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Benzalkonium chloride (BAC) is used as a preservative in eyedrops but induces subconjunctival fibrosis that can result in failure of glaucoma surgery. Tenon’s capsule fibroblasts in subconjunctival tissue interact with the corneal epithelium through tear fluid. With the use of a coculture system, we have now investigated the effect of human corneal epithelial (HCE) cells on myofibroblastic transdifferentiation of human Tenon fibroblasts (HTFs) induced by BAC (5 × 10−6%). Immunofluorescence and immunoblot analyses revealed that the BAC-induced expression of α smooth muscle actin (αSMA) in HTFs was suppressed by coculture of these cells with HCE cells (p p p p

Epithelial–mesenchymal transition (EMT) in retinal pigment epithelial (RPE) cells plays a key role in proliferative retinal diseases such as age-related macular degeneration by contributing to subretinal fibrosis. To investigate the potential role of retinoic acid receptor-α (RAR-α) signaling in this process, we have now examined the effects of the RAR-α agonist Am580 on EMT induced by transforming growth factor-β2 (TGF-β2) in primary mouse RPE cells cultured in a three-dimensional type I collagen gel as well as on subretinal fibrosis in a mouse model. We found that Am580 inhibited TGF-β2-induced collagen gel contraction mediated by RPE cells. It also attenuated the TGF-β2-induced expression of the mesenchymal markers α-smooth muscle actin, fibronectin, and collagen type I; production of pro-matrix metalloproteinase 2 and interleukin-6; expression of the focal adhesion protein paxillin; and phosphorylation of SMAD2 in the cultured RPE cells. Finally, immunofluorescence analysis showed that Am580 suppressed both the TGF-β2-induced translocation of myocardin-related transcription factor-A (MRTF-A) from the cytoplasm to the nucleus of cultured RPE cells as well as subretinal fibrosis triggered by laser-induced photocoagulation in a mouse model. Our observations thus suggest that RAR-α signaling inhibits EMT in RPE cells and might attenuate the development of fibrosis associated with proliferative retinal diseases.

Branch atheromatous disease (BAD) is often associated with corticospinal tract injury, and some patients develop early neurological deterioration (END) in the acute phase. This study investigated the progress of upper limb prognosis after BAD in the acute phases and examined the factors related to the prognosis of upper limb function.108 subjects diagnosed with BAD were included. Then subjects were classified into two groups: those with good recovery of upper limb function and those with poor recovery of upper limb function. Univariate and multivariate analyses were performed with the objective variable being good or poor upper limb function. The following factors were used as explanatory variables: age, the volume of infarction, initial Fugl-Meyer assessment (FMA) upper limb score, and presence of END.The univariate analysis showed significant differences in age and volume of infarction (p 0.05). Multivariate analysis showed the following finding: age;(OR 0.977,95%CI 0.917-0.997,p = 0.0061; volume of infarction;(OR 0.645,95%CI 0.461-0.902,p = 0.0104). A significant difference was found in the age and volume of the infarct.This study finding suggests that age and volume of infarction are associated with the prognosis of upper extremity paralysis in BAD.

Background Mechanical Thrombectomy (MT) is a recommended approach for post-cerebral ischemia in acute settings. Although a large amount of evidence suggests the use of MT, existing evidence has primarily focused on assessing lower limb performance or gait performance as an outcome measure. Methods This study was to investigate whether MT would be an effective approach for improving upper limb performance in post-stroke patients.This case control was divided into two groups: 154 patients as a control group only given conventional rehabilitation; and 25 patients as an intervention group given MT and conventional rehabilitation. Outcome variables were measured by calculating the change of Fugl-Meyer Assessment score at the last intervention compared with the beginning of the intervention. Result By comparing the FMA scores after, the propensity matching compared between before receiving therapy intervention and after, the intervention group showed as follows: 30.4 ± 26.4–44.3 ± 25.4, p = 0.0019, r = 0.59. The control group showed as follows: 39.9 ± 24.1–49.1 ± 21.3, p = 0.002, r = 0.69. Lastly, a comparison of the intervention group with the control group about their FMA score change indicates as follows: intervention group: 13.9 ± 19.4, control group 9.2 ± 10.0, p = 0.2967, r = 0.15. Conclusion This study indicated that there was no significant difference between MT and a conventional approach for improving UE function. However, this is the first study to investigate the course of recovery of UE function in the acute phase after MT, and this finding supports the need for further research.

BACKGROUND/AIM We have previously reported that treatment of pancreatic cancer cells with active hexose-correlated compound (AHCC), an extract of a basidiomycete mushroom, decreases the levels of tumor-associated proteins including heat-shock protein 27 (HSP27), heat shock factor 1 (HSF1) and sex-determining region Y-box 2 (SOX2). The transmembrane glycoprotein, CUB domain-containing protein 1 (CDCP1) has been reported to be up-regulated in various cancers, and be associated with invasion and metastasis. The aim of this study was to examine the effect of AHCC on the expression of CDCP1 in KLM1-R cells. MATERIALS AND METHODS Gemcitabine-resistant pancreatic cancer cells (KLM1-R) were treated with AHCC (10 mg/ml) for 48 h. Western blot analysis of cell extracts with anti-CDCP1 or anti-actin antibodies was performed to assess the expression of CDCP1. RESULTS Expression of CDCP1 was reduced by AHCC treatment of KLM1-R cells, whereas expression of actin was not affected. The ratio of intensities of CDCP1/actin in AHCC-treated KLM1-R cells was significantly suppressed (p

[This corrects the article DOI: 10.1371/journal.pone.0219405.].

Understanding how energy metabolism and related proteins influence neural progenitor cells in adult tissues is critical for developing new strategies in clinical tissue regeneration therapy. We have recently reported that a subtoxic concentration of glutamate-induced neural progenitor cells in the mature ex vivo rat retina. We herein explore changes in the metabolic pathways during the process. We firstly observed an increase in lactate and lactate dehydrogenase concentration in the glutamate-treated retina. We then investigated the levels of glycolytic enzymes and confirmed significant upregulation of pyruvate kinase M type (PKM), especially PKM2, enolase, phosphoglycerate mutase 1 (PGAM1), and inosine-5'-monophosphate dehydrogenase (IMPDH1) in the glutamate-treated retina compared to the untreated retina. An analysis of the subcellular localization of PKM2 revealed nuclear translocation in the treated retina, which has been reported to regulate cell cycle proliferation and glycolytic enzymes. Our findings indicate that the mature rat retina undergoes an increase in aerobic glycolysis. PKM2, both in the cytoplasm and in the nucleus, may thus play an important role during neural progenitor cell induction, as it does in other proliferating cells.

