Sample records for cells recognize grp78

  1. Receptor-recognized α₂-macroglobulin binds to cell surface-associated GRP78 and activates mTORC1 and mTORC2 signaling in prostate cancer cells.

    Directory of Open Access Journals (Sweden)

    Uma K Misra

    Full Text Available OBJECTIVE: Tetrameric α(2-macroglobulin (α(2M, a plasma panproteinase inhibitor, is activated upon interaction with a proteinase, and undergoes a major conformational change exposing a receptor recognition site in each of its subunits. Activated α(2M (α(2M* binds to cancer cell surface GRP78 and triggers proliferative and antiapoptotic signaling. We have studied the role of α(2M* in the regulation of mTORC1 and TORC2 signaling in the growth of human prostate cancer cells. METHODS: Employing immunoprecipitation techniques and Western blotting as well as kinase assays, activation of the mTORC1 and mTORC2 complexes, as well as down stream targets were studied. RNAi was also employed to silence expression of Raptor, Rictor, or GRP78 in parallel studies. RESULTS: Stimulation of cells with α(2M* promotes phosphorylation of mTOR, TSC2, S6-Kinase, 4EBP, Akt(T308, and Akt(S473 in a concentration and time-dependent manner. Rheb, Raptor, and Rictor also increased. α(2M* treatment of cells elevated mTORC1 kinase activity as determined by kinase assays of mTOR or Raptor immunoprecipitates. mTORC1 activity was sensitive to LY294002 and rapamycin or transfection of cells with GRP78 dsRNA. Down regulation of Raptor expression by RNAi significantly reduced α(2M*-induced S6-Kinase phosphorylation at T389 and kinase activity in Raptor immunoprecipitates. α(2M*-treated cells demonstrate about a twofold increase in mTORC2 kinase activity as determined by kinase assay of Akt(S473 phosphorylation and levels of p-Akt(S473 in mTOR and Rictor immunoprecipitates. mTORC2 activity was sensitive to LY294002 and transfection of cells with GRP78 dsRNA, but insensitive to rapamycin. Down regulation of Rictor expression by RNAi significantly reduces α(2M*-induced phosphorylation of Akt(S473 phosphorylation in Rictor immunoprecipitates. CONCLUSION: Binding of α(2M* to prostate cancer cell surface GRP78 upregulates mTORC1 and mTORC2 activation and promotes protein

  2. Phage display derived human monoclonal antibodies isolated by binding to the surface of live primary breast cancer cells recognize GRP78

    DEFF Research Database (Denmark)

    Jakobsen, Charlotte G; Rasmussen, Nicolaj; Laenkholm, Anne-Vibeke


    Clinical trials using monoclonal antibodies (mAb) against cell-surface markers have yielded encouraging therapeutic results in several cancer types. Generally, however, anticancer antibodies are only efficient against a subpopulation of cancers, and there is a strong need for identification of no...... therapies including mAb-based immunotherapy. Our results suggest that the human antibody Ab39 may be a useful starting point for further genetic optimization that could render it a useful diagnostic and therapeutic reagent for a variety of cancers...

  3. CRIPTO/GRP78 Signaling Maintains Fetal and Adult Mammary Stem Cells Ex Vivo

    Directory of Open Access Journals (Sweden)

    Benjamin T. Spike


    Full Text Available Little is known about the extracellular signaling factors that govern mammary stem cell behavior. Here, we identify CRIPTO and its cell-surface receptor GRP78 as regulators of stem cell behavior in isolated fetal and adult mammary epithelial cells. We develop a CRIPTO antagonist that promotes differentiation and reduces self-renewal of mammary stem cell-enriched populations cultured ex vivo. By contrast, CRIPTO treatment maintains the stem cell phenotype in these cultures and yields colonies with enhanced mammary gland reconstitution capacity. Surface expression of GRP78 marks CRIPTO-responsive, stem cell-enriched fetal and adult mammary epithelial cells, and deletion of GRP78 from adult mammary epithelial cells blocks their mammary gland reconstitution potential. Together, these findings identify the CRIPTO/GRP78 pathway as a developmentally conserved regulator of fetal and adult mammary stem cell behavior ex vivo, with implications for the stem-like cells found in many cancers.

  4. CRIPTO/GRP78 Signaling Maintains Fetal and Adult Mammary Stem Cells Ex Vivo (United States)

    Spike, Benjamin T.; Kelber, Jonathan A.; Booker, Evan; Kalathur, Madhuri; Rodewald, Rose; Lipianskaya, Julia; La, Justin; He, Marielle; Wright, Tracy; Klemke, Richard; Wahl, Geoffrey M.; Gray, Peter C.


    Summary Little is known about the extracellular signaling factors that govern mammary stem cell behavior. Here, we identify CRIPTO and its cell-surface receptor GRP78 as regulators of stem cell behavior in isolated fetal and adult mammary epithelial cells. We develop a CRIPTO antagonist that promotes differentiation and reduces self-renewal of mammary stem cell-enriched populations cultured ex vivo. By contrast, CRIPTO treatment maintains the stem cell phenotype in these cultures and yields colonies with enhanced mammary gland reconstitution capacity. Surface expression of GRP78 marks CRIPTO-responsive, stem cell-enriched fetal and adult mammary epithelial cells, and deletion of GRP78 from adult mammary epithelial cells blocks their mammary gland reconstitution potential. Together, these findings identify the CRIPTO/GRP78 pathway as a developmentally conserved regulator of fetal and adult mammary stem cell behavior ex vivo, with implications for the stem-like cells found in many cancers. PMID:24749068

  5. Ligation of cancer cell surface GRP78 with antibodies directed against its COOH-terminal domain up-regulates p53 activity and promotes apoptosis. (United States)

    Misra, Uma Kant; Mowery, Yvonne; Kaczowka, Steven; Pizzo, Salvatore Vincent


    Binding of activated α(2)-macroglobulin to GRP78 on the surface of human prostate cancer cells promotes proliferation by activating signaling cascades. Autoantibodies directed against the activated α(2)-macroglobulin binding site in the NH(2)-terminal domain of GRP78 are receptor agonists, and their presence in the sera of cancer patients is a poor prognostic indicator. We now show that antibodies directed against the GRP78 COOH-terminal domain inhibit [(3)H]thymidine uptake and cellular proliferation while promoting apoptosis as measured by DNA fragmentation, Annexin V assay, and clonogenic assay. These antibodies are receptor antagonists blocking autophosphorylation and activation of GRP78. Using 1-LN and DU145 prostate cancer cell lines and A375 melanoma cells, which express GRP78 on their cell surface, we show that antibodies directed against the COOH-terminal domain of GRP78 up-regulate the tumor suppressor protein p53. By contrast, antibody directed against the NH(2)-terminal domain of GRP78 shows negligible effects on p53 expression. PC-3 prostate cancer cells, which do not express GRP78 on their cell surface, are refractory to the effects of anti-GRP78 antibodies directed against either the COOH- or NH(2)-terminal domains. However, overexpression of GRP78 in PC-3 cells causes translocation of GRP78 to the cell surface and promotes apoptosis when these cells are treated with antibody directed against its COOH-terminal domain. Silencing GRP78 or p53 expression by RNA interference significantly blocked the increase in p53 induced by antibodies. Antibodies directed against the COOH-terminal domain may play a therapeutic role in cancer patients whose tumors trigger the production of autoantibodies directed against the NH(2)-terminal domain of GRP78.

  6. The Escherichia coli subtilase cytotoxin A subunit specifically cleaves cell-surface GRP78 protein and abolishes COOH-terminal-dependent signaling. (United States)

    Ray, Rupa; de Ridder, Gustaaf G; Eu, Jerry P; Paton, Adrienne W; Paton, James C; Pizzo, Salvatore V


    GRP78, a molecular chaperone with critical endoplasmic reticulum functions, is aberrantly expressed on the surface of cancer cells, including prostate and melanoma. Here it functions as a pro-proliferative and anti-apoptotic signaling receptor via NH(2)-terminal domain ligation. Auto-antibodies to this domain may appear in cancer patient serum where they are a poor prognostic indicator. Conversely, GRP78 COOH-terminal domain ligation is pro-apoptotic and anti-proliferative. There is no method to disrupt cell-surface GRP78 without compromising the total GRP78 pool, making it difficult to study cell-surface GRP78 function. We studied six cell lines representing three cancer types. One cell line per group expresses high levels of cell-surface GRP78, and the other expresses low levels (human hepatoma: Hep3B and HepG2; human prostate cancer: PC3 and 1-LN; murine melanoma: B16F0 and B16F1). We investigated the effect of Escherichia coli subtilase cytoxin catalytic subunit (SubA) on GRP78. We report that SubA specifically cleaves cell-surface GRP78 on HepG2, 1-LN, and B16F1 cells without affecting intracellular GRP78. B16F0 cells (GRP78(low)) have lower amounts of cleaved cell-surface GRP78. SubA has no effect on Hep3B and PC3 cells. The predicted 28-kDa GRP78 COOH-terminal fragment is released into the culture medium by SubA treatment, and COOH-terminal domain signal transduction is abrogated, whereas pro-proliferative signaling mediated through NH(2)-terminal domain ligation is unaffected. These experiments clarify cell-surface GRP78 topology and demonstrate that the COOH-terminal domain is necessary for pro-apoptotic signal transduction occurring upon COOH-terminal antibody ligation. SubA is a powerful tool to specifically probe the functions of cell-surface GRP78.

  7. GRP78 and Cripto Form a Complex at the Cell Surface and Collaborate To Inhibit Transforming Growth Factor β Signaling and Enhance Cell Growth▿ (United States)

    Shani, Gidi; Fischer, Wolfgang H.; Justice, Nicholas J.; Kelber, Jonathan A.; Vale, Wylie; Gray, Peter C.


    Cripto is a multifunctional cell surface protein with important roles in vertebrate embryogenesis and the progression of human tumors. While Cripto has been shown to modulate multiple signaling pathways, its binding partners do not appear to fully explain its molecular actions. Therefore, we conducted a screen aimed at identifying novel Cripto-interacting proteins. This screen led to our identification of glucose-regulated protein 78 (GRP78), an endoplasmic reticulum (ER) chaperone that is also expressed at the surfaces of tumor cells. Here we demonstrate that Cripto and GRP78 interact at the cell surfaces of multiple cell lines and that their interaction is independent of prior association within the ER. Interestingly, short hairpin RNA knockdown of endogenous GRP78 resulted in enhanced transforming growth factor β (TGF-β) signaling, indicating that like Cripto, GRP78 inhibits this pathway. We further show that when coexpressed, GRP78 and Cripto collaborate to antagonize TGF-β responses, including Smad phosphorylation and growth inhibition of prostate cancer cells grown under anchorage-dependent or -independent conditions. Finally, we provide evidence that cells coexpressing GRP78 and Cripto grow much more rapidly in soft agar than do cells expressing either protein individually. Together, our results indicate that these proteins bind at the cell surface to enhance tumor growth via the inhibition of TGF-β signaling. PMID:17991893

  8. Serum GRP78 as a Tumor Marker and Its Prognostic Significance in Non-Small Cell Lung Cancers: A Retrospective Study

    Directory of Open Access Journals (Sweden)

    Xiao Ma


    Full Text Available Introduction. Glucose-regulated protein 78 (78 kDa, GRP78, which is also known as immunoglobulin heavy chain binding protein (BIP, is a major chaperone in the endoplasmic reticulum (ER. The expression and clinical significance of GRP78 in the serum of non-small cell lung cancer patients have not yet been clearly described. The aims of the present study were to investigate the expression of GRP78 in the serum of non-small cell lung cancer patients, the relationships with clinicopathological parameters, and the potential implications for survival. Patients and Methods. A total of 163 peripheral blood samples from non-small cell lung cancer patients were prospectively collected at the Department of Thoracic Surgery, Fudan University Shanghai Cancer, China. Clinical characteristics data, including age, gender, stage, overall survival (OS time, and relapse-free survival (RFS time, were also collected. Serum GRP78 levels were measured using a commercially available ELISA kit. The associations between GRP78 levels and clinicopathological characteristics and survival were examined using Student’s t-test, Kaplan-Meier, or Cox regression analyses. Results. The mean ± standard error (SE value of GRP78 was 326.5 ± 49.77 pg/mL. This level was significantly lower compared with the level in late-stage non-small cell lung cancer patients (1227 ± 223.6, p=0.0001. There were no significant correlations with the clinicopathological parameters. No significant difference was found between high GRP78 expression and low GRP78 expression with regard to RFS (p=0.1585. However, the OS of patients with higher GRP78 expression was significantly poorer (p=0.0334. Conclusions. GRP78 was expressed in non-small cell lung cancer patients and was highly enriched in late-stage lung cancer. GRP78 may have an important role in the carcinogenesis of non-small cell lung cancer and may be a prognostic marker for non-small cell lung cancer.

  9. Knockdown of GRP78 promotes apoptosis in pancreatic acinar cells and attenuates the severity of cerulein and LPS induced pancreatic inflammation.

    Directory of Open Access Journals (Sweden)

    Yong Liu

    Full Text Available Acute pancreatitis (AP is a potentially lethal disease characterized by inflammation and parenchymal cell death; also, the severity of AP correlates directly with necrosis and inversely with apoptosis. However, mechanisms of regulating cell death in AP remain unclear. The endoplasmic reticulum (ER chaperone protein GRP78 has anti-apoptotic properties, in addition to modulating ER stress responses. This study used RNA interference (RNAi approach to investigate the potential role of GRP78 in regulating apoptosis during AP. In vitro models of AP were successfully developed by treating AR42J cells with cerulein or cerulein plus lipoplysaccharide (LPS. There was more pancreatic inflammation and less apoptosis with the cerulein plus LPS treatment. Furthermore, knockdown of GRP78 expression markedly promoted apoptosis and reduced necrosis in pancreatic acinar cells. This was accomplished by enhancing the activation of caspases and inhibiting the activity of X-linked inhibitor of apoptosis protein (XIAP, as well as a receptor interacting protein kinase-1(RIPK1, which is a key mediator of necrosis. This attenuated the severity of pancreatic inflammation, especially after cerulein plus LPS treatment. In conclusion, these findings indicate that GRP78 plays an anti-apoptotic role in regulating the cell death response during AP. Therefore, GRP78 is a potential therapeutic target for AP.

  10. Cancer-associated fibroblasts promote non-small cell lung cancer cell invasion by upregulation of glucose-regulated protein 78 (GRP78) expression in an integrated bionic microfluidic device. (United States)

    Yu, Ting; Guo, Zhe; Fan, Hui; Song, Jing; Liu, Yuanbin; Gao, Zhancheng; Wang, Qi


    The tumor microenvironment is comprised of cancer cells and various stromal cells and their respective cellular components. Cancer-associated fibroblasts (CAFs), a major part of the stromal cells, are a key determinant in tumor progression, while glucose-regulated protein (GRP)78 is overexpressed in many human cancers and is involved in tumor invasion and metastasis. This study developed a microfluidic-based three dimension (3D) co-culture device to mimic an in vitro tumor microenvironment in order to investigate tumor cell invasion in real-time. This bionic chip provided significant information regarding the role of GRP78, which may be stimulated by CAFs, to promote non-small cell lung cancer cell invasion in vitro. The data showed that CAF induced migration of NSCLC A549 and SPCA-1 cells in this three-dimensional invasion microdevice, which is confirmed by using the traditional Transwell system. Furthermore, CAF induced GRP78 expression in A549 and SPCA-1 cells to facilitate NSCLC cell migration and invasion, whereas knockdown of GRP78 expression blocked A549 and SPCA-1 cell migration and invasion capacity. In conclusion, these data indicated that CAFs might promote NSCLC cell invasion by up-regulation of GRP78 expression and this bionic chip microdevice is a robust platform to assess the interaction of cancer and stromal cells in tumor environment study.

  11. Differential requirement of GRP94 and GRP78 in mammary gland development (United States)

    Zhu, Genyuan; Wang, Miao; Spike, Benjamin; Gray, Peter C.; Shen, Jieli; Lee, Sung-Hyung; Chen, Si-Yi; Lee, Amy S.


    Glucose Regulated Protein (GRP) 94 and GRP78 are critical molecular chaperones and regulators of signaling. Conditional knockout mouse models have revealed tissue specific requirements for GRP94 and GRP78, including selection for allele retention in specific cell types. Here we report the consequences of mammary-targeted knockout of these GRPs. Our studies revealed that MMTV-Cre, Grp94f/f mammary glands, despite GRP94 deficiency, exhibited normal proliferation and ductal morphogenesis. Interestingly, MMTV-Cre, Grp78f/f mammary glands displayed only slightly reduced GRP78 protein levels, associating with the retention of the non-recombined Grp78 floxed alleles in isolated mammary epithelial cells and displayed phenotypes comparable to wild-type glands. In contrast, transduction of isolated Grp78f/f mammary epithelial stem/progenitor cells with adenovirus expressing GFP and Cre-recombinase was successful in GRP78 ablation, and the GFP sorted cells failed to give rise to repopulated mammary glands in de-epithelialized recipient mice. These studies imply GRP78, but not GRP94, is required for mammary gland development. PMID:24953136

  12. Transcriptional activation of endoplasmic reticulum chaperone GRP78 by HCMV IE1-72 protein

    Institute of Scientific and Technical Information of China (English)

    Derick Shi-Chen Ou; Sung-Bau Lee; Chi-Shuen Chu; Liang-Hao Chang; Bon-chu Chung; Li-Jung Juan


    Glucose-regulated protein 78 (GRP78), a key regulator of endoplasmic reticulum (ER) stress, facilitates cancer cell growth and viral replication. The mechanism leading to grp78 gene activation during viral infection is largely unknown, in this study, we show that the immediate-early 1 (IE1-72) protein of the human cytomegalovirus (HCMV) is essential for HCMV-mediated GRP78 activation. IE1-72 upregulated grp 78 gene expression depending on the ATPbinding site, the zinc-finger domain and the putative leucine-zipper motif of IE1-72, as well as the ER stress response elements (ERSEs) on the grp78 promoter. The purified IE1-72 protein bound to the CCAAT box within ERSE in vitro, whereas deletion mutants of IE1-72 deficient in grp78 promoter stimulation failed to do so. Moreover, IE1-72 binding to the grp78 promoter in infected cells accompanied the recruitment of TATA box-binding protein-associated factor 1 (TAF1), a histone acetyltransferase, and the increased level of acetylated histone H4, an indicator of activestate chromatin. These results provide evidence that HCMV IE1-72 activates grp78 gene expression through direct promoter binding and modulation of the local chromatin structure, indicating an active viral mechanism of cellular chaperone induction for viral growth.

  13. Enterovirus 71 induces dsRNA/PKR-dependent cytoplasmic redistribution of GRP78/BiP to promote viral replication (United States)

    Jheng, Jia-Rong; Wang, Shin-Chyang; Jheng, Chao-Rih; Horng, Jim-Tong


    GRP78/BiP is an endoplasmic reticulum (ER) chaperone protein with the important function of maintaining ER homeostasis, and the overexpression of GRP78/BiP alleviates ER stress. Our previous studies showed that infection with enterovirus 71 (EV71), a (+)RNA picornavirus, induced GRP78/BiP upregulation; however, ectopic GRP78/BiP overexpression in ER downregulates virus replication and viral particle formation. The fact that a virus infection increases GRP78/BiP expression, which is unfavorable for virus replication, is counterintuitive. In this study, we found that the GRP78/BiP protein level was elevated in the cytoplasm instead of in the ER in EV71-infected cells. Cells transfected with polyinosinic–polycytidylic acid, a synthetic analog of replicative double-stranded RNA (dsRNA), but not with viral proteins, also exhibited upregulation and elevation of GRP78/BiP in the cytosol. Our results further demonstrate that EV71 infections induce the dsRNA/protein kinase R-dependent cytosolic accumulation of GRP78/BiP. The overexpression of a GRP78/BiP mutant lacking a KDEL retention signal failed to inhibit both dithiothreitol-induced eIF2α phosphorylation and viral replication in the context of viral protein synthesis and viral titers. These data revealed that EV71 infection might cause upregulation and aberrant redistribution of GRP78/BiP to the cytosol, thereby facilitating virus replication. PMID:27004760

  14. Expression of GRP78 and caspase-12 in aortic vascular smooth muscle cells of hypertension rats and effects of enalapril on vascular remodeling%高血压大鼠血管平滑肌细胞GRP78和caspase-12表达的变化及依那普利的干预作用

    Institute of Scientific and Technical Information of China (English)

    郭丽; 赵连友; 刘静; 张志敏; 李雪; 丁璐


    AIM: To examine the effect of endoplasmic reticulum stress (ERS) on vascular remodeling induced by hypertension and to observe the effects of enalapril on the expressions of the correlation factors GRP78 and caspase-12 in vascular smooth muscle cells (VSMCs) and their relationship with vascular changes. METHODS: Forty adult male SD rats were randomly divided into sham operation group, sham + Ena group, AAB group and the AAB + Ena group. Four weeks later, MBP was measured through carotid artery incubation, the aortic media thickness was measured by image analyses software and the expressions of GRP78 and caspase-12 in the vascular smooth muscle cells were examined by immunohisto-chemistry. RESULTS: MAP of AAB group rats was significantly higher than those of rats in sham operation group, sham + Ena group and AAB + Ena group (P < 0. 05). The aortic media thickness of AAB group rats was significantly thicker than those of rats in sham operation group, sham + Ena group and the AAB + Ena group (P <0. 05). The expressions of GRP78 and caspase-12 of AAB group rats were significantly higher than those of rats in sham group, sham +Ena group and AAB +Ena group. CONCLUSION: High blood pressure resulting from abdominal aortic bounding causes endoplasmic reticulum stress response of vascular smooth muscle cells. Enalapril may exert some protective effects on VSMCs by inhibiting the endoplasmic reticulum stress via downregulation of the expression of caspase-12.%目的:观察高血压大鼠动脉血管平滑肌细胞(VSMCs)内质网应激(ERS)相关因子葡萄糖调节蛋白78(GRP78)和半胱氨酸门冬氨酸特异性蛋白酶12(caspase-12)表达的变化,并探讨高血压时VSMCs中ERS的分子机制,以及依那普利(enalapril,Ena)对血管重构的影响.方法:将40只健康雄性SD大鼠随机分为4组,即假手术(sham)组、sham+ Ena组,腹主动脉缩窄术(AAB)组及AAB+ Ena组,每组10只.4周后,通过颈动脉插管法测定大鼠血压和利用图像分析系

  15. Expression and Significance of GRP78 in Colon cancer%GRP78蛋白在结肠癌组织中的表达与意义

    Institute of Scientific and Technical Information of China (English)

    周臣敏; 董浦江; 付小利


    Objective To study the expression and significance of glucose regulated protein 78kD(GRP78) in colon cancer.Methods Western blot was used to detect the expression level of GRP78 in 82 cases of colon cancer tissue,17 cases of benign hyperplasia tissue and 33 cases of normal mucous membrane,meanwhile,vascular endothelial growth factor(VEGF),MTA1,Ezrin was detected in colon cancer tissue.The density of blood vessels in colon cancer was detected by immunohistochemistry SABC technique.Results The expression of GRP78 in the cancer tissue was significantly higher than that of the benign hyperplasia tissue and normal colorectal membrane(P<0.05).VEGF、MTA1 、Ezrin in GRP78-higher-expression group were higher than that in other two group(P<0.05),transfer rate of tumor cells and density of blood vessels in GRP78-higher-expression group were higher than that in other two group(P<0.05).Conclusion The expression level of GRP78 shows a increase trend when cancer develop from normal tissue,benign hyperplasia tissue to carcinoma.GRP78 is involved in the development of colorectal carcinoma.The GRP78 protein is highly expressed,which may play a key role in colon cancer cell metastasis.%目的 探讨葡萄耱调节蛋白78(GRP78)在结肠癌组织中的表达及意义.方法 用蛋白免疫印迹法(Wester blot)检测82例结肠癌组织、17例结肠良性增生组织、及33例正常肠组织的GRP78及癌组织中血管内皮生长因子(VEGF)、转移相关基因1(MTA1)、Ezrin的表达情况.免疫组化SABC法测定肿瘤组织微血管密度.结果 GRP78在结肠癌组织中的表达明显高于结肠良性增生组织及正常结肠组织(P<0.05),GRP78高表达组结肠癌组织的VEGF、MTA1、Ezrin表达量高于其他两组(P<0.05),GRP78高表达组结肠癌组织肿瘤细胞转移率及血管密度明显高于其他两组(P<0.05).结论 GRP78在结肠癌组织中的表达呈增高趋势,参与了结直肠腺癌的发生过程.GRP78高表达与结肠癌细胞转移有关.

  16. Heat shock at higher cell densities improves measles hemagglutinin translocation and human GRP78/BiP secretion in Saccharomyces cerevisiae. (United States)

    Zinkevičiūtė, Rūta; Bakūnaitė, Edita; Čiplys, Evaldas; Ražanskas, Raimundas; Raškevičiūtė, Jurgita; Slibinskas, Rimantas


    The yield of heterologous proteins is often limited by several bottlenecks in the secretory pathway of yeast Saccharomyces cerevisiae. It was shown earlier that synthesis of measles virus hemagglutinin (MeH) is inefficient mostly due to a bottleneck in the translocation of viral protein precursors into the endoplasmic reticulum (ER) of yeast cells. Here we report that heat shock with subsequent induction of MeH expression at 37°C improved translocation of MeH precursors when applied at higher cell densities. The amount of MeH glycoprotein increased by about 3-fold after heat shock in the late-log phases of both glucose and ethanol growth. The same temperature conditions increased both secretion titer and yield of another heterologous protein human GRP78/BiP by about 50%. Furthermore, heat shock at the late-log glucose growth phase also improved endogenous invertase yield by approximately 2.7-fold. In contrast, a transfer of yeast culture to lower temperature at diauxic shift followed by protein expression at 20°C almost totally inhibited translocation of MeH precursors. The difference in amounts of MeH glycoprotein under expression at 37°C and 20°C was about 80-fold, while amounts of unglycosylated MeH polypeptides were similar under both conditions. Comparative proteomic analysis revealed that besides over-expressed ER-resident chaperone Kar2, an increased expression of several cytosolic proteins (such as Hsp104, Hsp90 and eEF1A) may contribute to improved translocation of MeH.

  17. MDA-9 and GRP78 as potential diagnostic biomarkers for early detection of melanoma metastasis. (United States)

    Guan, Ming; Chen, Xiaofan; Ma, Yingyu; Tang, Lihua; Guan, Lei; Ren, Xuefeng; Yu, Bo; Zhang, Wei; Su, Bing


    Metastatic melanoma, the primary cause of skin cancer-related death, warrants new diagnostic and therapeutic approaches that target the regulatory machinery at molecular level. The heterogeneity and complexity of melanoma result in the difficulty to find biomarkers and targets for early detection and treatment. Here, we investigated metastasis-associated proteins by comparing the proteomic profiles of primary cutaneous melanomas to their matched lymph node metastases, which minimizes heterogeneity among samples from different patients. Results of two-dimensional gel electrophoresis (2-DE) followed by proteomic analysis revealed eight differentially expressed proteins. Among them, seven proteins (α-enolase, cofilin-1, LDH, m-β-actin, Nm23, GRP78, and MDA-9) showed increased and one (annexin A2) showed decreased expression in metastatic lymph node tissues than in primary melanomas. MDA-9 and GRP78 were the most highly expressed proteins in lymph node metastases, which was validated by immunohistochemical staining. Moreover, exosomes from serum samples of metastatic melanoma patients contained higher levels of MDA-9 and GRP78 than those of patients without metastases, indicating the potential of MDA-9 and GRP78 to be biomarkers for early detection of metastasis. Further, small interfering RNA (siRNA)-mediated knockdown confirmed a functional role for MDA-9 and GRP78 to promote cell invasion in the A375 cells. Finally, we showed that GRP78 co-localized with MDA-9 in 293T cells. Taken together, our findings support MDA-9, co-expressed with GRP78, as a melanoma protein associated with lymph node metastasis. Investigating how MDA-9 and GRP78 interact to contribute to melanoma metastasis and disease progression could reveal new potential avenues of targeted therapy and/or useful biomarkers for diagnosis and prognosis.

  18. Research Status of GRP7 8%GRP78研究新进展

    Institute of Scientific and Technical Information of China (English)

    张欣; 王玉明; 段勇


    葡萄糖调节蛋白78(GRP78)是1977年发现的一种分子伴侣,具有多种功能的内质网常驻蛋白。在肿瘤组织中, GRP78呈高表达及异位表达,参与内质网应激状态下肿瘤的发生发展,而细胞表面的 GRP78以受体形式参与信号转导功能。因此,GRP78作为肿瘤治疗靶点和生物分子标志物,具有重要的研究价值。近年,国内外学者发现,在非肿瘤疾病(病毒感染性疾病、心血管疾病、糖尿病、神经退行性疾病等)中,GRP78也呈高表达,预示了其应用前景。该文将从 GRP78的分子生物学特性、在肿瘤发病机制中的作用及治疗上的意义、在非肿瘤疾病中的应用前景等方面进行综述。%GRP78/Bip,which was first identified by scientists in 1977,is a major endoplasmic reticulum (ER)chaperone that has a variety of function.In tumor tissue,GRP78 is high expression and ectopic expression;participate in the development of tumor,ER-stress state and ectopic distribution on the cell surface,in the form of receptors involved in signal transduction. Therefore,GRP78 as an tumor therapeutic targets,biomarkers of tumor and prognosis of tumor biological markers,plays an important role in the tumor research.In recent years,domestic and foreign scholars have found that in the non-cancer disease (viral infectious disease cardiovascular disease diabetes neurodegenerative disease etc),GRP78 is high expression,indicate its application prospect.This article makes a brief overview on the molecular biological characteristics of GRP78,role in tumor pathogenesis and therapeutic significance and application prospect in non-tumor disease.

  19. Chikusetsu Saponin V Attenuates MPP+-Induced Neurotoxicity in SH-SY5Y Cells via Regulation of Sirt1/Mn-SOD and GRP78/Caspase-12 Pathways

    Directory of Open Access Journals (Sweden)

    Ding Yuan


    Full Text Available Studies have shown that saponins from Panax japonicus (SPJ possess neuroprotective effects. However, whether Chikusetsu saponin V (CsV, the most abundant member of SPJ, can exert neuroprotective effects against 1-methyl-4-phenylpyridinium ion (MPP+-induced cytotoxicity is not known. In this study, we aimed to investigate the neuroprotective effects of CsV on MPP+-induced cytotoxicity in human neuroblastoma SH-SY5Y cells and explore its possible mechanisms. Our results show that CsV attenuates MPP+-induced cytotoxicity, inhibits ROS accumulation, and increases mitochondrial membrane potential dose-dependently. We also found that levels of Sirt1 protein and Mn-SOD mRNA significantly decreased in MPP+-treated group but were restored with CsV treatment in a dose-dependent manner. Furthermore, GRP78 protein and Caspase-12 mRNA levels were elevated by MPP+ exposure but reversed by CsV treatment. CsV inhibited the MPP+-induced downregulation of Bcl-2 and up-regulation of Bax in a dose-dependent manner and, thus, increased the ratio of Bcl-2/Bax. Overall, these results suggest that Sirt1/Mn-SOD and GRP78/Caspase-12 pathways might be involved in the CsV-mediated neuroprotective effects.

  20. Elimination of head and neck cancer initiating cells through targeting glucose regulated protein78 signaling

    Directory of Open Access Journals (Sweden)

    Huang Chih-Yang


    Full Text Available Abstract Background Head and neck squamous cell carcinoma (HNSCC is a highly lethal cancer that contains cellular and functional heterogeneity. Previously, we enriched a subpopulation of highly tumorigenic head and neck cancer initiating cells (HN-CICs from HNSCC. However, the molecular mechanisms by which to govern the characteristics of HN-CICs remain unclear. GRP78, a stress-inducible endoplasmic reticulum chaperone, has been reported to play a crucial role in the maintenance of embryonic stem cells, but the role of GRP78 in CICs has not been elucidated. Results Initially, we recognized GRP78 as a putative candidate on mediating the stemness and tumorigenic properties of HN-CICs by differential systemic analyses. Subsequently, cells with GRP78 anchored at the plasma membrane (memGRP78+ exerted cancer stemness properties of self-renewal, differentiation and radioresistance. Of note, xenotransplantation assay indicated merely 100 memGRP78+ HNSCCs resulted in tumor growth. Moreover, knockdown of GRP78 significantly reduced the self-renewal ability, side population cells and expression of stemness genes, but inversely promoted cell differentiation and apoptosis in HN-CICs. Targeting GRP78 also lessened tumorigenicity of HN-CICs both in vitro and in vivo. Clinically, co-expression of GRP78 and Nanog predicted the worse survival prognosis of HNSCC patients by immunohistochemical analyses. Finally, depletion of GRP78 in HN-CICs induced the expression of Bax, Caspase 3, and PTEN. Conclusions In summary, memGRP78 should be a novel surface marker for isolation of HN-CICs, and targeting GRP78 signaling might be a potential therapeutic strategy for HNSCC through eliminating HN-CICs.

  1. The anti-cancer IgM monoclonal antibody PAT-SM6 binds with high avidity to the unfolded protein response regulator GRP78.

    Directory of Open Access Journals (Sweden)

    Zachary Rosenes

    Full Text Available The monoclonal IgM antibody PAT-SM6 derived from human tumours induces apoptosis in tumour cells and is considered a potential anti-cancer agent. A primary target for PAT-SM6 is the unfolded protein response regulator GRP78, over-expressed externally on the cell surface of tumour cells. Small angle X-ray scattering (SAXS studies of human GRP78 showed a two-domain dumbbell-shaped monomer, while SAXS analysis of PAT-SM6 revealed a saucer-shaped structure accommodating five-fold symmetry, consistent with previous studies of related proteins. Sedimentation velocity analysis of GRP78 and PAT-SM6 mixtures indicated weak complex formation characterized by dissociation constants in the high micromolar concentration range. In contrast, enzyme-linked immunosorbant assays (ELISAs showed strong and specific interactions between PAT-SM6 and immobilized GRP78. The apparent binding constant estimated from a PAT-SM6 saturation curve correlated strongly with the concentration of GRP78 used to coat the microtiter tray. Experiments using polyclonal antiGRP78 IgG antibodies or a monoclonal IgG derivative of PAT-SM6 did not show a similar dependence. Competition experiments with soluble GRP78 indicated more effective inhibition of PAT-SM6 binding at low GRP78 coating concentrations. These observations suggest an avidity-based binding mechanism that depends on the multi-point attachment of PAT-SM6 to GRP78 clustered on the surface of the tray. Analysis of ELISA data at high GRP78 coating concentrations yielded an apparent dissociation constant of approximately 4 nM. We propose that the biological action of PAT-SM6 in tumour cell apoptosis may depend on the multivalent nature of PAT-SM6 and the high avidity of its interaction with multiple GRP78 molecules clustered on the tumour cell surface.

  2. GRP78 as a regulator of liver steatosis and cancer progression mediated by loss of the tumor suppressor PTEN. (United States)

    Chen, W-T; Zhu, G; Pfaffenbach, K; Kanel, G; Stiles, B; Lee, A S


    Glucose-regulated protein 78 (GRP78), a molecular chaperone widely elevated in human cancers, is critical for endoplasmic reticulum (ER) protein folding, stress signaling and PI3K/AKT activation. Genetic knockout models of GRP78 revealed that GRP78 maintains homeostasis of metabolic organs, including liver, pancreas and adipose tissues. Hepatocellular carcinoma (HCC) and cholangiocarcinoma (CC) are the most common liver cancers. There is a lack of effective therapeutics for HCC and CC, highlighting the need to further understand liver tumorigenic mechanisms. PTEN (phosphatase and tenson homolog deleted on chromosome 10), a tumor suppressor that antagonizes the PI3K/AKT pathway, is inactivated in a wide range of tumors, including 40-50% of human liver cancers. To elucidate the role of GRP78 in liver cancer, we created a mouse model with biallelic liver-specific deletion of Pten and Grp78 mediated by Albumin-Cre-recombinase (cP(f/f)78(f/f)). Interestingly, in contrast to PTEN, deletion of GRP78 was progressive but incomplete. At 3 months, cP(f/f)78(f/f) livers showed hepatomegaly, activation of lipogenic genes, exacerbated steatosis and liver injury, implying that GRP78 protects the liver against PTEN-null-mediated pathogenesis. Furthermore, in response to liver injury, we observed increased proliferation and expansion of bile duct and liver progenitor cells in cP(f/f)78(f/f) livers. Strikingly, bile duct cells in cP(f/f)78(f/f) livers maintained wild-type (WT) GRP78 level, whereas adjacent areas showed GRP78 reduction. Analysis of signaling pathways revealed selective JNK activation, β-catenin downregulation, along with PDGFRα upregulation, which was unique to cP(f/f)78(f/f) livers at 6 months. Development of both HCC and CC was accelerated and was evident in cP(f/f)78(f/f) livers at 8-9 months, coinciding with intense GRP78 expression in the cancer lesions, and GRP78 expression in adjacent normal areas reverted back to the WT level. In contrast, c78(f/f) livers

  3. Structural insights from GRP78-NF-κB binding interactions: a computational approach to understand a possible neuroprotective pathway in brain injuries. (United States)

    Avila, Marco Fidel; Torrente, Daniel; Cabezas, Ricardo; Morales, Ludis; García-Segura, Luis Miguel; Gonzalez, Janneth; Barreto, George E


    GRP78 participates in multiple functions in the cell during normal and pathological conditions, controlling calcium homeostasis, protein folding and Unfolded Protein Response. GRP78 is located in the endoplasmic reticulum, but it can change its location under stress, hypoxic and apoptotic conditions. NF-κB represents the keystone of the inflammatory process and regulates the transcription of several genes related with apoptosis, differentiation, and cell growth. The possible relationship between GRP78-NF-κB could support and explain several mechanisms that may regulate a variety of cell functions, especially following brain injuries. Although several reports show interactions between NF-κB and Heat Shock Proteins family members, there is a lack of information on how GRP78 may be interacting with NF-κB, and possibly regulating its downstream activation. Therefore, we assessed the computational predictions of the GRP78 (Chain A) and NF-κB complex (IkB alpha and p65) protein-protein interactions. The interaction interface of the docking model showed that the amino acids ASN 47, GLU 215, GLY 403 of GRP78 and THR 54, ASN 182 and HIS 184 of NF-κB are key residues involved in the docking. The electrostatic field between GRP78-NF-κB interfaces and Molecular Dynamic simulations support the possible interaction between the proteins. In conclusion, this work shed some light in the possible GRP78-NF-κB complex indicating key residues in this crosstalk, which may be used as an input for better drug design strategy targeting NF-κB downstream signaling as a new therapeutic approach following brain injuries.

