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Background/aim Proteomics is an approach that can detect differentially expressed proteins between cancerous and non-cancerous tissue samples. Previously, we found that vinculin was predominantly expressed in pancreatic cancerous tissues compared to adjacent non-cancerous tissues by performing proteomic differential display analysis. However, the clinicopathological significance of vinculin in pancreatic cancer has not yet been documented. Materials and methods The GEPIA2 and the Human Protein Atlas databases were used to analyze vinculin expression levels in cancerous tissue samples and investigate whether its expression level is clinically associated with patient survival. Results Vinculin mRNA expression levels were solely increased in pancreatic cancer tissues, and increased expression was inversely related to patient survival. Higher levels of vinculin protein were found in pancreatic cancer tissues. In contrast, faint staining of vinculin was observed throughout the normal pancreatic tissues. Conclusion Vinculin may be an unfavorable prognostic indicator for patients with pancreatic cancer.

AHCC®, a standardized extract of cultured Lentinula edodes mycelia, enhances the therapeutic effects and reduces the adverse effects of chemotherapy. Our previous study reported that treatment with AHCC® downregulated the expression levels of tumor-associated proteins in the gemcitabine-resistant pancreatic cancer cell line, KLM1-R. However, to the best of our knowledge, the role of AHCC® in the inhibition of cell migration remains unexplored. Cortactin (CTTN), an actin nucleation-promoting factor, has been reported to be upregulated and correlated with migration, invasion and metastasis in pancreatic cancer cells. The present study aimed to investigate the effects of AHCC® on cell migration and the protein expression level of CTTN in KLM1-R cells. The Gene Expression Profiling Interactive Analysis (GEPIA2), an online bioinformatics platform, was used to analyze CTTN mRNA expression levels in pancreatic cancer tissues compared with normal pancreatic tissues. CTTN mRNA expression and its association with clinicopathological characteristics were assessed by using the GEPIA2 platform. Next, the effects of AHCC® on KLM1-R cell migration were investigated by in vitro wound-healing assay. The KLM1-R cells were treated with AHCC® at a concentration of 10 mg/ml for 48 h. Western blotting was performed on of cell lysates with anti-CTTN or anti-actin antibodies to assess the protein expression levels of CTTN. Bioinformatics analysis indicated that the mRNA expression level of CTTN increased in pancreatic cancer tissues. The increased mRNA expression levels of CTTN were inversely associated with clinicopathological characteristics, including disease stages and prolonged patient survival times. The administration of 10 mg/ml AHCC® significantly inhibited KLM1-R cells migration compared with controls. The protein expression levels of CTTN were significantly reduced in AHCC®-treated KLM1-R cells, whereas actin expression was not affected. The downregulation of CTTN indicated the anti-metastatic potential of AHCC® in pancreatic cancer cells. Overall, AHCC® may have the potential to be a complementary and alternative therapeutic approach in treating pancreatic cancer.

Background/Aim: Malignant pleural mesothelioma (MPM) is a rare but very aggressive tumor that is primarily pleural in origin. The 5-year overall survival rate of patients with MPM has not improved despite therapeutic advances. Therefore, biomarker discovery to identify premalignant or early malignant tumors of the mesothelium are crucial. PEA15 is a cytoplasmic protein that is involved in various human malignancies, including MPM. However, the clinicopathological involvement of PEA15 in MPM has not yet been documented. Materials and Methods: The Oncomine database and GEPIA2 platform were used to analyze PEA15 mRNA expression and patient survival in patients with MPM. Results: PEA15 was found to be significantly up-regulated in MPM, and this up-regulation inversely correlated with prolonged patient survival. Further, PEA15 expression was found to be increased in other cancer tissues without affecting overall survival. Conclusion: PEA15 may represent a new potential prognostic biomarker in MPM patients.

