched in genes methylated by overexpression of Dnmt3b and Dnmt3a, respectively. We also observed a large number of genes with Dnmt3b-dependent DNA methylation in primary cultures of mouse hepatocytes with adenoviral overexpression of Dnmt3, suggesting that Dnmt3b is an important DNA methyltransferase in primary mouse hepatocytes, targets specific genes, and potentially plays a role in vivo., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Insulin resistance is a pathological hallmark of type 2 diabetes mellitus and is characterized by defects in insulin signaling. Protein tyrosine phosphatase 1B (PTP1B) negatively regulates insulin signaling by tyrosine dephosphorylation of insulin receptor, and increased activity and expression of PTP1B is implicated in the pathogenesis of insulin resistance. Therefore, inhibition of PTP1B is anticipated to improve insulin resistance in type 2 diabetic subjects. Pyrroloquinoline quinone (PQQ), a redox cofactor for bacterial dehydrogenases, inhibits PTP1B to oxidatively modify the catalytic cysteine through its redox cycling activity. Here, we report that PQQ induces the ligand-independent activation of insulin signaling by inhibiting cellular PTP1B and enhances glucose uptake through the translocation of glucose transporter 4 in mouse C2C12 myotubes. Furthermore, we demonstrated that oral administration of PQQ improved impaired glucose tolerance in type 2 diabetic KK-A(y) mice. Our results strongly suggest that PQQ can be useful in anti-diabetic treatment for type 2 diabetic subjects., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Increased microvessel density in atherosclerotic plaques plays a major role in promoting plaque destabilization resulting in increased risk of stroke and myocardial infarction. Previously we have shown that expression of the inflammatory cytokine, Oncostatin-M (OSM), in human atherosclerotic plaques correlated with increased microvessel density, indicating a role for OSM in promoting plaque angiogenesis. The purpose of this study was to determine the mechanism by which OSM regulates Vascular Endothelial Growth Factor (VEGF) expression in human coronary artery smooth muscle cells. Using shRNA and overexpression studies, we have shown that the transcription factor, STAT-1 inhibited VEGF expression, while STAT-3 promoted the expression of VEGF. We further show that the mechanism by which STAT-1 and STAT-3 regulates VEGF expression is through modulation of Hypoxia Inducible Factor-1α (HIF-1α). STAT-1 suppresses HIF-1α expression, whereas STAT-3 positively regulates HIF-1α expression. These results provide evidence that activated STAT-1 and STAT-3 regulate VEGF expression indirectly, by modulating HIF-1α activity., (Published by Elsevier Inc.)

Dysfunction of Parkin, a RING-IBR-RING motif containing protein, causes autosomal recessive familial Parkinsonism. Biochemically, Parkin is a ubiquitin-ligating enzyme (E3) that catalyzes ubiquitin transfer from ubiquitin-activating and -conjugating enzymes (E1/E2) to a substrate. Recent studies have revealed that Parkin localizes in the cytoplasm and its E3 activity is repressed under steady-state conditions. In contrast, Parkin moves to mitochondria with low membrane potential, thereby activating the latent enzymatic activity of the protein, which in turn triggers Parkin-mediated ubiquitylation of numerous mitochondrial substrates. However, the mechanism of how Parkin-catalyzed ubiquitylation maintains mitochondrial integrity has yet to be determined. To begin to address this, we screened for novel Parkin substrate(s) and identified mitochondrial hexokinase I (HKI) as a candidate. Following a decrease in membrane potential, Parkin ubiquitylation of HKI leads to its proteasomal degradation. Moreover, most disease-relevant mutations of Parkin hinder this event and endogenous HKI is ubiquitylated upon dissipation of mitochondrial membrane potential in genuine-Parkin expressing cells, suggesting its physiological importance., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Transplantation of human cardiomyoblast-like cells (hCLCs) from human adipose tissue-derived multi-lineage progenitor cells improved left ventricular function and survival of rats with myocardial infarction. Here we examined the effect of intracoronary artery transplantation of human CLCs in a swine model of chronic heart failure. Twenty-four pigs underwent balloon-occlusion of the first diagonal branch followed by reperfusion, with a second balloon-occlusion of the left ascending coronary artery 1 week later followed by reperfusion. Four weeks after the second occlusion/reperfusion, 17 of the 18 surviving animals with severe chronic MI (ejection fraction <35% by echocardiography) were immunosuppressed then randomly assigned to receive either intracoronary artery transplantation of hCLCs hADMPCs or placebo lactic Ringer's solution with heparin. Intracoronary artery transplantation was followed by the distribution of DiI-stained hCLCs into the scarred myocardial milieu. Echocardiography at post-transplant days 4 and 8 weeks showed rescue and maintenance of cardiac function in the hCLCs transplanted group, but not in the control animals, indicating myocardial functional recovery by hCLCs intracoronary