  4. Repositioning of Verrucosidin, a purported inhibitor of chaperone protein GRP78, as an inhibitor of mitochondrial electron transport chain complex I.

    Directory of Open Access Journals (Sweden)

    Simmy Thomas

    Full Text Available Verrucosidin (VCD belongs to a group of fungal metabolites that were identified in screening programs to detect molecules that preferentially kill cancer cells under glucose-deprived conditions. Its mode of action was proposed to involve inhibition of increased GRP78 (glucose regulated protein 78 expression during hypoglycemia. Because GRP78 plays an important role in tumorigenesis, inhibitors such as VCD might harbor cancer therapeutic potential. We therefore sought to characterize VCD's anticancer activity in vitro. Triple-negative breast cancer cell lines MDA-MB-231 and MDA-MB-468 were treated with VCD under different conditions known to trigger increased expression of GRP78, and a variety of cellular processes were analyzed. We show that VCD was highly cytotoxic only under hypoglycemic conditions, but not in the presence of normal glucose levels, and VCD blocked GRP78 expression only when glycolysis was impaired (due to hypoglycemia or the presence of the glycolysis inhibitor 2-deoxyglucose, but not when GRP78 was induced by other means (hypoxia, thapsigargin, tunicamycin. However, VCD's strictly hypoglycemia-specific toxicity was not due to the inhibition of GRP78. Rather, VCD blocked mitochondrial energy production via inhibition of complex I of the electron transport chain. As a result, cellular ATP levels were quickly depleted under hypoglycemic conditions, and common cellular functions, including general protein synthesis, deteriorated and resulted in cell death. Altogether, our study identifies mitochondria as the primary target of VCD. The possibility that other purported GRP78 inhibitors (arctigenin, biguanides, deoxyverrucosidin, efrapeptin, JBIR, piericidin, prunustatin, pyrvinium, rottlerin, valinomycin, versipelostatin might act in a similar GRP78-independent fashion will be discussed.

  5. Cripto/GRP78 modulation of the TGF-β pathway in development and oncogenesis (United States)

    Gray, Peter C.; Vale, Wylie


    Cripto is a small, GPI-anchored signaling protein that regulates cellular survival, proliferation, differentiation and migration during normal developmental processes and tumorigenesis. Cripto functions as an obligatory co-receptor for the TGF-β ligands Nodal, GDF1 and GDF3 but attenuates signaling of others such as activin-A, activin-B and TGF-β1. Soluble, secreted forms of Cripto also activate Src, ras/raf/MAPK and PI3K/Akt pathways via a mechanism that remains largely obscure. This review describes the biological roles and signaling mechanisms of Cripto, highlighting our identification of Glucose Regulated Protein 78 (GRP78) as a cell surface receptor/co-factor required for Cripto signaling via both TGF-β and Src/MAPK/PI3K pathways. We discuss emerging evidence indicating that Cripto/GRP78 signaling regulates normal somatic stem cells and their tumorigenic counterparts. PMID:22306319

  6. Chop及Grp78基因在先天性脊柱裂大鼠胚胎12天神经管中的异常表达%Altered expression of Chop and Grp78 in neural tube of rat with spina bifida

    Institute of Scientific and Technical Information of China (English)

    吴迪; 刘波; 范洋


    目的 探讨Chop及Grp78基因在先天性脊柱裂大鼠胚胎12 d神经管及神经上皮干细胞中的表达情况.方法 取畸形大鼠12d胚胎神经管组织,荧光实时定量PCR考察Chop及Grp78基因表达.另取12 d正常大鼠胚胎,分离消化神经管行神经上皮干细胞培养.荧光实时定量PCR考察Chop及Grp78基因表达.结果 先天性脊柱裂大鼠胚胎12 d神经管内Chop基因及Grp78基因表达异常增高.神经上皮干细胞分化为神经元后Chop及Grp78基因的表达上调.结论 Chop及Grp78基因在先天性脊柱裂大鼠胚胎12d神经管中表达异常增高,二者在神经上皮干细胞分化为神经元后表达上调,可能与内质网应激相关的凋亡有关.神经上皮干细胞培养培养体系稳定、高效,可以为胚胎早期发育提供实验模型.%Chop and Grp78 genes are key genes in endoplasmic reticulum stress(ERS).To explore the gene alteration of Chop and Grp78 in embryo with spina bifida,we studied Chop and Grp78 gene expression by qRT-PCR in neural tube of rat with spina bifida in E12 and primary cultured neuroepithelial stem cells(NEP).We found higher expression of both Chop and Grp78 in neural tube of rat with spina bifida than normal rats.While Chop and Grp78 genes were up-regulated after NEP differentiated into neuron.The up-regulation of Chop and Grp78 genes in neural rube of rat and NEP might be involved in the abnormal apoptosis in spina bifida related with ERS.And we found the primary culture of NEP provided a good model for embryo early development research.

  7. Expression of GRP78 in Rats with Sleep Deprivation during High-altitude Environment and Intervention%高原环境下REM睡眠剥夺大鼠皮质GRP78的表达及药物干预

    Institute of Scientific and Technical Information of China (English)

    马亚杰; 杨金升; 杨晓; 石向群; 郑佳丽; 刘学娟


    目的:探讨高原环境下大鼠不同时间快速动眼睡眠(REM)期睡眠剥夺(SD)后皮层内质网应激(ERS)标志性分子葡萄糖调控蛋白78(GRP78)的动态变化,神经元凋亡影响及人参皂甙Rd(GS Rd)可能的神经保护作用.方法:采用小平台水环境法制作大鼠SD模型,应用免疫组织化学染色检测相关时间点GRP78的动态变化;TUNEL试剂盒检测凋亡细胞.结果:在平原(兰州)组,SD后1d皮层区GRP78蛋白表达开始增高,3d时达高峰,5d时与正常睡眠组相比无明显差别.GS Rd,组SD后1d、3d、5d GRP78的表达较生理盐水对照组增高.在对应时间点高原(可可西里)组GRP78的表达均高于平原组.结论:高原SD组大鼠GRP78的表达较平原组增高;GS Rd能够提高SD组大鼠GRP78表达;GS Rd可能会通过升高GRP78的表达保护内质网功能,以减轻脑损伤.%Objective To investigate the dynamic expression of endoplasmic reticulum stress(ERS)GRP78,neuron apoptosis in the rat cerebral contex at different stages of REM sleep deprivation in high altitude environment,and the possible neural protective effect of ginsenoside Rd. Methods The sleep deprivation of Wistar rats was induced by employing"flower pot"technique. Expression of GRP78 protein was check with immunohistochemistry staining. Apoptosis cell was studied with TUNEL. Results Immunohistochemistry staining results showed that the expressions of GRP78 protein was increased after one day of REM sleep deprivation compared with that in rats with normal steep in plain group(Lanzhou group) and reached the highest on the normal sleep group. After intervention of ginsenoside Rd,the expression of GRP78 was higher than physiological saline groups after first or third day of REM sleep deprivation. The expression of GRP78 protein was not different from physiological saline groups on the fifth day. At the same time the expression of GRP78 protein in plateau group(Kekexili group) was higher compared with plain group

  8. The expression and clinical significance of GRP78 and pERK in gastric adenocarcinoma, chronic atrophic gastritis and superifcial gastritis%GRP78和pERK在胃癌及非胃癌组织中的表达及临床意义

    Institute of Scientific and Technical Information of China (English)

    彭春雷; 杨书云; 季进锋; 徐薇薇; 季从飞; 王建红; 谭清和; 杨磊


    GRP78 was related to the prognosis of gastric adenocarcinoma. Conclusion:GRP78 and pERK may play an important role in the transition of normal gastric cells to malignant cells. The expression of these two genes enhances tumor progression. Overexpression of GRP78 and pERK is significantly correlated with poor prognosis in patients with gastric adenocarcinoma. The determination of the expression of GRP78 and pERK might be helpful for the prevention, early diagnosis of gastric carcinoma. Particularly, GRP78 is valuable for the judgement of prognosis, and might be a new target for the treatment of gastric adenocarcinoma.%背景与目的:在胃癌的发生、发展过程中,内质网应激、细胞的修复及凋亡是重要的病理生理过程,而GRP78与pERK在其中发挥了重要的作用。研究葡萄糖调节蛋白78(glucose-regulated proteins 78,GRP78)和磷酸化细胞外信号调节激酶(pERK)在胃腺癌、慢性萎缩性胃炎及浅表性胃炎组织中的表达,研究它们与胃癌发生、发展的关系。方法:RT-PCR法检测各25例胃腺癌、慢性萎缩性胃炎和浅表性胃炎新鲜组织中GRP78、pERK基因表达;免疫组化法分别检测各60例胃腺癌、慢性萎缩性胃炎和浅表性胃炎组织中GRP78、pERK蛋白表达,并分析其蛋白表达与临床病理参数间的相关性。结果:RT-PCR半定量结果显示,胃癌组织中GRP78及pERK mRNA的相对表达水平(1.26±0.18、2.35±0.36)均明显高于慢性萎缩性胃炎组织(0.89±0.25、1.18±0.25)及浅表性胃炎组织(0.29±0.09、0.68±0.10),差异有统计学意义(P<0.01)。免疫组织化学结果显示,GRP78及pERK蛋白在胃癌组织中的表达率(78.3%、88.3%)亦均高于慢性萎缩性胃炎(46.6%、43.3%)及浅表性胃炎组织(6.7%、5.0%),差异有统计学意义(P<0.01)。胃癌组织中GRP78及pERK蛋白表达与胃癌分化程度、分期、淋巴结转移等有明显相关性。GRP78及pERK基因及蛋白表达水平在

  9. Activated α2-macroglobulin binding to cell surface GRP78 induces T-loop phosphorylation of Akt1 by PDK1 in association with Raptor.

    Directory of Open Access Journals (Sweden)

    Uma Kant Misra

    Full Text Available PDK1 phosphorylates multiple substrates including Akt by PIP3-dependent mechanisms. In this report we provide evidence that in prostate cancer cells stimulated with activated α2-macroglobulin (α2M* PDK1 phosphorylates Akt in the T-loop at Thr(308 by using Raptor in the mTORC1 complex as a scaffold protein. First we demonstrate that PDK1, Raptor, and mTOR co-immunoprecipitate. Silencing the expression, not only of PDK1, but also Raptor by RNAi nearly abolished Akt phosphorylation at Akt(Thr308 in Raptor-immunoprecipitates of α2M*-stimulated prostate cancer cells. Immunodepleting Raptor or PDK from cell lysates of cells treated with α2M* drastically reduced Akt phosphorylation at Thr(308, which was recovered by adding the supernatant of Raptor- or PDK1-depleted cell lysates, respectively. Studies of insulin binding to its receptor on prostate cancer cells yielded similar results. We thus demonstrate that phosphorylating the T-loop Akt residue Thr(308 by PDK1 requires Raptor of the mTORC1 complex as a platform or scaffold protein.

  10. Association Between Promoter Polymorphisms of the GRP78 Gene and Risk of Type 2 Diabetes in a Chinese Han Population (United States)

    Liu, Shengyuan; Li, Tao; Xiong, Xingdong; Yao, Songpo; Chen, Zhongwei; Wang, Changyi


    There are large amounts of unfolding or misfolding protein accumulation in the endoplasmic reticulum in patients with type 2 diabetes (T2D), which in turn induces the expression of the glucose-regulated protein 78 (GRP78) that plays a key role in influencing insulin secretion and maintaining glucose homeostasis in pancreatic beta cells. The aim in the study is to analyze the potential association between single-nucleotide polymorphisms (SNPs) of GRP78 and the risk of T2D. To assess the association between GRP78 polymorphisms and T2D, a case–control study was conducted among 1058 consecutive unrelated subjects. Of the 1058 subjects, 523 of them were diagnosed with T2D and 535 of them were healthy controls. Four SNPs with R2>0.8 and the minor allele frequency>0.05 (rs391957, rs17840761, rs17840762, and rs11355458) in the GRP78 gene promoter were analyzed. Overall, no associations of GRP78 polymorphisms with T2D were observed in genotypic analyses. In addition, haplotypes combining those SNPs in the promoter in high linkage disequilibrium were also not associated with a T2D risk. However, the levels of fasting plasma glucose and HbA1c in patients with the −415AA/−180GG genotype were significantly lower than those of the patients with −415GG/−180deldel and −415AG/−180Gdel genotypes, and the level of fasting insulin in patients with the −415AA/−180GG genotype was significantly lower than that of the patients with −415GG/−180deldel. The study does not support a role for promoter polymorphisms of GRP78 in T2D in a Chinese Han population, but it does provide a clue for association between low levels of fasting plasma glucose, HbA1c and fasting insulin, and the −415AA/−180GG model. PMID:23402331

  11. The loss of glucose-regulated protein 78 (GRP78) during normal aging or from siRNA knockdown augments human alpha-synuclein (α-syn) toxicity to rat nigral neurons. (United States)

    Salganik, Maxim; Sergeyev, Valeriy G; Shinde, Vishal; Meyers, Craig A; Gorbatyuk, Marina S; Lin, Jonathan H; Zolotukhin, Sergey; Gorbatyuk, Oleg S


    Age-related structural changes and gradual loss of key enzymes significantly affect the ability of the endoplasmic reticulum (ER) to facilitate proper protein folding and maintain homeostasis. In this work, we present several lines of evidence supporting the hypothesis that the age-related decline in expression of the ER chaperone glucose-regulated protein 78 (GRP78) could be related to the development of Parkinson's disease. We first determined that old (24 months) rats exhibit significantly lower levels of GRP78 protein in the nigrostriatal system as compared with young (2 months) animals. Then using recombinant adeno-associate virus-mediated gene transfer, we found that GRP78 downregulation by specific small interfering RNAs (siRNAs) aggravates alpha-synuclein (α-syn) neurotoxicity in nigral dopamine (DA) neurons. Moreover, the degree of chaperone decline corresponds with the severity of neurodegeneration. Additionally, comparative analysis of nigral tissues obtained from old and young rats revealed that aging affects the capacity of nigral DA cells to upregulate endogenous GRP78 protein in response to human α-syn neurotoxicity. Finally, we demonstrated that a sustained increase of GRP78 protein over the course of 9 months protected aging nigral DA neurons in the α-syn-induced rat model of Parkinson's-like neurodegeneration. Our data indicate that the ER chaperone GRP78 may have therapeutic potential for preventing and/or slowing age-related neurodegeneration.

  12. Role of GRP78 in sevoflurane preconditioning-induced inhibition of apoptosis in cardiomyocytes of rats%GRP78在七氟醚预处理抑制大鼠心肌细胞凋亡中的作用

    Institute of Scientific and Technical Information of China (English)

    吴裕超; 孙志鹏; 钟良; 向强


    Objective To investigate the role of glucose-regulated protein 78 (GRP78) in sevoflurane preconditioning-induced inhibition of apoptosis in cardiomyocytes of rats.Methods The cultured cardiomyocytes of Sprague-Dawley rats were randomly divided into 5 groups (n =30 each) using a random number table:control group (group C),hypoxia-reoxgenation (H/R) group,sevoflurane preconditioning group (group S),siRNA-GRP78 group and siRNA control group.H/R was produced by 2 h exposure of cells to 95% N2-5% CO2 in an air-tight chamber at 37 ℃,followed by reoxygenation with 95% O2-5% CO2 in an air-tight chamber at 37 ℃ for 1 h.In group S,the cells were incubated with 2.5% sevoflurane for 20 min,followed by 10-min washout before H/R.In siRNA-GRP78 group,the cells were transfected with siRNA-GRP78 100 nmol/L,and 24 h later preconditioning with sevoflurane was performed and H/R was produced.In siRNA group,cells were transfected with siRNA,and the other treatments were similar to those previously described in siRNA-GRP78 group.After treatment in each group,the expression of GRP78 in myocardial cells and cytochrome c in cytoplasm and mitochondria was detected by Western blot.Lactic dehydrogenase (LDH) and creatine kinase (CK) activities in the culture medium of each group were determined by ELISA.The apoptosis in myocardial cells was assessed by flow cytometry.Apoptotic rate was calculated.Intracellular free Ca2+ concentration ([Ca2+]i) was measured with the fluorescent probe Fura-2/ AM.The opening of mPTP was measured by fluorescence spectrophotometry.Results Compared to group C,the expression of GRP78 in myocardial cells and cytochrome c in cytoplasm was significantly up-regulated,LDH and CK activities in the culture medium,apoptotic rate and [Ca2+]i were increased,and the expression of cytochrome c in mitochondria was down-regulated in H/R group.Compared to group H/R,the expression of GRP78 in myocardial cells and cytochrome c in mitochondria was significantly up

  13. Expression of GRP78 and capase-12 in rats with sleep deprivation during high-altitude environment and medicine intervention%高原环境下REM睡眠剥夺大鼠的GRP78及caspase-12表达及药物干预

    Institute of Scientific and Technical Information of China (English)

    马亚杰; 杨金升; 郑佳丽; 刘学娟


    Objective To observe the changes of GRP78 and caspase-12 expression in the rats' hippocampus on different stages of REMSD Ginsenoside Rd in plateau and plain. To discuss association between expression of the GRP78 and the caspase-12 and apoptosis as well as the possible neouruprotective effect of Ginsenoside Rd. Methods At this experiment, SD was induced in Wistar rats by employing " flower pot" technique. We checked out expression protein of GRP78 and caspase-12 with immunohistochemistry staining, and checked out apoptosis cell with TUNEL. Results Immunohistochemistry staining results showed the expressions of GRP78 and caspase-12 protein increased after one day of REM sleep deprivation compared with those in rats with normal sleep in Lanzhou groups.and reached the highest on the third day. While on the fifth day , the expressions of GRP78 and caspase-12 protein were not different from those of normal sleep group . After intervention of Ginsenoside Rd , the expression of CRP78 were higher than that of physiological saline groups after first or third day of REM sleep deprivation. The expression of GRP78 protein were not different from physiological saline groups on the fifth day. And after intervention of Ginsenoside Rd ,the expression of caspase-12 were lower than physiological saline groups after first or third day of REM sleep deprivation. The expression of caspase-12 protein were not different from physiological saline groups on the fifth day. At the same time the expression of GRP78 and caspase-12 protein were higher compared with Lanzhou group in the Kekexili group. Conclusion After endoplasmic reticulum stress , the stable regulation system was primed . But the state state regulation system will be lost after long-term serious endoplasmic reticulum stress , and induced apoptosis proceduring ,this is possiblely one of causes that damage brain. High-altitude environment exposure may aggravate endoplasmic reticulum stress. Perhaps Ginsenoside Rd lightens

  14. Effect of Electroacupuncture at Neiguan and Baihui Points on GRP78 and Caspase-12 Gene Expression in Rats with Ischemia-Reperfusion Injury%电针内关、百会穴对脑缺血/再灌注大鼠GRP78和Caspase-12基因表达的影响

    Institute of Scientific and Technical Information of China (English)

    沈菁; 雷晓明; 宋洋; 谭杏; 刘琴; 戴丽雯; 李文慧; 郁洁


    目的 观察电针内关、百会穴对脑缺血/再灌注(Ischemia/Reperfusion,I/R)大鼠脑GRP78和Caspase-12基因表达的影响,探讨针刺发挥脑保护作用是否与内质网应激凋亡通路有关. 方法 100只大鼠随机分为5组,每组20只,即正常组、假手术组、手术造模组、依达拉奉组和针刺干预组. 采用线栓法建立大鼠大脑中动脉阻塞(middle cerebral artery occlusion, MCAO) 脑 I/R 模型. 采用 TUNEL 染色法, 检测大鼠神经细胞凋亡指数; 采用实时定量多聚酶链式反应 (Real-Time polymerase chain reaction,RT-PCR),检测GRP78、Caspase-12 mRNA表达. 结果 与正常组和假手术组相比,手术造模组、依达拉奉组和针刺干预组大鼠细胞凋亡指数升高,GRP78和Caspase-12 mRNA表达增多,差异具有统计学意义(P0.05). 结论 针刺内关、百会穴可以有效抑制脑缺血神经元细胞凋亡,其保护机制可能上调内质网应激保护性GRP78表达,同时抑制促凋亡Caspase-12表达有关.%Objective To observe the effects of electro-acupuncture (EA) at Neiguan and Baihui point on GRP78 and Caspase-12 gene expression in rats with ischemia/ reperfusion (IR) injury and investigate protective effects of acupuncture wheather related to endocytoplasmic reticulum (ER) stressapoptosis passage. Methods 100 rats were randomly assigned to five groups, 20 in each group: the normal group, sham-operation group, operation model group, edaravone group and EA group. The IR model of middle cerebral artery occlusion (MCAO) was established by suture embolic method. The apoptosis index of nerve cells in rats were measured by TUNEL staining method. The mRNA expression of GRP78 and Caspase-12 were measure by Real-time polymerase chain reaction (RT-PCR). Results Compared with normal group and sham-operation group, the apoptosis indexes and mRNA expression of GRP78 and Caspase-12 in operation group, Edaravone group and EA group were increased, with statistical significance (P0

  15. Vasopressin increases expression of UT-A1, UT-A3, and ER chaperone GRP78 in the renal medulla of mice with a urinary concentrating defect. (United States)

    Cai, Qi; Nelson, Sarah K; McReynolds, Matthew R; Diamond-Stanic, Maggie Keck; Elliott, David; Brooks, Heddwen L


    Activation of V2 receptors (V2R) during antidiuresis increases the permeability of the inner medullary collecting duct to urea and water. Extracellular osmolality is elevated as the concentrating capacity of the kidney increases. Osmolality is known to contribute to the regulation of collecting duct water (aquaporin-2; AQP2) and urea transporter (UT-A1, UT-A3) regulation. AQP1KO mice are a concentrating mechanism knockout, a defect attributed to the loss of high interstitial osmolality. A V2R-specific agonist, deamino-8-D-arginine vasopressin (dDAVP), was infused into wild-type and AQP1KO mice for 7 days. UT-A1 mRNA and protein abundance were significantly increased in the medullas of wild-type and AQP1KO mice following dDAVP infusion. The mRNA and protein abundance of UT-A3, the basolateral urea transporter, was significantly increased by dDAVP in both wild-type and AQP1KO mice. Semiquantitative immunoblots revealed that dDAVP infusion induced a significant increase in the medullary expression of the endoplasmic reticulum (ER) chaperone GRP78. Immunofluorescence studies demonstrated that GRP78 expression colocalized with AQP2 in principal cells of the papillary tip of the renal medulla. Using immunohistochemistry and immunogold electron microscopy, we demonstrate that vasopressin induced a marked apical targeting of GRP78 in medullary principal cells. Urea-sensitive genes, GADD153 and ATF4 (components of the ER stress pathway), were significantly increased in AQP1KO mice by dDAVP infusion. These findings strongly support an important role of vasopressin in the activation of an ER stress response in renal collecting duct cells, in addition to its role in activating an increase in UT-A1 and UT-A3 abundance.

  16. Activation of Sonic Hedgehog Leads to Survival Enhancement of Astrocytes via the GRP78-Dependent Pathway in Mice Infected with Angiostrongylus cantonensis

    Directory of Open Access Journals (Sweden)

    Kuang-Yao Chen


    Full Text Available Angiostrongylus cantonensis infection may cause elevation of ROS and antioxidants in the CSF of infected mice. Astrocytes may protect the surrounding neurons from oxidative stress-induced cell death by secreting Sonic hedgehog (Shh via the PI3-K/AKT/Bcl-2 pathway. This study was conducted to determine the role of the Shh signaling pathway in A. cantonensis-infected BABL/c mice by coculturing astrocytes with living fifth-stage larvae or soluble antigens. The Shh pathway was activated with corresponding increases in the level of the Shh. Glial fibrillary acidic protein (GFAP and Shh were increased in astrocyte cocultured with living fifth-stage larvae or soluble antigens. The survival of astrocytes pretreated with Shh was significantly elevated in cocultures with the antigens but reduced by its inhibitor cyclopamine. The expression of GRP78 and Bcl-2 was significantly higher in astrocytes pretreated with recombinant Shh. These findings suggest that the expression of Shh may inhibit cell death by activating Bcl-2 through a GRP78-dependent pathway.

  17. Soluble tyrosinase is an endoplasmic reticulum (ER)-associated degradation substrate retained in the ER by calreticulin and BiP/GRP78 and not calnexin. (United States)

    Popescu, Costin I; Paduraru, Crina; Dwek, Raymond A; Petrescu, Stefana M


    Tyrosinase is a type I membrane protein regulating the pigmentation process in humans. Mutations of the human tyrosinase gene cause the tyrosinase negative type I oculocutaneous albinism (OCAI). Some OCAI mutations were shown to delete the transmembrane domain or to affect its hydrophobic properties, resulting in soluble tyrosinase mutants that are retained in the endoplasmic reticulum (ER). To understand the specific mechanisms involved in the ER retention of soluble tyrosinase, we have constructed a tyrosinase mutant truncated at its C-terminal end and investigated its maturation process. The mutant is retained in the ER, and it is degraded through the proteasomal pathway. We determined that the mannose trimming is required for an efficient degradation process. Moreover, this soluble ER-associated degradation substrate is stopped at the ER quality control checkpoint with no requirements for an ER-Golgi recycling pathway. Co-immmunoprecipitation experiments showed that soluble tyrosinase interacts with calreticulin and BiP/GRP78 (and not calnexin) during its ER transit. Expression of soluble tyrosinase in calreticulin-deficient cells resulted in the export of soluble tyrosinase of the ER, indicating the calreticulin role in ER retention. Taken together, these data show that OCAI soluble tyrosinase is an ER-associated degradation substrate that, unlike other albino tyrosinases, associates with calreticulin and BiP/GRP78. The lack of specificity for calnexin interaction reveals a novel role for calreticulin in OCAI albinism.

  18. Discovery of a novel target for the dysglycemic chromogranin A fragment pancreastatin: interaction with the chaperone GRP78 to influence metabolism.

    Directory of Open Access Journals (Sweden)

    Nilima Biswas

    Full Text Available RATIONALE: The chromogranin A-derived peptide pancreastatin (PST is a dysglycemic, counter-regulatory peptide for insulin action, especially in liver. Although previous evidence for a PST binding protein has been reported, such a receptor has not been identified or sequenced. METHODS AND RESULTS: We used ligand affinity to purify the PST target, with biotinylated human PST (hCHGA273-301-amide as "bait" and mouse liver homogenate as "prey", and identified GRP78 (a.k.a. "78 kDa Glucose Regulated Protein", HSPA5, BIP as a major interacting partner of PST. GRP78 belongs to the family of heat shock proteins (chaperones, involved in several cellular processes including protein folding and glucose metabolism. We analyzed expression of GRP78 in the absence of PST in a mouse knockout model lacking its precursor CHGA: hepatic transcriptome data revealed global over-expression of not only GRP78 but also other heat shock transcripts (of the "adaptive UPR" in CHGA(-/- mice compared to wild-type (+/+. By contrast, we found a global decline in expression of hepatic pro-apoptotic transcripts in CHGA(-/- mice. GRP78's ATPase enzymatic activity was dose-dependently inhibited by PST (IC50∼5.2 µM. PST also inhibited the up-regulation of GRP78 expression during UPR activation (by tunicamycin in hepatocytes. PST inhibited insulin-stimulated glucose uptake in adipocytes, and increased hepatic expression of G6Pase (the final step in gluconeogenesis/glycogenolysis. In hepatocytes not only PST but also other GRP78-ATPase inhibitors (VER-155008 or ADP increased G6Pase expression. GRP78 over-expression inhibited G6Pase expression in hepatocytes, with partial restoration by GRP78-ATPase inhibitors PST, VER-155008, or ADP. CONCLUSIONS: Our results indicate that an unexpected major hepatic target of PST is the adaptive UPR chaperone GRP78. PST not only binds to GRP78 (in pH-dependent fashion, but also inhibits GRP78's ATPase enzymatic activity, and impairs its biosynthetic

  19. Chelation of GRP78 with Lead and Its Localization Changes in the Astroglia of Rats Exposed to Lead

    Institute of Scientific and Technical Information of China (English)

    Ying ZHANG; Liping YE; Biao WANG; Yan LI; Liguang SUN


    remarkably increase when it transferred from ER to the cytosol around the nuclei 24 h after treatment with Pb. It is concluded that GRP78 in astroglia could strongly chelate with Pb ions and it might be a target protein of Pb.

  20. The Protective effect of the endoplasmic reticulum stress-related factors BiP/GRP78 and CHOP/Gadd153 on noise-induced hearing loss in guinea pigs

    Directory of Open Access Journals (Sweden)

    Qiuhong Xue


    Full Text Available Context: The audiological features and cochlear morphology of individuals with noise-induced hearing loss (NIHL are well characterized. However, the molecular processes in the cochlea are not well understood. Aims: To explore the role of the endoplasmic reticulum stress (ERS response in the guinea pig model of cochlear damage induced by exposure to intense noise. Settings and Design: A pilot case–control study. Subjects and Methods: Forty-eight guinea pigs were divided into four equal groups. At 1, 4, or 14 days (d post-exposure, the auditory brainstem responses (ABRs were tested before sacrificing the subjects. The expression levels of the binding immunoglobulin protein/glucose-regulated protein 78 (BiP/GRP78 and C/EBP-homologous protein/growth arrest and DNA damage-inducible gene 153 (CHOP/Gadd153 proteins were evaluated using immunohistochemistry and Western blotting. The number of cochlear hair cells with altered nuclei was counted using confocal fluorescence microscopy. Statistical analysis used: One-way analysis of variance (ANOVA and the least squares difference (LSD test. Results: The outer hair cells (OHCs showed changes of apoptosis, necrosis, and loss after noise exposure. In the 1- and 4-d groups, more apoptotic cells were found than necrotic cells (P < 0.01. The level of BiP/GRP78 was significantly higher in all three experimental groups compared to the control group (P < 0.01. The level of CHOP/Gadd153 was increased at 1 d post-exposure, achieving a peak that was maintained until 4 d, after which it returned to baseline levels by 14 d post-exposure. Conclusions: ERS response was activated by inducing the expression of BiP/GRP78 to lessen the extent of the resulting cellular damage and activating the CHOP/Gadd153 pathway to eliminate the most severely damaged cells.

  1. Relationship between GRP78 and diabetes mellitus-induced influence on myocardial protection provided by remifentanil postconditioning in vitro%葡萄糖调节蛋白78与糖尿病因素影响瑞芬太尼后处理心肌保护作用的关系:离体实验

    Institute of Scientific and Technical Information of China (English)

    陈满丽; 陈立建; 万俐娟; 张雷; 都建; 顾尔伟


    Objective To evaluate the relationship between glucose-regulated protein 78 (GRP78) and diabetes mellitus-induced influence on myocardial protection provided by remifentanil postconditioning in vitro.Methods H9c2 cells were cultured in DMEM/F12 culture medium supplemented with 10% fetal bovine serum.The cells were seeded in 96-well (100 μl/well) or 6-well (2 ml/well) plates at the density of l05 cells/ml.The cells were then randomly divided into 6 groups (n =24 each) using a random number table:normoglycemic control group (group NC),normoglycemic hypoxia/reoxygenation (H/R) group (group NHR),normoglycemic remifentanil postconditioning group (group NRP),hyperglycemic control group (group HC),hyperglycemic H/R group (group HHR),and hyperglycemic remifentanil postconditioning group (group HRP).In NC,NHR and NRP groups,the cells were cultured in normoglyccmic culture medium (5.5 mmol/L) for 48 h.In HC,HHR and HRP groups,the cells were incubated in hyperglycemic culture medium (25.0 mmol/L) for 48 h.In NHR,NRP,HHR and HRP groups,after changing the culture medium for Tyrode solution,the cells were exposed to 95% N2-5% CO2 in an incubator at 37 ℃ for 5 h.Subsequently,in NHR and HHR groups,the culture medium was changed to DMEM/F12 culture medium supplemented with 10% fetal bovine serum and glucose at the corresponding concentration,and the cells were incubated for 1 h;in NRP and HRP groups,the cells were incubated for 1 h in the DMEM culture medium containing remifentanil at the final concentration of 1 μmol/L.At 1 h of reoxygenation,the cells of 9 wells in each group were selected to measure the cell viability by CCK8 assay,the cells of 12 wells in each group were selected to determine the activity of lactic dehydrogenase (LDH) released in the supernatant using colorimetric method,and the cells of 3 wells in each group were selected to detect the expression of GRP78 by Western blot.Results Compared with group NC,the cell viability was significantly decreased

  2. Expression of heat shock proteins (HSP27, HSP60, HSP70, HSP90,GRP78, GRP94) in hepatitis B virus-related hepatocellular carcinomas and dysplastic nodules

    Institute of Scientific and Technical Information of China (English)

    Seung Oe Lim; Cheol Keun Park; Sung Gyoo Park; Jun-Hi Yoo; Young Min Park; Hie-Joon Kim; Kee-Taek Jang; Jae Won Cho; Byung Chul Yoo; Gu-Hung Jung


    AIM: Expression of heat shock proteins (HSPs) is frequently up-regulated in hepatocellular carcinoma (HCC), which evolves from dysplastic nodule (DN) and early HCC to advanced HCC. However, little is known about the differential expression of HSPs in multistep hepatocarcinogenesis. It was the purpose of this study to monitor the expression of HSPs in multistep hepatocarcinogenesis and to evaluate their prognostic significance in hepatitis B virus (HBV)related HCC.METHODS: Thirty-eight HCC and 19 DN samples were obtained from 52 hepatitis B surface antigen-positive Korean patients. Immunohistochemical and dot immunoblot analyses of HSP27, HSP60, HSP70, HSP90, glucoseregulated protein (GRP)78, and GRP94 were performed and their expression at different stages of HCC development was statistically analyzed.RESULTS: Expression of HSP27, HSP70, HSP90, GRP78, and GRP94 increased along with the stepwise progression of hepatocarcinogenesis. Strong correlation was found only in GRP78 (Spearman's r= 0.802). There was a positive correlation between the expressions of GRP78, GRP94, HSP90, or HSP70 and prognostic factors of HCC. Specifically, the expression of GRP78, GRP94, or HSP90 was associated significantly with vascular invasion and intrahepatic metastasis.CONCLUSION: The expressions of HSPs are commonly up-regulated in HBV-related HCCs and GRP78 might play an important role in the stepwise progression of HBVrelated hepatocarcinogenesis. GRP78, GRP94, and HSP90 may be important prognostic markers of HBV-related HCC, strongly suggesting vascular invasion and intrahepatic metastasis.

  3. Immune Cells in Blood Recognize Tumors (United States)

    NCI scientists have developed a novel strategy for identifying immune cells circulating in the blood that recognize specific proteins on tumor cells, a finding they believe may have potential implications for immune-based therapies.

  4. CotH3 mediates fungal invasion of host cells during mucormycosis. (United States)

    Gebremariam, Teclegiorgis; Liu, Mingfu; Luo, Guanpingsheng; Bruno, Vincent; Phan, Quynh T; Waring, Alan J; Edwards, John E; Filler, Scott G; Yeaman, Michael R; Ibrahim, Ashraf S


    Angioinvasion is a hallmark of mucormycosis. Previously, we identified endothelial cell glucose-regulated protein 78 (GRP78) as a receptor for Mucorales that mediates host cell invasion. Here we determined that spore coat protein homologs (CotH) of Mucorales act as fungal ligands for GRP78. CotH proteins were widely present in Mucorales and absent from noninvasive pathogens. Heterologous expression of CotH3 and CotH2 in Saccharomyces cerevisiae conferred the ability to invade host cells via binding to GRP78. Homology modeling and computational docking studies indicated structurally compatible interactions between GRP78 and both CotH3 and CotH2. A mutant of Rhizopus oryzae, the most common cause of mucormycosis, with reduced CotH expression was impaired for invading and damaging endothelial cells and CHO cells overexpressing GRP78. This strain also exhibited reduced virulence in a diabetic ketoacidotic (DKA) mouse model of mucormycosis. Treatment with anti-CotH Abs abolished the ability of R. oryzae to invade host cells and protected DKA mice from mucormycosis. The presence of CotH in Mucorales explained the specific susceptibility of DKA patients, who have increased GRP78 levels, to mucormycosis. Together, these data indicate that CotH3 and CotH2 function as invasins that interact with host cell GRP78 to mediate pathogenic host-cell interactions and identify CotH as a promising therapeutic target for mucormycosis.