Purpose Epithelial-mesenchymal transition (EMT) in retinal pigment epithelial (RPE) cells is related to the pathogenesis of subretinal fibrosis such as that associated with macular degeneration. The role of myocardin-related transcription factor A (MRTF-A) in EMT of RPE cells and subretinal fibrosis was investigated. Methods The migratory activity of human RPE-1 cells in culture was evaluated using a scratch assay. The subcellular distribution of MRTF-A in RPE-1 cells, as well as the extent of subretinal fibrosis in a mouse model, were determined by immunofluorescence analysis. Expression of α-smooth muscle actin (α-SMA), collagen type I (COL1), connective tissue growth factor (CTGF), and paxillin was examined by immunoblot analysis or reverse transcription and quantitative polymerase chain reaction analysis, whereas that of pro-matrix metalloproteinase-2 (MMP-2) was assessed by gelatin zymography. Results The MRTF-A signaling inhibitor CCG-1423 suppressed RPE-1 cell migration in a concentration-dependent manner. Transforming growth factor-beta (TGF-β2) induced MRTF-A translocation from the cytoplasm to the nucleus of RPE-1 cells, and this effect was attenuated by CCG-1423. TGF-β2 up-regulated the abundance of α-SMA, paxillin, and pro-MMP-2 proteins as well as the amounts of α-SMA, COL1, and CTGF mRNAs in a manner sensitive to inhibition by CCG-1423. Finally, intravitreal injection of CCG-1423 markedly attenuated the development of subretinal fibrosis induced by photocoagulation in vivo. Conclusions Our results implicate MRTF-A in EMT of RPE cells and in the development of subretinal fibrosis in vivo, suggesting that MRTF-A is a potential therapeutic target for retinal diseases characterized by subretinal fibrosis.

Necrosis is a form of cell death that results in rupture of the plasma membrane and the release of cellular contents, and it can give rise to sterile inflammation in the retina and other tissues. The secretion of vascular endothelial growth factor (VEGF) by retinal pigment epithelial (RPE) cells contributes to retinal homeostasis as well as to pathological angiogenesis. We have now examined the effect of a necrotic cell lysate prepared from human RPE cells (NLR) on the release of VEGF by healthy RPE cells. We found that NLR markedly increased the release of VEGF from RPE cells and that this effect was attenuated by nintedanib, a multiple receptor tyrosine kinase inhibitor, whereas it was unaffected by inhibitors of NF-κB signaling or of caspase-1. NLR also induced the phosphorylation of extracellular signal–regulated kinase (Erk) and signal transducer and activator of transcription 3 (Stat3) in a manner sensitive to inhibition by nintedanib, although inhibitors of Erk and Stat3 signaling pathways did not affect NLR-induced VEGF secretion. In addition, nintedanib attenuated the development of choroidal neovascularization in mice. Our results have thus shown that a necrotic lysate of RPE cells induced VEGF secretion from healthy RPE cells and that this effect was mediated by receptor tyrosine kinase signaling. They therefore suggest that VEGF secretion by healthy RPE cells is a potential therapeutic target for retinal diseases associated with sterile inflammation and pathological angiogenesis.

Microtubule-associated protein 1A/1B-light chain 3 (LC3)-II is essential for autophagosome formation and is widely used to monitor autophagic activity. We show that CGK733 induces LC3 II and LC3-puncta accumulation, which are not involved in the activation of autophagy. The treatment of CGK733 did not alter the autophagic flux and was unrelated to p62 degradation. Treatment with CGK733 activated the AMP-activated protein kinase (AMPK) and protein kinase RNA-like endoplasmic reticulum kinase/CCAAT-enhancer-binding protein homologous protein (PERK/CHOP) pathways and elevated the expression of p21Waf1/Cip1. Inhibition of both AMPK and PERK/CHOP pathways by siRNA or chemical inhibitor could block CGK733-induced p21Waf1/Cip1 expression as well as caspase-3 cleavage. Knockdown of LC3 B (but not LC3 A) abolished CGK733-triggered LC3 II accumulation and consequently diminished AMPK and PERK/CHOP activity as well as p21Waf1/Cip1 expression. Our results demonstrate that CGK733-triggered LC3 II formation is an initial event upstream of the AMPK and PERK/CHOP pathways, both of which control p21Waf1/Cip1 expression.

Gemcitabine (GEM) remains a major chemotherapeutic drug for pancreatic cancer, but resistance to GEM has been a big problem, as its response rate has been decreasing year by year. The effect of the histone deacetylase inhibitor (HDAI) valproic acid (VPA) was compared with tranilast and RI-1 as a combinatorial treatment with GEM in four pancreatic cancer cell lines, BxPC-3, PK45p, MiaPaCa-2 and PK59. Cell viability assays were carried out to check the cytotoxic effects, western blotting was carried out for DNA repair mechanisms, and localization was determined by immunofluorescence. The sensitization factors (i.e., the fold ratio of cell viability for GEM/GEM plus drug) reveal that VPA increases the cytotoxic sensitization to GEM at approximately 2.7-fold, 1.2-fold, 1.5-fold and 2.2-fold in BxPC-3, MiaPaCa-2, PK-45p and PK-59 cell lines, respectively. Moreover, GEM induces activation of the DNA repair protein H2AX proportional to the dosage. Interestingly, however, this effect can be abrogated by VPA. These results indicate that VPA enhances GEM-induced cytotoxicity in GEM-resistant pancreatic cancer cells, possibly through inhibition of DNA damage signaling and repair. Our study suggests VPA as a potential therapeutic agent for combinatorial treatment with GEM in pancreatic cancer.