transplantation. At 8 week post-transplantation, 7 of 8 hCLCs transplanted animals were still alive compared with only 1 of the 5 control (p=0.0147). Histological studies at week 12 post-transplantation demonstrated engraftment of the pre DiI-stained hCLCs into the scarred myocardium and their expression of human specific alpha-cardiac actin. Human alpha cardiac actin-positive cells also expressed cardiac nuclear factors; nkx2.5 and GATA-4. Our results suggest that intracoronary artery transplantation of hCLCs is a potentially effective therapeutic strategy for future cardiac tissue regeneration., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Lipopolysaccharide (LPS), an outer membrane component of Gram-negative bacteria, induces strong proinflammatory responses, including the release of cytokines and nitric oxide from macrophage. In this study, we found that a murine macrophage-derived line, RAW264.7, became multinuclear through cell-cell fusion after incubation with highly purified LPS or synthetic lipid A in the presence of Ca(2+). The same cell line is known to differentiate into multinuclear osteoclast, which expresses a specific proton pumping ATPase together with osteoclast markers on stimulation by the extracellular domain of receptor activator of nuclear factor κB ligand (Toyomura, T., Murata, Y., Yamamoto, A., Oka, T., Sun-Wada, G.-H., Wada, Y. and Futai, M., 2003). The LPS-induced multinuclear cells did not express osteoclast-specific enzymes including tartrate-resistant acid phosphatase and cathepsin K. During multinuclear cell formation, the cells internalized more and larger polystyrene beads (diameter 6-15 μm) than mononuclear cells and osteoclasts. The internalized beads were located in lysosome-marker positive organelles, which were probably phagolysosomes. The LPS-induced multinuclear cell could be a good model system to study phagocytosis of large foreign bodies., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Under fasting conditions, triacylglycerol in adipose tissue undergoes lipolysis to supply fatty acids as energy substrates. Such lipolysis is regulated by hormones, which activate lipases via stimulation of specific signalling cascades. We previously showed that macrophage-derived soluble protein, AIM induces obesity-associated lipolysis, triggering chronic inflammation in fat tissue which causes insulin resistance. However, the mechanism of how AIM mediates lipolysis remains unknown. Here we show that AIM induces lipolysis in a manner distinct from that of hormone-dependent lipolysis, without activation or augmentation of lipases. In vivo and in vitro, AIM did not enhance phosphorylation of hormone-sensitive lipase (HSL) in adipocytes, a hallmark of hormone-dependent lipolysis activation. Similarly, adipose tissue from obese AIM-deficient and wild-type mice showed comparable HSL phosphorylation. Consistent with the suppressive effect of AIM on fatty acid synthase activity, the amount of saturated and unsaturated fatty acids was reduced in adipocytes treated with AIM. This response ablated transcriptional activity of peroxisome proliferator-activated receptor (PPARγ), leading to diminished gene expression of lipid-droplet coating proteins including fat-specific protein 27 (FSP27) and Perilipin, which are indispensable for triacylglycerol storage in adipocytes. Accordingly, the lipolytic effect of AIM was overcome by a PPARγ-agonist or forced expression of FSP27, while it was synergized by a PPARγ-antagonist. Overall, distinct modes of lipolysis appear to take place in different physiological situations; one is a supportive response against nutritional deprivation achieved by enhancing lipase activity, and the other is a pathological consequence of obesity, causing subclinical inflammation and metabolic disorders, mediated by abolishing droplet-coating proteins., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Glucagon-like peptide-1 (GLP-1) and leptin are anorectic hormones produced in the small intestine and white adipose tissue, respectively. Investigating how these hormones act together as an integrated anorectic signal is important to elucidate a mechanism to maintain energy balance. In the present study, coadministration of subthreshold GLP-1 and leptin dramatically reduced feeding in rats. Although coadministration of GLP-1 with leptin did not enhance leptin signal transduction in the hypothalamus, it significantly decreased phosphorylation of AMP-activated protein kinase (AMPK). In addition, coadministration of GLP-1 with leptin significantly increased proopiomelanocortin (POMC) mRNA levels. Considering that α-melanocortin stimulating hormone (α-MSH) is derived from POMC and functions through the melanocortin-4-receptor (MC4-R) as a key molecule involved in feeding reduction, the interaction of GLP-1 and leptin on feeding reduction may be mediated through the α-MSH/MC4-R system. As expected, the interaction of GLP-1 and leptin was abolished by intracerebroventricular preadministration of the MC4-R antagonists agouti-related peptide and SHU9119. Taken together, GLP-1 and leptin cooperatively reduce feeding at least in part via inhibition of AMPK following binding of α-MSH to MC4-R., (Copyright © 2012 Elsevier Inc. All rights reserved.)