  5. Targeting Prostate Cancer Stemlike Cells through Cell Surface-Expressed GRP78 (United States)


    MgCl2, 0.5% NP40, and proteinase inhibitors ) on ice for 20 min, centrifuged, and the supernatants were collected as cytosolic protein lysates. The...residual pellets were washed with cytosolic lysis buffer once, and then incubated in nuclear lysis buffer (50 mM TRIS, 1% SDS, and proteinase inhibitors

  6. 牛磺酸对缺血/再灌注大鼠肾脏GRP78和Caspase-12表达的影响%Effect of taurine on expression of gene GRP78 and Caspase-12 of renal tissue at rats in ischemia reperfusion injury

    Institute of Scientific and Technical Information of China (English)

    龚慧; 万慧芳; 涂硕; 刘卓琦; 余乐涵; 万福生


    观察牛磺酸(Tau)对肾缺血/再灌注(I/R)大鼠肾脏GRP78、Caspase-12表达的影响及其意义.将Wistar 大鼠30只随机分为假手术组(对照组)、I/R组和I/R+TMP组.采用夹闭双侧肾蒂45 min再灌注24 h制备肾I/R模型.I/R+TMP组在手术前1h按200 mg· kg-1腹腔注射Tau,余操作同I/R组.检测血清尿素氮(BUN)和肌酐(Cr);光镜观察肾小管组织结构变化; RT-PCR和免疫组化检测GRP78、Caspase-12 mRNA和蛋白表达.与假手术组比较,I/R组大鼠血清BUN,Cr水平显著升高,肾组织损伤严重,GRP78和Caspase-12 mRNA和蛋白表达呈显著性增加(P<0.01);与I/R组比较,I/R+ Tau组血清BUN、Cr水平及GRP78、Caspase-12表达均有显著性下降(P<0.05),肾脏损伤明显减轻.牛磺酸对肾脏缺血/再灌注大鼠GRP78、Caspase-12过度表达升高有很好的抑制作用,这可能是它减轻肾脏缺血/再灌注损伤的重要机制之一.%The effects of taurine (Tau) on expressions of GRP78,Caspase-12 of the renal tissue at rats in renal ischemia /reperfusion (I/R) injury was observed. Thirty Wistar rats of either sex were randomly divided into sham operation group (control),I/R group and I/R+Tau group. The animal model of acute renal 1/ R injury was prepared by clamping bilateral renal pedicle for 45 mins and then reperfusion over 24 hours. 1/ R+Tau group was treated with intraperitoneal injection of 200 nag · kg-1 Tau solution 1 hour before surgery, and the rest operations followed the same as that in I/R group. The blood urea nitrogen (BUN) and creatinine (Cr) were detected. The histological changes of renal were observed by HE stainingtthe mRNA and protein expressions of GRP78. Caspase-12 in the renal tissue were analyzed by RT-PCR and immuno-histochemistry,respectively. Compared with the Sham group,the serum BUN.Cr in I/R group had significant increases ,the damage of renal tissue was much severer.the mRNA and protein expressions of GRP78 and Caspase-12 were significantly increased(p<0

  7. 惊厥持续状态幼年大鼠海马中葡萄糖调节蛋白78与半胱氨酸门冬氨酸特异性蛋白酶12的表达及依达拉奉对其影响%Effects of edravone on the expression of GRP78, Caspase-12, and neuron apoptosis in juvenile rat hippocampus after status convulsivus

    Institute of Scientific and Technical Information of China (English)

    李光乾; 王海萍; 蒋春明


    Objective To observe the expression of GRP78 (glucose regulated protein, GRP78),Caspase-12 and the change of neuron apoptosis in the juvenile rat hippocampus after status convulsivus ( SC), and to explore the effect of edaravone on them. Methods One hundred and ninety-five juvenile male Sprague-Dawley(SD) rats were randomly divided into normal saline control group (NS group), status convulsivus group(SC group)and edaravone treatment group( ED group). Each group was further divided into five subgroups in different executed time points after SC. The rats in status convulsivus group were kindled into epilepsy by lithium-pilocarpine method. Expression of GRP78 mRNA and caspase-12 mRNA was detected with reverse transcription-polymerase chain reaction (RT-PCR) method. Expressions of GRP78and caspase-12 protein were detected with immunohistochemical methods. The neuron gpoptosis was observed by TdT-mediated dUTP nick end labeling (TUNEL). Results (1) Measured by immunohistochemistry the value of OD of GRP78 ( 0. 1480 ± 0. 0164,0. 1682 ± 0. 0114, and 0. 1540 ±0. 0102,respectively, 12 h-48 h points ) and caspase-12 ( 0. 1325 ± 0. 0165,0. 1794 ± 0. 0213,0. 1525 ±0. 0423, and 0. 1309 ± 0. 0199, respectively, 12 h-72 h points)positive cells in the SC group increased, there was a significant difference compared with NS group (GRP78: 0. 1214 ± 0. 0147,0. 1272 ± 0. 0177, and 0. 1260 ±0. 0157, respectively, 12 h-72 h points. Caspase-12:0. 1050 ±0. 0121,0. 1041 ± 0. 0151,0. 1058±0. 0222, and 0. 1036 ± 0. 0186, respectively, 12 h-72 h points ) ( P < 0. 01, or P < 0. 05 ). By ED intervention GRP78 ( 0. 1550 ± 0. 0131, 0. 1886 ± 0. 0154, and 0. 1721 ± 0. 0151, respectively, 12 h-48 h points) positive cells value of the OD increased as compared with SC group (P < 0. 01, or P < 0. 05 ). and caspase-12(0. 1211 ±0.0184,0. 1545 ±0.0205,and 0. 1085 ± 0.0219,respectively, 12 h,24 h and 72 h points) positive cells value of the A decreased as compared with SC group

  8. The 78-kD Glucose-Regulated Protein Regulates Endoplasmic Reticulum Homeostasis and Distal Epithelial Cell Survival during Lung Development. (United States)

    Flodby, Per; Li, Changgong; Liu, Yixin; Wang, Hongjun; Marconett, Crystal N; Laird-Offringa, Ite A; Minoo, Parviz; Lee, Amy S; Zhou, Beiyun


    Bronchopulmonary dysplasia (BPD), a chronic lung disease of prematurity, has been linked to endoplasmic reticulum (ER) stress. To investigate a causal role for ER stress in BPD pathogenesis, we generated conditional knockout (KO) mice (cGrp78(f/f)) with lung epithelial cell-specific KO of Grp78, a gene encoding the ER chaperone 78-kD glucose-regulated protein (GRP78), a master regulator of ER homeostasis and the unfolded protein response (UPR). Lung epithelial-specific Grp78 KO disrupted lung morphogenesis, causing developmental arrest, increased alveolar epithelial type II cell apoptosis, and decreased surfactant protein and type I cell marker expression in perinatal lungs. cGrp78(f/f) pups died immediately after birth, likely owing to respiratory distress. Importantly, Grp78 KO triggered UPR activation with marked induction of the proapoptotic transcription factor CCAAT/enhancer-binding proteins (C/EBP) homologous protein (CHOP). Increased expression of genes involved in oxidative stress and cell death and decreased expression of genes encoding antioxidant enzymes suggest a role for oxidative stress in alveolar epithelial cell (AEC) apoptosis. Increased Smad3 phosphorylation and expression of transforming growth factor-β/Smad3 targets Cdkn1a (encoding p21) and Gadd45a suggest that interactions among the apoptotic arm of the UPR, oxidative stress, and transforming growth factor-β/Smad signaling pathways contribute to Grp78 KO-induced AEC apoptosis and developmental arrest. Chemical chaperone Tauroursodeoxycholic acid reduced UPR activation and apoptosis in cGrp78(f/f) lungs cultured ex vivo, confirming a role for ER stress in observed AEC abnormalities. These results demonstrate a key role for GRP78 in AEC survival and gene expression during lung development through modulation of ER stress, and suggest the UPR as a potential therapeutic target in BPD.

  9. A novel combination treatment for breast cancer cells involving BAPTA-AM and proteasome inhibitor bortezomib



    Glucose-regulated protein 78 kDa/binding immunoglobulin protein (GRP78/BIP) is a well-known endoplasmic reticulum (ER) chaperone protein regulating ER stress by facilitating protein folding, assembly and Ca2+ binding. GRP78 is also a member of the heat shock protein 70 gene family and induces tumor cell survival and resistance to chemotherapeutics. Bortezomib is a highly specific 26S proteasome inhibitor that has been approved as treatment for patients with multiple myeloma. The present study...

  10. Expression of GRP78 and GRP94 in rats cortex, hippocampus and medulla regions after rapid eye movement sleep deprivation%快速动眼睡眠剥夺后GRP78、GRP94在大鼠大脑皮质、海马及延髓中的表达

    Institute of Scientific and Technical Information of China (English)

    张红菊; 赵忠新; 张杰文; 索爱琴


    目的 研究不同时间快速动眼(REM)睡眠剥夺对大鼠皮质、海马及延髓葡萄糖调控蛋白(glucose-regulated protein,GRP)表达的影响.方法 大鼠随机分为睡眠剥夺1、3、5、7 d(SD 1 d,SD 3 d,SD 5 d,SD 7 d,n=10)组、剥夺7 d后恢复睡眠6、12 h(SD 7 d/RS 6 h,SD 7 d/RS 12 h,n=10)组、环境对照(TC,n=10)组和空白对照(CC,n=10)组.采用改良多平台睡眠剥夺法进行不同时间REM睡眠剥夺.应用免疫组织化学方法及半定量逆转录一聚合酶链反应(RT-PCR)技术检测脑组织标本中GRP78、GRP94表达分布规律及时空的变化.结果 睡眠剥夺d 1皮质、海马区GRP78、GRP94蛋白表达增多(与TC、CC组比较,P<0.05).d 3达高峰,d 5、7与TC、CC组比较无显著差异(P>0.05);延髓区无明显改变;恢复睡眠后只有延髓区GRP94表达增加.RT.PCR显示睡眠剥夺1 d GRP78转录增加,d 3达峰值,d 5、7转录无增加;GRP94转录在剥夺期间无增加;恢复睡眠后GRP78、GRP94mRNA增加.睡眠剥夺d 3 GRP蛋白表达改变皮质区最为明显(P<0.01),其次为海马区(P<0.05).结论 短时间睡眠剥夺后GRP78、GRP94表达渐升高,可能是内质网启动了自身稳定调节的保护功能;长时间睡眠剥夺导致内质网功能障碍,GRP78、GRP94表达下降.皮质区对睡眠剥夺引起内质网应激的保护性反应最为强烈.

  11. Dendritic Cells as Danger-Recognizing Biosensors

    Directory of Open Access Journals (Sweden)

    Seokmann Hong


    Full Text Available Dendritic cells (DCs are antigen presenting cells that are characterized by a potent capacity to initiate immune responses. DCs comprise several subsets with distinct phenotypes. After sensing any danger(s to the host via their innate immune receptors such as Toll-like receptors, DCs become mature and subsequently present antigens to CD4+ T cells. Since DCs possess the intrinsic capacity to polarize CD4+ helper cells, it is critical to understand the immunological roles of DCs for clinical applications. Here, we review the different DC subsets, their danger-sensing receptors and immunological functions. Furthermore, the cytokine reporter mouse model for studying DC activation is introduced.

  12. Effects of cerebral ischemia and postconditioning on signaling molecules PERK and GRP78 in hippocampus tissue of tree shrew during endoplas-mic reticulum stress%脑缺血及后适应对树鼩海马内质网应激信号分子 PERK 及 GRP78的影响

    Institute of Scientific and Technical Information of China (English)

    陈理军; 马艳; 李霞; 陈静; 李树清; 张颖


    [ ABSTRACT] AIM:To investigate the effects of cerebral ischemia and postconditioning on protein kinase R-like endoplasmic reticulum kinase (PERK) and glucose-regulated protein 78 (GRP78) in the hippocampus tissue of tree shrew during endoplasmic reticulum stress and the mechanism of post-conditioning protecting the brain from damage.METH-ODS:The focal cerebral ischemic model was duplicated by photochemical reaction in tree shrew and the postconditioning was induced by alternatively occluding and opening the carotid artery of ischemic side for 3 cycles (5 min each cycle) at 3.5 h after ischemia.The damage and ultrastructural changes of the hippocampal neurons were observed by HE staining. The expression of PERK and GRP78 at mRNA and protein levels in the hippocampal tissue at different time points after cer-ebral ischemia and postconditioning was determined by RT-PCR, immunohistochemistry and Western blot.RESULTS:The injuries of hippocampal neurons were aggravated with prolonged cerebral ischemia, which was most severe at 24 h after ischemia while the postconditioning alleviated these damages correspondingly.The expression of PERK at mRNA and pro-tein levels was upregulated at 4 h, 24 h and 72 h after ischemia (P lated the expressions of GRP78 at IP24 h group (P<0.05).CONCLUSION:The focal thrombotic cerebral ischemia ac-tivates the endoplasmic reticulum stress in ischemic hippocampus of tree shrews, leading to the changes in mRNA and pro-tein expression of PERK in the PERK/eIF2αsignal transduction pathway.The postconditioning treatment alleviates endo-plasmic reticulum stress and neuronal damages by downregulating PERK and upregulating GRP78, thereby protecting the brain from injury.%目的:探讨脑缺血及后适应对树鼩海马内质网应激信号分子蛋白激酶R样内质网激酶( PERK)及葡萄糖调节蛋白78(GRP78)的影响及后适应的脑保护机制。方法:光化学反应诱导树鼩局部血栓性脑缺血,于脑缺血后3.5 h

  13. Expression of endoplasmic reticulum stress-associated molecule GRP78 in testicular tissue of rats in differ-ent phases of morphine-dependence%内质网应激分子GRP78在不同时程吗啡依赖大鼠睾丸中的表达

    Institute of Scientific and Technical Information of China (English)

    吴明松; 罗素元; 郑翔; 涂平; 白威峰; 刘兴宇


    Objective To investigate expression level of endoplasmic reticulum stressmarker GRP78 in the testicular tissue in rats with different phases of morphine-dependence. To explore the role of ERS in morphine-de-pendence. Methods SD rats were divided into 6 groups: morphine (mor) -withdrawal group, mor-extinct group, mor-kindling group and their control groups, normal saline (NS)-withdrawal group, NS-extinct group, NS-kindling group. The experimental rats were injected with morphine subcutaneously on increasing dosage to establish the con-ditioned place preference (CPP) model. The rats in control groups were injected NS. Then the rats were suffered from withdrawal for 48 h, extinction and kindling by morphine, separately. The GRP78 expression level in testicular tissues of rats in the time point mentioned above were measured using Western Blot. Results The time of rats in the paired-box was (528.0 ± 81.0) s, which was significantly higher than that in the NS control group (P<0.001). It was (396.8 ± 116.9) s after extinctive phase, which was significantly higher than that the withdrawal phase of rats (P < 0.001). Also it was (396.8 ± 116.9) s after kindling with morphine which was significantly higher than that the extinctive phase of rats (P < 0.001). These changes of the time indicated that the animal models of extinction and kindling were established in the study. The GRP78 levels were down-regulated in 48 h after withdrawal (P <0.05), and increased a bit afterextinctive phase, but up-regulated highly after kindling with morphine (P < 0.01). Conclusion ERS may be related in the morphine dependence and it might play an important role of testicular dys-function in male drugabuser.%目的:通过观察内质网应激标志分子葡萄糖调节蛋白78(GRP78)在不同时程吗啡依赖大鼠睾丸中的表达变化,探索内质网应激在阿片类药物成瘾中的作用。方法:SD大鼠分为生理盐水(NS)戒断组、NS 消退组、NS 点燃组

  14. Peripheral leukocyte GRP78,CHOP and XBP1 mRNA levels in patients with chronic hepatitis B viral infection%慢性HBV感染者外周血白细胞GRP78、CHOP和XBP1 mRNA水平变化

    Institute of Scientific and Technical Information of China (English)

    潘高峰; 郜玉峰; 叶娇娇; 姜同翠; 沈玉君; 沈玉先


    Objective To investigate the changes of glucose regulated protein 78 (GRP78),a endoplasmic reticulum stress (ERS) related gene,C/ enhancer-binding protein homologous protein (CHOP) and X-box binding protein (XBP) 1 mRNA in peripheral leukocytes in patients with chronic hepatitis B viral infections. Methods The mRNA levels in peripheral leukocytes were measured by real time PCR in 43 individuals with asymptomatic hepatitis B virus carriers,47 patients with chronic hepatitis B,41 with hepatitis B related liver cirrhosis,35 with hepatitis B related hepatocellular carcinoma (HCC) and 40 normal healthy persons. Results The GRP78 mRNA levels in hepatitis B virus carriers,in patients with chronic hepatitis B,liver cirrhosis and hepatocellular carcinoma were higher than that in healthy controls[(0.58±0.53),(0.76±0.57),(0.59±0.67) and (0.50±0.64) vs.(0.01±0.61), P20000 IU/ml [(0.74 ±0.56),P1 ×105copies/ml)[(0.86 ±1.13) vs. (1.44±1.31),P20000IU/ml组患者,GRP78 mRNA水平分别为(0.48±0.59)、(0.76±0.56)和(0.74±0.56),三组间差异有统计学意义(P<0.05);HBV DNA水平为1×103~105copies/ml组XBP1 mRNA水平为(0.86±1.13),显著低于HBV DNA>1×105copies/ml组[(1.44±1.31),P<0.05];在不同转氨酶水平间、HBeAg阳性与阴性组间和胆红素正常与升高组间,以上三种基因 mRNA 水平差异均无统计学意义(P>0.05)。结论内质网应激可能与慢性HBV感染后的疾病进展相关。

  15. Cisplatin-induced regulation of signal transduction pathways and transcription factors in p53-mutated subclone variants of hepatoma cells: Potential application for therapeutic targeting. (United States)

    Kuo, Jinn-Rung; Shang, Hung-Sheng; Ho, Chun-Te; Lai, Kun-Goung; Liu, Tsan-Zon; Chen, Yin-Ju; Chiou, Jeng-Fong


    Cisplatin is commonly recognized as a DNA-damaging drug; however, its versatile antitumor effects have been demonstrated to extend beyond this narrow functional attribute. The present study determined how cisplatin regulates alternative pathways and transcription factors to exert its additional antitumor actions. Cisplatin was observed to be able to trigger an endoplasmic reticulum stress response through aggravated nitrosative stress coupled to perturbed mitochondrial calcium (Ca(2+)) homeostasis, which substantially downregulated glucose-regulated protein (GRP) 78 expression by suppressing the cleavage of activating transcription factor (ATF) 6α (90 kDa) to its active 50 kDa subunit. Concomitantly, the ATF4-ATF3-C/emopamil binding protein homologous protein axis was activated by cisplatin, which triggered cellular glutathione (GSH) depletion by strongly inhibiting γ-glutamylcysteine synthetase heavy chain (γ-GCSh), a key enzyme in GSH biosynthesis. The present study also demonstrated that cisplatin substantially inhibited β-catenin, causing a marked downregulation of survivin and B-cell lymphoma (Bcl)-2. Taken together, the present results uncovered a novel mechanism of cisplatin that could simultaneously trigger the inhibition of three prominent antiapoptotic effector molecules (Bcl-2, survivin and GRP78) and effectively promote GSH depletion by inhibiting γ-GCSh. These newly discovered functional attributes of cisplatin can provide an avenue for novel combined therapeutic strategies to kill hepatocellular carcinoma cells effectively.

  16. 葡萄糖调节蛋白78在PTSD样情感行为异常大鼠大脑皮质中的表达及意义%Expressions of GRP 78 in prefrontal cortex of rats with posttraumic stress disorder-like behavior

    Institute of Scientific and Technical Information of China (English)

    肖冰; 石玉秀; 韩芳


    Objective To observe the changes of GRP 78 in the prefrontal cortex neurons of PTSD rats may reveal part of the pathogensis. Methods The SPS-method was used to set up the rat PTSD models. A total of 60 male Wistar rats were randomly divided into ld,4d,7d groups of SPS and normal control group. The expression of GRP 78 were detected by using immunohistochemistry, western blotting and RT-PCR. Results The expression of GRP 78 in the prefrontal cortex gradually increased 1day after SPS, and reach the maximum at SPS 7d. The changes of GRP 78 mRNA was in accordance with the results of western blot. Conclusion The changes of expression of GRP 78 in prefrontal cortex of PTSD rats may play important roles in the pathogenesis of PTSD rats.%目的 观察创伤后应激障碍(PTSD)大鼠大脑皮质内质网应激标志物葡萄糖调节蛋白78(GRP78)的表达变化,探讨内质网分子伴侣在PTSD发病机制中的作用.方法 采用国际认定的SPS方法刺激建立大鼠PTSD模型,取成年健康雄性Wistar大鼠60只,随机分为SPS模型的1d、4d、7d组及正常对照组,采用免疫荧光法、免疫印迹和逆转录聚合酶链式反应检测大鼠前额皮质GRP 78的表达变化.结果 SPS刺激后大鼠前额皮质神经元细胞内GRP78于1d开始逐渐升高,7d时表达最多;GRP 78 mRNA的变化与之相一致.结论 大脑皮质GRP 78的表达变化,可能是PTSD大鼠情感行为异常的重要病理生理基础之一.

  17. An intronic variant in the GRP78, a stress-associated gene, improves prediction for liver cirrhosis in persistent HBV carriers.

    Directory of Open Access Journals (Sweden)

    Xiao Zhu

    Full Text Available BACKGROUND: Our previous study indicated that a common variant (rs430397 G>A in the intron 5 of glucose-regulated protein 78 (GRP78 gene was associated with risk and prognosis of primary hepatocellular carcinoma (HCC, including HBV- and cirrhosis-related HCC. rs430397 polymorphism may be a contributing factor or biomarker of HBV infection or HBV-related cirrhosis. METHODOLOGY/PRINCIPAL FINDINGS: 539 non-HBV-infected individuals, 205 self-limited infection and 496 persistent HBV infection were recruited between January 2001 and April 2005 from the hospitals in Southern China. Genomic DNA was genotyped for rs430397. The associations between the variation and susceptibility to liver cirrhosis (LC in persistent HBV infection were examined. We observed that individuals carrying allele rs430397A were more likely to become HBV-related LC. When persistently infected patients were divided into four subgroups, patients with phase IV had an increased allele A and genotype AG compared with phase I and/or phase III. Decreased serum albumin and prolonged plasma prothrombin time (PT were showed in LC patients carrying genotype AA. Furthermore, rs430397 genotype had an increased susceptibility to LC with dose-dependent manners (P-trend = 0.005, and the genotype did constitute a risk factor for the development of advanced LC (Child-Pugh classification C and B, P-trend = 0.021. CONCLUSIONS/SIGNIFICANCE: rs430397 polymorphism may be a contributing factor to LC in persistent HBV carriers.

  18. T cells recognizing a peptide contaminant undetectable by mass spectrometry

    DEFF Research Database (Denmark)

    Brezar, Vedran; Culina, Slobodan; Østerbye, Thomas


    Synthetic peptides are widely used in immunological research as epitopes to stimulate their cognate T cells. These preparations are never completely pure, but trace contaminants are commonly revealed by mass spectrometry quality controls. In an effort to characterize novel major histocompatibility...... complex (MHC) Class I-restricted ß-cell epitopes in non-obese diabetic (NOD) mice, we identified islet-infiltrating CD8+ T cells recognizing a contaminating peptide. The amount of this contaminant was so small to be undetectable by direct mass spectrometry. Only after concentration by liquid...... chromatography, we observed a mass peak corresponding to an immunodominant islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP)(206-214) epitope described in the literature. Generation of CD8+ T-cell clones recognizing IGRP(206-214) using a novel method confirmed the identity...

  19. Distinct Pattern of Human Vδ1 T Cells Recognizing MICA

    Institute of Scientific and Technical Information of China (English)

    Jianqiang Li; Lianxian Cui; Wei He


    γδ T cells represent one unique recognition pattern, the limited recognition, which distinguishes from the specific recognition for αβ T cells and pattern recognition for macrophages. Vδ1 γδ T cell is the major subset of human γδT cells, which predominates in mucosal tissue including the intestinal epithelia. Presently, a few antigens that human Vδ1TCR can recognize have been identified. Among them, MHC class Ⅰ chain-related molecules A (MICA)have been studied most intensively. Besides Vδ1TCR, MICA is also the ligand of NKG2D, a C-type lectin-like activating immunoreceptor. In human, only Vδ1 cells can simultaneously express both types of receptors of MICA while NK cells, αβ T cells and other subsets of γδ T cells likewise express NKG2D. Although the precise mechanisms are still enigmatic, this distinct pattern of Vδ1 cells recognizing MICA predicts unique biological significance of Vδ1 cells in immune defense. Recent years, some progresses have been made in this issue. In this review we summarize the related reports and put forward some novel views based on our group's studies.

  20. Total Synthesis of the GRP78-Downregulatory Macrolide (+)-Prunustatin A, the Immunosuppressant (+)-SW-163A, and a JBIR-04 Diastereoisomer That Confirms JBIR-04 Has Nonidentical Stereochemistry to (+)-Prunustatin A. (United States)

    Manaviazar, Soraya; Nockemann, Peter; Hale, Karl J


    A unified total synthesis of the GRP78-downregulator (+)-prunustatin A and the immunosuppressant (+)-SW-163A based upon [1 + 1 + 1 + 1]-fragment condensation and macrolactonization between O(4) and C(5) is herein described. Sharpless asymmetric dihydroxylation was used to set the C(2) stereocenter present in both targets. In like fashion, coupling of the (+)-prunustatin A macrolide amine with benzoic acid furnished a JBIR-04 diastereoisomer whose NMR spectra did not match those of JBIR-04, thus confirming that it has different stereochemistry than (+)-prunustatin A.

  1. Targeting the unfolded protein response in glioblastoma cells with the fusion protein EGF-SubA.

    Directory of Open Access Journals (Sweden)

    Antony Prabhu

    Full Text Available Rapidly growing tumors require efficient means to allow them to adapt to fluctuating microenvironments consisting of hypoxia, nutrient deprivation, and acidosis. The unfolded protein response (UPR represents a defense mechanism allowing cells to respond to these adverse conditions. The chaperone protein GRP78 serves as a master UPR regulator that is aberrantly expressed in a variety of cancers, including glioma. Therefore, cancer cells may be particularly reliant upon the adaptive mechanisms offered by the UPR and targeting GRP78 may represent a unique therapeutic strategy. Here we report that diffuse expression of GRP78 protein is present in Grade III-IV, but not Grade I-II glioma. To determine the role GRP78 plays in glioblastoma tumorigenesis, we explored the anti-tumor activity of the novel fusion protein EGF-SubA, which combines EGF with the cytotoxin SubA that has been recently shown to selectively cleave GRP78. EGF-SubA demonstrated potent tumor-specific proteolytic activity and cytotoxicity in glioblastoma lines and potentiated the anti-tumor activity of both temozolomide and ionizing radiation. To determine if the tumor microenvironment influences EGF-SubA activity, we maintained cells in acidic conditions that led to both UPR activation and increased EGF-SubA induced cytotoxicity. EGF-SubA was well tolerated in mice and led to a significant tumor growth delay in a glioma xenograft mouse model. The UPR is emerging as an important adaptive pathway contributing to glioma tumorigenesis. Targeting its primary mediator, the chaperone protein GRP78, through specific, proteolytic cleavage with the immunotoxin EGF-SubA represents a novel and promising multi-targeted approach to cancer therapy.

  2. On the Origin of Prostate Cancer Stem Cells through Transmissible ER Stress-Mediated Epithelial to Mesenchymal Transition (United States)


    UPR, Grp78, is a necessary component for prostate cancer tumorigenesis. UPR activation is often elicited by noxae in the tumor microenvironment...Collectively, these results suggest TERS priming may play an insidious role in protecting tumor cells from a variety of noxae , both tumor borne and therapeutic

  3. Machine learning in cell biology - teaching computers to recognize phenotypes. (United States)

    Sommer, Christoph; Gerlich, Daniel W


    Recent advances in microscope automation provide new opportunities for high-throughput cell biology, such as image-based screening. High-complex image analysis tasks often make the implementation of static and predefined processing rules a cumbersome effort. Machine-learning methods, instead, seek to use intrinsic data structure, as well as the expert annotations of biologists to infer models that can be used to solve versatile data analysis tasks. Here, we explain how machine-learning methods work and what needs to be considered for their successful application in cell biology. We outline how microscopy images can be converted into a data representation suitable for machine learning, and then introduce various state-of-the-art machine-learning algorithms, highlighting recent applications in image-based screening. Our Commentary aims to provide the biologist with a guide to the application of machine learning to microscopy assays and we therefore include extensive discussion on how to optimize experimental workflow as well as the data analysis pipeline.

  4. Invariant natural killer T cells recognize glycolipids from pathogenic Gram-positive bacteria. (United States)

    Kinjo, Yuki; Illarionov, Petr; Vela, José Luis; Pei, Bo; Girardi, Enrico; Li, Xiangming; Li, Yali; Imamura, Masakazu; Kaneko, Yukihiro; Okawara, Akiko; Miyazaki, Yoshitsugu; Gómez-Velasco, Anaximandro; Rogers, Paul; Dahesh, Samira; Uchiyama, Satoshi; Khurana, Archana; Kawahara, Kazuyoshi; Yesilkaya, Hasan; Andrew, Peter W; Wong, Chi-Huey; Kawakami, Kazuyoshi; Nizet, Victor; Besra, Gurdyal S; Tsuji, Moriya; Zajonc, Dirk M; Kronenberg, Mitchell


    Natural killer T cells (NKT cells) recognize glycolipid antigens presented by CD1d. These cells express an evolutionarily conserved, invariant T cell antigen receptor (TCR), but the forces that drive TCR conservation have remained uncertain. Here we show that NKT cells recognized diacylglycerol-containing glycolipids from Streptococcus pneumoniae, the leading cause of community-acquired pneumonia, and group B Streptococcus, which causes neonatal sepsis and meningitis. Furthermore, CD1d-dependent responses by NKT cells were required for activation and host protection. The glycolipid response was dependent on vaccenic acid, which is present in low concentrations in mammalian cells. Our results show how microbial lipids position the sugar for recognition by the invariant TCR and, most notably, extend the range of microbes recognized by this conserved TCR to several clinically important bacteria.

  5. Unraveling the Mystery of γδ T Cell Recognizing Lipid A

    Institute of Scientific and Technical Information of China (English)

    Yongchun Cui; Lianxian Cui; Wei He


    Traditionally, the materials which are regarded as antigens recognized by γδ T lymphocytes are protein and carbohydrate, not nucleic acid or lipid. Recently, it has been demonstrated that γδ T cells can recognize lipid A and directly induce immune responses that involve CD1 (cluster of differentiation type 1) family and Toll like receptors (TLRs). This is a review about the interacting-mechanism, immunological effect and clinical application of them.Cellular & Molecular Immunology.

  6. T Cell Receptors that Recognize the Tyrosinase Tumor Antigen | NCI Technology Transfer Center | TTC (United States)

    The National Cancer Institute, Surgery Branch, Tumor Immunology Section, is seeking statements of capability or interest from parties interested in collaborative research to further develop, evaluate, or commercialize T Cells Attacking Cancer: T Cell Receptors that Recognize the Tyrosinase Tumor Antigen

  7. Changes in the expression of proteins associated with aerobic glycolysis and cell migration are involved in tumorigenic ability of two glioma cell lines

    Directory of Open Access Journals (Sweden)

    Ramão Anelisa


    Full Text Available Abstract Background The most frequent and malignant brain cancer is glioblastoma multiforme (GBM. In gliomas, tumor progression and poor prognosis are associated with the tumorigenic ability of the cells. U87MG cells (wild-type p53 are known to be tumorigenic in nude mice, but T98G cells (mutant p53 are not tumorigenic. We investigated the proteomic profiling of these two cell lines in order to gain new insights into the mechanisms that may be involved in tumorigenesis. Results We found 24 differentially expressed proteins between T98G and U87MG cells. Gene Ontology supports the notion that over-representation of differentially expressed proteins is involved in glycolysis, cell migration and stress oxidative response. Among those associated with the glycolysis pathway, TPIS and LDHB are up-regulated in U87MG cells. Measurement of glucose consumption and lactate production suggests that glycolysis is more effective in U87MG cells. On the other hand, G6PD expression was 3-fold higher in T98G cells and this may indicate a shift to the pentose-phosphate pathway. Moreover, GRP78 expression was also three-fold higher in T98G than in U87MG cells. Under thapsigargin treatment both cell lines showed increased GRP78 expression and the effect of this agent was inversely correlated to cell migration. Quantitative RT-PCR and immunohistochemistry of GRP78 in patient samples indicated a higher level of expression of GRP78 in grade IV tumors compared to grade I and non-neoplastic tissues, respectively. Conclusions Taken together, these results suggest an important role of proteins involved in key functions such as glycolysis and cell migration that may explain the difference in tumorigenic ability between these two glioma cell lines and that may be extrapolated to the differential aggressiveness of glioma tumors.

  8. Identification of human T cell targets recognized during Chlamydia trachomatis genital infection

    DEFF Research Database (Denmark)

    Olsen, Anja Weinreich; Follmann, Frank; Højrup, Peter;


    were recognized in each specimen. Mass spectrometry analysis of the 30-42-kDa T cell-stimulating region identified 10 C. trachomatis proteins. Of these, CT583, CT603, and CT610 were identified as strong antigens that induced significantly higher levels of IFN- gamma secretion in PBMCs from case...

  9. Effects of serum containing natural cerebrolysin on glucose-regulated protein 78 and CCAAT enhancer-binding protein homologous protein expression in neuronal PC12 cells following tunicamycin-induced endoplasmic reticulum stress

    Institute of Scientific and Technical Information of China (English)

    Zhengzhi Wu; Ming Li; Andrew C.J. HuangO; Xiuqing Jia; Yinghong Li; Manyin Chen


    BACKGROUND: Glucose-regulated protein 78 (GRP78), a marker of endoplasmic reticulum stress, can prolong cell survival. Alternatively, CCAAT enhancer-binding protein homologous protein (CHOP), a transcription factor specific for endopiasmic reticulum stress, can cause cell cycle arrest and cell apoptosis.OBJECTIVE: To study the protective effects of serum containing natural cerebrolysin on endoplasmic reticulum stress in tunicamycin-induced neuronal PC12 cells, and analyze the influence on GRP78 and CHOP expressions.DESIGN, TIME AND SETTING: A parallel controlled study was performed at the Institute of Integrated Western and Traditional Chinese Medicine, Shenzhen Hospital, Southern Medical University, between March 2006 and August 2008.MATERIALS: Adult Sprague-Dawley rats were perfused with natural Cerebrelysin aqueous extract (0.185 g/kg/d) to produce serum containing natural Cerebrolysin. Physiological saline was used to produce blank serum. PC12 cell line was provided by Shanghai Institute of Cell Biology, Chinese Academy of Science. Tunicamycin was provided by Sigma (St. Louis, USA), and natural Cerebrolysin, containing ginseng, rhizoma gastrodiae, and gingko leaf (1:2:2), by Shengzhen Institute of Integrated Western and Traditional Chinese Medicine.METHODS: PC12 cells were treated with DMEM culture media containing 10% blank serum (normal control group), tunicamycin (1μg/mL; model group), and 5%, 10%, and 15% serum containing natural cerebrolysin and tunicamycin (1μg/mL; low-, moderate-, and high-dose serum containing natural cerebrelysin groups), for 2 hours.MAIN OUTCOME MEASURES: PC12 cells were treated with tunicamycin for 48 hours after which apoptosis was measured using the TUNEL method to calculate apoptotic index. GRP78 expression was detected using immunocytochemistry. After 24 hours of treatment with tunicamycin, GRP78 and CHOP mRNA expressions were measured using RT-PCR.RESULTS: The apoptotic index and CHOP mRNA expression were in the model group

  10. Regulating effect of endogenous carbon monoxide on brain damage induced by recurrent febrile seizures through GRP78-PERK-eIF2α-CHOP signal pathway%内源性一氧化碳对反复热性惊厥脑损伤大鼠内质网应激PERK信号通路的调控作用

    Institute of Scientific and Technical Information of China (English)

    伊文霞; 韩颖; 赵阳; 张静; 秦炯


    目的 观察内源性血红素加氧酶1(HO-1)/一氧化碳(CO)系统对反复热性惊厥(FS)脑损伤大鼠内质网应激PERK信号通路的调控作用.方法 21日龄雄性SD大鼠48只,随机分为正常对照组、FS组、FS+锌原卟啉(ZnPP-Ⅸ)组和FS+血红素(Hemin)组,每组12只;采用反复热水浴法诱导大鼠惊厥,隔日1次,共诱导10次;记录各组大鼠惊厥潜伏期、惊厥强度及惊厥持续时间;双分光光度计法间接测定皮质CO水平;原位缺口末端标记法(TUNEL)检测皮质神经元凋亡情况;免疫组织化学法测定各组大鼠皮质中HO-1、GRP78、p-PERK、p-eIF2α及CHOP蛋白表达.结果 FS组、FS+ ZnPP-Ⅸ组和FS+ Hemin组大鼠惊厥潜伏期、强度及持续时间比较差异均无统计学意义(P均>0.05);FS组大鼠大脑皮质CO水平较正常对照组增高(P<0.05),FS+ ZnPP-Ⅸ组较FS组皮质CO水平明显降低,FS+ Hemin组较FS组皮质CO水平明显升高,差异均有统计学意义(P均<0.05).FS组大鼠皮质神经元凋亡细胞数较正常对照组明显增多,FS+ ZnPP-Ⅸ组大鼠皮质神经元凋亡较FS组进一步增多,FS+ Hemin组神经元凋亡较FS组减少.免疫组织化学显示各组均有HO-1、GRP78、p-PERK、p-eIF2α及CHOP蛋白表达,与FS组比较,FS+ ZnPP-Ⅸ组HO-1、GRP78蛋白表达减少,p-PERK、p-eIF2α及CHOP蛋白表达增高,而FS+ Hemin组HO-1、GRP78表达增高,p-PERK、p-eIF2α及CHOP蛋白表达减少.结论 低浓度增加HO-1/CO的表达够减轻反复FS导致的脑损伤;HO-1/CO可能通过内质网GRP78-PERK-eIF2α-CHOP/GADD153通路发挥对反复FS脑损伤大鼠的保护作用.