Many fusion genes, which are the result of chromosomal translocation and work as an oncogene, have been recently identified, but their mode of actions is still unclear. Here, we performed a yeast mutant screening for oncogenes of Ewing's sarcoma to easily identify essential regions responsible for fusion protein functions using a yeast genetic system. Three kinds of oncogenes including EWS/FLI1, EWS/ERG, and EWS/E1AF exhibited growth inhibition in yeast. In this screening, we identified 13 single amino acid substitution mutants which could suppress growth inhibition by oncogenes. All of the point mutation positions of the EWS/ETS family proteins were located within the ETS domain, which is responsible for the interaction with a specific DNA motif. Eight-mutated residues within the ETS domain matched to 13 completely conserved amino acid residues in the human ETS domains. Moreover, mutants also showed reduced transcriptional activities on the DKK2 promoter, which is upregulated by the EWS/ETS family, compared to that of the wild type. These results suggest that the ETS domain in the EWS/ETS family proteins may be a primary target for growth inhibition of Ewing's sarcoma and that this yeast screening system can be applied for the functional screening of the oncogenes.

Background/Aim: From the standpoint of cancer therapy, it is valuable to enhance the anticancer effects of chemotherapy. Our previous reports revealed that upregulation of heat-shock protein 27 (HSP27) has been linked to gemcitabine resistance of pancreatic cancer cells. Enzyme-treated asparagus extract (ETAS) is an extract that is produced from asparagus. The purpose of this study was to investigate the effect of ETAS on the expression of HSP27 and other HSPs in the gemcitabine-resistant pancreatic cancer cell line KLM1-R. Materials and Methods: KLM1-R cells were treated with ETAS, and expression levels of HSPs, including HSP27, were investigated by western blotting. Results: ETAS down-regulated HSP27 and pHSP27 (serine 78) in KLM1-R cells, but, HSP70 and GRP78 levels were not altered. Conclusion: This study suggests the potential therapeutic benefit of ETAS in enhancing anticancer effects by its combination with gemcitabine for patients with pancreatic cancer., peer-reviewed

Numerous fixative solutions are available but many are not amenable to the histomorphological preservation of retinae. The investigators specifically focused on retinal histological studies, which rather than 4% formaldehyde (FA), often use Davidson’s fixative. However the latter has its limitations. The purpose of this study was to produce a new fixative which maintains retinae closer to the in vivo conditions. Experimental design. Four fixative formulations (4% paraformaldehyde, Davidson’s fixative, modified Davidson’s fixative and an in-house fixative – TB-Fix) were tested on retinae and the outcomes on histomorphology and immunohistochemical staining for selected antigenic markers was compared. TB-Fix markedly improved morphological detail following hematoxylin and eosin staining, most importantly eliminating the spongiform appearance in the plexiform layer and the swelling of somata (including Muller cells), when compared to FA, Davidson’s fixative and its modified version. Retinal samples fixed with TB-Fix or FA showed comparable results in immunohistological staining for neurons and glia in the retina. Importantly, while the whole eye fixed with FA collapsed in shape and induced artificial retinal detachment, the eye fixed with TB-Fix avoided deformation and detachment. Furthermore, we found that TB-Fix also prevented detachment from the culture plate when used to fix HEK293 cells, which are known to detach from the plate easily. It was demonstrated that TB-Fix provides an overall improvement in the preservation of retinal morphology and chemical composition.

We previously showed that dietary omega (ω)–3 long-chain polyunsaturated fatty acids (LCPUFAs) suppress inflammation in mice with experimental autoimmune uveitis (EAU). We have now investigated the role of antigen presenting cells (APCs) in this action of ω-3 LCPUFAs. C57BL/6 mice were fed a diet supplemented with ω-3 or ω-6 LCPUFAs for 2 weeks, after which splenocytes were isolated from the mice and cocultured with CD4+ T cells isolated from mice with EAU induced by injection of a human interphotoreceptor retinoid-binding protein peptide together with complete Freund’s adjuvant. The proliferation of and production of interferon-γ and interleukin-17 by T cells from EAU mice in vitro were attenuated in the presence of splenocytes from ω-3 LCPUFA–fed mice as compared with those from mice fed ω-6 LCPUFAs. Splenocyte fractionation by magnetic-activated cell sorting revealed that, among APCs, dendritic cells (DCs) were the target of ω-3 LCPUFAs. Adoptive transfer of DCs from mice fed ω-3 LCPUFAs attenuated disease progression in EAU mice as well as the production of pro-inflammatory cytokines by T cells isolated from these latter animals. The proliferation of T cells from control Balb/c mice was also attenuated in the presence of DCs from ω-3 LCPUFA–fed mice as compared with those from ω-6 LCPUFA–fed mice. Furthermore, T cell proliferation in such a mixed lymphocyte reaction was inhibited by prior exposure of DCs from mice fed an ω-6 LCPUFA diet to ω-3 LCPUFAs in vitro. Our results thus suggest that DCs mediate the anti-inflammatory action of dietary ω-3 LCPUFAs in EAU.

Aim As severe alcohol intoxication impairs memory function, a high concentration of ethanol (60 mmol L−1) acutely inhibits long-term potentiation (LTP), a cellular model of learning and memory, in rat hippocampal slices. Neurosteroids are involved in this LTP inhibition. We recently reported that the inhibitory effects of 60 mmol L−1 ethanol are blocked by 4-methylpyrazole (4MP), an inhibitor of alcohol dehydrogenase, suggesting that acetaldehyde locally generated within the hippocampus participates in LTP inhibition. We investigated whether acetaldehyde generated by ethanol metabolism contributes to neurosteroidogenesis and LTP inhibition. Results Like 60 mmol L−1 ethanol, we found that exogenous acetaldehyde enhanced neurosteroid immunostaining in CA1 pyramidal neurons, and that augmented neurosteroid immunostaining by high ethanol alone was blocked by 4MP, but not by inhibitors of other ethanol metabolism pathways. The inhibitory effects of 60 mmol L−1 ethanol on LTP were mimicked by a lower concentration of ethanol (20 mmol L−1) plus acetaldehyde (60 μmol L−1), although neither agent alone was effective at these concentrations, suggesting that 60 mmol L−1 ethanol inhibits LTP through multiple actions, one of which involves acetaldehyde and the other of which requires just 20 mmol L−1 ethanol. The effects of ethanol and acetaldehyde on neurosteroid staining and LTP were overcome by inhibition of neurosteroid synthesis and by blockade of N-methyl-D-aspartate receptors (NMDAR). Conclusion These observations show that acetaldehyde generated by local ethanol metabolism within the hippocampus serves as a signal for neurosteroid synthesis in pyramidal neurons, and participates in the synaptic dysfunction associated with severe alcohol intoxication.