The glial cells missing a (GCMa) transcription factor plays a pivotal role in the placental development by regulating the expression of several genes in the placenta that are responsible for the proper formation of the syncytiotrophoblast. It is well known that the function of GCMa is regulated at both transcriptional and post-translational levels by the cyclic AMP (cAMP)/protein kinase A (PKA)-dependent pathway, the activation of which increases the GCMa protein level and leads to trophoblast differentiation into the syncytiotrophoblast. However, little is known about the regulatory control of GCMa by PKC-dependent signaling mechanism(s). To investigate whether GCMa is regulated by PKC-dependent pathway, we treated the human choriocarcinoma JEG-3 cells with phorbol 12-myristate 13-acetate (PMA) and studied its effect on the GCMa protein using a monoclonal anti-GCMa antibody we prepared. PMA caused a transient decrease in the endogenous GCMa protein level in JEG-3 cells that was accompanied by an increase in GCMa phosphorylation. The phosphorylation and degradation of GCMa by PMA treatment was effectively reduced by pretreatment with protein kinase C (PKC) inhibitors and a mitogen-activated protein kinase (MAPK) kinase (MEK) inhibitor, indicating a PKC- and MEK-dependent mechanism. Furthermore, we identified the serine residues 328, 378 and 383 to be the phosphorylation sites on GCMa that are involved in the PMA-induced degradation of GCMa. Our data demonstrate for the first time that GCMa is phosphorylated by the PKC- and MEK/extracellular signal-regulated kinase (ERK)-dependent mechanism, and that this phosphorylation is involved in its degradation process., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Aryl hydrocarbon receptor (AHR) and AHR nuclear translocator (ARNT) are well-conserved transcription factors among species. However, there are a very limited number of reports on the physiological function of AHR, particularly on the regulation of AHR by endogenous compounds. We hence investigated the effects of all-trans retinoic acid (atRA) on cytochrome P450 (CYP) 1A1 gene transcription as a model of AHR-regulated transcription mechanisms in HepG2 cells, a human hepatoma cell line. Treatment with atRA significantly reduced transactivation and expression of CYP1A1 mRNA to less than half of its control value, and this inhibitory effect was mediated by RARα. The result of chromatin immunoprecipitation assay indicated that treatment with atRA at 1-100 nM drastically inhibited the recruitment of ARNT to DNA regions containing xenobiotic responsive elements. In conclusion, atRA at physiological concentrations could reduce AHR-mediated gene transcription via the inhibition of recruitment of ARNT to relevant DNA regions., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Double strand breaks (DSBs) are the most serious type of DNA damage. DSBs can be generated directly by exposure to ionizing radiation or indirectly by replication fork collapse. The DNA damage tolerance pathway, which is conserved from bacteria to humans, prevents this collapse by overcoming replication blockages. The INO80 chromatin remodeling complex plays an important role in the DNA damage response. The yeast INO80 complex participates in the DNA damage tolerance pathway. The mechanisms regulating yINO80 complex are not fully understood, but yeast INO80 complex are necessary for efficient proliferating cell nuclear antigen (PCNA) ubiquitination and for recruitment of Rad18 to replication forks. In contrast, the function of the mammalian INO80 complex in DNA damage tolerance is less clear. Here, we show that human INO80 was necessary for PCNA ubiquitination and recruitment of Rad18 to DNA damage sites. Moreover, the C-terminal region of human INO80 was phosphorylated, and overexpression of a phosphorylation-deficient mutant of human INO80 resulted in decreased ubiquitination of PCNA during DNA replication. These results suggest that the human INO80 complex, like the yeast complex, was involved in the DNA damage tolerance pathway and that phosphorylation of human INO80 was involved in the DNA damage tolerance pathway. These findings provide new insights into the DNA damage tolerance pathway in mammalian cells., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Dexamethasone (DEX), a potent glucocorticoid, increased the expression of T-cell death associated gene 8 (TDAG8), a proton-sensing G protein-coupled receptor, which is associated with the enhancement of acidic pH-induced cAMP accumulation, in peritoneal macrophages. We explored the role of increased TDAG8 expression in the anti-inflammatory actions of DEX. The treatment of macrophages with either DEX or acidic pH induced the cell death of macrophages; however, the cell death was not affected by TDAG8 deficiency. While DEX inhibited lipopolysaccharide-induced production of tumor necrosis factor-α, an inflammatory cytokine, which was independent of TDAG8, at neutral pH, the glucocorticoid enhanced the acidic pH-induced inhibition of tumor necrosis factor-α