BACKGROUND/AIM: Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) has been affecting Hokkaido, Japan since late February 2020 until present. The aim of this study was to report the relationship between anti-SARS-CoV-2 antibody-positive and SARS-CoV-2 PCR-positive cases by analyzing anti-SARS-CoV-2 antibodies (IgG and total-Ig). PATIENTS AND METHODS: Serum samples were collected from care workers and nurses in two nursing homes and two hospitals which underwent virus outbreak. All people were confirmed to be SARS-CoV-2-positive by RT-qPCR and their sera was analyzed for anti-SARS-CoV-2 antibodies (IgG and total-Ig). RESULTS: Although 34 out of 43 samples (79.1%) showed enough amount of anti-SARS-CoV-2 antibodies, 9 RT-qPCR -positive samples (20.9%) showed absence of anti-SARS-CoV-2 antibodies in their sera. CONCLUSION: The results that 20.9% of RT-qPCR-positive samples with SARS-CoV-2 showed absence of anti-SARS-CoV-2 antibodies provides a possibility that the innate immune reaction could eliminate the virus without activating adaptive immune reaction.

Pancreatic ductal adenocarcinoma (PDAC) is the most common form of pancreatic cancer with an abysmal prognosis rate over the last few decades. Early diagnosis and prevention could effectively combat this malignancy. Therefore, it is crucial to discover potential biomarkers to identify asymptomatic premalignant or early malignant tumors of PDAC. Gene expression analysis is a powerful technique to identify candidate biomarkers involved in disease progression. In the present study, five independent gene expression datasets, including 321 PDAC tissues and 208 adjacent non-cancerous tissue samples, were subjected to statistical and bioinformatics analysis. A total of 20 differentially expressed genes (DEGs) were identified in PDAC tissues compared to non-cancerous tissue samples. Gene ontology and pathway enrichment analysis showed that DEGs were mainly enriched in extracellular matrix (ECM), cell adhesion, ECM–receptor interaction, and focal adhesion signalling. The protein–protein interaction network was constructed, and the hub genes were evaluated. Collagen type XII alpha 1 chain (COL12A1), fibronectin 1 (FN1), integrin subunit alpha 2 (ITGA2), laminin subunit beta 3 (LAMB3), laminin subunit gamma 2 (LAMC2), thrombospondin 2 (THBS2), and versican (VCAN) were identified as hub genes. The correlation analysis revealed that identified hub genes were significantly interconnected. Wherein COL12A1, FN1, ITGA2, LAMB3, LAMC2, and THBS2 were significantly associated with PDAC pathological stages. The Kaplan–Meier survival plots revealed that ITGA2, LAMB3, and LAMC2 expression were inversely correlated with a prolonged patient survival period. Furthermore, the Human Protein Atlas database was used to validate the expression and cellular origins of hub genes encoded proteins. The protein expression of hub genes was higher in pancreatic cancer tissue than in normal pancreatic tissue samples, wherein ITGA2, LAMB3, and LAMC2 were exclusively expressed in pancreatic cancer cells. Pancreatic cancer cell-specific expression of these three proteins may play pleiotropic roles in cancer progression. Our results collectively suggest that ITGA2, LAMB3, and LAMC2 could provide deep insights into pancreatic carcinogenesis molecular mechanisms and provide attractive therapeutic targets., peer-reviewed

AHCC(®), a standardized extract of cultured Lentinula edodes mycelia, enhances the therapeutic effects and reduces the adverse effects of chemotherapy. Our previous study reported that treatment with AHCC(®) downregulated the expression levels of tumor-associated proteins in the gemcitabine-resistant pancreatic cancer cell line, KLM1-R. However, to the best of our knowledge, the role of AHCC(®) in the inhibition of cell migration remains unexplored. Cortactin (CTTN), an actin nucleation-promoting factor, has been reported to be upregulated and correlated with migration, invasion and metastasis in pancreatic cancer cells. The present study aimed to investigate the effects of AHCC(®) on cell migration and the protein expression level of CTTN in KLM1-R cells. The Gene Expression Profiling Interactive Analysis (GEPIA2), an online bioinformatics platform, was used to analyze CTTN mRNA expression levels in pancreatic cancer tissues compared with normal pancreatic tissues. CTTN mRNA expression and its association with clinicopathological characteristics were assessed by using the GEPIA2 platform. Next, the effects of AHCC(®) on KLM1-R cell migration were investigated by in vitro wound-healing assay. The KLM1-R cells were treated with AHCC(®) at a concentration of 10 mg/ml for 48 h. Western blotting was performed on of cell lysates with anti-CTTN or anti-actin antibodies to assess the protein expression levels of CTTN. Bioinformatics analysis indicated that the mRNA expression level of CTTN increased in pancreatic cancer tissues. The increased mRNA expression levels of CTTN were inversely associated with clinicopathological characteristics, including disease stages and prolonged patient survival times. The administration of 10 mg/ml AHCC(®) significantly inhibited KLM1-R cells migration compared with controls. The protein expression levels of CTTN were significantly reduced in AHCC(®)-treated KLM1-R cells, whereas actin expression was not affected. The downregulation of CTTN indicated the anti-metastatic potential of AHCC(®) in pancreatic cancer cells. Overall, AHCC(®) may have the potential to be a complementary and alternative therapeutic approach in treating pancreatic cancer.