  11. Naphthoquinone Derivative PPE8 Induces Endoplasmic Reticulum Stress in p53 Null H1299 Cells

    Directory of Open Access Journals (Sweden)

    Jin-Cherng Lien


    Full Text Available Endoplasmic reticulum (ER plays a key role in synthesizing secretory proteins and sensing signal function in eukaryotic cells. Responding to calcium disturbance, oxidation state change, or pharmacological agents, ER transmembrane protein, inositol-regulating enzyme 1 (IRE1, senses the stress and triggers downstream signals. Glucose-regulated protein 78 (GRP78 dissociates from IRE1 to assist protein folding and guard against cell death. In prolonged ER stress, IRE1 recruits and activates apoptosis signal-regulating kinase 1 (ASK1 as well as downstream JNK for cell death. Naphthoquinones are widespread natural phenolic compounds. Vitamin K3, a derivative of naphthoquinone, inhibits variant tumor cell growth via oxygen uptake and oxygen stress. We synthesized a novel naphthoquinone derivative PPE8 and evaluated capacity to induce ER stress in p53 null H1299 and p53 wild-type A549 cells. In H1299 cells, PPE8 induced ER enlargement, GRP78 expression, and transient IER1 activation. Activated IRE1 recruited ASK1 for downstream JNK phosphorylation. IRE1 knockdown by siRNA attenuated PPE8-induced JNK phosphorylation and cytotoxicity. Prolonged JNK phosphorylation may be involved in PPE8-induced cytotoxicity. Such results did not arise in A549 cells, but p53 knockdown by siRNA restored PPE8-induced GRP78 expression and JNK phosphorylation. We offer a novel compound to induce ER stress and cytotoxicity in p53-deficient cancer cells, presenting an opportunity for treatment.

  12. Inhibitory Effects of Verrucarin A on Tunicamycin-Induced ER Stress in FaO Rat Liver Cells. (United States)

    Bae, Eun Young; Lee, Seung Woong; Seong, Sin; Cho, Wonjun; Ahn, Jong Seog; Cho, Hyun-Sug


    Endoplasmic reticulum (ER) stress is linked with development and maintenance of cancer, and serves as a therapeutic target for treatment of cancer. Verrucarin A, isolated from the broth of Fusarium sp. F060190, showed potential inhibitory activity on tunicamycin-induced ER stress in FaO rat liver cells. In addition, the compound decreased tunicamycin-induced GRP78 promoter activity in a dose dependent manner without inducing significant inhibition of luciferase activity and cell growth for 6 and 12 h. Moreover, the compound decreased the expression of GRP78, CHOP, XBP-1, and suppressed XBP-1, and reduced phosphorylation of IRE1α in FaO rat liver cells. This evidence suggests for the first time that verrucarin A inhibited tunicamycin-induced ER stress in FaO rat liver cells.

  13. How T-cells use large deviations to recognize foreign antigens

    CERN Document Server

    Zint, Natali; Hollander, Frank den


    A stochastic model for the activation of T-cells is analysed. T-cells are part of the immune system and recognize foreign antigens against a background of the body's own molecules. The model under consideration is a slight generalization of a model introduced by Van den Berg, Rand and Burroughs in 2001, and is capable of explaining how this recognition works on the basis of rare stochastic events. With the help of a refined large deviation theorem and numerical evaluation it is shown that, for a wide range of parameters, T-cells can distinguish reliably between foreign antigens and self-antigens.

  14. A Novel Agent Enhances the Chemotherapeutic Efficacy of Doxorubicin in MCF-7 Breast Cancer Cells (United States)

    Wang, Liang; Chan, Judy Y.; Zhou, Xinhua; Cui, Guozhen; Yan, Zhixiang; Wang, Li; Yan, Ru; Di, Lijun; Wang, Yuqiang; Hoi, Maggie P.; Shan, Luchen; Lee, Simon M.


    We have previously demonstrated that DT-010, a novel conjugate of danshensu (DSS) and tetramethylpyrazine (TMP), displays anti-tumor effects in breast cancer cells both in vitro and in vivo. In the present study, we investigated whether DT-010 enhances the chemotherapeutic effect of doxorubicin (Dox) in MCF-7 breast cancer cells and exerts concurrent cardioprotective benefit at the same time. Our findings showed that DT-010 was more potent than TMP, DSS, or their combination in potentiating Dox-induced toxicity in MCF-7 cells. Co-treatment with DT-010 and Dox increased apoptosis in MCF-7 cells relative to Dox alone. Further study indicated that glycolytic capacity, glycolytic reserve and lactate level of MCF-7 cells were significantly inhibited after DT-010 treatment. DT-010 also increased the expression of the pro-survival protein GRP78, which was inhibited by co-treatment with Dox. Both endoplasmic reticulum stress inhibitor 4-PBA and knockdown of the expression of GRP78 protein potentiated DT-010-mediated apoptosis in MCF-7 cells. Moreover, DT-010 inhibited Dox-induced cardiotoxicity in H9c2 myoblasts. In conclusion, DT-010 and Dox confer synergistic anti-tumor effect in MCF-7 breast cancer cells through downregulation of the glycolytic pathway and inhibition of the expression of GRP78. Meanwhile, DT-010 also protects against Dox-induced cardiotoxicity. PMID:27559313

  15. Identification of beryllium-dependent peptides recognized by CD4+ T cells in chronic beryllium disease. (United States)

    Falta, Michael T; Pinilla, Clemencia; Mack, Douglas G; Tinega, Alex N; Crawford, Frances; Giulianotti, Marc; Santos, Radleigh; Clayton, Gina M; Wang, Yuxiao; Zhang, Xuewu; Maier, Lisa A; Marrack, Philippa; Kappler, John W; Fontenot, Andrew P


    Chronic beryllium disease (CBD) is a granulomatous disorder characterized by an influx of beryllium (Be)-specific CD4⁺ T cells into the lung. The vast majority of these T cells recognize Be in an HLA-DP–restricted manner, and peptide is required for T cell recognition. However, the peptides that stimulate Be-specific T cells are unknown. Using positional scanning libraries and fibroblasts expressing HLA-DP2, the most prevalent HLA-DP molecule linked to disease, we identified mimotopes and endogenous self-peptides that bind to MHCII and Be, forming a complex recognized by pathogenic CD4⁺ T cells in CBD. These peptides possess aspartic and glutamic acid residues at p4 and p7, respectively, that surround the putative Be-binding site and cooperate with HLA-DP2 in Be coordination. Endogenous plexin A peptides and proteins, which share the core motif and are expressed in lung, also stimulate these TCRs. Be-loaded HLA-DP2–mimotope and HLA-DP2–plexin A4 tetramers detected high frequencies of CD4⁺ T cells specific for these ligands in all HLADP2+ CBD patients tested. Thus, our findings identify the first ligand for a CD4⁺ T cell involved in metal-induced hypersensitivity and suggest a unique role of these peptides in metal ion coordination and the generation of a common antigen specificity in CBD.

  16. Effect of berberine on endoplasmic reticulum stress PERK apoptosis pathway in HK-2 cells by high fructose

    Directory of Open Access Journals (Sweden)

    Yong ZHANG


    Full Text Available Objective To investigate the effect of berberine on endoplasmic reticulum stress PERK apoptosis pathway in HK-2 cells by high fructose. Methods HK-2 cells were grown in DMEM/F12, containing 10% fetal bovine serum (FBS and divided randomly into four groups: normal control group (Group C; Fructose group (Group F: it contains 25mmol/L fructose culture; Berberine group (Group B: 25mmol/L fructose + 10μmol/L berberine treatment group; TUDCA group (Group T: 25mmol/L fructose +2μmol/L TUDCA culture group; Cells were collected after culturing 24h. The expression of glucose-regulated protein 78 (GRP78, CHOP protein and the phosphorylation levels of PERK, eIF2α were tested by Western blotting. The cell cycles were detected by flow cytometry and the apoptosis of cells were detected by TUNEL staining. Results Western blotting showed that the expression of GRP78 and CHOP protein in group F was significantly higher than that in group C, and the levels of p-PERK and p-eIF2α in group F were significantly higher than those in group F. Compared with group F, GRP78, CHOP, p-PERK and p-eIF2α in group B and T were significantly lower (P0.05. Conclusion Persistent high fructose can activate the intracellular PERK pathway in HK-2 cells, causing endoplasmic reticulum stress. Berberine can inhibit the fructose-induced PERK and eIF2α phosphorylation, down-regulated the expression of GRP78, CHOP protein, thus by regulating PERK Pathways to alleviate cell cycle arrest and reduce cell apoptosis. DOI: 10.11855/j.issn.0577-7402.2017.01.02

  17. Glucosamine-induced Sp1 O-GlcNAcylation ameliorates hypoxia-induced SGLT dysfunction in primary cultured renal proximal tubule cells. (United States)

    Suh, Han Na; Lee, Yu Jin; Kim, Mi Ok; Ryu, Jung Min; Han, Ho Jae


    The aim of this study is to determine whether GlcN could recover the endoplasmic reticulum (ER) stress-induced dysfunction of Na(+) /glucose cotransporter (SGLT) in renal proximal tubule cells (PTCs) under hypoxia. With the rabbit model, the renal ischemia induced tubulointerstitial abnormalities and decreased SGLTs expression in tubular brush-border, which were recovered by GlcN. Thus, the protective mechanism of GlcN against renal ischemia was being examined by using PTCs. Hypoxia decreased the level of protein O-GlcNAc and the expression of O-GlcNAc transferase (OGT) while increased O-GlcNAcase (OGA) and these were reversed by GlcN. Hypoxia also decreased the expression of SGLTs (SGLT1 and 2) and [(14) C]-α-methyl-D-glucopyranoside (α-MG) uptake which were recovered by GlcN and PUGNAc (OGA inhibitor). Hypoxia enhanced reactive oxygen species (ROS) and then ER stress proteins, glucose-regulated protein 78 (GRP78), and C/EBP-homologous protein (CHOP). However, the expression of GRP78 increased till 6 h and then decreased whereas CHOP increased gradually. Moreover, decreased GRP78 and increased CHOP were reversed by NAC (antioxidant) and GlcN. GlcN ameliorated hypoxia-induced decrease of O-GlcNAc modification of Sp1 but OGT or Sp1 siRNAs blocked the recovery effect of GlcN on SGLT expression and α-MG uptake. In addition, hypoxia-decreased GRP78 and HIF-1α expression was reversed by GlcN but OGT siRNA or Sp1 siRNA ameliorated the effect of GlcN. When PTCs were transfected with GRP78 siRNA or HIF-1α siRNA, SGLT expression and α-MG uptake was decreased. Taken together, these data suggest that GlcN-induced O-GlcNAc modified Sp1 with stimulating GRP78 and HIF-1α activity ameliorate hypoxia-induced SGLT dysfunction in renal PTCs. J. Cell. Physiol. 229: 1557-1568, 2014. © 2014 Wiley Periodicals, Inc.

  18. Effect of BSA-induced ER stress on SGLT protein expression levels and alpha-MG uptake in renal proximal tubule cells. (United States)

    Lee, Yu Jin; Suh, Han Na; Han, Ho Jae


    Recent studies demonstrated that endoplasmic reticulum (ER) stress regulates glucose homeostasis and that ER stress preconditioning which induces an adaptive, protective unfolded protein response (UPR) offers cytoprotection against nephrotoxins. Thus the aim of the present study was to use renal proximal tubule cells (PTCs) to further elucidate the link between the BSA-induced ER stress and alpha-methyl-d-glucopyranoside (alpha-MG) uptake and to identify related signaling pathways. Among ER stress inducers such as high glucose, BSA, H2O2, or tumicamycin, BSA pretreatment ameliorated the reduction of Na(+)-glucose cotransporter (SGLT) expression and alpha-MG uptake by gentamicin or cyclosporine A. Immunofluorescence studies revealed that BSA (10 mg/ml) stimulated the expression of glucose-regulated protein 78 (GRP78), an ER stress biomarker. In addition, BSA increased levels of GRP78 protein expression and eukaryotic initiation factor 2alpha (eIF2alpha) phosphorylation in a time-dependent manner. Furthermore, transfection with a GRP78-specific small interfering RNA (siRNA) inhibited BSA-stimulated SGLT expression and alpha-MG uptake. In experiments designed to unravel the mechanisms underlying BSA-induced ER stress, BSA stimulated the production of cellular reactive oxygen species (ROS), and antioxidants such as ascorbic acid or N-acetylcysteine (NAC) blocked BSA-induced increases in GRP78 activation, eIF2alpha phosphorylation, SGLT expression, and alpha-MG uptake. Moreover, the cells upregulated peroxisome proliferator-activated receptor-gamma (PPARgamma) mRNA levels in response to BSA or troglitazone (a PPARgamma agonist), but BSA was ineffective in the presence of GW9662 (a PPARgamma antagonist). In addition, both BSA and troglitazone stimulated GRP78 and eIF2alpha activation, SGLT expression, and alpha-MG uptake, whereas GW9662 inhibited the effects of BSA. BSA also stimulated phosphorylation of JNK and NF-kappaB, and GW9662 or GRP78 siRNA attenuated this

  19. Mast cell sarcoma: a rare and potentially under-recognized diagnostic entity with specific therapeutic implications. (United States)

    Ryan, Russell J H; Akin, Cem; Castells, Mariana; Wills, Marcia; Selig, Martin K; Nielsen, G Petur; Ferry, Judith A; Hornick, Jason L


    Mast cell sarcoma is a rare, aggressive neoplasm composed of cytologically malignant mast cells presenting as a solitary mass. Previous descriptions of mast cell sarcoma have been limited to single case reports, and the pathologic features of this entity are not well known. Here, we report three new cases of mast cell sarcoma and review previously reported cases. Mast cell sarcoma has a characteristic morphology of medium-sized to large epithelioid cells, including bizarre multinucleated cells, and does not closely resemble either normal mast cells or the spindle cells of systemic mastocytosis. One of our three cases arose in a patient with a remote history of infantile cutaneous mastocytosis, an association also noted in one previous case report. None of our three cases were correctly diagnosed as mast cell neoplasms on initial pathological evaluation, suggesting that this entity may be under-recognized. Molecular testing of mast cell sarcoma has not thus far detected the imatinib-resistant KIT D816V mutation, suggesting that recognition of these cases may facilitate specific targeted therapy.

  20. 未折叠蛋白反应在强噪声致豚鼠耳蜗细胞损伤过程中的作用%Participation of the Unfolded Protein Response in the Guinea Pig Cochlea Cell Damage after Intense Noise Exposure

    Institute of Scientific and Technical Information of China (English)

    薛秋红; 陈小林; 龚树生; 谢静; 陈佳; 何坚


    Objective To study the unfolded protein glucose-regulated protein 78 (GRP78) expression level after intense noise exposure,and to find out the relationship between UPR and the intense noise induced cochlea cell damage. Methods Forty-eight guinea pigs were randomly divided into 6 groups(8 guinea pigs/group). The guinea pigs in the experiment groups were exposed to 4 kHz narrow band noise at 120 dB SPL for 4 housr while aninals in control group received no noise exprsure. Auditory brainstem response(ABR) of the guinea pigs in experiment and control groups were tested at 3 hours, 1, 4, 14,30 days post noise exposure. Four guinea pig's cochleas from each group were used for paraffin sectioning, and the rest was used for the total protein extraction. Expression of Bip/GRP78 was studied by immunohistochemistry sectioning and western blot. Results There were significantly higher expressions of Tunel-Positve cells in the OHC,SGC and SV in experiment groups compared with those in the controi group (P<0.01). Protein levels of Bip/GRP78 were significantly increased after noise exposure compared with those in the control group (P<0.01). Conclusion After intense noise exposure, UPR protection mechanisms were initiated and by upregulating the expression of molecular chaperones Bip/GRP78, folded proteins were correctly guided, thus reducing cell damage. This may be one of the endogenous protective mechanisms in the guinea pig cochlea.%目的 探讨未折叠蛋白反应(unfolded protein response,UPR)标志物葡萄糖调节蛋白78(Bip/GRP78)在强噪声致豚鼠耳蜗细胞损伤中的作用.方法 48只豚鼠随机分为6组,分别为健康对照组(不给噪声暴露)和强噪声暴露后3 h、1 d、4 d、14 d、30 d组,每组8只,噪声暴露的5组豚鼠在120 dB SPL、4 kHz窄带噪声环境暴露4 h后,各组豚鼠于相应时间点处死前及对照组均测试听性脑干反应(ABR),然后每组各取4只豚鼠耳蜗作石蜡切片,余4只豚鼠提取耳蜗

  1. Proteomic analysis of endothelial cell autoantigens recognized by anti-dengue virus nonstructural protein 1 antibodies. (United States)

    Cheng, Hsien-Jen; Lin, Chiou-Feng; Lei, Huan-Yao; Liu, Hsiao-Sheng; Yeh, Trai-Ming; Luo, Yueh-Hsia; Lin, Yee-Shin


    We previously showed the occurrence of autoimmune responses in dengue virus (DV) infection, which has potential implications for the pathogenesis of dengue hemorrhagic syndrome. In the present study, we have used a proteomic analysis to identify several candidate proteins on HMEC-1 endothelial cells recognized by anti-DV nonstructural protein 1 (NS1) antibodies. The target proteins, including ATP synthase beta chain, protein disulfide isomerase, vimentin, and heat shock protein 60, co-localize with anti-NS1 binding sites on nonfixed HMEC-1 cells using immunohistochemical double staining and confocal microscopy. The cross-reactivity of anti-target protein antibodies with HMEC-1 cells was inhibited by NS1 protein pre-absorption. Furthermore, a cross-reactive epitope on NS1 amino acid residues 311-330 (P311-330) was predicted using homologous sequence alignment. The reactivity of dengue hemorrhagic patient sera with HMEC-1 cells was blocked by synthetic peptide P311-330 pre-absorption. Taken together, our results identify putative targets on endothelial cells recognized by anti-DV NS1 antibodies, where NS1 P311-330 possesses the shared epitope.

  2. Acellbia® and Mabtera® are recognize CD20-positive cells with equal efficiency

    Directory of Open Access Journals (Sweden)

    V. A. Misyurin


    Full Text Available In present study were compared characteristics of rituximab produced by Hoffmann–La Roche (Mabtera® and first domestic biosimilar Acellbia® from Biocad Company. Concentration of protein was measured using Bradford, s method. According to our results, protein concentration in formulations was the same. We electrophoresed formulations in denaturing conditions. Protein from formulations was denatured into fragments. Heavy and light chains of immunoglobulin were observed in gel. Finally, we performed flow cytometry where rituximab was used as primary antibody to detect CD20-positive B-cells of patients with B-cell chronic lymphocytic leukemia. Both Mabtera® and Acellbia® recognized the same number of cells. Thus, assays performed in vitro submitted identity of Mabtera® and Acellbia® characteristics.

  3. Tumor-infiltrating B lymphocytes as an efficient source of highly specific immunoglobulins recognizing tumor cells

    Directory of Open Access Journals (Sweden)

    Pelliccia Angela


    Full Text Available Abstract Background There is much evidence that tumor cells elicit a humoral immune response in patients. In most cases, the presence of antibodies in peripheral blood is detected only in small proportion of patients with tumors overexpressing the corresponding antigen. In the present study, we analyzed the significance of local humoral response provided by tumor-infiltrating lymphocytes in breast cancer patients. Methods The ability of a patient's immune system to produce specific antibodies inside tumor tissue, capable of recognizing tumor cells, was explored through analysis of the oligoclonality of antibodies derived from tumor-infiltrating lymphocytes and construction of a series of recombinant antibody libraries in scFv format, derived from breast tumor-infiltrating B lymphocytes. These libraries and one from peripheral blood lymphocytes of a single breast cancer patient were panned against three purified surface tumor antigens, such as CEA, MUC1 and ED-B domain, and against intact MCF7 breast carcinoma cells. Results Application of novel display vector, pKM19, allowed isolation of a large panel of breast cancer-specific antibodies against known tumor antigens, as well as against breast carcinoma cells. Reactivity of novel scFvs was confirmed by ELISA, immunohistochemistry, fluorescence staining and flow cytometry. We demonstrated that seven of ten primary breast tumor specimens, obtained using discarded surgical material, could be exploited as an appropriate source for generation of phage display libraries, giving highly specific antitumor antibodies which recognize heterologous tumor cells. Conclusion Local humoral immune response within tumor tissue in breast cancer patients frequently has an oligoclonal character. Efficient selection of specific antitumor antibodies from recombinant antibody libraries, derived from such oligoclonal tumor-infiltrated B lymphocytes, indicates the presence of natural immune response against tumor antigens

  4. A novel combination treatment for breast cancer cells involving BAPTA-AM and proteasome inhibitor bortezomib (United States)



    Glucose-regulated protein 78 kDa/binding immunoglobulin protein (GRP78/BIP) is a well-known endoplasmic reticulum (ER) chaperone protein regulating ER stress by facilitating protein folding, assembly and Ca2+ binding. GRP78 is also a member of the heat shock protein 70 gene family and induces tumor cell survival and resistance to chemotherapeutics. Bortezomib is a highly specific 26S proteasome inhibitor that has been approved as treatment for patients with multiple myeloma. The present study first examined the dose- and time-dependent effects of bortezomib on GRP78 expression levels in the highly metastatic mouse breast cancer 4T1 cell line using western blot analysis. The analysis results revealed that GRP78 levels were significantly increased by bortezomib at a dose as low as 10 nM. Time-dependent experiments indicated that the accumulation of GRP78 was initiated after a 24 h incubation period following the addition of 10 nM bortezomib. Subsequently, the present study determined the half maximal inhibitory concentration of intracellular calcium chelator BAPTA-AM (13.6 µM) on 4T1 cells. The combination effect of BAPTA-AM and bortezomib on the 4T1 cells was investigated using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and WST-1 assays and an iCELLigence system. The results revealed that the combination of 10 nM bortezomib + 5 µM BAPTA-AM is more cytotoxic compared with monotherapies, including 10 nM bortezomib, 1 µM BAPTA-AM and 5 µM BAPTA-AM. In addition, the present results revealed that bortezomib + BAPTA-AM combination causes cell death through the induction of apoptosis. The present results also revealed that bortezomib + BAPTA-AM combination-induced apoptosis is associated with a clear increase in the phosphorylation of stress-activated protein kinase/Jun amino-terminal kinase SAPK/JNK. Overall, the present results suggest that bortezomib and BAPTA-AM combination therapy may be a novel therapeutic strategy for breast cancer treatment

  5. Durable Complete Response from Metastatic Melanoma after Transfer of Autologous T Cells Recognizing 10 Mutated Tumor Antigens. (United States)

    Prickett, Todd D; Crystal, Jessica S; Cohen, Cyrille J; Pasetto, Anna; Parkhurst, Maria R; Gartner, Jared J; Yao, Xin; Wang, Rong; Gros, Alena; Li, Yong F; El-Gamil, Mona; Trebska-McGowan, Kasia; Rosenberg, Steven A; Robbins, Paul F


    Immunotherapy treatment of patients with metastatic cancer has assumed a prominent role in the clinic. Durable complete response rates of 20% to 25% are achieved in patients with metastatic melanoma following adoptive cell transfer of T cells derived from metastatic lesions, responses that appear in some patients to be mediated by T cells that predominantly recognize mutated antigens. Here, we provide a detailed analysis of the reactivity of T cells administered to a patient with metastatic melanoma who exhibited a complete response for over 3 years after treatment. Over 4,000 nonsynonymous somatic mutations were identified by whole-exome sequence analysis of the patient's autologous normal and tumor cell DNA. Autologous B cells transfected with 720 mutated minigenes corresponding to the most highly expressed tumor cell transcripts were then analyzed for their ability to stimulate the administered T cells. Autologous tumor-infiltrating lymphocytes recognized 10 distinct mutated gene products, but not the corresponding wild-type products, each of which was recognized in the context of one of three different MHC class I restriction elements expressed by the patient. Detailed clonal analysis revealed that 9 of the top 20 most prevalent clones present in the infused T cells, comprising approximately 24% of the total cells, recognized mutated antigens. Thus, we have identified and enriched mutation-reactive T cells and suggest that such analyses may lead to the development of more effective therapies for the treatment of patients with metastatic cancer. Cancer Immunol Res; 4(8); 669-78. ©2016 AACR.

  6. The Petal-Specific InMYB1 Promoter Functions by Recognizing Petaloid Cells. (United States)

    Azuma, Mirai; Mitsuda, Nobutaka; Goto, Koji; Oshima, Yoshimi; Ohme-Takagi, Masaru; Otagaki, Shungo; Matsumoto, Shogo; Shiratake, Katsuhiro


    The InMYB1 gene in Japanese morning glory (Ipomoea nil) is a member of the MYB transcription factor family. The promoter of InMYB1 has been reported to induce petal-specific gene expression in Arabidopsis and Eustoma, and has the same function in several other dicotyledonous plants. Most flowers consist of sepals, petals, stamens and a carpel, whose identity establishment is explained by the ABC model. The establishment of the identity of petals is determined by the expression of class A and B genes in whorl 2. The aim of this study was to clarify whether the InMYB1 promoter functions by recognizing whorl position or petal identity by examining its activity in various mutant and transgenic Arabidopsis thaliana plants in which genes related to the ABC model have been modified. In plants defective in class C gene function, the InMYB1 promoter functioned not only in petals generated in whorl 2 but also in petaloid organs generated in whorl 3; while in the plants defective in class B gene function, the InMYB1 promoter did not function in the sepaloid organs generated in whorl 2. Plants overexpressing class A, B and E genes set flowers with petaloid sepals in whorl 1, i.e. the lateral parts were white and looked like petals, while the central parts were green and looked like sepals. The InMYB1 promoter functioned in the lateral white parts but not in the central green parts. These results show that the InMYB1 promoter functions by recognizing petal identity at the cellular level rather than the whorl position. The petal-specific function of the InMYB1 promoter could be used as a marker to identify petaloid cells.

  7. Nephritogenic lupus antibodies recognize glomerular basement membrane-associated chromatin fragments released from apoptotic intraglomerular cells. (United States)

    Kalaaji, Manar; Mortensen, Elin; Jørgensen, Leif; Olsen, Randi; Rekvig, Ole Petter


    Antibodies to dsDNA represent a classification criterion for systemic lupus erythematosus. Subpopulations of these antibodies are involved in lupus nephritis. No known marker separates nephritogenic from non-nephritogenic anti-dsDNA antibodies. It is not clear whether specificity for glomerular target antigens or intrinsic antibody-affinity for dsDNA or nucleosomes is a critical parameter. Furthermore, it is still controversial whether glomerular target antigen(s) is constituted by nucleosomes or by non-nucleosomal glomerular structures. Previously, we have demonstrated that antibodies eluted from murine nephritic kidneys recognize nucleosomes, but not other glomerular antigens. In this study, we determined the structures that bind nephritogenic autoantibodies in vivo by transmission electron microscopy, immune electron microscopy, and colocalization immune electron microscopy using experimental antibodies to dsDNA, to histones and transcription factors, or to laminin. The data obtained are consistent and point at glomerular basement membrane-associated nucleosomes as target structures for the nephritogenic autoantibodies. Terminal deoxynucleotidyl-transferase-mediated dUTP nick end-labeling or caspase-3 assays demonstrate that lupus nephritis is linked to intraglomerular cell apoptosis. The data suggest that nucleosomes are released by apoptosis and associate with glomerulus basement membranes, which may then be targeted by pathogenic anti-nucleosome antibodies. Thus, apoptotic nucleosomes may represent both inducer and target structures for nephritogenic autoantibodies in systemic lupus erythematosus.

  8. The monoclonal antibody ER-BMDM1 recognizes a macrophage and dendritic cell differentiation antigen with aminopeptidase activity

    NARCIS (Netherlands)

    P.J. Leenen (Pieter); M.L. Melis (Marleen); G. Kraal (Georg); A.T. Hoogeveen (Andre); W. van Ewijk (Willem)


    markdownabstractAbstract Here we describe the reactivity of monoclonal antibody (mAb) ER-BMDM1, directed against a 160-kDa cell membrane-associated antigen (Ag) with aminopeptidase activity. The aminopeptidase recognized by ER-BMDM1 is present on various mouse macrophage (MΦ) and dendritic cell (D

  9. Let's Talk about the Needs of African American Children with Sickle Cell Disease: A Recognized "Other Health Impairment." (United States)

    Dooley, Elizabeth A.; Perkins, Nechelle

    Children who inherit sickle cell disease, primarily African Americans and Hispanics, are at risk for serious medical conditions and require special care both at home and in school. Sickle cell disease is recognized as an "Other Health Impairment" and identified students may be eligible for special education services under the Individuals…

  10. TIR-only protein RBA1 recognizes a pathogen effector to regulate cell death in Arabidopsis (United States)

    Anderson, Ryan G.; Cherkis, Karen A.; Law, Terry F.; Liu, Qingli L.; Machius, Mischa; Nimchuk, Zachary L.; Yang, Li; Chung, Eui-Hwan; El Kasmi, Farid; Hyunh, Michael; Sondek, John E.; Dangl, Jeffery L.


    Detection of pathogens by plants is mediated by intracellular nucleotide-binding site leucine-rich repeat (NLR) receptor proteins. NLR proteins are defined by their stereotypical multidomain structure: an N-terminal Toll–interleukin receptor (TIR) or coiled-coil (CC) domain, a central nucleotide-binding (NB) domain, and a C-terminal leucine-rich repeat (LRR). The plant innate immune system contains a limited NLR repertoire that functions to recognize all potential pathogens. We isolated Response to the bacterial type III effector protein HopBA1 (RBA1), a gene that encodes a TIR-only protein lacking all other canonical NLR domains. RBA1 is sufficient to trigger cell death in response to HopBA1. We generated a crystal structure for HopBA1 and found that it has similarity to a class of proteins that includes esterases, the heme-binding protein ChaN, and an uncharacterized domain of Pasteurella multocida toxin. Self-association, coimmunoprecipitation with HopBA1, and function of RBA1 require two previously identified TIR–TIR dimerization interfaces. Although previously described as distinct in other TIR proteins, in RBA1 neither of these interfaces is sufficient when the other is disrupted. These data suggest that oligomerization of RBA1 is required for function. Our identification of RBA1 demonstrates that “truncated” NLRs can function as pathogen sensors, expanding our understanding of both receptor architecture and the mechanism of activation in the plant immune system. PMID:28137883

  11. Low-dose radiation induces endoplasmic reticulum stress and activates PERK-CHOP signaling pathway in mouse testicular cells%低剂量电离辐射诱导小鼠睾丸细胞内质网应激及PERK-CHOP信号通路的激活

    Institute of Scientific and Technical Information of China (English)

    方芳; 龚平生; 宋祥福; 龚守良; 王志成


    and the activation of the PERK-CHOP signaling pathway in mouse testicular cells. Methods: Healthy Kunming mice were randomly assigned to time-effect (0,3,6, 12 and 24 h of irradiation at 75 mGy) and dose-effect (12 h of irradiation at 0, 50, 75, 100 and 200 mGy) groups. The contents of H2O2 and MDA were measured by colorimetry with the agent kits, the expressions of GRP78, PERK and CHOP mRNA detected by quantitative RT-PCR, and the levels of GRP78, PERK, phosphorylated PERK (pho-PERK) and CHOP proteins determined by Western blotting and image analysis. Results: After whole-body irradiation of the mice with 75 mGy, the content of H2O2 in the testis tissue was increased with time prolongation, while that of MDA decreased slightly at 3 and 6 h and then increased with the lengthening of time, both increased significantly at 12 and 24 h as compared with those at 0 h (P < 0. 05 , P < 0. 01). Apart from reduced levels of GRP78 mRNA at 3 and 24 h and GRP78 protein at 6 h after irradiation, significant increases were found in the mRNA expressions of GRP78 at 12 h, PERK at 3, 6, 12 and 24 h and CHOP at 12 and 24 h (P < 0.05, P < 0.01) , as well as in the protein levels of GRP78 at 12 and 24 h, pho-PERK at 3 , 12 and 24 h and CHOP at 3 , 6, 12 and 24 h in comparison with those at 0 h (P < 0. 05 , P < 0.01). No obvious regularity was observed in the change of the PERK protein expression. After 12 h of whole-body irradiation, the content of H2O2 was increased at 50, 75 and 100 mGy, but decreased slightly at 200 mGy, while that of MDA was increased with dose increasing, with significant increases in the content of H2O2 at 75 and 100 mGy and in that of MDA at 75 , 100 and 200 mGy as compared with the 0 mGy group. Apart from the reduced levels of GRP78 mRNA at 50 and 200 mGy, significant increases were found in the mRNA expressions of PERK at 75 , 100 and 200 mGy and CHOP at 50, 75 , 100 and 200 ( P < 0. 05 , P < 0. 01) as well as in the protein levels of GRP78 at 100 and 200 m

  12. Cellular Immune Responses for Squamous Cell Carcinoma Antigen Recognized by T Cells 3 in Patients with Hepatocellular Carcinoma (United States)

    Kaji, Kiichiro; Mizukoshi, Eishiro; Yamashita, Tatsuya; Arai, Kuniaki; Sunagozaka, Hajime; Fushimi, Kazumi; Nakagawa, Hidetoshi; Yamada, Kazutoshi; Terashima, Takeshi; Kitahara, Masaaki; Kaneko, Shuichi


    Background & Aims Squamous cell carcinoma antigen recognized by T cells 3 (SART3), a tumor-associated antigen expressed in many cancers, functions in tumor rejection. In this study, we investigated its usefulness as an immunotherapeutic target in hepatocellular carcinoma (HCC). Methods The expression of SART3 in hepatoma cell lines and HCC tissues was investigated by immunofluorescence and immunohistochemical analyses. Two peptides derived from SART3 (SART3109 and SART3315) were used for immunological analysis. T-cell responses were investigated by interferon-gamma (IFN-γ) enzyme-linked immunospot and cytotoxic T lymphocyte (CTL) assays using peripheral blood mononuclear cells (PBMCs) in 47 patients, and tumor-infiltrating lymphocytes in 8 of 47 patients with HCC. The safety of immunotherapy using a SART3-derived peptide was investigated by vaccinations of SART3109 in 12 patients with HCC (trial registration: UMIN000005677). Results The immunofluorescence and immunohistochemical analyses showed that SART3 was expressed in six HCC cell lines, and in HCC tissues including of alpha-fetoprotein-negative individuals. SART3-specific CTLs were generated by stimulating PBMCs with the peptides, and they showed cytotoxicity against HCC cells expressing the protein. Of the 47 HCC patients, 25.5% and 10.6% showed significant responses to SART3109 and SART3315, respectively. The infiltration of SART3109-specific IFN-γ-producing CTLs into the tumor site was confirmed. In the vaccination study, no severe adverse events were observed, and the peptide-specific CTLs were newly induced in four of five patients tested. Conclusions SART3 is an immunotherapeutic candidate, and peptides from this antigen may be applied in HCC immunotherapy. Trial Registration UMIN000005677 PMID:28114424

  13. Identification of DRG-1 As a Melanoma-Associated Antigen Recognized by CD4+ Th1 Cells.

    Directory of Open Access Journals (Sweden)

    Yukiko Kiniwa

    Full Text Available Immunotherapy has emerged as a promising strategy for the treatment of metastatic melanoma. Clinical studies have demonstrated the feasibility of cancer immunotherapy using tumor antigens recognized by CD8(+ T cells. However, the overall immune responses induced by these antigens are too weak and transient to induce tumor regression in the majority of patients who received immunization. A growing body of evidence suggests that CD4(+ T helper (Th cells play an important role in antitumor immunity. Therefore, the identification of MHC class II-restricted tumor antigens capable of stimulating CD4(+ T cells may provide opportunities for developing effective cancer vaccines. To this end, we describe the identification of developmentally regulated GTP-binding protein 1 (DRG-1 as a melanoma-associated antigen recognized by HLA-DR11-restricted CD4(+ Th1 cells. Epitope mapping analysis showed that the DRG1248-268 epitope of DRG-1 was required for T cell recognition. Reverse transcription-polymerase chain reaction revealed that DRG-1 was highly expressed in melanoma cell lines but not in normal tissues. DRG-1 knockdown by lentiviral-based shRNA suppressed melanoma cell proliferation and soft agar colony formation. Taken together, these data suggest that DRG-1 plays an important role in melanoma cell growth and transformation, indicating that DRG1 may represent a novel target for CD4(+ T cell-mediated immunotherapy in melanoma.

  14. GM-CSF production allows the identification of immunoprevalent antigens recognized by human CD4+ T cells following smallpox vaccination.

    Directory of Open Access Journals (Sweden)

    Valeria Judkowski

    Full Text Available The threat of bioterrorism with smallpox and the broad use of vaccinia vectors for other vaccines have led to the resurgence in the study of vaccinia immunological memory. The importance of the role of CD4+ T cells in the control of vaccinia infection is well known. However, more CD8+ than CD4+ T cell epitopes recognized by human subjects immunized with vaccinia virus have been reported. This could be, in part, due to the fact that most of the studies that have identified human CD4+ specific protein-derived fragments or peptides have used IFN-γ production to evaluate vaccinia specific T cell responses. Based on these findings, we reasoned that analyzing a large panel of cytokines would permit us to generate a more complete analysis of the CD4 T cell responses. The results presented provide clear evidence that TNF-α is an excellent readout of vaccinia specificity and that other cytokines such as GM-CSF can be used to evaluate the reactivity of CD4+ T cells in response to vaccinia antigens. Furthermore, using these cytokines as readout of vaccinia specificity, we present the identification of novel peptides from immunoprevalent vaccinia proteins recognized by CD4+ T cells derived from smallpox vaccinated human subjects. In conclusion, we describe a "T cell-driven" methodology that can be implemented to determine the specificity of the T cell response upon vaccination or infection. Together, the single pathogen in vitro stimulation, the selection of CD4+ T cells specific to the pathogen by limiting dilution, the evaluation of pathogen specificity by detecting multiple cytokines, and the screening of the clones with synthetic combinatorial libraries, constitutes a novel and valuable approach for the elucidation of human CD4+ T cell specificity in response to large pathogens.