production in a manner dependent on TDAG8. In conclusion, the DEX-induced increase in TDAG8 expression is in part involved in the glucocorticoid-induced anti-inflammatory actions through the inhibition of inflammatory cytokine production under the acidic pH environment. On the other hand, the role of TDAG8 in the DEX-induced cell death is questionable., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Epigallocatechin-3-gallate (EGCG), the major polyphenolic component of green tea, has been demonstrated to possess anti-inflammatory, antioxidant, anti-mutagenic and anti-carcinogenic properties. The anti-melanoma effect of EGCG has been previously suggested, but no clear mechanism of action has been established. In this study, we demonstrated that EGCG inhibits melanoma cell growth at physiological doses (0.1-1 μM). In the search for mechanisms of EGCG-mediated melanoma cell suppression, we found that NF-κB was inhibited, and that reduced NF-κB activity was associated with decreased IL-1β secretion from melanoma cells. Since inflammasomes are involved in IL-1β secretion, we investigated whether IL-1β suppression was mediated by inflammasomes, and found that EGCG treatment led to downregulation of the inflammasome component, NLRP1, and reduced caspase-1 activation. Furthermore, silencing the expression of NLRP1 abolished EGCG-induced inhibition of tumor cell proliferation both in vitro and in vivo, suggesting a key role of inflammasomes in EGCG efficacy. This paper provides a novel mechanism for EGCG-induced melanoma inhibition: inflammasome downregulation→decreased IL-1β secretion→decreased NF-κB activities→decreased cell growth. In addition, it suggests inflammasomes and IL-1β could be potential targets for future melanoma therapeutics., (Published by Elsevier Inc.)

Ligands for L-selectin, a leukocyte adhesion molecule, are expressed in high endothelial venules (HEVs) in lymph nodes and extravascular tissues, such as renal tubules. Here, we report that the binding of L-selectin to its vascular and extravascular ligands is differentially regulated by pH. The optimal L-selectin-dependent binding of leukocytes to HEVs was observed at pH 7.4, a physiological pH in the blood. In contrast, the optimal binding of leukocytes to the renal tubules was observed at pH 5.6. Consistently, optimal binding of soluble recombinant L-selectin to a major vascular ligand, 6-sulfo sialyl Lewis X, was observed at pH 7.4. Binding to extravascular ligands, such as chondroitin sulfate (CS) B, CS E and heparan sulfate, occurred at pH 5.6. Under physiological shear stress ranging from 1 to 2 dynes/cm(2), maximal leukocyte rolling on vascular ligands was observed at pH 6.8 to 7.4, and no rolling was detected at pH conditions below 5.6. These findings suggest that the pH environment is one important factor that determines leukocyte trafficking under physiological and pathological conditions., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Asthma is characterized by airway inflammation, hyper-responsiveness and remodeling. Extracellular acidification is known to be associated with severe asthma; however, the role of extracellular acidification in airway remodeling remains elusive. In the present study, the effects of acidification on the expression of connective tissue growth factor (CTGF), a critical factor involved in the formation of extracellular matrix proteins and hence airway remodeling, were examined in human airway smooth muscle cells (ASMCs). Acidic pH alone induced a substantial production of CTGF, and enhanced transforming growth factor (TGF)-β-induced CTGF mRNA and protein expression. The extracellular acidic pH-induced effects were inhibited by knockdown of a proton-sensing ovarian cancer G-protein-coupled receptor (OGR1) with its specific small interfering RNA and by addition of the G(q/11) protein-specific inhibitor, YM-254890, or the inositol-1,4,5-trisphosphate (IP(3)) receptor antagonist, 2-APB. In conclusion, extracellular acidification induces CTGF production through the OGR1/G(q/11) protein and inositol-1,4,5-trisphosphate-induced Ca(2+) mobilization in human ASMCs., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Familial hypercholesterolemia (FH) is an autosomal codominant disease characterized by high concentrations of proatherogenic lipoproteins secondary to deficiency in low-density lipoprotein (LDL) receptor. We reported recently the use of in situ stem cell therapy of human adipose tissue-derived multilineage progenitor cells (hADMPCs) in lowering serum total cholesterol in the homozygous Watanabe heritable hyperlipidemic (WHHL) rabbits, an animal model of homozygous FH. Here we demonstrate that pravastatin, an HMG-CoA reductase inhibitor, augmented the cholesterol-lowering effect of transplanted hADMPCs and enhanced LDL clearance in homozygous WHHL rabbit. The results suggest the potential beneficial effects of in situ stem cell therapy in concert with appropriately selected pharmaceutical agents, in regenerative medicine., (Copyright © 2011 Elsevier Inc. All rights reserved.)