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

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.

Background: Recent studies have revealed that the disruption of the blood–brain barrier (BBB) might contribute to the induction of neurodegeneration in the progressive stage of multiple sclerosis (MS). Objective: We investigated a potential target for the serum auto-antibodies responsible for the BBB impairment in patients with secondary progressive MS (SPMS). Methods: We identified undetermined target antigens in human brain microvascular endothelial cells (BMECs) that reacted with auto-antibodies in sera from SPMS patients using a proteomic approach. In addition, we examined how the identified auto-antibodies compromise the BBB integrity. Results: We found that 10 of 11 SPMS sera had auto-antibodies against galectin-3, although the patients with other neurological diseases did not have these antibodies. Downregulation of galectin-3 led to elevated intercellular adhesion molecule-1 (ICAM-1) and phospho-nuclear factor-kappa (NFκ) B p65 expression in the BMECs. Exposure to SPMS patients’ sera also increased the protein levels of ICAM-1 and phospho-NFκB p65 in BMECs, but these effects induced by anti-galectin-3 immunoreactivity were canceled by the downregulation of galectin-3. Conclusion: Galectin-3 is a possible immunological target molecule of the pathogenic auto-antibodies and contributes to the persistent BBB breakdown in patients with SPMS. These antibodies may also serve as a novel biomarker for SPMS.

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.

Slingshot-1L (SSH1L), a cofilin-phosphatase, plays a role in actin dynamics and cell migration by reactivating cofilin-1. However, the expression of SSH1L in malignant diseases is poorly understood. The overexpression of SSH1L in cancerous tissue compared to the matched surrounding non-cancerous tissues from patients with late stages (III-IV) of PC was detected in 90% (9/10) of cases by western blotting. The expression of SSH1L was shown to be upregulated in tumor cells from 10.7% (11/102) of patients with pancreatic cancer (PC) by immunohistochemistry (IHC). The positive rate of SSH1L in patients with PC at stage VI (TNM) categorized as grade 3 was of 50% (2/4) and 15% (6/40), respectively. Moreover, SSH1L expression was shown to be up-regulated in the PC cell lines (KLM1, PANC-1 and MIAPaCa-2) with high metastatic potential. Loss of SSH1L expression was associated with an increase in the phosphorylation of cofilin-1 at serine-3 and further inhibited cell migration (but not proliferation) in KLM1, PANC-1 and MIAPaCa-2. Actin polymerization inhibitor cytochalasin-D was sufficient to abrogate cell migration of PC without changing SSH1L expression. These results reveal that SSH1L is upregulated in a subset of PCs and that the SSH1L/cofilin-1 signal pathway is associated positively in PC with cell migration. Our study may thus provide potential targets to prevent and/or treat PC invasion and metastasis in patients with SSH1L-positive PC.

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.