Consistent with the ability of severe alcohol intoxication to impair memory, high concentrations of ethanol (60 mM) acutely inhibit long-term potentiation (LTP) in the CA1 region of rat hippocampal slices. To account for this, we hypothesized that local metabolism to acetaldehyde may contribute to the effects of high ethanol on synaptic function. However, sodium azide, a catalase inhibitor, and allyl sulfide, an inhibitor of cytochrome P450 2E1 (CYP2E1), failed to overcome LTP inhibition by 60 mM ethanol. In contrast, LTP was successfully induced in the presence of ethanol plus 4-methylpyrazole (4MP), an inhibitor of alcohol dehydrogenase, suggesting that local metabolism via alcohol dehydrogenase contributes to synaptic effects. Furthermore, exogenously administered acetaldehyde overcame the effects of 4MP on LTP inhibition mediated by ethanol. These observations indicate that acetaldehyde generated by local metabolism within the hippocampus participates in the synaptic dysfunction associated with severe alcohol intoxication.

LY294002 and wortmannin are chemical compounds that act as potent inhibitors of phosphoinositide 3-kinases (PI3Ks). Both of them are generally used to inhibit cell proliferation as cancer treatment by inhibiting the PI3K/protein kinase B (AKT) signaling pathway. In this study, LY294002 (but not wortmannin) showed an abnormal ability to enhance AKT phosphorylation (at Ser472) specifically in gemcitabine (GEM)-resistant pancreatic cancer (PC) cell lines PK59 and KLM1-R. LY294002 was shown to activate AKT and accumulate phospho-AKT at the intracellular membrane in PK59, which was abolished by treatment with AKTi-1/2 or wortmannin. Inhibiting AKT phosphorylation by treatment with AKTi-1/2 or wortmannin further enhanced LY294002-induced cell death in PK59 and KLM1-R cells. In addition, treatment with wortmannin alone failed to inhibit cell proliferation in both PK59 and KLM1-R cells. Thus, our results reveal that LY294002 displays the opposite effect on PI3K-dependent AKT phosphorylation, which maintains cell survival from the cytotoxicity introduced by LY294002 itself in GEM-resistant pancreatic cancer cells. We suggest that targeting the PI3K/AKT signaling pathway with inhibitors may be counterproductive for patients with PC who have acquired GEM-resistance.

Children with neurofibromatosis type 1 (NF1) are prone to learning and behavioral abnormalities, including problems with spatial learning and attention. The molecular etiology for these deficits is unclear, as previous studies have implicated defective dopamine, cyclic adenosine monophosphate (cAMP), and Ras homeostasis. Using behavioral, electrophysiological, and primary culture, we now demonstrate that reduced dopamine signaling is responsible for cAMP-dependent defects in neuron function and learning. Collectively, these results establish defective dopaminergic function as a contributing factor underlying impaired spatial learning and memory in children and adults with NF1, and support the use of treatments that restore normal dopamine homeostasis for select individuals.

Using an antibody against 5α-reduced neurosteroids, predominantly allopregnanolone, we found that immunostaining in the CA1 region of rat hippocampal slices was confined to pyramidal neurons. This neurosteroid staining was increased following 15 min administration of 60 mm but not 20 mm ethanol, and the enhancement was blocked by finasteride and dutasteride, selective inhibitors of 5α-reductase, a key enzyme required for allopregnanolone synthesis. Consistent with a prior report indicating that N-methyl-D-aspartate (NMDA) receptor (NMDAR) activation can promote steroid production, we observed that D-2-amino-5-phosphonovalerate (APV), a competitive NMDAR antagonist, blocked the effects of 60 mm ethanol on staining. We previously reported that 60 mm ethanol inhibits the induction of long-term potentiation (LTP), a cellular model for memory formation, in the CA1 region. In the present study, LTP inhibition by 60 mm ethanol was also overcome by both the 5α-reductase inhibitors and by APV. Furthermore, the effects of ethanol on neurosteroid production and LTP were mimicked by a low concentration of NMDA (1 μm), and the ability of NMDA to inhibit LTP and to enhance neurosteroid staining was reversed by finasteride and dutasteride, as well as by APV. These results indicate that ethanol paradoxically enhances GABAergic neurosteroid production by activation of unblocked NMDARs and that acute LTP inhibition by ethanol represents a form of NMDAR-mediated metaplasticity.

Low-level activation of N-methyl-d-aspartate receptors (NMDARs) results in a decrease in the ability of tetanic stimulation to induce long-term potentiation (LTP). This NMDAR-mediated LTP inhibition is observed with low micromolar concentrations of NMDA or chelation of ambient extracellular zinc. In rat hippocampal slices, we examined whether LTP inhibition by 1 muM NMDA and zinc chelation share common mechanisms. We found that both forms of LTP inhibition involve nitric oxide (NO) synthase (NOS) and calcineurin. Furthermore, both forms of LTP inhibition are overcome by block of p38 mitogen-activated protein kinase (MAPK), but not by inhibition of extracellular signal-regulated kinase 1/2 or c-Jun-N-terminal kinase. A p38 antagonist also overcame the block of LTP by sodium nitroprusside, an agent that releases NO, suggesting that NO release occurs upstream of MAPK activation. Despite the involvement of p38 MAPK in NMDAR-mediated LTP inhibition, p38 antagonism did not enhance LTP induction in response to weak tetanic stimulation under baseline conditions. These results indicate that p38 MAPK is part of a complex NMDAR-driven signaling pathway involving calcineurin and NO that helps to regulate synaptic plasticity in the CA1 region.