L-type amino-acid transporter 1 (LAT1) is the first identified light chain of CD98 molecule, disulfide-linked to a heavy chain of CD98. Following cDNA cloning of chicken full-length LAT1, we have constructed targeting vectors for the disruption of chicken LAT1 gene from genomic DNA of chicken LAT1 consisting of 5.4kb. We established five homozygous LAT1-disrupted (LAT1(-/-)) cell clones, derived from a heterozygous LAT1(+/-) clone of DT40 chicken B cell line. Reactivity of anti-chicken CD98hc monoclonal antibody (mAb) with LAT1(-/-) DT40 cells was markedly decreased compared with that of wild-type DT40 cells. All LAT1(-/-) cells were deficient in L-type amino-acid transporting activity, although alternative-splice variant but not full-length mRNA of LAT1 was detected in these cells. LAT1(-/-) DT40 clones showed outstandingly slow growth in liquid culture and decreased colony-formation capacity in soft agar compared with wild-type DT40 cells. Cell-cycle analyses indicated that LAT1(-/-) DT40 clones have prolonged cell-cycle phases compared with wild-type or LAT1(+/-) DT40 cells. Knockdown of human LAT1 by small interfering RNAs resulted in marked in vitro cell-growth inhibition of human cancer cells, and in vivo tumor growth of HeLa cells in athymic mice was significantly inhibited by anti-human LAT1 mAb. All these results indicate essential roles of LAT1 in the cell proliferation and occurrence of malignant phenotypes and that LAT1 is a promising candidate as a molecular target of human cancer therapy., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Matrix metalloproteinase (MMP) plays an important role in homeostatic regulation of the extracellular environment and degradation of matrix. During liver fibrosis, several MMPs, including MMP-2, are up-regulated in activated hepatic stellate cells, which are responsible for exacerbation of liver cirrhosis. However, it remains unclear how loss of MMP-2 influences molecular dynamics associated with fibrogenesis in the liver. To explore the role of MMP-2 in hepatic fibrogenesis, we employed two fibrosis models in mice; toxin (carbon tetrachloride, CCl4)-induced and cholestasis-induced fibrosis. In the chronic CCl4 administration model, MMP-2 deficient mice exhibited extensive liver fibrosis as compared with wild-type mice. Several molecules related to activation of hepatic stellate cells were up-regulated in MMP-2 deficient liver, suggesting that myofibroblastic change of hepatic stellate cells was promoted in MMP-2 deficient liver. In the cholestasis model, fibrosis in MMP-2 deficient liver was also accelerated as compared with wild type liver. Production of tissue inhibitor of metalloproteinase 1 increased in MMP-2 deficient liver in both models, while transforming growth factor β, platelet-derived growth factor receptor and MMP-14 were up-regulated only in the CCl4 model. Our study demonstrated, using 2 experimental murine models, that loss of MMP-2 exacerbates liver fibrosis, and suggested that MMP-2 suppresses tissue inhibitor of metalloproteinase 1 up-regulation during liver fibrosis., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Ectodermal organs, such as the tooth, salivary gland, hair, and mammary gland, develop through reciprocal epithelial-mesenchymal interactions. Tooth morphologies are defined by the crown width and tooth length (macro-morphologies), and by the number and locations of the cusp and roots (micro-morphologies). In our current study, we report that the crown width of a bioengineered molar tooth, which was reconstructed using dissociated epithelial and mesenchymal cells via an organ germ method, can be regulated by the contact area between epithelial and mesenchymal cell layers. We further show that this is associated with cell proliferation and Sonic hedgehog (Shh) expression in the inner enamel epithelium after the germ stage has formed a secondary enamel knot. We also demonstrate that the cusp number is significantly correlated with the crown width of the bioengineered tooth. These findings suggest that the tooth micro-morphology, i.e. the cusp formation, is regulated after the tooth width, or macro-morphology, is determined. These findings also suggest that the spatiotemporal patterning of cell proliferation and the Shh expression areas in the epithelium regulate the crown width and cusp formation of the developing tooth., (Copyright © 2011 Elsevier Inc. All rights reserved.)