  15. Analysis of the role of nerve growth factor in promoting cell survival during endoplasmic reticulum stress in PC12 cells. (United States)

    Shimoke, Koji; Sasaya, Harue; Ikeuchi, Toshihiko


    Nerve growth factor (NGF) was first described by Rita Levi-Montalcini in the early 1960s from her studies of peripheral neurons. It has since been reported that NGF has the potential to elongate neurites or to prevent apoptosis via specific intracellular mechanisms. It has further been reported that as a component of these mechanisms, NGF binds to a specific receptor, TrkA, and thereby contributes to peripheral nerve cell functions or neuronal functions. It is noteworthy in this regard that pheochromocytoma 12 (PC12) cells express TrkA and respond to neurite outgrowth or anti-apoptotic signals by binding to NGF. Hence, PC12 cells have been used as an in vitro model system for the study of neuronal functions. It has been reported that endoplasmic reticulum (ER) stress is involved in neurodegenerative disorders, including Alzheimer's, Parkinson's, and Huntington's disease. The common link with regard to ER stress is that the neuronal cells die in these pathologies via specific intracellular mechanisms. This type of cell death, if it is apoptotic in nature, is termed ER stress-mediated apoptosis. In the process of ER stress-mediated apoptosis, the cleavage of pro-caspase-12 residing on the ER and the expression of glucose-regulated protein 78 (GRP78) can be observed. The expression of GRP78 protein is a characteristic of an unfolded protein response (UPR) via specific signal transduction pathways mediated by the unfolded protein response element (UPRE) in the upstream region of the grp78 gene so on. In ER stress-mediated apoptosis, a caspase cascade is also observed. To further clarify the mechanisms underlying ER stress-mediated apoptosis, a better understanding of the UPR is therefore important. In our current study, we describe a method for detecting gene induction via the UPR, focusing on GRP78 and caspase activities as the measurement end-points. The information generated by our method will accelerate our understanding of the pathophysiological processes leading

  16. Celiac lesion T cells recognize epitopes that cluster in regions of gliadins rich in proline residues

    DEFF Research Database (Denmark)

    Arentz-Hansen, Helene; McAdam, Stephen N; Molberg, Øyvind


    BACKGROUND & AIMS: Celiac disease is a gluten-induced enteropathy that shows a strong association with HLA-DQ2 and -DQ8. Gluten-specific T cells, invariably restricted by DQ2 or DQ8, can be isolated from celiac lesions. Such gut-derived T cells have a preference for recognition of gluten that has...

  17. The T-cell accessory molecule CD4 recognizes a monomorphic determinant on isolated Ia

    DEFF Research Database (Denmark)

    Gay, D; Buus, S; Pasternak, J;


    The membrane protein CD4 is commonly found on mature T cells specific for antigen in association with class II major histocompatibility complex (MHC; Ia) proteins. This correlation has led to the suggestion that CD4 binds to a monomorphic region of the Ia molecule on the antigen-presenting cell...... proteins into a planar membrane system, we show that different Ia molecules can greatly enhance the ability of a CD4+ but not a CD4- variant of this class I-restricted T hybrid to respond to isolated class I molecules. T-cell responses can be strongly augmented by the concurrent expression of CD4 on the T...... cell and any of four different Ia proteins on planar membranes, thus supporting the idea that CD4 binds to a monomorphic region of the Ia molecule and increases the avidity with which the T cell can interact with its target....

  18. Initiation and regulation of CD8+T cells recognizing melanocytic antigens in the epidermis: implications for the pathophysiology of vitiligo. (United States)

    Steitz, Julia; Wenzel, Jörg; Gaffal, Evelyn; Tüting, Thomas


    Antigen-specific CD8+T lymphocytes play an important role in defense against cutaneous microbial infection and skin cancer as well as in the pathophysiology of autoimmune skin disease such as lupus erythematodes and vitiligo. We have explored the role of CD8+ cytotoxic T lymphocytes (CTL) in an experimental mouse model of vitiligo, a pigmentation disorder characterized by focal loss of melanocytes in the skin. Using genetic immunization techniques we found that pigment cells in the epidermis can be destroyed by CD8+ T cells specifically recognizing a single H2-Kb-binding peptide derived from the model melanocytic self antigen tyrosinase-related protein 2 (TRP2), a melanosomal enzyme involved in pigment synthesis. Experimental evidence suggests that peripheral tolerance of pigment cell-specific cytotoxic CD8+T cells is regulated in two steps. In the induction phase, stimulation and expansion of these T cells in vivo strictly depends on CD4+ T cell help. In the effector phase, autoimmune destruction of melanocytes in the skin depends on local inflammation facilitating the migration of T cells into the epidermis and supporting effector functions. Our results suggest that accidental stimulation of CD8+ CTL recognizing MHC class I-binding peptides derived from melanocytic proteins in the context of an inflammatory skin disease may play an important role in the pathophysiology of vitiligo. Further investigations will address the role of chemokines, chemokine receptors and adhesion molecules in this experimental system and will reveal the role of keratinocytes and Langerhans cells in regulating cutaneous CD8+ T cell responses.

  19. Salvianolic acid B protects endothelial cells from oxidant-mediated damage

    Institute of Scientific and Technical Information of China (English)

    LI Xue-jun


    Objective To investigate the protective effects of Salvianolic acid B(Sal B) on hydrogen peroxide (H2O2)-induced injury in human umbilical vein endothelial cells (HUVECs). Sal B is considered as one of the most active anti-oxidant and the major pharmacological component of the herb, Salvia miltiorrhiza. Its beneficial effects include hepatoprotection, elicitation of endothelium-dependent vasodilation, lowering blood pressure in hypertension, inhibition of HIV-1 replication and suppressing inflammatory cytokine- stimulated endothelial adhesiveness to human monocytie cells by its strong antioxidant activities. Methods Treatment with H2O2 significantly decreased the cell viability and increased the lactate dehydrogenase (LDH) leakage that is an apoptotic feature. Pretreatment with Sal B prevented significantly from H2O2-induced cell apoptosis and other damages in a concentration-dependent manner. The mechanism of Sal B protection was studied with two-dimensional gel electrophoresis (2-DE) coupled to hybrid quadrupole time-of-flight mass spectrometry (Q-TOF) mass spectrometer. Results Data base searching implicated glucose-regulated protein 78 (GRP78), a central regulator for ER stress, was up-regulated in Sal B-exposed HUVECs. After exposure to Sal B, the level of activating transcription factor 4 (ATF4) was raised, with a transient phosphorylation of the α subunit of eukaryotic translation initiation factor (eIF2α). Knock-down of GRP78 by siRNA significantly reduced protective effects of Sal B. Conclusions These results suggest that Sal B-induced GRP78 upregulation via phosphorylation of eIF2α and resultant translation of ATF4. And up-regulation of ER chaperones induced by Sal B may play an important role in protecting human endothelial cells from oxidative stress-induced cellular damage.

  20. Neocytolysis on descent from altitude: a newly recognized mechanism for the control of red cell mass (United States)

    Rice, L.; Ruiz, W.; Driscoll, T.; Whitley, C. E.; Tapia, R.; Hachey, D. L.; Gonzales, G. F.; Alfrey, C. P.


    BACKGROUND: Studies of space-flight anemia have uncovered a physiologic process, neocytolysis, by which young red blood cells are selectively hemolyzed, allowing rapid adaptation when red cell mass is excessive for a new environment. OBJECTIVES: 1) To confirm that neocytolysis occurs in another situation of acute plethora-when high-altitude dwellers with polycythemia descend to sea level; and 2) to clarify the role of erythropoietin suppression. DESIGN: Prospective observational and interventional study. SETTING: Cerro de Pasco (4380 m) and Lima (sea level), Peru. PARTICIPANTS: Nine volunteers with polycythemia. INTERVENTIONS: Volunteers were transported to sea level; three received low-dose erythropoietin. MEASUREMENTS: Changes in red cell mass, hematocrit, hemoglobin concentration, reticulocyte count, ferritin level, serum erythropoietin, and enrichment of administered(13)C in heme. RESULTS: In six participants, red cell mass decreased by 7% to 10% within a few days of descent; this decrease was mirrored by a rapid increase in serum ferritin level. Reticulocyte production did not decrease, a finding that establishes a hemolytic mechanism.(13)C changes in circulating heme were consistent with hemolysis of young cells. Erythropoietin was suppressed, and administration of exogenous erythropoietin prevented the changes in red cell mass, serum ferritin level, and(13)C-heme. CONCLUSIONS: Neocytolysis and the role of erythropoietin are confirmed in persons with polycythemia who descend from high altitude. This may have implications that extend beyond space and altitude medicine to renal disease and other situations of erythropoietin suppression, hemolysis, and polycythemia.

  1. The value of recognizing suspect diagnoses in the triple diagnosis of giant cell tumor of bone

    Directory of Open Access Journals (Sweden)

    Kotru Mrinalini


    Full Text Available Giant cell tumor (GCT of bone is the most frequently over-diagnosed neoplasm in orthopedic pathology because giant cells are a common component of many neoplastic and nonneoplastic conditions of bone. Triple diagnosis, requiring substantial individual and collective inputs by orthopedic surgeons, radiologists and pathologists, is the preferred method for the workup of patients with suspected bone neoplasms. At each stage in triple diagnosis, deviations from the typical must be regarded as clues to alternate diagnoses: the greater the deviation, the more a diagnosis of GCT must be considered suspect. A suspect diagnosis must trigger renewed analysis of the available data and a diligent search to exclude alternate diagnoses.

  2. Identification of Theileria lestoquardi Antigens Recognized by CD8+ T Cells (United States)

    Ngugi, Daniel; Lizundia, Regina; Hostettler, Isabel; Woods, Kerry; Ballingall, Keith; MacHugh, Niall D.; Morrison, W. Ivan; Weir, Willie; Shiels, Brian; Werling, Dirk


    As part of an international effort to develop vaccines for Theileria lestoquardi, we undertook a limited screen to test T. lestoquardi orthologues of antigens recognised by CD8+ T lymphocyte responses against T. annulata and T. parva in cattle. Five MHC defined sheep were immunized by live T. lestoquardi infection and their CD8+ T lymphocyte responses determined. Thirteen T. lestoquardi orthologues of T. parva and T. annulata genes, previously shown to be targets of CD8+ T lymphocyte responses of immune cattle, were expressed in autologous fibroblasts and screened for T cell recognition using an IFNγ assay. Genes encoding T. lestoquardi antigens Tl8 (putative cysteine proteinase, 349 aa) or Tl9 (hypothetical secreted protein, 293 aa) were recognise by T cells from one animal that displayed a unique MHC class I genotype. Antigenic 9-mer peptide epitopes of Tl8 and Tl9 were identified through peptide scans using CD8+ T cells from the responding animal. These experiments identify the first T. lestoquardi antigens recognised by CD8+ T cell responses linked to specific MHC class I alleles. PMID:27611868

  3. Problem-Solving Test: Analysis of DNA Damage Recognizing Proteins in Yeast and Human Cells (United States)

    Szeberenyi, Jozsef


    The experiment described in this test was aimed at identifying DNA repair proteins in human and yeast cells. Terms to be familiar with before you start to solve the test: DNA repair, germline mutation, somatic mutation, inherited disease, cancer, restriction endonuclease, radioactive labeling, [alpha-[superscript 32]P]ATP, [gamma-[superscript…

  4. The characterization of equine herpes virus-1-infected cell polypeptides recognized by equine lymphocytes. (United States)

    Bridges, C G; Ledger, N; Edington, N


    Ponies, without evidence of previous exposure to Equine herpes virus-1 (EHV-1), were experimentally infected with EHV-1 subtype 2 and investigated for lymphocyte transformation to virus-infected cell polypeptides, as shown by separation with gel electrophoresis. Animals made significant responses to Western blot fractions that corresponded to molecular weights of approximately 30,000, 40,000-45,000, 60,000-65,000, 80,000-95,000 and 100,000-140,000 MW. These molecular weight ranges correlated with the positions of major EHV-1 subtype 2 glycoproteins that were found at migration distances approximating to 137,000, 111,000, 90,000, 65,000 and 47,000 MW. Responses were also made to a subset of similar points on the subtype 1 profile. Hyperimmune equine serum precipitated numerous infected-cell proteins of both subtypes; in particular the recognition of polypeptides with MW of 142,000, 132,000, 114,000, and 46,000 was in agreement with the mitogenic responses. Labelling with 125I indicated that immunoprecipitated greater than 250,000, 182,000, 142,000, 132,000, 75,000, 46,000 and 32,000/34,000 MW products were exposed on the surface of infected cells.

  5. Cerebrospinal fluid T-regulatory cells recognize Borrelia burgdorferi NAPA in chronic Lyme borreliosis. (United States)

    Amedei, A; Codolo, G; Ozolins, D; Ballerini, C; Biagioli, T; Jaunalksne, I; Zilevica, A; D Elios, S; De Bernard, M; D' Elios, M M


    The NapA protein of B. burgdorferi is essential for the persistence of spirochetes in ticks. One of the most intriguing aspects of NapA is its potential to interfere with the host immune system. Here, we investigated the role of the acquired immune responses induced by NapA in the cerebrospinal fluids (CSF) of patients with chronic Lyme borreliosis. We evaluated the cytokine profile induced in microglia cells and CSF T cells following NapA stimulation. We report here that NapA induced a regulatory T (Treg) response in the CSF of patients with chronic Lyme borreliosis and it is able to expand this suppressive response by promoting the production of TGF-beta and IL-10 by microglia cells. Collectively, these data strongly support a central role of NapA in promoting both Treg response and immune suppression in the CSF of patients with chronic Lyme borreliosis and suggest that NapA and the Treg pathway may represent novel therapeutic targets for the prevention and treatment of the disease.

  6. Characterization of monoclonal antibodies recognizing 130 kDa surface proteins on human embryonic stem cells and cancer cell lines. (United States)

    Kim, Jum-Ji; Choi, Hong Seo; Lee, Mi-Young; Ryu, Chun Jeih


    To study cell surface proteins expressed on human embryonic stem cells (hESCs), we generated a panel of monoclonal antibodies (MAbs) against undifferentiated hESCs by a decoy immunization strategy in a previous study. Two of the MAbs, 63-B6 and 246-D7, bound to human pluripotent stem cells but not to human primary cells such as human peripheral blood mononuclear cells and human lung fibroblasts. They did not bind to either mouse embryonic stem cells or mouse embryonic fibroblasts. The two MAbs had similar binding profiles for many various cancer cells, with few exceptions. Expression of antigens recognized by the two MAbs was rapidly decreased during embryoid body formation of hESCs and gradually increased after initial decrease. The MAbs recognized approximately 130 kDa proteins on the surface of hESCs. Cloning and sequence analysis of antibody genes showed that although the MAbs had exactly the same light chain sequences, they had different heavy chain sequences. Taken together, the results suggest that the two MAbs may recognize two different epitopes of the same or different 130 kDa surface proteins involved in regulating the early differentiation of human pluripotent stem cells and cancer cells.

  7. Binding domains of Bacillus anthracis phage endolysins recognize cell culture age-related features on the bacterial surface. (United States)

    Paskaleva, Elena E; Mundra, Ruchir V; Mehta, Krunal K; Pangule, Ravindra C; Wu, Xia; Glatfelter, Willing S; Chen, Zijing; Dordick, Jonathan S; Kane, Ravi S


    Bacteriolytic enzymes often possess a C-terminal binding domain that recognizes specific motifs on the bacterial surface and a catalytic domain that cleaves covalent linkages within the cell wall peptidoglycan. PlyPH, one such lytic enzyme of bacteriophage origin, has been reported to be highly effective against Bacillus anthracis, and can kill up to 99.99% of the viable bacteria. The bactericidal activity of this enzyme, however, appears to be strongly dependent on the age of the bacterial culture. Although highly bactericidal against cells in the early exponential phase, the enzyme is substantially less effective against stationary phase cells, thus limiting its application in real-world settings. We hypothesized that the binding domain of PlyPH may differ in affinity to cells in different Bacillus growth stages and may be primarily responsible for the age-restricted activity. We therefore employed an in silico approach to identify phage lysins differing in their specificity for the bacterial cell wall. Specifically we focused our attention on Plyβ, an enzyme with improved cell wall-binding ability and age-independent bactericidal activity. Although PlyPH and Plyβ have dissimilar binding domains, their catalytic domains are highly homologous. We characterized the biocatalytic mechanism of Plyβ by identifying the specific bonds cleaved within the cell wall peptidoglycan. Our results provide an example of the diversity of phage endolysins and the opportunity for these biocatalysts to be used for broad-based protection from bacterial pathogens.

  8. Inhibition of Histone Deacetylation and DNA Methylation Improves Gene Expression Mediated by the Adeno-Associated Virus/Phage in Cancer Cells

    Directory of Open Access Journals (Sweden)

    Amin Hajitou


    Full Text Available Bacteriophage (phage, viruses that infect bacteria only, have become promising vectors for targeted systemic delivery of genes to cancer, although, with poor efficiency. We previously designed an improved phage vector by incorporating cis genetic elements of adeno-associated virus (AAV. This novel AAV/phage hybrid (AAVP specifically targeted systemic delivery of therapeutic genes into tumors. To advance the AAVP vector, we recently introduced the stress-inducible Grp78 tumor specific promoter and found that this dual tumor-targeted AAVP provides persistent gene expression, over time, in cancer cells compared to silenced gene expression from the CMV promoter in the parental AAVP. Herein, we investigated the effect of histone deacetylation and DNA methylation on AAVP-mediated gene expression in cancer cells and explored the effect of cell confluence state on AAVP gene expression efficacy. Using a combination of AAVP expressing the GFP reporter gene, flow cytometry, inhibitors of histone deacetylation, and DNA methylation, we have demonstrated that histone deacetylation and DNA methylation are associated with silencing of gene expression from the CMV promoter in the parental AAVP. Importantly, inhibitors of histone deacetylases boost gene expression in cancer cells from the Grp78 promoter in the dual tumor-targeted AAVP. However, cell confluence had no effect on AAVP-guided gene expression. Our findings prove that combination of histone deacetylase inhibitor drugs with the Grp78 promoter is an effective approach to improve AAVP-mediated gene expression in cancer cells and should be considered for AAVP-based clinical cancer gene therapy.

  9. Selection and characterization of novel DNA aptamers specifically recognized by Singapore grouper iridovirus-infected fish cells. (United States)

    Li, Pengfei; Wei, Shina; Zhou, Lingli; Yang, Min; Yu, Yepin; Wei, Jingguang; Jiang, Guohua; Qin, Qiwei


    Singapore grouper iridovirus (SGIV) is a major viral pathogen of grouper aquaculture, and has caused heavy economic losses in China and South-east Asia. In this study, we generated four ssDNA aptamers against SGIV-infected grouper spleen (GS) cells using SELEX (systematic evolution of ligands by exponential enrichment) technology. Four aptamers exhibited high affinity to SGIV-infected GS cells, in particular the Q2 aptamer. Q2 had a binding affinity of 12.09 nM, the highest of the four aptamers. These aptamers also recognized SGIV-infected tissues with high levels of specificity. Protease treatment and flow cytometry analysis of SGIV-infected cells revealed that the target molecules of the Q3, Q4 and Q5 aptamers were trypsin-sensitive proteins, whilst the target molecules of Q2 might be membrane lipids or surface proteins that were not trypsin-sensitive. The generated aptamers appeared to inhibit SGIV infection in vitro. Aptamer Q2 conferred the highest levels of protection against SGIV and was able to inhibit SGIV infection in a dose-dependent manner. In addition, Q2 was efficiently internalized by SGIV-infected GS cells and localized at the viral assembly sites. Our results demonstrated that the four novel aptamers we generated were specific for SGIV-infected cells and could potentially be applied as rapid molecular diagnostic test reagents or therapeutic drugs targeting SGIV.

  10. Canine and feline parvoviruses preferentially recognize the non-human cell surface sialic acid N-glycolylneuraminic acid

    Energy Technology Data Exchange (ETDEWEB)

    Löfling, Jonas [Departments of Medicine and Cellular and Molecular Medicine, Glycobiology Research and Training Center, Center for Academic Research and Training in Anthropogeny, 9500 Gilman Drive, University of California, San Diego, La Jolla, CA 92093 (United States); Michael Lyi, Sangbom; Parrish, Colin R. [Baker Institute for Animal Health, Department of Microbiology and Immunology, College of Veterinary Medicine, Cornell University, Ithaca, NY 14853 (United States); Varki, Ajit, E-mail: [Departments of Medicine and Cellular and Molecular Medicine, Glycobiology Research and Training Center, Center for Academic Research and Training in Anthropogeny, 9500 Gilman Drive, University of California, San Diego, La Jolla, CA 92093 (United States)


    Feline panleukopenia virus (FPV) is a pathogen whose canine-adapted form (canine parvovirus (CPV)) emerged in 1978. These viruses infect by binding host transferrin receptor type-1 (TfR), but also hemagglutinate erythrocytes. We show that hemagglutination involves selective recognition of the non-human sialic acid N-glycolylneuraminic acid (Neu5Gc) but not N-acetylneuraminic acid (Neu5Ac), which differs by only one oxygen atom from Neu5Gc. Overexpression of α2-6 sialyltransferase did not change binding, indicating that both α2-3 and α2-6 linkages are recognized. However, Neu5Gc expression on target cells did not enhance CPV or FPV infection in vitro. Thus, the conserved Neu5Gc-binding preference of these viruses likely plays a role in the natural history of the virus in vivo. Further studies must clarify relationships between virus infection and host Neu5Gc expression. As a first step, we show that transcripts of CMAH (which generates Neu5Gc from Neu5Ac) are at very low levels in Western dog breed cells. - Highlights: ► Feline and canine parvoviruses recognize Neu5Gc but not Neu5Ac, which differ by one oxygen atom. ► The underlying linkage of these sialic acids does not affect recognition. ► Induced Neu5Gc expression on target cells that normally express Neu5Ac did not enhance infection. ► Thus, the conserved binding preference plays an important yet unknown role in in vivo infections. ► Population and breed variations in Neu5Gc expression occur, likely by regulating the gene CMAH.

  11. The majority of human memory B cells recognizing RhD and tetanus resides in IgM+ B cells. (United States)

    Della Valle, Luciana; Dohmen, Serge E; Verhagen, Onno J H M; Berkowska, Magdalena A; Vidarsson, Gestur; Ellen van der Schoot, C


    B cell memory to T cell-dependent (TD) Ags are considered to largely reside in class-switched CD27(+) cells. However, we previously observed that anti-RhD (D) Igs cloned from two donors, hyperimmunized with D(+) erythrocytes, were predominantly of the IgM isotype. We therefore analyzed in this study the phenotype and frequency of D- and tetanus toxoid-specific B cells by culturing B cells in limiting dilution upon irradiated CD40L-expressing EL4.B5 cells and testing the culture supernatant. Most Ag-specific B cells for both TD Ags were found to reside in the IgM-expressing B cells, including CD27(-) B cells, in both hyperimmunized donors and nonhyperimmunized volunteers. Only shortly after immunization a sharp increase in Ag-specific CD27(+)IgG(+) B cells was observed. Next, B cells were enriched with D(+) erythrocyte ghosts and sorted as single cells. Sequencing of IGHV, IGLV, IGKV, and BCL6 genes from these D-specific B cell clones demonstrated that both CD27(-)IgM(+) and CD27(+)IgM(+) B cells harbored somatic mutations, documenting their Ag-selected nature. Furthermore, sequencing revealed a clonal relationship between the CD27(-)IgM(+), CD27(+)IgM(+), and CD27(+)IgG(+) B cell subsets. These data strongly support the recently described multiple layers of memory B cells to TD Ags in mice, where IgM(+) B cells represent a memory reservoir which can re-enter the germinal center and ensure replenishment of class-switched memory CD27(+) B cells from Ag-experienced precursors.

  12. Endoplasmic reticulum stress causes autophagy and apoptosis leading to cellular redistribution of the autoantigens Ro/Sjögren's syndrome-related antigen A (SSA) and La/SSB in salivary gland epithelial cells. (United States)

    Katsiougiannis, S; Tenta, R; Skopouli, F N


    The aim of this study was to examine the levels of endoplasmic reticulum (ER) stress in minor salivary glands, to investigate the interplay between ER stress-induced autophagy and apoptosis in human salivary gland (HSG) cells and to test the effect of ER stress-induced apoptosis on the cellular redistribution of the two major Sjögren's syndrome (SS) autoantigens Ro/Sjögren's syndrome-related antigen A (SSA) and La/Sjögren's syndrome-related antigen B (SSB). Minor salivary gland biopsies from SS patients and sicca controls were examined by immunohistochemistry for the expression of 78 kDa glucose-regulated protein/binding immunoglobulin protein (GRP78/BiP) as an indicator of unfolded protein response (UPR). HSG cells were treated with thapsigargin (TG) and cell viability, autophagy and apoptosis were assessed. Immunoblot was applied to detect the conversion of LC3I to LC3II and the protein levels of GRP78/BiP and X-box binding protein-1 (XBP-1). Apoptosis was evaluated by a single-stranded DNA enzyme-linked immunosorbent assay (ELISA). Ro/SSA and La/SSB localization was visualized using immunofluorescence. GRP78/BiP was expressed by acinar and ductal epithelial cells in salivary glands of patients and sicca controls. TG treatment induced autophagy, as indicated by enhanced protein expression of LC3II. The protein levels of UPR marker XBP-1 were increased after TG treatment, while GRP78/BiP levels were decreased. TG treatment resulted in induction of HSG apoptosis. Ro/SSA and La/SSB autoantigens were localized predominantly to the cytoplasm in resting cells, while they were redistributed to cell membrane and blebs in the apoptotic cells. In conclusion, ER stress is activated in minor salivary gland epithelial cells from SS patients and controls. ER stress-induced apoptosis in HSG cells leads to cell surface and apoptotic blebs relocalization of Ro/SSA and La/SSB autoantigens.

  13. Dendritic cell are able to differentially recognize Sporothrix schenckii antigens and promote Th1/Th17 response in vitro. (United States)

    Verdan, F F; Faleiros, J C; Ferreira, L S; Monnazzi, L G S; Maia, D C G; Tansine, A; Placeres, M C P; Carlos, I Z; Santos-Junior, R R


    Sporotrichosis is a disease caused by the dimorphic fungus Sporothrix schenckii. The main clinical manifestations occur in the skin, however the number of systemic and visceral cases has increased, especially in immunocompromised patients. Dendritic cells (DCs) are highly capable to recognize the fungus associated data and translate it into differential T cells responses both in vivo and in vitro. Although, the mechanisms involved in the interaction between DCs and S. schenckii are not fully elucidated. The present study investigated the phenotypic and functional changes in bone marrow dendritic cells (BMDCs) stimulated in vitro with the yeast form of S. schenckii or exoantigen (ExoAg) and its ability to trigger a cellular immune response in vitro. Our results demonstrated that the live yeast of S. schenckii and its exoantigen, at a higher dose, were able to activate BMDCs and made them capable of triggering T cell responses in vitro. Whereas the yeast group promoted more pronounced IFN-γ production rather than IL-17, the Exo100 group generated similar production of both cytokines. The exoantigen stimulus suggests a capability to deviate the immune response from an effector Th1 to an inflammatory Th17 response. Interestingly, only the Exo100 group promoted the production of IL-6 and a significant increase of TGF-β, in addition to IL-23 production. Interestingly, only Exo100 group was capable to promote the production of IL-6 and a significant increase on TGF-β, in addition with IL-23 detection. Our results demonstrated the plasticity of DCs in translating the data associated with the fungus S. schenckii and ExoAg into differential T cell responses in vitro. The possibility of using ex vivo-generated DCs as vaccinal and therapeutic tools for sporotrichosis is a challenge for the future.

  14. FOXO3-mediated up-regulation of Bim contributes to rhein-induced cancer cell apoptosis. (United States)

    Wang, Jiao; Liu, Shu; Yin, Yancun; Li, Mingjin; Wang, Bo; Yang, Li; Jiang, Yangfu


    The anthraquinone compound rhein is a natural agent in the traditional Chinese medicine rhubarb. Preclinical studies demonstrate that rhein has anticancer activity. Treatment of a variety of cancer cells with rhein may induce apoptosis. Here, we report that rhein induces atypical unfolded protein response in breast cancer MCF-7 cells and hepatoma HepG2 cells. Rhein induces CHOP expression, eIF2α phosphorylation and caspase cleavage, while it does not induce glucose-regulated protein 78 (GRP78) expression in both MCF-7 and HepG2 cells. Meanwhile, rhein inhibits thapsigargin-induced GRP78 expression and X box-binding protein 1 splicing. In addition, rhein inhibits Akt phosphorylation and stimulates FOXO transactivation activity. Rhein induces Bim expression in MCF-7 and HepG2 cells, which can be abrogated by FOXO3a knockdown. Knockdown of FOXO3a or Bim abrogates rhein-induced caspase cleavage and apoptosis. The chemical chaperone 4-phenylbutyrate acid antagonizes the induction of FOXO activation, Bim expression and caspase cleavage by rhein, indicating that protein misfolding may be involved in triggering these deleterious effects. We conclude that FOXO3a-mediated up-regulation of Bim is a key mechanism underlying rhein-induced cancer cells apoptosis.

  15. Generation of human scFvs antibodies recognizing a prion protein epitope expressed on the surface of human lymphoblastoid cells

    Directory of Open Access Journals (Sweden)

    Imperiale Valentina


    Full Text Available Abstract Background A hallmark of prion disease is the transformation of normal cellular prion protein (PrPc into an infectious disease-associated isoform, (PrPsc. Anti-prion protein monoclonal antibodies are invaluable for structure-function studies of PrP molecules. Furthermore recent in vitro and in vivo studies indicate that anti-PrP monoclonal antibodies can prevent the incorporation of PrPc into propagating prions. In the present article, we show two new human phage antibodies, isolated on recombinant hamster prion protein (rHaPrP. Results We adopted an antibody phage display strategy to isolate specific human antibodies directed towards rHaPrP which has been used as a bait for panning the synthetic ETH-2 antibody phage library. Two phage antibodies clones named MA3.B4 and MA3.G3 were isolated and characterized under genetic biochemical and immunocytochemical aspects. The clones were found to recognize the prion protein in ELISA studies. In flow-cytometry studies, these human single chain Fragment variable (scFv phage-antibodies show a well defined pattern of reactivity on human lymphoblastoid and myeloid cells. Conclusion Sequence analysis of the gene encoding for the antibody fragments and antigen recognition patterns determined by flow-cytometry analysis indicate that the isolated scFvs recognize novel epitopes in the PrPc molecule. These new anti PrPc human antibodies are unique reagents for prion protein detection and may represent a biologic platform to develop new reagents to treat PrPsc associated disease.

  16. Canine and feline parvoviruses preferentially recognize the non-human cell surface sialic acid N-glycolylneuraminic acid. (United States)

    Löfling, Jonas; Lyi, Sangbom Michael; Parrish, Colin R; Varki, Ajit


    Feline panleukopenia virus (FPV) is a pathogen whose canine-adapted form (canine parvovirus (CPV)) emerged in 1978. These viruses infect by binding host transferrin receptor type-1 (TfR), but also hemagglutinate erythrocytes. We show that hemagglutination involves selective recognition of the non-human sialic acid N-glycolylneuraminic acid (Neu5Gc) but not N-acetylneuraminic acid (Neu5Ac), which differs by only one oxygen atom from Neu5Gc. Overexpression of α2-6 sialyltransferase did not change binding, indicating that both α2-3 and α2-6 linkages are recognized. However, Neu5Gc expression on target cells did not enhance CPV or FPV infection in vitro. Thus, the conserved Neu5Gc-binding preference of these viruses likely plays a role in the natural history of the virus in vivo. Further studies must clarify relationships between virus infection and host Neu5Gc expression. As a first step, we show that transcripts of CMAH (which generates Neu5Gc from Neu5Ac) are at very low levels in Western dog breed cells.

  17. Effects of phycoerythrin from Gracilaria lemaneiformis in proliferation and apoptosis of SW480 cells. (United States)

    Li, Peizhen; Ying, Jun; Chang, Qingli; Zhu, Wen; Yang, Guangjian; Xu, Teng; Yi, Huiguang; Pan, Ruowang; Zhang, Enyong; Zeng, Xiaofeng; Yan, Chunxia; Bao, Qiyu; Li, Shengbin


    We studied phycoerythrin (PE) in human SW480 tumor cells and the underlying molecular mechanisms of action. PE inhibited cell proliferation as evidenced by CCK-8 assay. The IC50 values of phycoerythrin were 48.2 and 27.4 µg/ml for 24 and 48 h of exposure, respectively. PE induced apoptosis and cell cycle arrest in SW480 cells as observed under electron microscopy and with flow cytometry. Apoptosis increased from 5.1 (controls) to 39.0% in 80.0 µg/ml PE-treated cells. Differences in protein expression were identified using proteomic techniques. Protein spots (1018±60 and 1010±60) were resolved in PE-treated and untreated group. Forty differential protein spots were analyzed with MALDI-TOF-MS, including GRP78 and NPM1. The expression as measured by qPCR and western blotting agreed with data from two-dimensional electrophoresis. GRP78, NPM1, MTHSP75, Ezrin and Annexin A2 were decreased and HSP60 was increased after PE treatment, indicating that PE may target multiple proteins to induce apoptosis.

  18. A sensor kinase recognizing the cell-cell signal BDSF (cis-2-dodecenoic acid) regulates virulence in Burkholderia cenocepacia

    DEFF Research Database (Denmark)

    McCarthy, Y.; Yang, Liang; Twomey, K.B.;


    the input domain of RpfC was active in BDSF signal perception when expressed in X. campestris. Mutation of BCAM0227 gave rise to reduced cytotoxicity to Chinese hamster ovary cells and reduced virulence to Wax moth larvae and in the agar-bead mouse model of pulmonary infection. The findings identify BCAM...

  19. Endoplasmic Reticulum Stress Participates in Intense Noise-Induced Cochlea Spiral Ganglion Cell Apoptosis in Guinea Pig%内质网应激反应参与强噪声诱导豚鼠耳蜗螺旋神经节细胞凋亡的研究

    Institute of Scientific and Technical Information of China (English)

    薛秋红; 何坚; 陈佳; 龚树生; 谢静


    Objective: To explore whether intense noise can induce spiral ganglion cell (SGC) death in guinea pig's cochlea via the apoptotic pathway, to investigate the expression changes of signal conduct protein Caspase-12, endoplasmic reticulum-related factor Bip/Grp78 and CHOP/Gaddl53 after intense noise expose, and to find out the relationship between endoplasmic reticulum stress (ERS) and cochlea cell damage after intense noise expose. Methods: Twenty guinea pigs were randomly divided into four groups. The guinea pigs in the experiment groups were exposed to 4 kHz narrow band noise at 120 dB SPL for 4 h. Auditory brainstem response (ABR) were tested after the noise expose for 1,4, and 14 day(s) respectively. Apoptosis was assayed or observed by TUNEL and transmission electron microscopy, and expression of Caspase-12, Bip/Grp78, and CHOP/Gaddl53 were determined by immunohistochemical method. Results: The observation by electron microscopy showed the features of apoptotic cells in the SGCs in all experiment groups, while no apoptotic cell in the control was found. There were more TUNEL-positive cells in the SGCs in all the experiment groups than in control group (all P<0. 01). Protein levels of Bip/Grp78, Caspase-12 and CHOP/Gaddl53 were risen up after noise expose in all the three experiment groups (all P<0. 01). Conclusion; Intense noise causes SGC damage by inducing apoptosis, and Caspase-12 induced ER stress-related apoptosis plays an important role in the procedure of apoptosis.%目的:探讨强噪声能否诱导豚鼠耳蜗螺旋神经节细胞(SGC)的凋亡,及观察凋亡蛋白酶Caspase-12、内质网应激相关因子Bip/Grp78和CHOP/Gadd153在强噪声暴露后豚鼠耳蜗SGC中的表达,探讨内质网应激在强噪声暴露后豚鼠耳蜗SGC损伤中的作用.方法:选用健康雄性白色豚鼠20只,将实验组豚鼠暴露在4 kHz窄带噪声120 dB SPL噪声环境中4h,噪声刺激停止后1,4,14d组及对照组在处死前测ABR(每组各5只).通过透

  20. TLR4 and DC-SIGN receptors recognized Mycobacterium scrofulaceum promoting semi-activated phenotype on bone marrow dendritic cells. (United States)

    Cruz-Aguilar, Marisa; Castillo-Rodal, Antonia I; Schcolnik-Cabrera, Alejandro; Bonifaz, Laura C; Molina, Gabriela; López-Vidal, Yolanda


    Nontuberculous mycobacteria (NTM) are recognized as emerging pathogens and their immune regulatory mechanisms are not well described yet. From them, Mycobacterium avium is known to be a weak activator of dendritic cells (DCs) that impairs the response induced by BCG vaccine. However, whether other NTM such as Mycobacterium scrofulaceum may modulate the activation of DCs, has not been extensively studied. Here, we exposed bone marrow-derived DCs (BMDCs) to M. scrofulaceum and we analyzed the effect on the activation of DCs. We found that M. scrofulaceum has a comparable ability to induce a semi-mature DC phenotype, which was produced by its interaction with DC-SIGN and TLR4 receptors in a synergic effect. BMDCs exposed to M. scrofulaceum showed high expression of PD-L2 and production of IL-10, as well as low levels of co-stimulatory molecules and pro-inflammatory cytokines. In addition to immunophenotype induced on DCs, changes in morphology, re-organization of cytoskeleton and decreased migratory capacity are consistent with a semi-mature phenotype. However, unlike other pathogenic mycobacteria, the DC-semi-mature phenotype induced by M. scrofulaceum was reversed after re-exposure to BCG, suggesting that modulation mechanisms of DC-activation used by M. scrofulaceum are different to other known pathogenic mycobacteria. This is the first report about the immunophenotypic characterization of DC stimulated by M. scrofulaceum.