The isolation and application of auxotrophic mutants for gene manipulations, such as genetic transformation, mating selection and tetrad analysis, form the basis of yeast genetics. For the development of these genetic methods in the thermotolerant fermentative yeast Kluyveromyces marxianus, we isolated a series of auxotrophic mutants with defects in amino acid or nucleic acid metabolism. To identify the mutated genes, linear DNA fragments of nutrient biosynthetic pathway genes were amplified from Saccharomyces cerevisiae chromosomal DNA and used to directly transform the K. marxianus auxotrophic mutants by random integration into chromosomes through non-homologous end joining (NHEJ). The appearance of transformant colonies indicated that the specific S. cerevisiae gene complemented the K. marxianus mutant. Using this interspecific complementation approach with linear PCR-amplified DNA, we identified auxotrophic mutations of ADE2, ADE5,7, ADE6, HIS2, HIS3, HIS4, HIS5, HIS6, HIS7, LYS1, LYS2, LYS4, LYS9, LEU1, LEU2, MET2, MET6, MET17, TRP3, TRP4 and TRP5 without the labour-intensive requirement of plasmid construction. Mating, sporulation and tetrad analysis techniques for K. marxianus were also established. With the identified auxotrophic mutant strains and S. cerevisiae genes as selective markers, NHEJ-mediated integrative transformation with PCR-amplified DNA is an attractive system for facilitating genetic analyses in the yeast K. marxianus. Copyright © 2013 John Wiley & Sons, Ltd.

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.

SUMMARY Helicobacter pylori, a common pathogen that causes chronic gastritis and cancer, has evolved to establish persistent infections in the human stomach. Epidemiological evidence suggests that H. pylori with both highly active vacuolating cytotoxin A (VacA) and cytotoxin-associated gene A (CagA), the major virulence factors, has an advantage in adapting to the host environment. However, the mechanistic relationship between VacA and CagA remains obscure. Here, we report that CagA interferes with eukaryotic endocytosis, as revealed by genome-wide screening in yeast. Moreover, CagA suppresses pinocytic endocytosis and the cytotoxicity of VacA in gastric epithelial cells without affecting clathrin-dependent endocytosis. Our data suggest that H. pylori secretes VacA to attack distant host cells while injecting CagA into the gastric epithelial cells to which the bacteria are directly attached, thereby protecting these attached host cells from the cytotoxicity of VacA and creating a local ecological niche. This mechanism might allow H. pylori to balance damage to one population of host cells with the preservation of another, allowing for persistent infection.

An exposure test was conducted on test specimens simulating marine structures to which cathodic protection was applied. Test specimens of seawater resistant stainless steel plates and those of carbon steel plates exposed to the tidal zone environment were analyzed. We measured the density of cathodic current flowing into the test specimens, corrosion rate of the specimens and weight loss of sacrificial anode. The cathodic current density was influenced by the reduction of rust and calcareous deposits. Weight loss of the anode calculated using cathodic current was found to be approximately the same as that obtained from the experiment. Weight loss of the anode for seawater resistant stainless steel as a test specimen was approximately 60% for those of carbon steel plates. Furthermore, we investigated the design method for cathodic protection taking into account the current flowing into the plate placed at the tidal zone.

The plant pathogenic bacterium Ralstonia solanacearum injects more than 70 effector proteins (virulence factors) into the host plant cells via the needle-like structure of a type III secretion system. The type III secretion system effector proteins manipulate host regulatory networks to suppress defense responses with diverse molecular activities. Uncovering the molecular function of these effectors is essential for a mechanistic understanding of R. solanacearum pathogenicity. However, few of the effectors from R. solanacearum have been functionally characterized, and their plant targets remain largely unknown. Here, we show that the ChaC domain-containing effector RipAY/RSp1022 from R. solanacearum exhibits γ-glutamyl cyclotransferase (GGCT) activity to degrade the major intracellular redox buffer, glutathione. Heterologous expression of RipAY, but not other ChaC family proteins conserved in various organisms, caused growth inhibition of yeast Saccharomyces cerevisiae, and the intracellular glutathione level was decreased to ∼30% of the normal level following expression of RipAY in yeast. Although active site mutants of GGCT activity were non-toxic, the addition of glutathione did not reverse the toxicity, suggesting that the toxicity might be a consequence of activity against other γ-glutamyl compounds. Intriguingly, RipAY protein purified from a bacterial expression system did not exhibit any GGCT activity, whereas it exhibited robust GGCT activity upon its interaction with eukaryotic thioredoxins, which are important for intracellular redox homeostasis during bacterial infection in plants. Our results suggest that RipAY has evolved to sense the host intracellular redox environment, which triggers its enzymatic activity to create a favorable environment for R. solanacearum infection., This work was supported by Japan Society for the Promotion of Science KAKENHI Grants 22580095 and 25450104 and also, in part, by the General Research Grant of the Institute of Fermentation, Osaka.