Oseltamivir (Tamiflu) is now being stockpiled by several governments as a first line treatment for an anticipated outbreak of avian influenza caused by H5N1. However, abnormal behaviors and death associated with the use of Tamiflu have developed into a major issue in Japan where Tamiflu is often prescribed for seasonal influenza. Thus, it is critical to determine neuropsychiatric effects of oseltamivir and to establish methods for safe administration. Using juvenile rats and rat hippocampal slices, we investigated whether oseltamivir has adverse effects on the central nervous system. Systemic injection of oseltamivir (50mg/kg i.p.) produced no change in behavior within 2h. However, prior injection of oseltamivir significantly altered the duration of loss of lightning reflex following ethanol injection (3.3g/kg, i.p.). Ethanol injection in the presence of oseltamivir also resulted in enhanced hypothermia. In the CA1 region of hippocampal slices, oseltamivir (100 microM) induced paired-pulse facilitation in population spikes without changes in excitatory postsynaptic potentials. Similarly, 3 microM oseltamivir carboxylate, the active metabolite of oseltamivir, facilitated neuronal firing, though the facilitation did not involve GABAergic disinhibition. Moreover, oseltamivir carboxylate produced further facilitation following administration of 60mM ethanol. These findings indicate that oseltamivir has effects on the central nervous system, especially when combined with other agents.

We previously found that ethanol has complex effects on hippocampal synaptic plasticity, inhibiting long-term potentiation (LTP) and long-term depression by different mechanisms. The block of long-term depression appears to be mediated by effects on N-methyl-d-aspartate receptors, whereas the block of LTP involves augmented inhibition via gamma-aminobutyric acid-A receptors (GABA(A)Rs). To pursue factors contributing to effects on LTP, we examined the ability of various concentrations of ethanol to block LTP in the CA1 region of rat hippocampal slices. Complete LTP block required 60 mm ethanol. LTP block was enhanced at lower ethanol concentrations in the presence of (3alpha5alpha)-3-hydroxypregnan-20-one, a GABA(A)R-potentiating neurosteroid, suggesting that neurosteroids may be important contributors to the effects of ethanol on LTP. Consistent with this, we found that block of LTP by 60 mm ethanol was overcome by coadministration of a cyclodextrin that binds and removes lipophilic neurosteroids. More specifically, treatment of slices with finasteride, an agent that inhibits the synthesis of 5alpha-reduced neurosteroids, or with an agent that inhibits the effects of 5alpha-reduced neurosteroids on GABA(A)Rs overcame the effects of 60 mm ethanol on LTP. Taken together, these results indicate that acute production of GABA(A)R-enhancing neurosteroids plays a key role in mediating the effects of ethanol on LTP.

To determine how acute ethanol intoxication may alter memory processing, we examined the effects of stepwise increases in ethanol on long-term potentiation (LTP) in rat hippocampal slices. LTP was inhibited by acute administration of 60 mM ethanol, but was readily induced if ethanol was increased gradually to 60 mM over 75 min. Administration of 2-amino-5 phosphonovalerate (APV), an N-methyl-D-aspartate receptor (NMDAR) antagonist, during the stepwise increase in ethanol inhibited LTP, suggesting involvement of NMDARs in the development of tolerance. However, APV and nifedipine, an inhibitor of L-type calcium channels, failed to inhibit LTP when administered following the slow increase in ethanol. Ethanol-tolerant LTP was inhibited by thapsigargin, suggesting a major role for intracellular calcium release in this form of plasticity. The unique properties of ethanol-tolerant LTP suggest that memories formed during binge drinking are not acquired by standard synaptic mechanisms and that acute tolerance may involve the induction of novel mechanisms to maintain function.

Active hexose-correlated compound (AHCC) is an extract of a basidiomycete mushroom that enhances the therapeutic effects and reduces the side-effects of chemotherapy. Our previous studies demonstrated that heat-shock protein 27 (HSP27) was involved in gemcitabine-resistance of pancreatic cancer cells and it was down-regulated by AHCC-treatment. However, how AHCC down-regulated HSP27 is unknown. In the present study, we focused on two transcription factors reported to induce HSP27, heat shock factor 1 (HSF1) and high-mobility group box 1 (HMGB1) and investigated the effect of AHCC on their expression.KLM1-R cells were treated with AHCC and the protein expression of HSF1 and HMGB1 were analyzed by western blotting.The protein expression of HSF1 in KLM1-R was down-regulated by AHCC treatment. On the other hand, the protein expression of HMGB1 was not reduced in KLM1-R cells after AHCC treatment.The possibility that AHCC down-regulated HSP27 through down-regulation of the HSF1, was herein shown.

Results of our previous studies demonstrated that the expression of heat-shock protein 27 (HSP27) was increased and HSP27 was phosphorylated in the GEM-resistant pancreatic cancer cell line, KLM1-R. The expression of HSP27 is regulated mainly by heat-shock factor 1, but other transcription factors or kinases have been reported to activate HSP27. High-mobility group box 1 (HMGB1) is a nuclear transcription factor. It has been reported that HMGB1 regulates HSP27 gene expression. Mitogen-activated protein kinase-activated protein kinase-2 (MAPKAPK2) phosphorylates HSP27. In the present study, we investigated the expression of HMGB1 and MAPKAPK2 in KLM1-R cells.The expression levels of HMGB1 and MAPKAPK2 were compared between KLM1 and KLM1-R cells by western blotting.The protein expression of both HMGB1 and MAPKAPK2 were increased in KLM1-R cells compared to KLM1 cells.The increase of both HMGB1 and MAPKAPK2 in KLM1-R cells compared to KLM1 suggest the possibility of the activation of the pathway of HSP27 by HMGB1 and MAPKAPK2 in gemcitabine-resistant KLM1-R cells.

Tumor progression is one of the most serious issues to overcome cancer disease. As a model of inflammation-induced tumor progression, we used the regressive murine fibrosarcoma cell clone QR-32 and the progressive malignant clone QRsP-11, that was derived from QR-32. Heat shock protein beta-1 (Hspb1) is a molecular chaperone. Hspb1 plays roles in not only cell protection but also chemo-resistance, tumorigenicity and protection from apoptosis. In a recent study, we showed that Hspb1 was up-regulated in QRsP-11 compared to QR-32.We compared the expression levels of Hspb1, Hsf1 and Sox2 in QR-32 and QRsP-11 cells by means of western blotting.Hsf1, a transcription factor for Hspb1 was not increased in QRsP-11. Sex determining region Y-box 2 (Sox2) is a transcription factor, reported to interact with Hspb1. Sox2 was up-regulated in QRsP-11 compared to QR-32.These results suggest that Sox2-Hspb1 signaling is a possible pathway responsible to tumor progression of QRsP-11.