  1. Dectin-2 Recognizes Mannosylated O-antigens of Human Opportunistic Pathogens and Augments Lipopolysaccharide Activation of Myeloid Cells* (United States)

    Wittmann, Alexandra; Lamprinaki, Dimitra; Bowles, Kristian M.; Katzenellenbogen, Ewa; Knirel, Yuriy A.; Whitfield, Chris; Nishimura, Takashi; Matsumoto, Naoki; Yamamoto, Kazuo; Iwakura, Yoichiro; Saijo, Shinobu; Kawasaki, Norihito


    LPS consists of a relatively conserved region of lipid A and core oligosaccharide and a highly variable region of O-antigen polysaccharide. Whereas lipid A is known to bind to the Toll-like receptor 4 (TLR4)-myeloid differentiation factor 2 (MD2) complex, the role of the O-antigen remains unclear. Here we report a novel molecular interaction between dendritic cell-associated C-type lectin-2 (Dectin-2) and mannosylated O-antigen found in a human opportunistic pathogen, Hafnia alvei PCM 1223, which has a repeating unit of [-Man-α1,3-Man-α1,2-Man-α1,2-Man-α1,2-Man-α1,3-]. H. alvei LPS induced higher levels of TNFα and IL-10 from mouse bone marrow-derived dendritic cells (BM-DCs), when compared with Salmonella enterica O66 LPS, which has a repeat of [-Gal-α1,6-Gal-α1,4-[Glc-β1,3]GalNAc-α1,3-GalNAc-β1,3-]. In a cell-based reporter assay, Dectin-2 was shown to recognize H. alvei LPS. This binding was inhibited by mannosidase treatment of H. alvei LPS and by mutations in the carbohydrate-binding domain of Dectin-2, demonstrating that H. alvei LPS is a novel glycan ligand of Dectin-2. The enhanced cytokine production by H. alvei LPS was Dectin-2-dependent, because Dectin-2 knock-out BM-DCs failed to do so. This receptor cross-talk between Dectin-2 and TLR4 involved events including spleen tyrosine kinase (Syk) activation and receptor juxtaposition. Furthermore, another mannosylated LPS from Escherichia coli O9a also bound to Dectin-2 and augmented TLR4 activation of BM-DCs. Taken together, these data indicate that mannosylated O-antigens from several Gram-negative bacteria augment TLR4 responses through interaction with Dectin-2. PMID:27358401

  2. A novel antibody for human induced pluripotent stem cells and embryonic stem cells recognizes a type of keratan sulfate lacking oversulfated structures. (United States)

    Kawabe, Keiko; Tateyama, Daiki; Toyoda, Hidenao; Kawasaki, Nana; Hashii, Noritaka; Nakao, Hiromi; Matsumoto, Shogo; Nonaka, Motohiro; Matsumura, Hiroko; Hirose, Yoshinori; Morita, Ayaha; Katayama, Madoka; Sakuma, Makoto; Kawasaki, Nobuko; Furue, Miho Kusuda; Kawasaki, Toshisuke


    We have generated a monoclonal antibody (R-10G) specific to human induced pluripotent stem (hiPS)/embryonic stem (hES) cells by using hiPS cells (Tic) as an antigen, followed by differential screening of mouse hybridomas with hiPS and human embryonal carcinoma (hEC) cells. Upon western blotting with R-10G, hiPS/ES cell lysates gave a single but an unusually diffuse band at a position corresponding to >250 kDa. The antigen protein was isolated from the induced pluripotent stem (iPS) cell lysates with an affinity column of R-10G. The R-10G positive band was resistant to digestion with peptide N-glycanase F (PNGase F), neuraminidase, fucosidase, chondrotinase ABC and heparinase mix, but it disappeared almost completely on digestion with keratanase, keratanase II and endo-β-galactosidase, indicating that the R-10G epitope is a keratan sulfate. The carrier protein of the R-10G epitope was identified as podocalyxin by liquid chromatography/mass spectrometry (LC/MS/MS) analysis of the R-10G positive-protein band material obtained on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The R-10G epitope is a type of keratan sulfate with some unique properties. (1) The epitope is expressed only on hiPS/ES cells, i.e. not on hEC cells, unlike those recognized by the conventional hiPS/ES marker antibodies. (2) The epitope is a type of keratan sulfate lacking oversulfated structures and is not immunologically cross-reactive with high-sulfated keratan sulfate. (3) The R-10G epitope is distributed heterogeneously on hiPS cells, suggesting that a single colony of undifferentiated hiPS cells consists of different cell subtypes. Thus, R-10G is a novel antibody recognizing hiPS/ES cells, and should be a new molecular probe for disclosing the roles of glycans on these cells.

  3. Involvement of necroptosis, a newly recognized cell death type, in steroid-induced osteonecrosis in a rabbit model (United States)

    Ichiseki, Toru; Ueda, Shusuke; Ueda, Yoshimichi; Tuchiya, Masanobu; Kaneuji, Ayumi; Kawahara, Norio


    We investigated the role of programmed necrosis (necroptosis), a newly recognized form of cell necrosis that has been implicated in the development of steroid-induced osteonecrosis. We used an osteonecrosis model in which 30 Japanese white rabbits each weighing 3.5kg were injected once with methylprednisolone at 20 mg/kg body weight into the right gluteal muscle. Ten animals killed 14 days thereafter were designated as S14d groups, while another 10 animals injected with necroptosis, a specific inhibitor of necrostatin-1 i.v. at 1.65mg/kg on the same day as the steroid were also killed on the 14th day and designated as SN14d group. As a control, 10 animals injected only with physiological saline were studied as N group. After the animals were sacrificed the bilateral femoral bone was examined histopathologically and the presence of osteonecrosis determined. Furthermore, animals subjected to the same treatment and killed on the 3rd day after drug administration were set up as S3d group and SN3d group, and Western blotting of Receptor-interacting protein ( RIP ) 1 and RIP3 in femoral bone performed. The osteonecrosis rate was 70% in S14d group, and 0% in both N and SN groups. In 2 of 10 animals in SN group fatty marrow was found. On Western blotting significantly increased expression of both RIP1 and RIP3 was noted in S3d group, confirming that Nec-1 was suppressed. Necroptosis mediated by RIP1 and RIP3 expression was thought to be implicated in the development of steroid-induced osteonecrosis. Also, by suppressing expression of RIP1 and 3 with the administration of Nec-1 the osteonecrosis rate was significantly decreased. These results suggest that necroptosis may have potential as a novel target for both elucidating the mechanisms underlying steroid-induced osteonecrosis and establishing more effective prophylactic countermeasures. PMID:28260985

  4. Arctigenin suppresses unfolded protein response and sensitizes glucose deprivation-mediated cytotoxicity of cancer cells. (United States)

    Sun, Shengrong; Wang, Xiong; Wang, Changhua; Nawaz, Ahmed; Wei, Wen; Li, Juanjuan; Wang, Lijun; Yu, De-Hua


    The involvement of unfolded protein response (UPR) activation in tumor survival and resistance to chemotherapies suggests a new anticancer strategy targeting UPR pathway. Arctigenin, a natural product, has been recently identified for its antitumor activity with selective toxicity against cancer cells under glucose starvation with unknown mechanism. Here we found that arctigenin specifically blocks the transcriptional induction of two potential anticancer targets, namely glucose-regulated protein-78 (GRP78) and its analog GRP94, under glucose deprivation, but not by tunicamycin. The activation of other UPR pathways, e.g., XBP-1 and ATF4, by glucose deprivation was also suppressed by arctigenin. A further transgene experiment showed that ectopic expression of GRP78 at least partially rescued arctigenin/glucose starvation-mediated cell growth inhibition, suggesting the causal role of UPR suppression in arctigenin-mediated cytotoxicity under glucose starvation. These observations bring a new insight into the mechanism of action of arctigenin and may lead to the design of new anticancer therapeutics.

  5. Chaperone-Targeting Cytotoxin and Endoplasmic Reticulum Stress-Inducing Drug Synergize to Kill Cancer Cells

    Directory of Open Access Journals (Sweden)

    Joseph M. Backer


    Full Text Available Diverse physiological and therapeutic insults that increase the amount of unfolded or misfolded proteins in the endoplasmic reticulum (ER induce the unfolded protein response, an evolutionarily conserved protective mechanism that manages ER stress. Glucose-regulated protein 78/immunoglobulin heavy-chain binding protein (GRP78/BiP is an ER-resident protein that plays a central role in the ER stress response and is the only known substrate of the proteolytic A subunit (SubA of a novel bacterial AB5 toxin. Here, we report that an engineered fusion protein, epidermal growth factor (EGF-SubA, combining EGF and SubA, is highly toxic to growing and confluent epidermal growth factor receptor-expressing cancer cells, and its cytotoxicity is mediated by a remarkably rapid cleavage of GRP78/BiP. Systemic delivery of EGF-SubA results in a significant inhibition of human breast and prostate tumor xenografts in mouse models. Furthermore, EGF-SubA dramatically increases the sensitivity of cancer cells to the ER stress-inducing drug thapsigargin, and vice versa, demonstrating the first example of mechanism-based synergism in the action of a cytotoxin and an ER-targeting drug.

  6. Amaranthus leucocarpus lectin recognizes a moesin-like O-glycoprotein and costimulates murine CD3-activated CD4+ T cells (United States)

    Arenas-Del Ángel, Maria; Legorreta-Herrera, Martha; Mendoza-Hernández, Guillermo; Garfias, Yonathan; Chávez, Raul; Zenteno, Edgar; Lascurain, Ricardo


    The Galβ1,3GalNAcα1,O-Ser/Thr specific lectin from Amaranthus leucocarpus (ALL) binds a ∼70 kDa glycoprotein on murine T cell surface. We show that in the absence of antigen presenting cells, murine CD4+ T cells activated by an anti-CD3 antibody plus ALL enhanced cell proliferation similar to those cells activated via CD3/CD28 at 48 h of culture. Moreover, ALL induced the production of IL-4, IL-10, TNF-alpha, and TGF-beta in CD3-activated cells. Proteomic assay using two-dimensional electrophoresis and far-Western blotting, ALL recognized two prominent proteins associated to the lipid raft microdomains in CD3/CD28-activated CD4+ T cells. By mass spectrometry, the peptide fragments from ALL-recognized proteins showed sequences with 33% homology to matricin (gi|347839 NCBInr) and 41% identity to an unnamed protein related to moesin (gi|74186081 NCBInr). Confocal microscopy analysis of CD3/CD28-activated CD4+ T cells confirmed that staining by ALL colocalized with anti-moesin FERM domain antibody along the plasma membrane and in the intercellular contact sites. Our findings suggest that a moesin-like O-glycoprotein is the ALL-recognized molecule in lipid rats, which induces costimulatory signals on CD4+ T cells. PMID:26417436

  7. Sulindac sulfide inhibits sarcoendoplasmic reticulum Ca2+ ATPase, induces endoplasmic reticulum stress response, and exerts toxicity in glioma cells: relevant similarities to and important differences from celecoxib. (United States)

    White, M C; Johnson, G G; Zhang, W; Hobrath, J V; Piazza, G A; Grimaldi, M


    Malignant gliomas have low survival expectations regardless of current treatments. Nonsteroidal anti-inflammatory drugs (NSAIDs) prevent cell transformation and slow cancer cell growth by mechanisms independent of cyclooxygenase (COX) inhibition. Certain NSAIDs trigger the endoplasmic reticulum stress response (ERSR), as revealed by upregulation of molecular chaperones such as GRP78 and C/EBP homologous protein (CHOP). Although celecoxib (CELE) inhibits the sarcoendoplasmic reticulum Ca(2+) ATPase (SERCA), an effect known to induce ERSR, sulindac sulfide (SS) has not been reported to affect SERCA. Here, we investigated these two drugs for their effects on Ca(2+) homeostasis, ERSR, and glioma cell survival. Our findings indicate that SS is a reversible inhibitor of SERCA and that both SS and CELE bind SERCA at its cyclopiazonic acid binding site. Furthermore, CELE releases additional Ca(2+) from the mitochondria. In glioma cells, both NSAIDS upregulate GRP78 and activate ER-associated caspase-4 and caspase-3. Although only CELE upregulates the expression of CHOP, it appears that CHOP induction could be associated with mitochondrial poisoning. In addition, CHOP induction appears to be uncorrelated with the gliotoxicity of these NSAIDS in our experiments. Our data suggest that activation of ERSR is primarily responsible for the gliotoxic effect of these NSAIDS. Because SS has good brain bioavailability, has lower COX-2 inhibition, and has no mitochondrial effects, it represents a more appealing molecular candidate than CELE to achieve gliotoxicity via activation of ERSR.

  8. Identification of prostate-specific G-protein coupled receptor as a tumor antigen recognized by CD8(+ T cells for cancer immunotherapy.

    Directory of Open Access Journals (Sweden)

    Satoko Matsueda

    Full Text Available BACKGROUND: Prostate cancer is the most common cancer among elderly men in the US, and immunotherapy has been shown to be a promising strategy to treat patients with metastatic castration-resistant prostate cancer. Efforts to identify novel prostate specific tumor antigens will facilitate the development of effective cancer vaccines against prostate cancer. Prostate-specific G-protein coupled receptor (PSGR is a novel antigen that has been shown to be specifically over-expressed in human prostate cancer tissues. In this study, we describe the identification of PSGR-derived peptide epitopes recognized by CD8(+ T cells in an HLA-A2 dependent manner. METHODOLOGY/PRINCIPAL FINDINGS: Twenty-one PSGR-derived peptides were predicted by an immuno-informatics approach based on the HLA-A2 binding motif. These peptides were examined for their ability to induce peptide-specific T cell responses in peripheral blood mononuclear cells (PBMCs obtained from either HLA-A2(+ healthy donors or HLA-A2(+ prostate cancer patients. The recognition of HLA-A2 positive and PSGR expressing LNCaP cells was also tested. Among the 21 PSGR-derived peptides, three peptides, PSGR3, PSGR4 and PSGR14 frequently induced peptide-specific T cell responses in PBMCs from both healthy donors and prostate cancer patients. Importantly, these peptide-specific T cells recognized and killed LNCaP prostate cancer cells in an HLA class I-restricted manner. CONCLUSIONS/SIGNIFICANCE: We have identified three novel HLA-A2-restricted PSGR-derived peptides recognized by CD8(+ T cells, which, in turn, recognize HLA-A2(+ and PSGR(+ tumor cells. The PSGR-derived peptides identified may be used as diagnostic markers as well as immune targets for development of anticancer vaccines.

  9. Inhibition of HSP90 by AUY922 Preferentially Kills Mutant KRAS Colon Cancer Cells by Activating Bim through ER Stress. (United States)

    Wang, Chun Yan; Guo, Su Tang; Wang, Jia Yu; Liu, Fen; Zhang, Yuan Yuan; Yari, Hamed; Yan, Xu Guang; Jin, Lei; Zhang, Xu Dong; Jiang, Chen Chen


    Oncogenic mutations of KRAS pose a great challenge in the treatment of colorectal cancer. Here we report that mutant KRAS colon cancer cells are nevertheless more susceptible to apoptosis induced by the HSP90 inhibitor AUY922 than those carrying wild-type KRAS. Although AUY922 inhibited HSP90 activity with comparable potency in colon cancer cells irrespective of their KRAS mutational statuses, those with mutant KRAS were markedly more sensitive to AUY922-induced apoptosis. This was associated with upregulation of the BH3-only proteins Bim, Bik, and PUMA. However, only Bim appeared essential, in that knockdown of Bim abolished, whereas knockdown of Bik or PUMA only moderately attenuated apoptosis induced by AUY922. Mechanistic investigations revealed that endoplasmic reticulum (ER) stress was responsible for AUY922-induced upregulation of Bim, which was inhibited by a chemical chaperone or overexpression of GRP78. Conversely, siRNA knockdown of GRP78 or XBP-1 enhanced AUY922-induced apoptosis. Remarkably, AUY922 inhibited the growth of mutant KRAS colon cancer xenografts through activation of Bim that was similarly associated with ER stress. Taken together, these results suggest that AUY922 is a promising drug in the treatment of mutant KRAS colon cancers, and the agents that enhance the apoptosis-inducing potential of Bim may be useful to improve the therapeutic efficacy.

  10. Th1-like human T-cell clones recognizing Leishmania gp63 inhibit Leishmania major in human macrophages

    DEFF Research Database (Denmark)

    Kemp, M; Hey, A S; Bendtzen, K


    The major surface protease of Leishmania major, gp63, has been suggested as a vaccine candidate for cutaneous leishmaniasis. In this study gp63 was purified from L. major promastigotes. A panel of human T-cell clones recognizing this protein were generated from individuals who had previously had...... self-healing cutaneous leishmaniasis. The T-cell clones expressed CD4, and the alpha chain of the T-cell antigen receptor. GP63 reactive T-cell clones activated by antigen or by immobilized anti-CD3 antibody released relative large amounts of interferon-gamma and no or little interleukin-4, thereby...

  11. γδ T cells recognize the insulin B:9-23 peptide antigen when it is dimerized through thiol oxidation. (United States)

    Aydintug, M Kemal; Zhang, Li; Wang, Chao; Liang, Dongchun; Wands, J M; Michels, Aaron W; Hirsch, Brooke; Day, Brian J; Zhang, Gongyi; Sun, Deming; Eisenbarth, George S; O'Brien, Rebecca L; Born, Willi K


    The insulin peptide B:9-23 is a natural antigen in the non-obese diabetic (NOD) mouse model of type 1 diabetes (T1D). In addition to αβ T cells and B cells, γδ T cells recognize the peptide and infiltrate the pancreatic islets where the peptide is produced within β cells. The peptide contains a cysteine in position 19 (Cys19), which is required for the γδ but not the αβ T cell response, and a tyrosine in position 16 (Tyr16), which is required for both. A peptide-specific mAb, tested along with the T cells, required neither of the two amino acids to bind the B:9-23 peptide. We found that γδ T cells require Cys19 because they recognize the peptide antigen in an oxidized state, in which the Cys19 thiols of two peptide molecules form a disulfide bond, creating a soluble homo-dimer. In contrast, αβ T cells recognize the peptide antigen as a reduced monomer, in complex with the MHCII molecule I-A(g7). Unlike the unstructured monomeric B:9-23 peptide, the γδ-stimulatory homo-dimer adopts a distinct secondary structure in solution, which differs from the secondary structure of the corresponding portion of the native insulin molecule. Tyr16 is required for this adopted structure of the dimerized insulin peptide as well as for the γδ response to it. This observation is consistent with the notion that γδ T cell recognition depends on the secondary structure of the dimerized insulin B:9-23 antigen.

  12. Primary B Lymphocytes Infected with Kaposi's Sarcoma-Associated Herpesvirus Can Be Expanded In Vitro and Are Recognized by LANA-Specific CD4+ T Cells (United States)

    Nicol, Samantha M.; Sabbah, Shereen; Brulois, Kevin F.; Jung, Jae U.; Bell, Andrew I.


    ABSTRACT Kaposi's sarcoma-associated herpesvirus (KSHV) has tropism for B lymphocytes, in which it establishes latency, and can also cause lymphoproliferative disorders of these cells manifesting as primary effusion lymphoma (PEL) and multicentric Castleman disease (MCD). T cell immunity is vital for the control of KSHV infection and disease; however, few models of B lymphocyte infection exist to study immune recognition of such cells. Here, we developed a model of B lymphocyte infection with KSHV in which infected tonsillar B lymphocytes were expanded by providing mitogenic stimuli and then challenged with KSHV-specific CD4+ T cells. The infected cells expressed viral proteins found in PELs, namely, LANA and viral IRF3 (vIRF3), albeit at lower levels, with similar patterns of gene expression for the major latency, viral interleukin 6 (vIL-6), and vIRF3 transcripts. Despite low-level expression of open reading frame 50 (ORF50), transcripts for the immune evasion genes K3 and K5 were detected, with some downregulation of cell surface-expressed CD86 and ICAM. The vast majority of infected lymphocytes expressed IgM heavy chains with Igλ light chains, recapitulating the features seen in infected cells in MCD. We assessed the ability of the infected lymphocytes to be targeted by a panel of major histocompatibility complex (MHC) class II-matched CD4+ T cells and found that LANA-specific T cells restricted to different epitopes recognized these infected cells. Given that at least some KSHV latent antigens are thought to be poor targets for CD8+ T cells, we suggest that CD4+ T cells are potentially important effectors for the in vivo control of KSHV-infected B lymphocytes. IMPORTANCE KSHV establishes a latent reservoir within B lymphocytes, but few models exist to study KSHV-infected B cells other than the transformed PEL cell lines, which have likely accrued mutations during the transformation process. We developed a model of KSHV-infected primary B lymphocytes that

  13. In type 1 diabetes a subset of anti-coxsackievirus B4 antibodies recognize autoantigens and induce apoptosis of pancreatic beta cells.

    Directory of Open Access Journals (Sweden)

    Caterina Bason

    Full Text Available Type 1 diabetes is characterized by autoimmune destruction of pancreatic beta cells. The role played by autoantibodies directed against beta cells antigens in the pathogenesis of the disease is still unclear. Coxsackievirus B infection has been linked to the onset of type 1 diabetes; however its precise role has not been elucidated yet. To clarify these issues, we screened a random peptide library with sera obtained from 58 patients with recent onset type 1 diabetes, before insulin therapy. We identified an immunodominant peptide recognized by the majority of individual patients'sera, that shares homology with Coxsackievirus B4 VP1 protein and with beta-cell specific autoantigens such as phogrin, phosphofructokinase and voltage-gated L-type calcium channels known to regulate beta cell apoptosis. Antibodies against the peptide affinity-purified from patients' sera, recognized the viral protein and autoantigens; moreover, such antibodies induced apoptosis of the beta cells upon binding the L-type calcium channels expressed on the beta cell surface, suggesting a calcium dependent mechanism. Our results provide evidence that in autoimmune diabetes a subset of anti-Coxsackievirus antibodies are able to induce apoptosis of pancreatic beta cells which is considered the most critical and final step in the development of autoimmune diabetes without which clinical manifestations do not occur.


    NARCIS (Netherlands)



    Mice were immunized with purified infectious bronchitis virus (IBV), strain M41. Spleen cells, expanded in vitro by stimulation with M41, were immortalized by fusion to obtain T-cell hybridomas, and two major histocompatability complex (MHC) class II (I-E)-restricted T-cell hybridomas were selected

  15. Cutting edge: Multiple sclerosis-like lesions induced by effector CD8 T cells recognizing a sequestered antigen on oligodendrocytes. (United States)

    Saxena, Amit; Bauer, Jan; Scheikl, Tanja; Zappulla, Jacques; Audebert, Marc; Desbois, Sabine; Waisman, Ari; Lassmann, Hans; Liblau, Roland S; Mars, Lennart T


    CD8 T cells are emerging as important players in multiple sclerosis (MS) pathogenesis, although their direct contribution to tissue damage is still debated. To assess whether autoreactive CD8 T cells can contribute to the pronounced loss of oligodendrocytes observed in MS plaques, we generated mice in which the model Ag influenza hemagglutinin is selectively expressed in oligodendrocytes. Transfer of preactivated hemagglutinin-specific CD8 T cells led to inflammatory lesions in the optic nerve, spinal cord, and brain. These lesions, associating CD8 T cell infiltration with focal loss of oligodendrocytes, demyelination, and microglia activation, were very reminiscent of active MS lesions. Thus, our study demonstrates the potential of CD8 T cells to induce oligodendrocyte lysis in vivo as a likely consequence of direct Ag-recognition. These results provide new insights with regard to CNS tissue damage mediated by CD8 T cells and for understanding the role of CD8 T cells in MS.

  16. Yellow fever vaccination elicits broad functional CD4+ T cell responses that recognize structural and nonstructural proteins. (United States)

    James, Eddie A; LaFond, Rebecca E; Gates, Theresa J; Mai, Duy T; Malhotra, Uma; Kwok, William W


    Yellow fever virus (YFV) can induce acute, life-threatening disease that is a significant health burden in areas where yellow fever is endemic, but it is preventable through vaccination. The live attenuated 17D YFV strain induces responses characterized by neutralizing antibodies and strong T cell responses. This vaccine provides an excellent model for studying human immunity. While several studies have characterized YFV-specific antibody and CD8(+) T cell responses, less is known about YFV-specific CD4(+) T cells. Here we characterize the epitope specificity, functional attributes, and dynamics of YFV-specific T cell responses in vaccinated subjects by investigating peripheral blood mononuclear cells by using HLA-DR tetramers. A total of 112 epitopes restricted by seven common HLA-DRB1 alleles were identified. Epitopes were present within all YFV proteins, but the capsid, envelope, NS2a, and NS3 proteins had the highest epitope density. Antibody blocking demonstrated that the majority of YFV-specific T cells were HLA-DR restricted. Therefore, CD4(+) T cell responses could be effectively characterized with HLA-DR tetramers. Ex vivo tetramer analysis revealed that YFV-specific T cells persisted at frequencies ranging from 0 to 100 cells per million that are detectable years after vaccination. Longitudinal analysis indicated that YFV-specific CD4(+) T cells reached peak frequencies, often exceeding 250 cells per million, approximately 2 weeks after vaccination. As frequencies subsequently declined, YFV-specific cells regained CCR7 expression, indicating a shift from effector to central memory. Cells were typically CXCR3 positive, suggesting Th1 polarization, and produced gamma interferon and other cytokines after reactivation in vitro. Therefore, YFV elicits robust early effector CD4(+) T cell responses that contract, forming a detectable memory population.

  17. Tissue-resident memory CD8+ T cells continuously patrol skin epithelia to quickly recognize local antigen. (United States)

    Ariotti, Silvia; Beltman, Joost B; Chodaczek, Grzegorz; Hoekstra, Mirjam E; van Beek, Anna E; Gomez-Eerland, Raquel; Ritsma, Laila; van Rheenen, Jacco; Marée, Athanasius F M; Zal, Tomasz; de Boer, Rob J; Haanen, John B A G; Schumacher, Ton N


    Recent work has demonstrated that following the clearance of infection a stable population of memory T cells remains present in peripheral organs and contributes to the control of secondary infections. However, little is known about how tissue-resident memory T cells behave in situ and how they encounter newly infected target cells. Here we demonstrate that antigen-specific CD8(+) T cells that remain in skin following herpes simplex virus infection show a steady-state crawling behavior in between keratinocytes. Spatially explicit simulations of the migration of these tissue-resident memory T cells indicate that the migratory dendritic behavior of these cells allows the detection of antigen-expressing target cells in physiologically relevant time frames of minutes to hours. Furthermore, we provide direct evidence for the identification of rare antigen-expressing epithelial cells by skin-patrolling memory T cells in vivo. These data demonstrate the existence of skin patrol by memory T cells and reveal the value of this patrol in the rapid detection of renewed infections at a previously infected site.

  18. Interferon-¿ and interleukin-4 production by human T cells recognizing Leishmania donovani antigens separated by SDS-PAGE

    DEFF Research Database (Denmark)

    Bahrenscheer, J; Kemp, M; Kurtzhals, J A


    of proliferation and interferon-gamma (IFN-gamma) production in cultures of peripheral blood mononuclear cells (PBMC) from individuals who had recovered from visceral leishmaniasis caused by L. donovani. The release of interleukin-4 (IL-4) by PBMC stimulated with the isolated L. donovani antigen fractions...... was infrequently observed. The results show that T cells from individuals who have been cured of visceral leishmaniasis recognize and respond to a wide range of leishmanial antigens. There was no evidence of particular fractions constantly giving either IFN-gamma or IL-4-producing responses....

  19. The four serotypes of dengue recognize the same putative receptors in Aedes aegypti midgut and Ae. albopictus cells

    Directory of Open Access Journals (Sweden)

    Camacho-Nuez Minerva


    Full Text Available Abstract Background Dengue viruses (DENV attach to the host cell surface and subsequently enter the cell by receptor-mediated endocytosis. Several primary and low affinity co-receptors for this flavivirus have been identified. However, the presence of these binding molecules on the cell surface does not necessarily render the cell susceptible to infection. Determination of which of them serve as bona fide receptors for this virus in the vector may be relevant to treating DENV infection and in designing control strategies. Results (1 Overlay protein binding assay showed two proteins with molecular masses of 80 and 67 kDa (R80 and R67. (2 Specific antibodies against these two proteins inhibited cell binding and infection. (3 Both proteins were bound by all four serotypes of dengue virus. (4 R80 and R67 were purified by affinity chromatography from Ae. aegypti mosquito midguts and from Ae albopictus C6/36 cells. (5 In addition, a protein with molecular mass of 57 kDa was purified by affinity chromatography from the midgut extracts. (6 R80 and R67 from radiolabeled surface membrane proteins of C6/36 cells were immunoprecipitated by antibodies against Ae. aegypti midgut. Conclusion Our results strongly suggest that R67 and R80 are receptors for the four serotypes of dengue virus in the midgut cells of Ae. aegypti and in C6/36 Ae. albopictus cells.

  20. Innate invariant NKT cells recognize Mycobacterium tuberculosis-infected macrophages, produce interferon-gamma, and kill intracellular bacteria.

    Directory of Open Access Journals (Sweden)

    Isabel Sada-Ovalle


    Full Text Available Cellular immunity to Mycobacterium tuberculosis (Mtb requires a coordinated response between the innate and adaptive arms of the immune system, resulting in a type 1 cytokine response, which is associated with control of infection. The contribution of innate lymphocytes to immunity against Mtb remains controversial. We established an in vitro system to study this question. Interferon-gamma is produced when splenocytes from uninfected mice are cultured with Mtb-infected macrophages, and, under these conditions, bacterial replication is suppressed. This innate control of bacterial replication is dependent on CD1d-restricted invariant NKT (iNKT cells, and their activation requires CD1d expression by infected macrophages as well as IL-12 and IL-18. We show that iNKT cells, even in limiting quantities, are sufficient to restrict Mtb replication. To determine whether iNKT cells contribute to host defense against tuberculosis in vivo, we adoptively transferred iNKT cells into mice. Primary splenic iNKT cells obtained from uninfected mice significantly reduce the bacterial burden in the lungs of mice infected with virulent Mtb by the aerosol route. Thus, iNKT cells have a direct bactericidal effect, even in the absence of synthetic ligands such as alpha-galactosylceramide. Our finding that iNKT cells protect mice against aerosol Mtb infection is the first evidence that CD1d-restricted NKT cells mediate protection against Mtb in vivo.

  1. The influence of physical factors on recognizing blood cells in the computer microscopy systems of acute leukemia diagnosis (United States)

    Nikitaev, V. G.; Pronichev, A. N.; Polyakov, E. V.; Dmitrieva, V. V.; Tupitsyn, N. N.; Frenkel, M. A.; Mozhenkova, A. V.


    The work investigated the effect of the choice of color space component on blood cell detection based on the calculation of texture attributes of blood cells nuclei in bone marrow. The study identified the most informative color space and texture characteristics of blood cells, designed for components of these spaces. Significance ratio was introduced to assess the quality of features. We offered features that have enabled to divide lymphocytes from lymphoblasts. The selection of the features was based on the results of the data analysis.

  2. Protective effect of catechin in type I Gaucher disease cells by reducing endoplasmic reticulum stress

    Energy Technology Data Exchange (ETDEWEB)

    Lee, Yea-Jin [Department of Biotechnology, Hoseo University, Baebang, Asan, Chungnam, 336-795 (Korea, Republic of); Kim, Sung-Jo, E-mail: [Department of Biotechnology, Hoseo University, Baebang, Asan, Chungnam, 336-795 (Korea, Republic of); Heo, Tae-Hwe, E-mail: [College of Pharmacy, The Catholic University of Korea, Bucheon 420-743 (Korea, Republic of)


    Highlights: {yields} Catechin reduces the expression level of ER stress marker protein in type I Gaucher disease cells. {yields} Catechin induces the proliferation rate of GD cells similar levels to normal cells. {yields} Catechin improves wound healing activity. {yields} Catechin-mediated reductions in ER stress may be associated with enhanced cell survival. {yields} We identified catechin as a protective agent against ER stress in GD cells. -- Abstract: Gaucher disease (GD) is the most common lysosomal storage disorder (LSD) and is divided into three phenotypes, I, II, and III. Type I is the most prevalent form and has its onset in adulthood. The degree of endoplasmic reticulum (ER) stress is one of the factors that determine GD severity. It has recently been reported that antioxidants reduce ER stress and apoptosis by scavenging the oxidants that cause oxidative stress. For this report, we investigated the possibility that catechin can act on type I GD patient cells to alleviate the pathogenic conditions of GD. We treated GD cells with catechin and examined the expression level of GRP78/BiP (an ER stress marker) by western blots and fluorescence microscopy, the proliferation rate of GD cells, and scratch-induced wound healing activity. Our results show that catechin reduces the expression level of GRP78/BiP, leads to cell proliferation rates of GD cells similar levels to normal cells, and improves wound healing activity. We conclude that catechin protects against ER stress in GD cells and catechin-mediated reductions in ER stress may be associated with enhanced cell survival.

  3. Members of the Candida parapsilosis complex and Candida albicans are differentially recognized by human peripheral blood mononuclear cells

    Directory of Open Access Journals (Sweden)

    Eine eEstrada-Mata


    Full Text Available The systemic infections caused by members of the Candida parapsilosis complex are currently associated to high mobility and mortality rates, and are considered as relevant as those caused by Candida albicans. Since the fungal cell wall is the first point of contact with the host cells, here we performed a comparison of this organelle in members of the C. parapsilosis complex, and its relevance during interaction with human peripheral blood mononuclear cells. We found that the wall of the C. parapsilosis complex members is similar in composition, but differs to that from C. albicans, with less mannan content and more β-glucan and porosity levels. Furthermore, lectin-based analysis showed increased chitin and β1,3-glucan exposure at the surface of C. parapsilosis sensu lato when compared to C. albicans. Yeast cells of members of the C. parapsilosis complex stimulated more cytokine production by human peripheral blood mononuclear cells than C. albicans cells; and this significantly changed upon removal of O-linked mannans, indicating this wall component plays a significant role in cytokine stimulation by C. parapsilosis sensu lato. When inner wall components were exposed on the wall surface, C. parapsilosis sensu stricto and C. metapsilosis, but not C. orthopsilosis, stimulated higher cytokine production. Moreover, we found a strong dependency on β1,3-glucan recognition for the members of the C. parapsilosis complex, but not for live C. albicans cells; whereas TLR4 was required for TNFα production by the three members of the complex, and stimulation of IL-6 by C. orthopsilosis. Mannose receptor had a significant role during TNF and IL-1β stimulation by members of the complex. Finally, we demonstrated that purified N- and O-mannans from either C. parapsilosis sensu lato or C. albicans are capable to block the recognition of these pathogens by human peripheral blood mononuclear cells. Together; our results suggest that the innate immune

  4. MHC-I-restricted epitopes conserved among variola and other related orthopoxviruses are recognized by T cells 30 years after vaccination

    DEFF Research Database (Denmark)

    Tang, Sheila Tuyet; Wang, M.; Lamberth, K.;


    It is many years since the general population has been vaccinated against smallpox virus. Here, we report that human leukocyte antigen (HLA) class I restricted T cell epitopes can be recognized more than 30 years after vaccination. Using bioinformatic methods, we predicted 177 potential cytotoxic...... to a better understanding of poxvirus immunity and may aid in the development of new improved vaccines and diagnostic tools.......It is many years since the general population has been vaccinated against smallpox virus. Here, we report that human leukocyte antigen (HLA) class I restricted T cell epitopes can be recognized more than 30 years after vaccination. Using bioinformatic methods, we predicted 177 potential cytotoxic T...... lymphocyte epitopes. Eight epitopes were confirmed to stimulate IFN-gamma release by T cells in smallpox-vaccinated subjects. The epitopes were restricted by five supertypes (HLA-A1, -A2, -A24 -A26 and -B44). Significant T cell responses were detected against 8 of 45 peptides with an HLA class I affinity...

  5. Glycans from avian influenza virus are recognized by chicken dendritic cells and are targets for the humoral immune response in chicken. (United States)

    de Geus, Eveline D; Tefsen, Boris; van Haarlem, Daphne A; van Eden, Willem; van Die, Irma; Vervelde, Lonneke


    To increase our understanding of the interaction between avian influenza virus and its chicken host, we identified receptors for putative avian influenza virus (AIV) glycan determinants on chicken dendritic cells. Chicken dendritic cells (DCs) were found to recognize glycan determinants containing terminal αGalNAc, Galα1-3Gal, GlcNAcβ1-4GlcNAcβ1-4GlcNAcβ (chitotriose) and Galα1-2Gal. Infection of chicken dendritic cells with either low pathogenic (LP) or highly pathogenic (HP) AIV results in elevated mRNA expression of homologs of the mouse C-type lectins DEC205 and macrophage mannose receptor (MMR), whereas expression levels of the human dendritic cell-specific intercellular adhesion molecule-3-grabbing non-integrin (DC-SIGN) homolog remained unchanged. Following uptake and subsequent presentation of avian influenza virus by DCs, adaptive immunity, including humoral immune responses are induced. We have investigated the antibody responses against virus glycan epitopes after avian influenza virus infection. Using glycan micro-array analysis we showed that chicken contained antibodies that predominantly recognize terminal Galα1-3Gal-R, chitotriose and Fucα1-2Galβ1-4GlcNAc-R (H-type 2). After influenza-infection, glycan array analysis showed that both levels and repertoire of glycan-recognizing antibodies decreased. However, analysis of the sera by ELISA indicated that the levels of different isotypes of anti-glycan Abs against specific glycan antigens was increased after influenza-infection, suggesting that the presentation of the glycan antigens and iso-type of the Abs are critical parameters to take into account when measuring anti-glycan Abs. This novel approach in avian influenza research may contribute to the development of a broad spectrum vaccine and improves our mechanistic understanding of innate and adaptive responses to glycans.