The cytolethal distending toxins (CDTs) are secreted virulence proteins produced by several bacterial pathogens, and the subunit CdtB has the ability to create DNA lesions, primarily DNA single-strand breaks (SSBs) in vitro, and cause cell cycle arrest, cellular distension, and cell death in both mammalian and yeast cells. To elucidate the components of the mechanisms underlying the response to CdtB-induced DNA lesions, a CdtB expression plasmid was transformed into a series of diploid yeast strains harboring deletions in 4,708 nonessential genes. A total of 4,706 of these clones were successfully transformed, which we have now designated as a systematic transformation array (STA), and were subsequently screened. We identified 61 sensitive strains from the STA whose deleted genes can be categorized into a number of groups, including DNA metabolism, chromosome segregation, vesicular traffic, RNA catabolism, protein translation, morphogenesis, and nuclear transport, as well as one unknown open reading frame. However, only 28 of these strains were found to be sensitive to HO endonuclease, which is known to create a DNA double-strand break (DSB), suggesting that CdtB-induced DNA lesion is not similar to the direct DSB. Amazingly, CdtB expression elicits severe growth defects in haploid yeast cells, but only marginal defects in diploid yeast cells. The presence and absence of genes known to be involved in DNA repair in these genome-wide data reveal that CdtB-induced DNA damage is specifically repaired well in the diploid by homologous recombination but not by other repair mechanisms. Our present results provide insights into how CdtB pathogenesis is linked to eukaryotic cellular functions.

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.

Micro-environment plays a crucial role in determining the phenotypes within a tumor.In order to understand how the micro-environment affects pancreatic cancer, KLM1 cells were cultured under growth factor stress by culturing in foetal bovine serum (FBS)-free and reduced (1%) medium over several passages to mimic the core of a solid tumor with low vascularisation.Proteomic analysis on these conditioned pancreatic cancer cells, called KLM1-S, compared to the parent cell line KLM1 revealed that a number of proteins including α-enolase, GAPDH, GRP78, HSP60 and STIP-1 were dysregulated. Additionally, KLM1-S cells exhibited a 250-fold increase in half-maximal inhibitory concentration (IC50) over the parent cell line KLM1.By decreasing their replication rate and levels of intracellular reactive oxygen species (ROS), KLM1-S cells are able to resist gemcitabine (GEM). The results obtained suggest that in KLM1 different phenotypes are a result of cellular plasticity rather than a committed transformation.

It is known that cancers adopt different strategies to cope with stress and overcome adverse micro-environmental conditions. Such strategies are also applicable to chemo-therapeutic treatment, which could subsequently result in chemo-resistance.In order to investigate known stress-evasion strategies observed in pancreatic cancer, the stress-resistant KLM1-derived cell lines KLM1-R (Gemcitabine (GEM)-induced stress) and KLM1-S (growth factor restriction-induced stress) were employed. Comparative proteomics were employed between for the two cell lines that were also compared against the parent cell line KLM1.Proteomic analysis revealed changes in the expression levels of 6 proteins, namely: transitional endoplasmic reticulum ATPase, lamin A/C, PDZ and LIM protein 1, calmodulin, heat shock protein 60 and alpha enolase. Resistance to GEM of KLM1-R and KLM1-S was found to be comparable, with KLM1-S cells exhibiting close to 1.5-fold higher half-maximal inhibitory concentration (IC50) compared to KLM1-R cells.These results suggest that KLM1-R can be used as a model of directly-acquired chemoresistance (responding directly to evade GEM treatment), while KLM1-S is a good model of indirectly-acquired chemoresistance (formed in response to having to survive with less availability of growth factors), additionally gaining a selective advantage upon GEM treatment.