Glutamate has been shown to induce neural progenitor cells in the adult vertebrate retina. However, protein dynamics during progenitor cell induction by glutamate are not fully understood. To identify specific proteins involved in the process, we employed two-dimensional electrophoresis-based proteomics on glutamate untreated and treated retinal ex vivo sections. Rat retinal tissues were incubated with 1 mM glutamate for 1 h, followed by incubation in glutamate-free media for a total of 24 h. Consistent with prior reports, it was found that mitotic cells appeared in the outer nuclear layer without any histological damage. Immunohistological evaluations and immunoblotting confirmed the emergence of neuronal progenitor cells in the mature retina treated with glutamate. Proteomic analysis revealed the up-regulation of dihydropyrimidinase-related protein 3 (DRP-3), DRP-2 and stress-induced-phosphoprotein 1 (STIP1) during neural progenitor cell induction by glutamate. Moreover, mRNA expression of DRP-3, especially, its long isoform, robustly increased in the treated retina compared to that in the untreated retina. These results may indicate that glutamate induces neural progenitor cells in the mature rat retina by up-regulating the proteins which mediate cell mitosis and neurite growth.

// Yufeng Wang 1 , Yasuhiro Kuramitsu 1 , Byron Baron 1 , Takao Kitagawa 1 , Junko Akada 1 , Kazuhiro Tokuda 1 , Dan Cui 2 , Kazuyuki Nakamura 1, 3 1 Department of Biochemistry and Functional Proteomics, Yamguchi University Graduate School of Medicine, Ube, Japan 2 Department of Pathology, Yamguchi University Graduate School of Medicine, Ube, Japan 3 Centre of Clinical Laboratories in Tokuyama Medical Association Hospital, Shunan, Japan Correspondence to: Yasuhiro Kuramitsu, e-mail: climates@yamaguchi-u.ac.jp Keywords: CGK733, calcium sequestration, PERK/CHOP, ER stress, non-apoptotic death Received: April 02, 2015 Accepted: June 29, 2015 Published: July 10, 2015 ABSTRACT Calcium ions (Ca 2+ ) are indispensable for the physiology of organisms and the molecular regulation of cells. We observed that CGK733, a synthetic chemical substance, induced non-apoptotic cell death and stimulated reversible calcium sequestration by vesicles in pancreatic cancer cells. The endoplasmic reticulum (ER) stress eukaryotic translation initiation factor 2-alpha kinase 3/C/EBP homologous protein (PERK/CHOP) signaling pathway was shown to be activated by treatment with CGK733. Ionomycin, an ER stress drug and calcium ionophore, can activate PERK/CHOP signaling and accelerate CGK733-induced calcium sequestration. Knockdown of CHOP diminished CGK733-induced vesicular calcium sequestration, but had no effects on the cell death. Proteomic analysis demonstrated that the ER-located calcium-binding proteins, calumenin and protein S100-A11, were altered in CGK733-treated cells compared to non-treated controls. Our study reveals that CGK733-induced intracellular calcium sequestration is correlated with the PERK/CHOP signaling pathway and may also be involved in the dysregulations of calcium-binding proteins.

Purpose: We investigated whether artificial aqueous humours are adequate incubation media compared with artificial cerebrospinal fluid (aCSF), and evaluated the retinal toxicity of two vital stains − trypan blue (TB) and indocyanine green (ICG) − and triamcinolone acetonide (TA) using isolated rat retinas incubated in artificial aqueous humours. Methods: In experiment 1, retinal segments were isolated and incubated in aCSF, BSS plus®, Opeguard® Neo Kit, or phosphate-buffered saline (PBS). In experiment 2, retinal tissues were exposed to one of the agents and incubated in BSS plus®. Retinal damage was assessed by morphological examination and biochemical assay, which measured lactate dehydrogenase (LDH) in the medium once every hour. Results: In experiment 1, BSS plus® was confirmed as a suitable incubation medium. In experiment 2, there were no significant changes in the retinas exposed to TB or TA. Tissues exposed to ICG showed damage in every retinal layer and significantly higher release of LDH. Conclusion: Exposure to ICG caused retinal damage in isolated rat retina tissue in our experimental model (in vitro).

Subretinal fibrosis contributes to the loss of vision associated with age-related macular degeneration (AMD). Retinal pigment epithelial (RPE) cells play a key role in the pathogenesis of AMD including the fibrotic reaction. We examined the role of retinoic acid receptor-γ (RAR-γ) in the epithelial-mesenchymal transition (EMT) and other fibrosis-related processes in mouse RPE cells cultured in a type I collagen gel. Transforming growth factor-β2 (TGF-β2)-induced collagen gel contraction mediated by the RPE cells was inhibited by the RAR-γ agonist R667 in a concentration- and time-dependent manner. Expression of the mesenchymal markers α-smooth muscle actin and fibronectin, the release of interleukin-6, and the phosphorylation of paxillin, mitogen-activated protein kinases (ERK, p38, and JNK), Smad2, and AKT induced by TGF-β2 were also suppressed by the RAR-γ agonist. Furthermore, gelatin zymography and immunoblot analysis revealed that the TGF-β2-induced release of matrix metalloproteinase (MMP)-2, MMP-3, MMP-8, and MMP-9 from RPE cells was inhibited by R667, and the MMP inhibitor GM6001 attenuated TGF-β2-induced RPE cell contraction. Finally, immunohistofluorescence analysis with antibodies to glial fibrillary acidic protein showed that R667 inhibited the development of subretinal fibrosis in a mouse model in vivo. Our results thus suggest that RAR-γ agonists may prove effective for the treatment of subretinal fibrosis associated with AMD.RAR-γ agonist R667 suppressed collagen gel contraction mediated by RPE cells. Epithelial-mesenchymal transition (EMT) in RPE cells was inhibited by RAR-γ agonist R667. RAR-γ agonist R667 inhibited fibrosis-related processes in RPE cells. RAR-γ agonists may attenuate AMD-associated fibrosis.