  6. Members of the Candida parapsilosis Complex and Candida albicans are Differentially Recognized by Human Peripheral Blood Mononuclear Cells. (United States)

    Estrada-Mata, Eine; Navarro-Arias, María J; Pérez-García, Luis A; Mellado-Mojica, Erika; López, Mercedes G; Csonka, Katalin; Gacser, Attila; Mora-Montes, Héctor M


    The systemic infections caused by members of the Candida parapsilosis complex are currently associated to high morbility and mortality rates, and are considered as relevant as those caused by Candida albicans. Since the fungal cell wall is the first point of contact with the host cells, here we performed a comparison of this organelle in members of the C. parapsilosis complex, and its relevance during interaction with human peripheral blood mononuclear cells (PBMCs). We found that the wall of the C. parapsilosis complex members is similar in composition, but differs to that from C. albicans, with less mannan content and more β-glucan and porosity levels. Furthermore, lectin-based analysis showed increased chitin and β1,3-glucan exposure at the surface of C. parapsilosis sensu lato when compared to C. albicans. Yeast cells of members of the C. parapsilosis complex stimulated more cytokine production by human PBMCs than C. albicans cells; and this significantly changed upon removal of O-linked mannans, indicating this wall component plays a significant role in cytokine stimulation by C. parapsilosis sensu lato. When inner wall components were exposed on the wall surface, C. parapsilosis sensu stricto and C. metapsilosis, but not C. orthopsilosis, stimulated higher cytokine production. Moreover, we found a strong dependency on β1,3-glucan recognition for the members of the C. parapsilosis complex, but not for live C. albicans cells; whereas TLR4 was required for TNFα production by the three members of the complex, and stimulation of IL-6 by C. orthopsilosis. Mannose receptor had a significant role during TNFα and IL-1β stimulation by members of the complex. Finally, we demonstrated that purified N- and O-mannans from either C. parapsilosis sensu lato or C. albicans are capable to block the recognition of these pathogens by human PBMCs. Together; our results suggest that the innate immune recognition of the members of the C. parapsilosis complex is differential

  7. Dengue virus-specific, human CD4+ CD8- cytotoxic T-cell clones: multiple patterns of virus cross-reactivity recognized by NS3-specific T-cell clones.


    Kurane, I; Brinton, M.A.; Samson, A L; Ennis, F A


    Thirteen dengue virus-specific, cytotoxic CD4+ CD8- T-cell clones were established from a donor who was infected with dengue virus type 3. These clones were examined for virus specificity and human leukocyte antigen (HLA) restriction in cytotoxic assays. Six patterns of virus specificities were determined. Two serotype-specific clones recognized only dengue virus type 3. Two dengue virus subcomplex-specific clones recognized dengue virus types 2, 3, and 4, and one subcomplex-specific clone re...

  8. A HLA-A2 restricted human CTL line recognizes a novel tumor cell expressed p53 epitope

    DEFF Research Database (Denmark)

    Würtzen, Peter A; Claesson, Mogens H


    , the CTL line, which expressed relatively low affinity for the HLA-A2/peptide complex, was able to kill 3 different HLA-A2(+) p53 mutated tumor cell lines. The present and our previous observations expand the number of p53-derived peptides suitable for vaccination protocols for cancer patients with p53......A p53 peptide-specific CTL line was generated through stimulation with autologous monocyte-derived dendritic cells (DC) pulsed with wild-type HLA-A2 binding p53 derived peptides. A p53 peptide-specific CD8(+) CTL line was established from a healthy HLA-A2 positive donor. The CTL line...... was characterized with respect to specificity, affinity and killing of cell lines derived from p53 mutated spontaneous tumors. The CTL line demonstrated lysis of p53(139-147) pulsed target cells and cold target inhibition experiments as well as antibody blocking confirmed that the killing was epitope-specific, HLA...

  9. ROS accumulation by PEITC selectively kills ovarian cancer cells via UPR-mediated apoptosis

    Directory of Open Access Journals (Sweden)

    Yoon-hee eHong


    Full Text Available Unfolded protein response (UPR is crucial for both survival and death of mammalian cells, which is regulated by reactive oxygen species (ROS and nutrient depletion. In this study, we demonstrated the effect of ROS-accumulation, induced by β-phenethyl isothiocyanate (PEITC, on UPR mediated apoptosis in ovarian cancer cells. We used ovarian cancer cell lines, PA-1 and SKOV-3, with different p53 status (wild- and null- type, respectively. PEITC caused increased ROS-accumulation and inhibited proliferation selectively in ovarian cancer cells, and glutathione (GSH depletion in SKOV-3. However, PEITC did not cause any effect in normal ovarian epithelial cells and peripheral blood mononuclear cells. After 48 h of PEITC treatment (5 µM, apoptotic cell death was shown to increase significantly in the ovarian cancer cells and not in the normal cells. The key regulator of UPR-mediated apoptosis, CHOP/GADD153 and ER resident chaperone BiP/GRP78 were parallely up-regulated with activation of two major sensors of the UPR (PERK and ATF-6 in PA-1; PERK, and IRE1α in SKOV-3 in response to ROS accumulation induced by PEITC (5 µM. ROS scavenger, N-acetyl-cysteine (NAC, attenuated the effect of PEITC on UPR signatures (P-PERK, IRE1α, CHOP/GADD153, and BiP/GRP78, suggesting the involvement of ROS in UPR-mediated apoptosis. Altogether, PEITC induces UPR-mediated apoptosis in ovarian cancer cells via accumulation of ROS in a cancer-specific manner.

  10. Cis-hydroxyproline-induced inhibition of pancreatic cancer cell growth is mediated by endoplasmic reticulum stress

    Institute of Scientific and Technical Information of China (English)

    Christoph Mueller; Joerg Emmrich; Robert Jaster; Dagmar Braun; Stefan Liebe; Gisela Sparmann


    AIM: To investigate the biological effects of cishydroxyproline (CHP) on the rat pancreatic carcinoma cell line DSL6A, and to examine the underlying molecular mechanisms.METHODS: The effect of CHP on DSL6A cell proliferation was assessed by using BrdU incorporation. The expression of focal adhesion kinase (FAK) was characterized by Western blotting and immunofluorescence.Induction of endoplasmic reticulum (ER) stress was investigated by using RT-PCR and Western blotting for the glucose-related protein-78 (GRP78) and growth arrest and DNA inducible gene (GADD153). Cell viability was determined through measuring the metabolic activity based on the reduction potential of DSL6A cells. Apoptosis was analyzed by detection of caspase-3 activation and cleavage of poly(ADP-ribose) polymerase (PARP) as well as DNA laddering.RESULTS: In addition to inhibition of proliferation,incubation with CHP induced proteolytic cleavage of FAK and a delocalisation of the enzyme from focal adhesions,followed by a loss of cell adherence. Simultaneously,we could show an increased expression of GRP78 and GADD153, indicating a CHP-mediated activation of the ER stress cascade in the DSL6A cell line. Prolonged incubation of DSL6A cells with CHP finally resulted in apoptotic cell death. Beside L-proline, the inhibition of intracellular proteolysis by addition of a broad spectrum protease inhibitor could abolish the effects of CHP on cellular functions and the molecular processes. In contrast, impeding the activity of apoptosis-executing caspases had no influence on CHP-mediated cell damage.CONCLUSION: Our data suggest that the initiation of ER stress machinery by CHP leads to an activation of intracellular proteolytic processes, including caspaseindependent FAK degradation, resulting in damaging pancreatic carcinoma cells.

  11. Lysosome dysfunction enhances oxidative stress-induced apoptosis through ubiquitinated protein accumulation in Hela cells. (United States)

    Yu, Chunyan; Huang, Xiaowei; Xu, Ye; Li, Hongyan; Su, Jing; Zhong, Jiateng; Kang, Jinsong; Liu, Yuhe; Sun, Liankun


    The role of lysosomal system in oxidative stress-induced apoptosis in cancer cells is not fully understood. Menadione is frequently used as oxidative stress model. It is indicated that menadione could induce autophagy in Hela cells. In the present study, we examined whether the lysosomal inhibitor, ammonium chloride (NH(4)Cl) could prevent the autophagy flux by inhibiting the fusion of autophagosomes with lysosomes and enhance apoptosis induced by menadione via mitochondrial pathway. The results demonstrated generation and accumulation of reactive oxygen species and increased levels of ubiquitinated proteins and GRP78 in cells treated with both menadione and NH(4)Cl. Our data indicates that lysosomal system through autophagy plays an important role in preventing menadione-induced apoptosis in Hela cells by clearing misfolded proteins, which alleviates endoplasmic reticulum stress.

  12. Identifying protective Streptococcus pyogenes vaccine antigens recognized by both B and T cells in human adults and children

    DEFF Research Database (Denmark)

    Mortensen, Rasmus; Nissen, Thomas Nørrelykke; Fredslund, Sine


    No commercial vaccine exists against Group A streptococci (GAS; Streptococcus pyogenes) and only little is known about anti-GAS protective immunity. In our effort to discover new protective vaccine candidates, we selected 21 antigens based on an in silico evaluation. These were all well......-conserved among different GAS strains, upregulated in host-pathogen interaction studies, and predicted to be extracellular or associated with the surface of the bacteria. The antigens were tested for both antibody recognition and T cell responses in human adults and children. The antigenicity of a selected group...

  13. Shared fine specificity between T-cell receptors and an antibody recognizing a peptide/major histocompatibility class I complex

    DEFF Research Database (Denmark)

    Stryhn, A; Andersen, P S; Pedersen, L O;


    -MHC complexes have been less well characterized. We have used a complete set of singly substituted analogs of a mouse MHC class I, Kk-restricted peptide, influenza hemagglutinin (Ha)255-262, to address the binding specificity of this MHC molecule. Using the same peptide-MHC complexes we determined the fine...... specificity of two Ha255-262-specific, Kk-restricted T cells, and of a unique antibody, pSAN, specific for the same peptide-MHC complex. Independently, a model of the Ha255-262-Kk complex was generated through homology modeling and molecular mechanics refinement. The functional data and the model corroborated...

  14. How legumes recognize rhizobia. (United States)

    Via, Virginia Dalla; Zanetti, María Eugenia; Blanco, Flavio


    Legume plants have developed the capacity to establish symbiotic interactions with soil bacteria (known as rhizobia) that can convert N2 to molecular forms that are incorporated into the plant metabolism. The first step of this relationship is the recognition of bacteria by the plant, which allows to distinguish potentially harmful species from symbiotic partners. The main molecular determinant of this symbiotic interaction is the Nod Factor, a diffusible lipochitooligosaccharide molecule produced by rhizobia and perceived by LysM receptor kinases; however, other important molecules involved in the specific recognition have emerged over the years. Secreted exopolysaccharides and the lipopolysaccharides present in the bacterial cell wall have been proposed to act as signaling molecules, triggering the expression of specific genes related to the symbiotic process. In this review we will briefly discuss how transcriptomic analysis are helping to understand how multiple signaling pathways, triggered by the perception of different molecules produced by rhizobia, control the genetic programs of root nodule organogenesis and bacterial infection. This knowledge can help to understand how legumes have evolved to recognize and establish complex ecological relationships with particular species and strains of rhizobia, adjusting gene expression in response to identity determinants of bacteria.

  15. Is the Framework of Cohn's 'Tritope Model' for How T Cell Receptors Recognize Peptide/Self-MHC Complexes and Allo-MHC Plausible? (United States)

    Bretscher, Peter A


    Cohn has developed the tritope model to describe how distinct domains of the T cell receptor (TcR) recognize peptide/self-MHC complexes and allo-MHC. He has over the years employed this model as a framework for considering how the TcR might mediate various signals [1-5]. In a recent publication [5], Cohn employs the Tritope Model to propose a detailed mechanism for the T cell receptor's involvement in positive thymic selection [5]. During a review of this proposal, I became uneasy over the plausibility of the underlying framework of the Tritope Model. I outline here the evolutionary considerations making me question this framework. I also suggest that the proposed framework underlying the Tritope Model makes strong predictions whose validity can most probably be assessed by considering observations reported in the literature.

  16. Live cell imaging with protein domains capable of recognizing phosphatidylinositol 4,5-bisphosphate; a comparative study

    Directory of Open Access Journals (Sweden)

    Lemmon Mark A


    Full Text Available Abstract Background Phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5P2] is a critically important regulatory phospholipid found in the plasma membrane of all eukaryotic cells. In addition to being a precursor of important second messengers, PtdIns(4,5P2 also regulates ion channels and transporters and serves the endocytic machinery by recruiting clathrin adaptor proteins. Visualization of the localization and dynamic changes in PtdIns(4,5P2 levels in living cells is critical to understanding the biology of PtdIns(4,5P2. This has been mostly achieved with the use of the pleckstrin homology (PH domain of PLCδ1 fused to GFP. Here we report on a comparative analysis of several recently-described yeast PH domains as well as the mammalian Tubby domain to evaluate their usefulness as PtdIns(4,5P2 imaging tools. Results All of the yeast PH domains that have been previously shown to bind PtdIns(4,5P2 showed plasma membrane localization but only a subset responded to manipulations of plasma membrane PtdIns(4,5P2. None of these domains showed any advantage over the PLCδ1PH-GFP reporter and were compromised either in their expression levels, nuclear localization or by causing peculiar membrane structures. In contrast, the Tubby domain showed high membrane localization consistent with PtdIns(4,5P2 binding and displayed no affinity for the soluble headgroup, Ins(1,4,5P3. Detailed comparison of the Tubby and PLCδ1PH domains showed that the Tubby domain has a higher affinity for membrane PtdIns(4,5P2 and therefore displays a lower sensitivity to report on changes of this lipid during phospholipase C activation. Conclusion These results showed that both the PLCδ1PH-GFP and the GFP-Tubby domain are useful reporters of PtdIns(4,5P2 changes in the plasma membrane, with distinct advantages and disadvantages. While the PLCδ1PH-GFP is a more sensitive reporter, its Ins(1,4,5P3 binding may compromise its accuracy to measure PtdIns(4,5P2changes. The Tubby domain

  17. FDA Recognized Consensus Standards (United States)

    U.S. Department of Health & Human Services — This database consists of those national and international standards recognized by FDA which manufacturers can declare conformity to and is part of the information...

  18. Recognizing teen depression (United States)

    ... Recognizing teen depression To use the sharing features on this page, ... life. Be Aware of the Risk for Teen Depression Your teen is more at risk for depression ...

  19. An antibody to the lutheran glycoprotein (Lu recognizing the LU4 blood type variant inhibits cell adhesion to laminin α5.

    Directory of Open Access Journals (Sweden)

    Yamato Kikkawa

    Full Text Available BACKGROUND: The Lutheran blood group glycoprotein (Lu, an Ig superfamily (IgSF transmembrane receptor, is also known as basal cell adhesion molecule (B-CAM. Lu/B-CAM is a specific receptor for laminin α5, a major component of basement membranes in various tissues. Previous reports have shown that Lu/B-CAM binding to laminin α5 contributes to sickle cell vaso-occlusion. However, as there are no useful tools such as function-blocking antibodies or drugs, it is unclear how epithelial and sickled red blood cells adhere to laminin α5 via Lu/B-CAM. METHODOLOGY/PRINCIPAL FINDINGS: In this study, we discovered a function-blocking antibody that inhibits Lu binding to laminin α5 using a unique binding assay on tissue sections. To characterize the function-blocking antibody, we identified the site on Lu/B-CAM recognized by this antibody. The extracellular domain of Lu/B-CAM contains five IgSF domains, D1-D2-D3-D4-D5. The antibody epitope was localized to D2, but not to the D3 domain containing the major part of the laminin α5 binding site. Furthermore, mutagenesis studies showed that Arg(175, the LU4 blood group antigenic site, was crucial for forming the epitope and the antibody bound sufficiently close to sterically hinder the interaction with α5. Cell adhesion assay using the antibody also showed that Lu/B-CAM serves as a secondary receptor for the adhesion of carcinoma cells to laminin α5. CONCLUSION/SIGNIFICANCE: This function-blocking antibody against Lu/B-CAM should be useful for not only investigating cell adhesion to laminin α5 but also for developing drugs to inhibit sickle cell vaso-occlusion.

  20. A chicken influenza virus recognizes fucosylated α2,3 sialoglycan receptors on the epithelial cells lining upper respiratory tracts of chickens. (United States)

    Hiono, Takahiro; Okamatsu, Masatoshi; Nishihara, Shoko; Takase-Yoden, Sayaka; Sakoda, Yoshihiro; Kida, Hiroshi


    Influenza viruses recognize sialoglycans as receptors. Although viruses isolated form chickens preferentially bind to sialic acid α2,3 galactose (SAα2,3Gal) glycans as do those of ducks, chickens were not experimentally infected with viruses isolated from ducks. A chicken influenza virus, A/chicken/Ibaraki/1/2005 (H5N2) (Ck/IBR) bound to fucose-branched SAα2,3Gal glycans, whereas the binding towards linear SAα2,3Gal glycans was weak. On the epithelial cells of the upper respiratory tracts of chickens, fucose-branched SAα2,3Gal glycans were detected, but not linear SAα2,3Gal glycans. The growth of Ck/IBR in MDCK-FUT cells, which were genetically prepared to express fucose-branched SAα2,3Gal glycans, was significantly higher than that in the parental MDCK cells. The present results indicate that fucose-branched SAα2,3Gal glycans existing on the epithelial cells lining the upper respiratory tracts of chickens are critical for recognition by Ck/IBR.

  1. ER-mediated stress induces mitochondrial-dependent caspases activation in NT2 neuron-like cells. (United States)

    Arduino, Daniela M; Esteves, A Raquel; Domingues, A Filipa; Pereira, Claudia M F; Cardoso, Sandra M; Oliveira, Catarina R


    Recent studies have revealed that endoplasmic reticulum (ER) disturbance is involved in the pathophysiology of neurodegenerative disorders, contributing to the activation of the ER stress-mediated apoptotic pathway. Therefore, we investigated here the molecular mechanisms underlying the ER-mitochondria axis, focusing on calcium as a potential mediator of cell death signals. Using NT2 cells treated with brefeldin A or tunicamycin, we observed that ER stress induces changes in the mitochondrial function, impairing mitochondrial membrane potential and distressing mitochondrial respiratory chain complex Moreover, stress stimuli at ER level evoked calcium fluxes between ER and mitochondria. Under these conditions, ER stress activated the unfolded protein response by an overexpression of GRP78, and also caspase-4 and-2, both involved upstream of caspase-9. Our findings show that ER and mitochondria interconnection plays a prominent role in the induction of neuronal cell death under particular stress circumstances.

  2. Distribution of cells labelled by a novel somatic stem cell-recognizing antibody (A3) in pulmonary genesis and bleomycin induced pulmonary fibrosis in rats. (United States)

    Hori, M; Juniantito, V; Izawa, T; Ichikawa, C; Tanaka, M; Tanaka, K; Takenaka, S; Kuwamura, M; Yamate, J


    Stem cells play important roles in organogenesis and remodelling after tissue injury. A monoclonal antibody (A3) has been produced against rat somatic stem cells. The present study investigated the distribution of cells labelled by A3 in the lung of fetal, neonatal and adult rats, as well as in the lung of rats with bleomycin (BLM) induced pulmonary fibrosis. In developing fetal lungs, A3(+) interstitial cells were present around the bronchi/bronchioles and arterioles, while in neonatal and adult lungs, the A3 reactivity of the interstitial cells gradually disappeared and instead, vascular endothelial cells in alveolar capillaries and arterioles expressed A3. By double immunofluorescence labelling, the A3(+) interstitial cells also expressed vimentin (a mesenchymal marker) and CD34 (a marker of immature mesenchymal cells), indicating that the interstitial cells were immature mesenchymal cells concentrated in organs as precursors to cells of connective tissues. A3(+)endothelial cells were co-expressed RECA-1 (a marker of rat endothelial cells) and A3 was localized to the cell membrane and cytoplasm of these cells by immunoelectron microscopy. In BLM induced fibrotic lesions, there were many A3(+) cells, which also expressed vimentin or RECA-1 by dual immunofluorescence labelling. There were few CD34(+)/A3(+) double positive cells. No cells co-expressed A3 and α-smooth muscle actin (a marker of well-differentiated myofibroblastic cells). Although the detailed properties of cells labelled by A3 remain to be discovered, A3 would appear to be a useful marker of immature mesenchymal cells and vascular endothelial cells in developing lungs and in pulmonary fibrosis.

  3. Urotensin II Induces ER Stress and EMT and Increase Extracellular Matrix Production in Renal Tubular Epithelial Cell in Early Diabetic Mice

    Directory of Open Access Journals (Sweden)

    Xin-Xin Pang


    Full Text Available Background/Aims: Urotensin II (UII and its receptor are highly expressed in the kidney tissue of patients with diabetic nephropathy (DN. The aim of this study is to examine the roles of UII in the induction of endoplasmic reticulum stress (ER stress and Epithelial-mesenchymal transition (EMT in DN in vivo and in vitro. Methods: Kidney tissues were collected from patients with DN. C57BL/6 mice and mice with UII receptor knock out were injected with two consecutive doses of streptozotocin to induce diabetes and were sacrificed at 3th week for in vivo study. HK-2 cells in vitro were cultured and treated with UII. Markers of ER stress and EMT, fibronectin and type IV collagen were detected by immunohistochemistry, real time PCR and western blot. Results: We found that the expressions of protein of UII, GRP78, CHOP, ALPHA-SMA, fibronectin and type IV collagen were upregulated while E-cadherin protein was downregulated as shown by immunohistochemistry or western blot analysis in kidney of diabetic mice in comparison to normal control; moreover expressions of GRP78, CHOP, ALPHA-SMA, fibronectin and type IV collagen were inhibited while E-caherin expression was enhanced in kidney in diabetic mice with UII receptor knock out in comparison to C57BL/6 diabetic mice. In HK-2 cells, UII induced upregulation of GRP78, CHOP, ALPHA-SMA, fibroblast-specifc protein 1(FSP-1, fibronectin and type collagen and downregulation of E-cadherin. UII receptor antagonist can block UII-induced ER stress and EMT; moreover, 4-PBA can inhibit the mRNA expression of ALPHA-SMA and FSP1 induced by UII in HK-2 cells. Conclusions: We are the first to verify UII induces ER stress and EMT and increase extracellular matrix production in renal tubular epithelial cell in early diabetic mice. Moreover, UII may induce renal tubular epithelial EMT via triggering ER stress pathway in vitro, which might be the new pathogenic pathway for the development of renal fibrosis in DN.

  4. 3-O-sulfated heparan sulfate recognized by the antibody HS4C3 contributes [corrected] to the differentiation of mouse embryonic stem cells via fas signaling.

    Directory of Open Access Journals (Sweden)

    Kazumi Hirano

    Full Text Available Maintenance of self-renewal and pluripotency in mouse embryonic stem cells (mESCs is regulated by the balance between several extrinsic signaling pathways. Recently, we demonstrated that heparan sulfate (HS chains play important roles in the maintenance and differentiation of mESCs by regulating extrinsic signaling. Sulfated HS structures are modified by various sulfotransferases during development. However, the significance of specific HS structures during development remains unclear. Here, we show that 3-O-sulfated HS structures synthesized by HS 3-O-sulfotransferases (3OSTs and recognized by the antibody HS4C3 increase during differentiation of mESCs. Furthermore, expression of Fas on the cell surface of the differentiated cells also increased. Overexpression of the HS4C3-binding epitope in mESCs induced apoptosis and spontaneous differentiation even in the presence of LIF and serum. These data showed that the HS4C3-binding epitope was required for differentiation of mESCs. Up-regulation of the HS4C3-binding epitope resulted in the recruitment of Fas from the cytoplasm to lipid rafts on the cell surface followed by activation of Fas signaling. Indeed, the HS4C3-binding epitope interacted with a region that included the heparin-binding domain (KLRRRVH of Fas. Reduced self-renewal capability in cells overexpressing 3OST resulted from the degradation of Nanog by activated caspase-3, which is downstream of Fas signaling, and was rescued by the inhibition of Fas signaling. We also found that knockdown of 3OST and inhibition of Fas signaling reduced the potential for differentiation into the three germ layers during embryoid body formation. This is the first demonstration that activation of Fas signaling is mediated by an increase in the HS4C3-binding epitope and indicates a novel signaling pathway for differentiation in mESCs.

  5. Human lung epithelial cells contain Mycobacterium tuberculosis in a late endosomal vacuole and are efficiently recognized by CD8⁺ T cells.

    Directory of Open Access Journals (Sweden)

    Melanie J Harriff

    Full Text Available Mycobacterium tuberculosis (Mtb is transmitted via inhalation of aerosolized particles. While alveolar macrophages are thought to play a central role in the acquisition and control of this infection, Mtb also has ample opportunity to interact with the airway epithelium. In this regard, we have recently shown that the upper airways are enriched with a population of non-classical, MR1-restricted, Mtb-reactive CD8⁺ T cells (MAIT cells. Additionally, we have demonstrated that Mtb-infected epithelial cells lining the upper airways are capable of stimulating IFNγ production by MAIT cells. In this study, we demonstrate that airway epithelial cells efficiently stimulate IFNγ release by MAIT cells as well as HLA-B45 and HLA-E restricted T cell clones. Characterization of the intracellular localization of Mtb in epithelial cells indicates that the vacuole occupied by Mtb in epithelial cells is distinct from DC in that it acquires Rab7 molecules and does not retain markers of early endosomes such as Rab5. The Mtb vacuole is also heterogeneous as there is a varying degree of association with Lamp1 and HLA-I. Although the Mtb vacuole shares markers associated with the late endosome, it does not acidify, and the bacteria are able to replicate within the cell. This work demonstrates that Mtb infected lung epithelial cells are surprisingly efficient at stimulating IFNγ release by CD8⁺ T cells.

  6. Hardware bitstream sequence recognizer


    Karpin, Oleksandr; Sokil, Volodymyr


    This paper describes how to implement in hardware a bistream sequence recognizer using the PSoC Pseudo Random Sequence Generator (PRS) User Module. The PRS can be used in digital communication systems with the serial data interface for automatic preamble detection and extraction, control words selection, etc.

  7. Antibodies recognizing Mycobacterium avium paratuberculosis epitopes cross-react with the beta-cell antigen ZnT8 in Sardinian type 1 diabetic patients.

    Directory of Open Access Journals (Sweden)

    Speranza Masala

    Full Text Available The environmental factors at play in the pathogenesis of type 1 diabetes (T1D remain enigmatic. Mycobacterium avium subspecies paratuberculosis (MAP is transmitted from dairy herds to humans through food contamination. MAP causes an asymptomatic infection that is highly prevalent in Sardinian T1D patients compared with type 2 diabetes (T2D and healthy controls. Moreover, MAP elicits humoral responses against several mycobacterial proteins. We asked whether antibodies (Abs against one of these proteins, namely MAP3865c, which displays a sequence homology with the β-cell protein zinc transporter 8 (ZnT8 could be cross-reactive with ZnT8 epitopes. To this end, Ab responses against MAP3865c were analyzed in Sardinian T1D, T2D and healthy subjects using an enzymatic immunoassay. Abs against MAP3865c recognized two immunodominant transmembrane epitopes in 52-65% of T1D patients, but only in 5-7% of T2D and 3-5% of healthy controls. There was a linear correlation between titers of anti-MAP3865c and anti-ZnT8 Abs targeting these two homologous epitopes, and pre-incubation of sera with ZnT8 epitope peptides blocked binding to the corresponding MAP3865c peptides. These results demonstrate that Abs recognizing MAP3865c epitopes cross-react with ZnT8, possibly underlying a molecular mimicry mechanism, which may precipitate T1D in MAP-infected individuals.

  8. CD4+ T Cells Recognizing PE/PPE Antigens Directly or via Cross Reactivity Are Protective against Pulmonary Mycobacterium tuberculosis Infection. (United States)

    Sayes, Fadel; Pawlik, Alexandre; Frigui, Wafa; Gröschel, Matthias I; Crommelynck, Samuel; Fayolle, Catherine; Cia, Felipe; Bancroft, Gregory J; Bottai, Daria; Leclerc, Claude; Brosch, Roland; Majlessi, Laleh


    Mycobacterium tuberculosis (Mtb), possesses at least three type VII secretion systems, ESX-1, -3 and -5 that are actively involved in pathogenesis and host-pathogen interaction. We recently showed that an attenuated Mtb vaccine candidate (Mtb Δppe25-pe19), which lacks the characteristic ESX-5-associated pe/ppe genes, but harbors all other components of the ESX-5 system, induces CD4+ T-cell immune responses against non-esx-5-associated PE/PPE protein homologs. These T cells strongly cross-recognize the missing esx-5-associated PE/PPE proteins. Here, we characterized the fine composition of the functional cross-reactive Th1 effector subsets specific to the shared PE/PPE epitopes in mice immunized with the Mtb Δppe25-pe19 vaccine candidate. We provide evidence that the Mtb Δppe25-pe19 strain, despite its significant attenuation, is comparable to the WT Mtb strain with regard to: (i) its antigenic repertoire related to the different ESX systems, (ii) the induced Th1 effector subset composition, (iii) the differentiation status of the Th1 cells induced, and (iv) its particular features at stimulating the innate immune response. Indeed, we found significant contribution of PE/PPE-specific Th1 effector cells in the protective immunity against pulmonary Mtb infection. These results offer detailed insights into the immune mechanisms underlying the remarkable protective efficacy of the live attenuated Mtb Δppe25-pe19 vaccine candidate, as well as the specific potential of PE/PPE proteins as protective immunogens.

  9. A two-step stimulus-response cell-SELEX method to generate a DNA aptamer to recognize inflamed human aortic endothelial cells as a potential in vivo molecular probe for atherosclerosis plaque detection. (United States)

    Ji, Kaili; Lim, Wee Siang; Li, Sam Fong Yau; Bhakoo, Kishore


    Aptamers are single-stranded oligonucleotides that are capable of binding wide classes of targets with high affinity and specificity. Their unique three-dimensional structures present numerous possibilities for recognizing virtually any class of target molecules, making them a promising alternative to antibodies used as molecular probes in biomedical analysis and clinical diagnosis. In recent years, cell-systematic evolution of ligands by exponential enrichment (SELEX) has been used extensively to select aptamers for various cell targets. However, aptamers that have evolved from cell-SELEX to distinguish the "stimulus-response cell" have not previously been reported. Moreover, a number of cumbersome and time-consuming steps involved in conventional cell-SELEX reduce the efficiency and efficacy of the aptamer selection. Here, we report a "two-step" methodology of cell-SELEX that successfully selected DNA aptamers specifically against "inflamed" endothelial cells. This has been termed as stimulus-response cell-SELEX (SRC-SELEX). The SRC-SELEX enables the selection of aptamers to distinguish the cells activated by stimulus of healthy cells or cells isolated from diseased tissue. We report a promising aptamer, N55, selected by SRC-SELEX, which can bind specifically to inflamed endothelial cells both in cell culture and atherosclerotic plaque tissue. This aptamer probe was demonstrated as a potential molecular probe for magnetic resonance imaging to target inflamed endothelial cells and atherosclerotic plaque detection.

  10. Recognizing occluded MSTAR targets (United States)

    Bhanu, Bir; Jones, Grinnell, III


    This paper presents an approach for recognizing occluded vehicle targets in Synthetic Aperture Radar (SAR) images. Using quasi-invariant local features, SAR scattering center locations and magnitudes, a recognition algorithm is presented that successfully recognizes highly occluded versions of actual vehicles from the MSTAR public data. Extensive experimental results are presented to show the effect of occlusion on recognition performance in terms of Probability of Correct Identification, Receiver Operating Characteristic (ROC) curves and confusion matrices. The effect of occlusion on performance of this recognition algorithm is accurately predicted. Combined effects such as occlusion and measured positional noise, as well as occlusion and other observed extended operating conditions (e.g., articulation) are also addressed. Although excellent forced recognition results can be achieved at very high (70%) occlusion, practical limitations are found due to the similarity of unoccluded confuser vehicles to highly occluded targets.

  11. Large, but not small, antigens require time- and temperature-dependent processing in accessory cells before they can be recognized by T cells

    DEFF Research Database (Denmark)

    Buus, S; Werdelin, O


    of antigen presentation we used the proliferative response of appropriately primed T cells during a co-culture with the paraformaldehyde-fixed and antigen-exposed presenting cells. We demonstrate that the large synthetic polypeptide antigen, dinitrophenyl-poly-L-lysine, requires processing. After an initial...... time-lag of 30 min this antigen is fully processed within 2 to 4 of culture at 37 degrees C. In contrast, the immunogenic heptapeptide, angiotensin III, can be presented by pre-fixed accessory cells, viz. without any prior processing. Antigen processing was found to be temperature...

  12. FAD-linked Presenilin-1 V97L mutation impede tranport regulation and intracellular Ca(2+) homeostasis under ER stress. (United States)

    Shao, Yankun; Li, Miao; Wu, Miao; Shi, Kai; Fang, Boyan; Wang, Jie


    We report a PS1 gene mutation (Val 97Leu) in a Chinese familial Alzheimer's disease (FAD) pedigree and a cell model of FAD built by transfecting PS1 v97L mutants into human neuroblastoma SH-SY5Y cells. To test our hypothesis that the PS1 v97L mutation is pathogenic, we investigated possible alterations in transport regulation and intracellular Ca(2+) homeostasis in endoplasmic reticulum (ER). Grp78 is an ER-resident chaperone mediating the unfolded protein response (UPR) and is a key regulator of ER stress transducers. KDEL is a 4-amino-acid retention sequence made of Lys-Asp-Glu-Leu-COO. KDEL is a "resident" sequence as protein residence in ER is consistently associated with KDEL at the C-extremity. Our group used KDEL recognizing anti-Grp78 monoclonal antibody to detect the level of Grp78. We found increased KDEL level in all the transfected cells including cells transfected with PS1 V97L genes, wild-type and the mock. However cells with PS1 V97L mutation expressed a relatively lower KDEL compared with the wild-type and the mock, and a significantly lower Grp78 level compared with the wild-type, the mock and control. These results suggest that PS1 V97L mutation impedes intracellular transport regulation in ER. PS1 V97L mutation mediates increased ER Ca(2+) content in human neuroblastoma SH-SY5Y cells. The increased intracellular Ca(2+) release is due to depleted Ca(2+) storing content of ER but not due to extracellular environment as capacitative Ca(2+) entry (CCE) is invariant. PS1 V97L mutation interferes with intracellular Ca(2+) homeostasis. Abnormal transport regulation and Ca(2+) homeostasis attributed to PS1 V97L mutation may be associated with the pathology of Chinese familial FAD.

  13. Identification of novel amelogenin-binding proteins by proteomics analysis.

    Directory of Open Access Journals (Sweden)

    Takao Fukuda

    Full Text Available Emdogain (enamel matrix derivative, EMD is well recognized in periodontology. It is used in periodontal surgery to regenerate cementum, periodontal ligament, and alveolar bone. However, the precise molecular mechanisms underlying periodontal regeneration are still unclear. In this study, we investigated the proteins bound to amelogenin, which are suggested to play a pivotal role in promoting periodontal tissue regeneration. To identify new molecules that interact with amelogenin and are involved in osteoblast activation, we employed coupling affinity chromatography with proteomic analysis in fractionated SaOS-2 osteoblastic cell lysate. In SaOS-2 cells, many of the amelogenin-interacting proteins in the cytoplasm were mainly cytoskeletal proteins and several chaperone molecules of heat shock protein 70 (HSP70 family. On the other hand, the proteomic profiles of amelogenin-interacting proteins in the membrane fraction of the cell extracts were quite different from those of the cytosolic-fraction. They were mainly endoplasmic reticulum (ER-associated proteins, with lesser quantities of mitochondrial proteins and nucleoprotein. Among the identified amelogenin-interacting proteins, we validated the biological interaction of amelogenin with glucose-regulated protein 78 (Grp78/Bip, which was identified in both cytosolic and membrane-enriched fractions. Confocal co-localization experiment strongly suggested that Grp78/Bip could be an amelogenin receptor candidate. Further biological evaluations were examined by Grp78/Bip knockdown analysis with and without amelogenin. Within the limits of the present study, the interaction of amelogenin with Grp78/Bip contributed to cell proliferation, rather than correlate with the osteogenic differentiation in SaOS-2 cells. Although the biological significance of other interactions are not yet explored, these findings suggest that the differential effects of amelogenin-derived osteoblast activation could be of

  14. Recombinant Newcastle disease virus (rL-RVG)triggers apoptosis by inducing endoplasmic reticulum stress in gastric carcinoma cell%重组新城疫病毒rL-RV G诱导人胃腺癌细胞内质网应激和凋亡

    Institute of Scientific and Technical Information of China (English)

    赵英海; 严玉兰; 李米; 王穆彬; 步雪峰


    Objective:To investigate the effects of newcastle disease virus (NDV)and recombinant avirulent NDV LaSota strain expressing the rabies virus glycoprotein(rL-RVG)on endoplasmic reticulum stress and apoptosis of HGC cells.Methods:The cells were divided into three groups at random,control group,NDV group,and rL-RVG group,which infected with PBS,NDV and rL-RVG,respectively.The expression of endoplasmic reticulum stress related proteins,glucose-regulated protein 78 (GRP78 ), CCAAT/enhancer-binding protein homologous protein(CHOP)were analyzed by western blotting and im-munofluorescence.The HGC cells were divided into four groups at random,control group,4-PBA group, rL-RVG group and 4-PBA+rL-RVG group.4-PBA group and 4-PBA+rL-RVG group were treated with sodium phenylbutyrate(4-PBA)for 4 h,then rL-RVG group and 4-PBA+rL-RVG group were infected with rL-RVG,while rL-RVG were replaced with PBS in control group and 4-PBA group.The expression of caspase-3 protein was detected with western blotting.The rate of cell early apoptosis were detected by flow cytometry.Results:After infected 24 hours,compared with the control group,NDV group and rL-RVG group had high levels of GRP78 and CHOP protein expression (P<0.05 ),and rL-RVG group showed higher levels than NDV group(P<0.01).The expression of GRP78,CHOP,caspase-3 protein and the rate of apoptosis in 4-PBA+rL-RVG group was lower than rL-RVG group after pretreated with 4-PBA (P<0.05).Conclusion:The rL-RVG could induce stronger endoplasmic reticulum stress and re-lated apoptosis than the NDV.%目的:观察新城疫病毒(newcastle disease virus,NDV)及稳定表达狂犬病毒糖蛋白的重组新城疫病毒(re-combinant avirulent NDV LaSota strain expressing the rabies virus glycoprotein,rL-RVG)对胃腺癌HGC细胞内质网应激及凋亡的影响。方法:将HGC细胞随机分为对照组、NDV组和rL-RVG组,分别转染PBS、NDV和rL-RVG 24 h,免疫印迹法和免疫荧光检测糖调节蛋白78

  15. Amino acid substitutions in the melanoma antigen recognized by T cell 1 peptide modulate cytokine responses in melanoma-specific T cells

    DEFF Research Database (Denmark)

    Nielsen, M B; Kirkin, A F; Loftus, D


    enhances the production of mRNA for interleukin (IL)-5, IL-10, IL-13, IL-15, and interferon-gamma and significantly enhances release of IL-13 and IL-10 from anti-MART-1 cytotoxic T cells. Another heteroclitic peptide, 1L, with an A to L substitution in MART-1(27-35), also enhances the tyrosine...