Active hexose-correlated compound (AHCC) is an extract of basidiomycete mushroom. It has been used as health food due to its efficacy of enhancing antitumor effects and reducing adverse effects of chemotherapy. Our previous research showed that AHCC down-regulated heat-shock protein (HSP)-27 and exhibited cytotoxic effects against gemcitabine-resistant pancreatic cancer cells. Sex-determining region Y-box 2 (SOX2) is reported to be up-regulated in other kinds of cancer cells and involved in carcinogenesis and malignancy. The aim of this study was to investigate the effects of AHCC on protein expression of SOX2 in the gemcitabine-resistant pancreatic cancer cell line KLM1-R.AHCC was applied to KLM1-R cells and expression of SOX2 was analyzed by western blotting.AHCC down-regulated SOX2 in KLM1-R cells. Nanog and Oct4, co-workers of SOX2 in maintaining pluripotency, did not exhibit any significant change in protein expression.We showed the potential of AHCC to be a candidate for combinatorial therapy in anticancer drug regimens. This result suggests that the target of AHCC in expressing therapeutic efficacy was not the pluripotent cells such as cancer stem cells (CSCs) but SOX2-specific.

Lactoylglutathione lyase (GLO1), a ubiquitously expressed methylglyoxal (MG) detoxification enzyme, is implicated in the progression of various human malignant diseases. However, the role of GLO1 in the development or progression of murine fibrosarcoma is still unclear. We performed proteomic analysis to identify differences in the intracellular proteins of the regressive tumor cell line QR-32 and the inflammatory cell-promoting progressive tumor cell line QRsP-11 of murine fibrosarcoma by 2DE combined with MS. Seven upregulated proteins were identified in QRsP-11 compared to QR-32 cells, namely, GLO1, annexin A1, adenylate kinase isoenzyme 1, transcription factor BTF3, myosin light polypeptide 6, low molecular weight phosphotyrosine protein phosphatase and nucleoside diphosphate kinase B. Heat shock protein beta-1 (HspB1), a methylglyoxal-adducted protein, is concomitantly over-expressed in QRsP-11 as compared to QR-32 cells. We also found out that GLO1 is translocated into the nucleus to a higher extent in QRsP-11 compared to QR-32 cells, which can be reversed by using a MEK inhibitor (U0126). Moreover, U0126 and GLO1 siRNA can inhibit cell proliferation and migration in QRsP-11 cells. Our data suggest that overexpression and nuclear translocation of GLO1 might be associated with tumor progression in murine fibrosarcoma.

Gemcitabine (2'-deoxy-2'-difluorodeoxycytidine) is the only clinically effective drug for pancreatic cancer. However, high levels of inherent and acquired tumor resistance to gemcitabine lead to difficulty of chemotherapy for pancreatic cancer. We have reported on a proteomic study of gemcitabine-sensitive KLM1 and -resistant KLM1-R pancreatic cancer cells, and identified some proteins which were shown to be up-regulated in KLM1-R compared to KLM1 cells. In those proteomic studies, peroxiredoxin-2 was listed as an up-regulated protein in KLM1-R cells. Peroxiredoxin-2 is a member of a family of peroxiredoxins providing a protective role for redox damage. In this study, the expression of peroxiredoxin-2 in KLM1 and KLM1-R cells was compared. It was found that peroxiredoxin-2 was significantly up-regulated in KLM1-R cells compared to KLM1 cells (p0.001). However, peroxiredoxin-1 expression was significantly down-regulated in KLM1-R cells (p0.001). These results suggest that peroxiredoxin-2 is a possible candidate biomarker for predicting the response of patients with pancreatic cancer to treatment with gemcitabine.

Lactoylglutathione lyase (GLO1), a ubiquitously expressed methylglyoxal (MG) detoxification enzyme, is implicated in the progression of various human malignant diseases. However, the role of GLO1 in the development or progression of murine fibrosarcoma is still unclear. We performed proteomic analysis to identify differences in the intracellular proteins of the regressive tumor cell line QR-32 and the inflammatory cell-promoting progressive tumor cell line QRsP-11 of murine fibrosarcoma by 2DE combined with MS. Seven upregulated proteins were identified in QRsP-11 compared to QR-32 cells, namely, GLO1, annexin A1, adenylate kinase isoenzyme 1, transcription factor BTF3, myosin light polypeptide 6, low molecular weight phosphotyrosine protein phosphatase and nucleoside diphosphate kinase B. Heat shock protein beta-1 (HspB1), a methylglyoxal-adducted protein, is concomitantly over-expressed in QRsP-11 as compared to QR-32 cells. We also found out that GLO1 is translocated into the nucleus to a higher extent in QRsP-11 compared to QR-32 cells, which can be reversed by using a MEK inhibitor (U0126). Moreover, U0126 and GLO1 siRNA can inhibit cell proliferation and migration in QRsP-11 cells. Our data suggest that overexpression and nuclear translocation of GLO1 might be associated with tumor progression in murine fibrosarcoma.

QR-32 is a regressive murine fibrosarcoma cell clone which cannot grow when they are transplanted in mice; QRsP-11 is a progressive malignant tumor cell clone derived from QR-32 which shows strong tumorigenicity. A recent study showed there to be differentially expressed up-regulated and down-regulated proteins in these cells, which were identified by proteomic differential display analyses by using two-dimensional gel electrophoresis and mass spectrometry. Cofilins are small proteins of less than 20 kDa. Their function is the regulation of actin assembly. Cofilin-1 is a small ubiquitous protein, and regulates actin dynamics by means of binding to actin filaments. Cofilin-1 plays roles in cell migration, proliferation and phagocytosis. Cofilin-2 is also a small protein, but it is mainly expressed in skeletal and cardiac muscles. There are many reports showing the positive correlation between the level of cofilin-1 and cancer progression. We have also reported an increased expression of cofilin-1 in pancreatic cancer tissues compared to adjacent paired normal tissues. On the other hand, cofilin-2 was significantly less expressed in pancreatic cancer tissues. Therefore, the present study investigated the comparison of the levels of cofilin-1 and cofilin-2 in regressive QR-32 and progressive QRsP-11cells by western blotting. Cofilin-2 was significantly up-regulated in QRsP-11 compared to QR-32 cells (p0.001). On the other hand, the difference of the intensities of the bands of cofilin-1 (18 kDa) in QR-32 and QRsP-11 was not significant. However, bands of 27 kDa showed a quite different intensity between QR-32 and QRsP-11, with much higher intensities in QRsP-11 compared to QR-32 (p0.001). These results suggested that the 27-kDa protein recognized by the antibody against cofilin-1 is a possible biomarker for progressive tumor cells.

Intrinsic or acquired resistance of pancreatic cancer to gemcitabine (2'-deoxy-2'-difluorodeoxycytidine) is an important factor in the failure of gemcitabine treatment. Proteomic analysis of gemcitabine-sensitive KLM1 pancreatic cancer cells and -resistant KLM1-R cells identified heat-shock protein-27(HSP27) as a biomarker protein which is involved in gemcitabine resistance. However, a knock-down experiment showed that HSP27 was not the only protein implicated with gemcitabine-resistance. Finding further candidate proteins is necessary for achieving effective gemcitabine therapy for patients with pancreatic cancer. DDX39 is an Asp-Glu-Ala-Asp (DEAD)-box RNA helicase reported to be overexpressed in tumor cells, such as lung squamous cell cancer, gastrointestinal stromal tumor, urinary bladder cancer and malignant pleural mesothelioma. In urinary bladder cancer cells, overexpression of this protein is intimately bound with tumorigenesis and poor prognosis. In the present study, the expression of DDX39 in gemcitabine-sensitive KLM1 and -resistant KLM1-R cells was compared. It was found that DDX39 was significantly up-regulated in gemcitabine-resistant KLM1-R cells compared to sensitive KLM1 cells. The ratio of expression of DDX39 to that of actin was significantly up-regulated in KLM1-R cells compared to KLM1 cells (p=0.0072 by Student's t-test). These results suggest that DDX39 is a possible candidate biomarker for predicting the response of patients with pancreatic cancer to treatment with gemcitabine.

Three clones with differential metastatic potential were established from the parental SN12C human renal cell carcinoma (HRCC). We previously reported that in the two high metastatic SN12C clones, two isoforms of ubiquitin carboxyl-terminal hydrolase isozyme L1 (UCH‑L1) showed decreased expression by using two-dimensional electrophoresis (2‑DE) covering a pH range (pH 3.0‑10.0) followed by liquid chromatography‑tandem mass spectrometry. However, in the case of the low metastatic clone, the spot volume for UCH‑L1 was almost the same as for the parental SN12C. In the present study, we found one protein spot which was correlated with the metastatic potential of SN12C clones by using 2‑DE over a narrow pH range (pH 4.0‑7.0). The protein glyoxalase 1 (GLO1) appeared to be directly proportional to the metastatic potential of the SN12C clones. GLO1 was the only protein which consistently varied according to the metastatic potentials of SN12C clones. GLO1 was increased in high metastatic cell lines by western blot analysis. These findings suggest that GLO1 is associated with the metastatic potential of SN12C HRCC clones. We expanded our experimental range to include clones of scirrhous gastric cancer cell lines (OCUM‑2M, OCUM‑2D and OCUM‑2MLN) and similar results were obtained, thereby further strengthening our original findings.

Malignant pleural mesothelioma (MPM) is a malignant tumor originating from mesothelial cells existing in pleura. Since its incidence, it is closely related to the amount and time of exposure to asbestos, and the latency period after exposure to asbestos is very long, the incidence may increase over the next two decades. Since early detection is very difficult and there is no standard curative therapy, it is important to understand the biology of MPM, and to find biomarkers and molecular targets for its therapy. DDX39 is one of the Asp-Glu-Ala-Asp (DEAD)-box RNA helicases, which are required for the export of mRNA out of the nucleus, and transcription, splicing and transport of mRNA. Some reports have shown differential expression of DDX39 in tumor cells or tissues such as lung squamous cell cancer, gastrointestinal stromal tumor and urinary bladder cancer. In the present study, the protein levels of DDX39 in the human MPM cell lines NCI-H28, NCI-H2052 and NCI-H2452, and the human pleural mesothelial cell line MeT-5A were investigated by western blotting. The protein levels of DDX39 were found to be higher in NCI-H28, NCI-H2052 and NCI-H2452 compared to MeT-5A. The intensity of the bands of DDX39 in NCI-H28, NCI-H2052 and NCI-H2452 cells were increased by 1.351-, 1.887- and 2.024-fold, respectively, compared to MPM cells. These results suggest that DDX39 is a possible candidate biomarker for molecular-targeting of MPM.

Children with neurofibromatosis type 1 (NF1) frequently have cognitive and behavioral deficits. Some of these deficits have been successfully modeled in Nf1 genetically-engineered mice that develop optic gliomas (Nf1 OPG mice). In the current study, we show that abnormal motivational influences affect the behavior of Nf1 OPG mice, particularly with regard to their response to novel environmental stimuli. For example, Nf1 OPG mice made fewer spontaneous alternations in a Y-maze and fewer arm entries relative to WT controls. However, analysis of normalized alternation data demonstrated that these differences were not due to a spatial working memory deficit. Other reported behavioral results (e.g., open-field test, below) suggest that differential responses to novelty and/or other motivational influences may be more important determinants of these kinds of behavior than simple differences in locomotor activity/spontaneous movements. Importantly, normal long-term depression was observed in hippocampal slices from Nf1 OPG mice. Results from elevated plus maze testing showed that differences in exploratory activity between Nf1 OPG and WT control mice may be dependent on the environmental context (e.g., threatening or non-threatening) under which exploration is being measured. Nf1 OPG mice also exhibited decreased exploratory hole poking in a novel holeboard and showed abnormal olfactory preferences, although L-dopa (50 mg/kg) administration resolved the abnormal olfactory preference behaviors. Nf1 OPG mice displayed an attenuated response to a novel open field in terms of decreased ambulatory activity and rearing but only during the first 10 min of the session. Importantly, Nf1 OPG mice demonstrated investigative rearing deficits with regard to a novel hanging object suspended on one side of the field which were not rescued by L-dopa administration. Collectively, our results provide new data important for evaluating therapeutic treatments aimed at ameliorating NF1-associated cognitive/behavioral deficits.