  16. Recognizing outstanding achievements (United States)

    Speiss, Fred

    One function of any professional society is to provide an objective, informed means for recognizing outstanding achievements in its field. In AGU's Ocean Sciences section we have a variety of means for carrying out this duty. They include recognition of outstanding student presentations at our meetings, dedication of special sessions, nomination of individuals to be fellows of the Union, invitations to present Sverdrup lectures, and recommendations for Macelwane Medals, the Ocean Sciences Award, and the Ewing Medal.Since the decision to bestow these awards requires initiative and judgement by members of our section in addition to a deserving individual, it seems appropriate to review the selection process for each and to urge you to identify those deserving of recognition.

  17. Amino acid sequences recognized by T cells: studies on a merozoite surface antigen from the FCQ-27/PNG isolate of Plasmodium falciparum. (United States)

    Rzepczyk, C M; Csurhes, P A; Baxter, E P; Doran, T J; Irving, D O; Kere, N


    Twenty-six overlapping peptides, spanning the entire FCQ-27/PNG sequence of the Plasmodium falciparum antigen known as merozoite surface antigen 2 were screened for their ability to induce the proliferation of peripheral blood lymphocytes (PBL) obtained from 12 donors living in Honiara, Solomon Islands where P. falciparum is endemic. A recombinant (r) form of MSA2, known as Ag 1609 was also screened in these assays and tetanus toxoid (TT) antigen was included as a control. The location of the predicted T cell determinants within MSA2 was examined using the algorithm, AMPHI and by scanning MSA2 for amino acid sequences showing the Rothbard motif. There were 13 predicted amphipathic helical sites and five examples of Rothbard sequences in the antigen. The location of these with regard to the peptides tested is shown. Nine of the 12 individuals responded to TT with high stimulation indices (greater than 4) being obtained in the majority of donors. Only three individuals responded to r-MSA2 with the stimulation indices (SI) in the range of 2.4-4.1. Peptides from both the constant and variable regions of MSA2 were recognized in the proliferative assays. However, the majority of the positive proliferative responses were to peptides which spanned the central variable region which included the two copies of the 32-amino-acid repeat occurring in the antigen. High SI comparable to those obtained to TT were seen in some individuals with some peptides. There was considerable variation between donors in number and nature of the peptides recognised and two donors did not respond to any of the antigens tested. The significance of these findings to vaccine development is discussed.

  18. B-cell epitopes in NTS-DBL1α of PfEMP1 recognized by human antibodies in Rosetting Plasmodium falciparum. (United States)

    Albrecht, Letusa; Angeletti, Davide; Moll, Kirsten; Blomqvist, Karin; Valentini, Davide; D'Alexandri, Fabio Luiz; Maurer, Markus; Wahlgren, Mats


    Plasmodium falciparum is the most lethal of the human malaria parasites. The virulence is associated with the capacity of the infected red blood cell (iRBC) to sequester inside the deep microvasculature where it may cause obstruction of the blood-flow when binding is excessive. Rosetting, the adherence of the iRBC to uninfected erythrocytes, has been found associated with severe malaria and found to be mediated by the NTS-DBL1α-domain of Plasmodium falciparum Erythrocyte Membrane Protein 1 (PfEMP1). Here we show that the reactivity of plasma of Cameroonian children with the surface of the FCR3S1.2-iRBC correlated with the capacity to disrupt rosettes and with the antibody reactivity with a recombinant PfEMP1 (NTS-DBL1α of IT4var60) expressed by parasite FCR3S1.2. The plasma-reactivity in a microarray, consisting of 96 overlapping 15-mer long peptides covering the NTS-DBL1α domain from IT4var60 sequence, was compared with their capacity to disrupt rosettes and we identified five peptides where the reactivity were correlated. Three of the peptides were localized in subdomain-1 and 2. The other two peptide-sequences were localized in the NTS-domain and in subdomain-3. Further, principal component analysis and orthogonal partial least square analysis generated a model that supported these findings. In conclusion, human antibody reactivity with short linear-peptides of NTS-DBL1α of PfEMP1 suggests subdomains 1 and 2 to hold anti-rosetting epitopes recognized by anti-rosetting antibodies. The data suggest rosetting to be mediated by the variable areas of PfEMP1 but also to involve structurally relatively conserved areas of the molecule that may induce biologically active antibodies.

  19. Definition of the region on NS3 which contains multiple epitopes recognized by dengue virus serotype-cross-reactive and flavivirus-cross-reactive, HLA-DPw2-restricted CD4+ T cell clones. (United States)

    Okamoto, Y; Kurane, I; Leporati, A M; Ennis, F A


    The epitopes recognized by six CD4+ CD8- cytotoxic T lymphocyte (CTL) clones established from a dengue-3 virus-immune donor were defined. (i) Three CTL clones, JK10, JK34 and JK39, were cross-reactive for dengue virus types 1-4. (ii) One clone, JK28, was cross-reactive for dengue virus types 1-4 and West Nile virus. (iii) Two clones, JK26 and JK49, were cross-reactive for dengue virus types 1-4, West Nile virus and yellow fever virus. The clones, except for JK49, recognized the same epitope on NS3 in an HLA-DPw2-restricted fashion. The smallest synthetic peptide recognized by the five CTL clones was a 10 aa peptide which comprises aa 255-264 on dengue virus NS3. JK49 recognized the overlapping epitope which comprises aa 257-266 in an HLA-DPw2-restricted fashion. Analysis of T cell receptor (TCR) usage by these T cell clones revealed that (i) JK10 and JK34 use V alpha11, and JK34 and JK28 use V beta23, and (ii) the amino acid sequences of the V(D)J junctional region of the TCR were different among these five CTL clones. There were, however, single amino acid conservations among TCRs of some of these T cell clones. These results indicate that the region on NS3 which comprises aa 255-266 contains multiple epitopes recognized by dengue serotype-cross-reactive and flavivirus-cross-reactive CD4+ CTL in an HLA-DPw2-restricted fashion and that a single epitope can be recognized by T cells which have heterogeneous virus specificities.

  20. Melatonin-Mediated Intracellular Insulin during 2-Deoxy-d-glucose Treatment Is Reduced through Autophagy and EDC3 Protein in Insulinoma INS-1E Cells

    Directory of Open Access Journals (Sweden)

    Han Sung Kim


    Full Text Available 2-DG triggers glucose deprivation without altering other nutrients or metabolic pathways and then activates autophagy via activation of AMPK and endoplasmic reticulum (ER stress. We investigated whether 2-DG reduced intracellular insulin increased by melatonin via autophagy/EDC3 in insulinoma INS-1E cells. p-AMPK and GRP78/BiP level were significantly increased by 2-DG in the presence/absence of melatonin, but IRE1α level was reduced in 2-DG treatment. Levels of p85α, p110, p-Akt (Ser473, Thr308, and p-mTOR (Ser2481 were also significantly reduced by 2-DG in the presence/absence of melatonin. Mn-SOD increased with 2-DG plus melatonin compared to groups treated with/without melatonin alone. Bcl-2 was decreased and Bax increased with 2-DG plus melatonin. LC3II level increased with 2-DG treatment in the presence/absence of melatonin. Intracellular insulin production increased in melatonin plus 2-DG but reduced in treatment with 2-DG with/without melatonin. EDC3 was increased by 2-DG in the presence/absence of melatonin. Rapamycin, an mTOR inhibitor, increased GRP78/BiP and EDC3 levels in a dose-dependent manner and subsequently resulted in a decrease in intracellular production of insulin. These results suggest that melatonin-mediated insulin synthesis during 2-DG treatment involves autophagy and EDC3 protein in rat insulinoma INS-1E cells and subsequently results in a decrease in intracellular production of insulin.

  1. MnCl2引起SH-SY5Y细胞内质网应激及Sigma-1受体的保护作用%Protection of sigma-1 receptor in the endoplasmic reticulum stress induced by MnCI2 in the SH-SY5Y cells

    Institute of Scientific and Technical Information of China (English)

    米岚; 李振宁; 赵鹏; 王婷; 傅娟玲; 姚碧云; 周宗灿


    Objective To explore whether endoplasmic reticulum stress (ERS) and ERS-mediated apoptosis are involved in the mechanisms of manganese-induced neurotoxicity, further exploring the role of Sigma-IR in the manganese-induced neurotoxicity. Methods Using SH-SY5Y cells as the model of dopaminergic neuron, MTT colorimetry test was used to detect the survival state of SH-SY5Y cells in various doses of manganese chloride; apoptosis of SH-SY5Y cells was detected by flow cytometry (FCM); the expression of endoplasmic reticulum stress chaperone Bip(Grp78) , Sigma-IR, CHOP and Caspase-4 were detected by Western Blot;After Sigma-IR agonist BD1047 inhibit its activity for 4 and 24 hours, apoptosis of SH-SY5Y cells was detected by FCM. Results MnCl2 could dose and time-dependently suppress the viability of SH-SY5Y cells and induce SH-SY5Y cells apoptosis. MnCl2 also could promote the expression of ER stress chaperone Bip(Grp78) , Sigma-IR and apoptosis related protein CHOP, Caspase-4. BD1047 can inhibit the activity of Sigma-IR, and MnCl2 can promote the cell apoptosis largely. Conclusion MnCl2 could cause endoplasmic reticulum stress in SH-SY5Y cells and reduce apoptosis via the pathway of ERS by increasing the expression of Sigma-IR. So, Sigma-IR is likely to be involved in protection of the manganese-induced neurotoxicity.%目的 探讨内质网应激(ERS)及其介导的细胞凋亡在锰引起的神经毒性中的作用,进一步探讨Sigma-1R在锰致神经系统损害中的作用.方法 以人神经母细胞瘤SH-SY5Y细胞为细胞模型,通过MTT比色法检测0.5、1、2、4、8和24 h处MnCl2对细胞存活的影响,流式细胞技术(FCM)检测上述时间段MnCl2对细胞凋亡的影响,Westernblot检测MnCl2作用上述时间后,细胞ERS分子伴侣GRP78、Sigma-1受体(Sigma-1R)、CHOP及凋亡相关蛋白Caspase-4表达的变化,Sigma-1R拮抗剂BD1047抑制其活性4和24 h后,检测细胞凋亡的情况.结果 MnCl2可呈时间、剂量依赖性地降低

  2. Amaranthus leucocarpus lectin recognizes a moesin-like O-glycoprotein and costimulates murine CD3-activated CD4+ T cells


    Arenas-Del Ángel, Maria; Legorreta-Herrera, Martha; Mendoza-Hernández, Guillermo; Garfias, Yonathan; Chávez, Raul; Zenteno, Edgar; Lascurain,Ricardo


    The Galβ1,3GalNAcα1,O-Ser/Thr specific lectin from Amaranthus leucocarpus (ALL) binds a ∼70 kDa glycoprotein on murine T cell surface. We show that in the absence of antigen presenting cells, murine CD4+ T cells activated by an anti-CD3 antibody plus ALL enhanced cell proliferation similar to those cells activated via CD3/CD28 at 48 h of culture. Moreover, ALL induced the production of IL-4, IL-10, TNF-alpha, and TGF-beta in CD3-activated cells. Proteomic assay using two-dimensional electroph...

  3. Trichodermin induces cell apoptosis through mitochondrial dysfunction and endoplasmic reticulum stress in human chondrosarcoma cells

    Energy Technology Data Exchange (ETDEWEB)

    Su, Chen-Ming [Graduate Institute of Basic Medical Science, China Medical University, Taichung, Taiwan (China); Wang, Shih-Wei [Department of Medicine, Mackay Medical College, New Taipei City, Taiwan (China); Lee, Tzong-Huei [Graduate Institute of Pharmacognosy, Taipei Medical University, Taipei, Taiwan (China); Tzeng, Wen-Pei [Graduate Institute of Sports and Health, National Changhua University of Education, Changhua, Taiwan (China); Hsiao, Che-Jen [School of Respiratory Therapy, College of Medicine, Taipei Medical University, Taipei, Taiwan (China); Liu, Shih-Chia [Department of Orthopaedics, Mackay Memorial Hospital, Taipei, Taiwan (China); Tang, Chih-Hsin, E-mail: [Graduate Institute of Basic Medical Science, China Medical University, Taichung, Taiwan (China); Department of Pharmacology, School of Medicine, China Medical University, Taichung, Taiwan (China); Department of Biotechnology, College of Health Science, Asia University, Taichung, Taiwan (China)


    Chondrosarcoma is the second most common primary bone tumor, and it responds poorly to both chemotherapy and radiation treatment. Nalanthamala psidii was described originally as Myxosporium in 1926. This is the first study to investigate the anti-tumor activity of trichodermin (trichothec-9-en-4-ol, 12,13-epoxy-, acetate), an endophytic fungal metabolite from N. psidii against human chondrosarcoma cells. We demonstrated that trichodermin induced cell apoptosis in human chondrosarcoma cell lines (JJ012 and SW1353 cells) instead of primary chondrocytes. In addition, trichodermin triggered endoplasmic reticulum (ER) stress protein levels of IRE1, p-PERK, GRP78, and GRP94, which were characterized by changes in cytosolic calcium levels. Furthermore, trichodermin induced the upregulation of Bax and Bid, the downregulation of Bcl-2, and the dysfunction of mitochondria, which released cytochrome c and activated caspase-3 in human chondrosarcoma. In addition, animal experiments illustrated reduced tumor volume, which led to an increased number of terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL)-positive cells and an increased level of cleaved PARP protein following trichodermin treatment. Together, this study demonstrates that trichodermin is a novel anti-tumor agent against human chondrosarcoma cells both in vitro and in vivo via mitochondrial dysfunction and ER stress. - Highlights: • Trichodermin induces chondrosarcoma apoptosis. • ER stress is involved in trichodermin-induced cell death. • Trichodermin induces chondrosarcoma death in vivo.


    Directory of Open Access Journals (Sweden)

    E. P. Starikova


    Full Text Available Abstract. The aim of study was to compare the influence of lipopolysaccharide (LPS component from Gram-negative bacteria (E. coli 055:B5, and a lysate of Gram-positive bacterium (Streptococcus pyogenes, type M1, strain 40/58 upon transendothelial migration rates of monocyte-like cells (THP-1 strain. Both LPS and lysate of Streptococcus pyogenes acted as chemoattractants for THP-1 cells. he studied components of Streptococcus pyogenes proved to be more active stimulants of transendothelial THP-1 cell migration, than LPS from E. coli. During spontaneous transmigration of THP-1 cells through a monolayer of endothelial cells, augmented levels of chemokines (RANTES, MCP-1, IL-8, IP-10 were noticed, that were more pronounced in presence of LPS. Upon spontaneous transmigration of THP-1 cells through endothelial monolayer, the levels of proinflammatory cytokines (TNFα, IL-1β and IL-6 in cultural medium were found to be rather low. The transmigration-associated secretion of these cytokines increased in presence of LPS and Streptococcus pyogenes lysate. Incubation with these bacterial constituents did increase cytokine levels both in monoculture of THP-1 cells and in transmigration model. Our results suggest that the levels of THP-1 transendothelial migration depend mainly on activation of monocyte-like cells influenced by PRR-ligands from Streptococcus pyogenes lysate. (Med. Immunol., vol. 10, N 6, pp 571-576.

  5. The dendritic cell-specific C-type lectin DC-SIGN is a receptor for Schistosoma mansoni egg antigens and recognizes the glycan antigen Lewis x.

    NARCIS (Netherlands)

    Die, van I.M.; Vliet, van SJ; Nyame, AK; Cummings, RD; Bank, CM; Appelmelk, B.J.; Geijtenbeek, T.B.H.; Kooijk, van Y.


    Schistosoma mansoni soluble egg antigens (SEAs) are crucially involved in modulating the host immune response to infection by S. mansoni. We report that human dendritic cells bind SEAs through the C-type lectin dendritic cell-specific ICAM-3-grabbing nonintegrin (DC-SIGN). Monoclonal antibodies agai

  6. Production of interferon-¿ and interleukin-4 by human T cells recognizing Leishmania lipophosphoglycan-associated protein

    DEFF Research Database (Denmark)

    Kemp, M; Kurtzhals, J A; Christensen, C B


    The Leishmania protein LPGAP which is co-isolated with lipophosphoglycan is a specific activator of T cells from individuals who have recovered from American leishmaniasis. We have tested the effect of LPGAP on peripheral blood mononuclear cells (PBMC) from Kenyan donors cured from L. donovani...

  7. Bacteriocin protein BacL1 of Enterococcus faecalis targets cell division loci and specifically recognizes L-Ala2-cross-bridged peptidoglycan. (United States)

    Kurushima, Jun; Nakane, Daisuke; Nishizaka, Takayuki; Tomita, Haruyoshi


    Bacteriocin 41 (Bac41) is produced from clinical isolates of Enterococcus faecalis and consists of two extracellular proteins, BacL1 and BacA. We previously reported that BacL1 protein (595 amino acids, 64.5 kDa) is a bacteriolytic peptidoglycan D-isoglutamyl-L-lysine endopeptidase that induces cell lysis of E. faecalis when an accessory factor, BacA, is copresent. However, the target of BacL1 remains unknown. In this study, we investigated the targeting specificity of BacL1. Fluorescence microscopy analysis using fluorescent dye-conjugated recombinant protein demonstrated that BacL1 specifically localized at the cell division-associated site, including the equatorial ring, division septum, and nascent cell wall, on the cell surface of target E. faecalis cells. This specific targeting was dependent on the triple repeat of the SH3 domain located in the region from amino acid 329 to 590 of BacL1. Repression of cell growth due to the stationary state of the growth phase or to treatment with bacteriostatic antibiotics rescued bacteria from the bacteriolytic activity of BacL1 and BacA. The static growth state also abolished the binding and targeting of BacL1 to the cell division-associated site. Furthermore, the targeting of BacL1 was detectable among Gram-positive bacteria with an L-Ala-L-Ala-cross-bridging peptidoglycan, including E. faecalis, Streptococcus pyogenes, or Streptococcus pneumoniae, but not among bacteria with alternate peptidoglycan structures, such as Enterococcus faecium, Enterococcus hirae, Staphylococcus aureus, or Listeria monocytogenes. These data suggest that BacL1 specifically targets the L-Ala-L-Ala-cross-bridged peptidoglycan and potentially lyses the E. faecalis cells during cell division.

  8. Inability to induce consistent T-cell responses recognizing conserved regions within HIIV-1 antigens: a potential mechanism for lack of vaccine efficacy in the step study

    Energy Technology Data Exchange (ETDEWEB)

    Korber, Bette [Los Alamos National Laboratory; Szinger, James [Los Alamos National Laboratory


    T cell based vaccines are based upon the induction of CD8+ T cell memory responses that would be effective in inhibiting infection and subsequent replication of an infecting HIV-1 strain, a process that requires a high probability of matching the epitope induced by vaccination with the infecting viral strain. We compared the frequency and specificity of the CTL epitopes elicited by the replication defective AdS gag/pol/nef vaccine used in the STEP trial with the likelihood of encountering those epitopes among recently sequenced Clade B isolates of HIV-1. On average vaccination elicited only one epitope per gene. Importantly, the highly conserved epitopes in gag, pol, and nef (> 80% of strains in the current collection of the Los Alamos database []) were rarely elicited by vaccination. Moreover there was a statistically significant skewing of the T cell response to relative variable epitopes of each gene; only 20% of persons possessed > 3 T cell responses to epitopes likely to be found in circulating strains in the CladeB populations in which the Step trial was conducted. This inability to elicit T cell responses likely to be found in circulating viral strains is a likely factor in the lack of efficacy of the vaccine utilized in the STEP trial. Modeling of the epitope specific responses elicited by vaccination, we project that a median of 8-10 CD8+ T cell epitopes are required to provide >80% likelihood of eliciting at least 3 CD8+ T cell epitopes that would be found on a circulating population of viruses. Development of vaccine regimens which elicit either a greater breadth of responses or elicit responses to conserved regions of the HIV-1 genome are needed to fully evaluate the concept of whether induction of T cell immunity can alter HIV-1 in vivo.

  9. Differential impact of bortezomib on HL-60 and K562 cells. (United States)

    Kliková, Katarína; Štefaniková, Andrea; Pilchová, Ivana; Hatok, Jozef; Chudý, Peter; Chudej, Juraj; Dobrota, Dušan; Račay, Peter


    Bortezomib (PS-341, or Velcade), reversible inhibitor of 20S proteasome approved for the treatment of multiple myeloma and mantle cell lymphoma, exhibited a cytotoxic effect toward other malignancies including leukaemia. In this study, we have documented that incubation of both HL-60 and K562 leukaemia cells with nanomolar concentrations of bortezomib is associated with the death of HL-60 cells observed within 24 hours of incubation with bortezomib and the death of K562 cells that were observed after 72 hours of incubation with bortezomib. The relative resistance of K562 cells to bortezomib correlated well with significantly higher expression of HSP27, HSP70, HSP90α, HSP90β and GRP75 in these cells. Incubation of both HL-60 and K562 cells with bortezomib induced a cleavage of HSP90β as well as expression of HSP70 and HSP90β but bortezomib did not affect levels of HSP27, HSP90α, GRP75 and GRP78. The death of both types of cells was accompanied with proteolytic activation of caspase 3 that was observed in HL-60 cells and proteolytic degradation of procaspase 3 in K562 cells. Our study has also pointed to essential role of caspase 8 in bortezomib-induced cleavage of HSP90β in both HL-60 and K562 cells. Finally, we have shown that bortezomib induced activation of caspase 9/caspase 3 axis in HL-60 cells, while the mechanism of death of K562 cells remains unknown.

  10. Structural Mechanism Underpinning Cross-reactivity of a CD8+ T-cell Clone That Recognizes a Peptide Derived from Human Telomerase Reverse Transcriptase* (United States)

    Cole, David K.; van den Berg, Hugo A.; Lloyd, Angharad; Crowther, Michael D.; Beck, Konrad; Ekeruche-Makinde, Julia; Miles, John J.; Bulek, Anna M.; Dolton, Garry; Schauenburg, Andrea J.; Wall, Aaron; Fuller, Anna; Clement, Mathew; Laugel, Bruno; Rizkallah, Pierre J.; Wooldridge, Linda; Sewell, Andrew K.


    T-cell cross-reactivity is essential for effective immune surveillance but has also been implicated as a pathway to autoimmunity. Previous studies have demonstrated that T-cell receptors (TCRs) that focus on a minimal motif within the peptide are able to facilitate a high level of T-cell cross-reactivity. However, the structural database shows that most TCRs exhibit less focused antigen binding involving contact with more peptide residues. To further explore the structural features that allow the clonally expressed TCR to functionally engage with multiple peptide-major histocompatibility complexes (pMHCs), we examined the ILA1 CD8+ T-cell clone that responds to a peptide sequence derived from human telomerase reverse transcriptase. The ILA1 TCR contacted its pMHC with a broad peptide binding footprint encompassing spatially distant peptide residues. Despite the lack of focused TCR-peptide binding, the ILA1 T-cell clone was still cross-reactive. Overall, the TCR-peptide contacts apparent in the structure correlated well with the level of degeneracy at different peptide positions. Thus, the ILA1 TCR was less tolerant of changes at peptide residues that were at, or adjacent to, key contact sites. This study provides new insights into the molecular mechanisms that control T-cell cross-reactivity with important implications for pathogen surveillance, autoimmunity, and transplant rejection. PMID:27903649

  11. Strategies for the identification of T cell-recognized tumor antigens in hematological malignancies for improved graft-versus-tumor responses after allogeneic blood and marrow transplantation. (United States)

    Zilberberg, Jenny; Feinman, Rena; Korngold, Robert


    Allogeneic blood and marrow transplantation (allo-BMT) is an effective immunotherapeutic treatment that can provide partial or complete remission for patients with hematological malignancies. Mature donor T cells in the donor inoculum play a central role in mediating graft-versus-tumor (GVT) responses by destroying residual tumor cells that persist after conditioning regimens. Alloreactivity towards minor histocompatibility antigens (miHA), which are varied tissue-related self-peptides presented in the context of major histocompatibility complex (MHC) molecules on recipient cells, some of which may be shared on tumor cells, is a dominant factor for the development of GVT. Potentially, GVT can also be directed to tumor-associated antigens or tumor-specific antigens that are more specific to the tumor cells themselves. The full exploitation of allo-BMT, however, is greatly limited by the development of graft-versus-host disease (GVHD), which is mediated by the donor T cell response against the miHA expressed in the recipient's cells of the intestine, skin, and liver. Because of the significance of GVT and GVHD responses in determining the clinical outcome of patients, miHA and tumor antigens have been intensively studied, and one active immunotherapeutic approach to separate these two responses has been cancer vaccination after allo-BMT. The combination of these two strategies has an advantage over vaccination of the patient without allo-BMT because his or her immune system has already been exposed and rendered unresponsive to the tumor antigens. The conditioning for allo-BMT eliminates the patient's existing immune system, including regulatory elements, and provides a more permissive environment for the newly developing donor immune compartment to selectively target the malignant cells. Utilizing recent technological advances, the identities of many human miHA and tumor antigenic peptides have been defined and are currently being evaluated in clinical and basic

  12. Cross-clade conservation of HIV type 1 Nef immunodominant regions recognized by CD8+ T cells of HIV type 1 CRF02_AG-infected Ivorian (West Africa). (United States)

    Inwoley, André; Recordon-Pinson, Patricia; Dupuis, Marion; Gaston, Jessintha; Genête, Mathieu; Minga, Albert; Letourneur, Franck; Rouet, François; Choppin, Jeannine; Fleury, Hervé; Guillet, Jean-Gérard; Andrieu, Muriel


    Most HIV vaccine trials in the world are conducted with clade B while most circulating viral strains in Africa are non-B subtypes. We determined whether CD8+ T cells from HIV-1 intersubtype CRF02_AG-infected Ivorian individuals were able to recognize clade B epitopes. CD8+ T cell responses of nine HIV-1 intersubtype CRF02_AG-infected Ivorian patients and nine HIV-1 subtype B-infected French patients were studied using pools of HIV-1 clade B peptides (110 well-defined HIV CD8+ T cell epitopes) in an ELISPOT IFN-gamma assay. There was no difference in the number of recognized peptide pools between Ivorian and French cohorts (mean of four pools in both cases). Ivorian individuals had generated CD8+ T cell responses cross-reactive against HIV-1 subtype B and some individual peptides had been identified. Furthermore, sequence analysis of nef HIV genes of the Ivorian patients and nef cloning in two patients revealed very few variations between HIV- 1 intersubtype CRF02_AG and subtype B in nef immunodominant regions included in HIV clade B lipopeptide vaccines, currently tested in France.

  13. The preferential homing of a platelet derived growth factor receptor-recognizing macromolecule to fibroblast-like cells in fibrotic tissue

    NARCIS (Netherlands)

    Beljaars, L.; Weert, B; Geerts, Albert; Meijer, D.K F; Poelstra, Klaas


    Platelet derived growth factor (PDGF) is a key factor in the induction and progression of fibrotic diseases with the activated fibroblast as its target cell. Drug targeting to the PDGF-receptor is explored as a new approach to treat this disease. Therefore, we constructed a macromolecule with affini

  14. Spontaneous human squamous cell carcinomas are killed by a human cytotoxic T lymphocyte clone recognizing a wild-type p53-derived peptide

    DEFF Research Database (Denmark)

    Röpke, M; Hald, J; Guldberg, Per


    A cytotoxic T lymphocyte (CTL) clone generated in vitro from the peripheral blood of a healthy HLA-A2-positive individual against a synthetic p53 protein-derived wild-type peptide (L9V) was shown to kill squamous carcinoma cell lines derived from two head and neck carcinomas, which expressed mutant...

  15. Three novel NY-ESO-1 epitopes bound to DRB1*0803, DQB1*0401 and DRB1*0901 recognized by CD4 T cells from CHP-NY-ESO-1-vaccinated patients. (United States)

    Mizote, Yu; Taniguchi, Taku; Tanaka, Kei; Isobe, Midori; Wada, Hisashi; Saika, Takashi; Kita, Shoichi; Koide, Yukari; Uenaka, Akiko; Nakayama, Eiichi


    Three novel NY-ESO-1 CD4 T cell epitopes were identified using PBMC obtained from patients who were vaccinated with a complex of cholesterol-bearing hydrophobized pullulan (CHP) and NY-ESO-1 protein (CHP-NY-ESO-1). The restriction molecules were determined by antibody blocking and using various EBV-B cells with different HLA alleles as APC to present peptides to CD4 T cells. The minimal epitope peptides were determined using various N- and C-termini truncated peptides deduced from 18-mer overlapping peptides originally identified for recognition. Those epitopes were DRB1*0901-restricted NY-ESO-1 87-100, DQB1*0401-restricted NY-ESO-1 95-107 and DRB1*0803-restricted NY-ESO-1 124-134. CD4 T cells used to determine those epitope peptides recognized EBV-B cells or DC that were treated with recombinant NY-ESO-1 protein or NY-ESO-1-expressing tumor cell lysate, suggesting that the epitope peptides are naturally processed. These CD4 T cells showed a cytokine profile with Th1 characteristics. Furthermore, NY-ESO-1 87-100 peptide/HLA-DRB1*0901 tetramer staining was observed. Multiple Th1-type CD4 T cell responses are beneficial for inducing effective anti-tumor responses after NY-ESO-1 protein vaccination.

  16. Porcine B-cells recognize epitopes that are conserved between the structural proteins of American- and European-type porcine reproductive and respiratory syndrome virus

    DEFF Research Database (Denmark)

    Oleksiewicz, Martin B.; Bøtner, Anette; Normann, Preben


    By selecting phage display libraries with immune sera from experimentally infected pigs, porcine B-cell epitopes in the open reading frame (ORF) 2, 3, 5 and 6 proteins of European-type porcine reproductive and respiratory syndrome virus (PRRSV) were identified. The sequences of all the epitopes...... were well conserved in European-type PRRSV and even between European- and American-type PRRSV. Accordingly, sera from pigs infected with American-type PRRSV cross-reacted with the European-type epitopes. Thus, this study showed, for the first time, the presence of highly conserved epitopes...... epitopes was subjected to closer scrutiny. A heptad motif, VSRRIYQ, which is present in a single copy in ORF2 and 3 proteins, was identified; this arrangement is completely conserved in all European-type PRRSV sequences available. The VSRRIYQ repeat motif colocalized closely with one of the ORF2 epitopes...

  17. Recognizing Prefixes in Scientific Quantities (United States)

    Sokolowski, Andrzej


    Although recognizing prefixes in physical quantities is inherent for practitioners, it might not be inherent for students, who do not use prefixes in their everyday life experiences. This deficiency surfaces in AP Physics exams. For example, readers of an AP Physics exam reported "a common mistake of incorrectly converting nanometers to…

  18. Recognizing the adolescent drug abuser. (United States)

    MacKenzie, R G; Jacobs, E A


    Adolescents are at high risk for using and abusing illicit drugs. Guidelines for recognizing drug abusers are presented as well as a staging process for progression of drug use. The family physician is in an ideal position to identify young users/abusers and to assist them and their families in obtaining much needed assistance.

  19. Imaging of non-small cell lung cancers with a monoclonal antibody, KC-4G3, which recognizes a human milk fat globule antigen

    Energy Technology Data Exchange (ETDEWEB)

    Dienhart, D.G.; Schmelter, R.F.; Lear, J.L.; Miller, G.J.; Glenn, S.D.; Bloedow, D.C.; Kasliwal, R.; Moran, P.; Seligman, P.; Murphy, J.R. (Univ. of Colorado Cancer Center, Denver (USA))


    To determine the role of lung cancer tumor imaging with monoclonal antibodies directed against high molecular weight human milk fat globule antigens, we administered i.v. 111In-KC-4G3 to 24 patients with advanced non-small cell lung cancer. One mg of 111In-KC-4G3 was mixed with 0, 9, 49, 99, or 499 mg of unlabeled KC-4G3 and infused i.v. over 1 to 5 h. The mean 111In-KC-4G3 radiochemical purity was greater than 97% and the resultant immunoreactivity averaged 62%. Successful imaging of cancer sites was accomplished in 92% of 24 patients, and 57% of 91 total lesions were visualized. Successful localization of tumor sites related to size (P less than 0.001), with 81% of lesions greater than 3.0 cm in diameter, 50% of lesions 1.5 to 3 cm, and 6% of lesions less than 1.5 cm successfully imaging, and to location (P less than 0.05), with 69% of pulmonary lesions, 80% of soft tissue lesions, and only 32% of bone metastases being visualized. Nonspecific reticulo-endothelial uptake of radioactivity was a major problem. Approximately 35% of 111In was chelated to serum transferrin by 24 and 48 h after infusion. The mean t 1/2 beta for plasma radioisotope and immunoreactive KC-4G3 was 29 and 27 h, respectively. There was no correlation between total infused antibody dose and imaging success or between total dose and effect on 111In and KC-4G3 kinetics. Circulating free KC-4 antigen was measurable in all but one patient before study. Tumor biopsy following infusion could demonstrate antibody presence but not saturable antigen binding. We conclude that (a) 111In-KC-4G3 demonstrates successful tumor localization in non-small cell lung cancers bearing generally high expression of its antigen and (b) further investigations to diminish nonspecific radioactivity for imaging and utilization of high dose radiolabeled antibody for therapeutic intent are warranted.

  20. Implication of unfolded protein response in resveratrol-induced inhibition of K562 cell proliferation

    Energy Technology Data Exchange (ETDEWEB)

    Liu, Bao-Qin; Gao, Yan-Yan; Niu, Xiao-Fang [Department of Biochemistry and Molecular Biology, China Medical University, Shenyang 110001 (China); Xie, Ji-Sheng [Youjiang Medical College for Nationalities, Guangxi 533000 (China); Meng, Xin; Guan, Yifu [Department of Biochemistry and Molecular Biology, China Medical University, Shenyang 110001 (China); Wang, Hua-Qin, E-mail: [Department of Biochemistry and Molecular Biology, China Medical University, Shenyang 110001 (China)


    Resveratrol (RES), a natural plant polyphenol, is an effective inducer of cell cycle arrest and apoptosis in a variety of carcinoma cell types. In addition, RES has been reported to inhibit tumorigenesis in several animal models suggesting that it functions as a chemopreventive and anti-tumor agent in vivo. The chemopreventive and chemotherapeutic properties associated with resveratrol offer promise for the design of new chemotherapeutic agents. However, the mechanisms by which RES mediates its effects are not yet fully understood. In this study, we showed that RES caused cell cycle arrest and proliferation inhibition via induction of unfolded protein response (UPR) in human leukemia K562 cell line. Treatment of K562 cells with RES induced a number of signature UPR markers, including transcriptional induction of GRP78 and CHOP, phosphorylation of eukaryotic initiation factor 2{alpha} (eIF2{alpha}), ER stress-specific XBP-1 splicing, suggesting the induction of UPR by RES. RES inhibited proliferation of K562 in a concentration-dependent manner. Flow cytometric analyses revealed that K562 cells were arrested in G1 phase upon RES treatment. Salubrinal, an eIF2{alpha} inhibitor, or overexpression of dominant negative mutants of PERK or eIF2{alpha}, effectively restored RES-induced cell cycle arrest, underscoring the important role of PERK/eIF2{alpha} branch of UPR in RES-induced inhibition of cell proliferation.

  1. Recognizing species, present and past. (United States)

    Tattersall, Ian


    Nobody disputes that nature is meaningfully "packaged" in some way. But debate persists over exactly how (and even whether) the boundaries dividing taxa should (can) be drawn. At one end of the scale, some zealots abstrusely deny real existence to higher taxa.(1) At the other, laborers at the taxonomic rock-face confront genuine challenges in recognizing and delineating the species that systematists agree constitute the most fundamental unit of taxonomic analysis.

  2. OASIS/CREB3L1 is induced by endoplasmic reticulum stress in human glioma cell lines and contributes to the unfolded protein response, extracellular matrix production and cell migration.

    Directory of Open Access Journals (Sweden)

    Ravi N Vellanki

    Full Text Available OASIS is a transcription factor similar to ATF6 that is activated by endoplasmic reticulum stress. In this study we investigated the expression of OASIS in human glioma cell lines and the effect of OASIS knock-down on the ER stress response and cell migration. OASIS mRNA was detected in three distinct glioma cell lines (U373, A172 and U87 and expression levels were increased upon treatment with ER stress-inducing compounds in the U373 and U87 lines. OASIS protein, which is glycosylated on Asn-513, was detected in the U373 and U87 glioma lines at low levels in control cells and protein expression was induced by ER stress. Knock-down of OASIS in human glioma cell lines resulted in an attenuated unfolded protein response to ER stress (reduced GRP78/BiP and GRP94 induction and decreased expression of chondroitin sulfate proteoglycan extracellular matrix proteins, but induction of the collagen gene Col1a1 was unaffected. Cells in which OASIS was knocked-down exhibited altered cell morphology and reduced cell migration. These results suggest that OASIS is important for the ER stress response and maintenance of some extracellular matrix proteins in human glioma cells.

  3. Distinct subpopulations of head and neck cancer cells with different levels of intracellular reactive oxygen species exhibit diverse stemness, proliferation, and chemosensitivity. (United States)

    Chang, Ching-Wen; Chen, Yu-Syuan; Chou, Shiu-Huey; Han, Chia-Li; Chen, Yu-Ju; Yang, Cheng-Chieh; Huang, Chih-Yang; Lo, Jeng-Fan


    Head and neck squamous cell carcinoma (HNSCC) is driven by cancer-initiating cells (CIC), but their maintenance mechanisms are obscure. For hematopoietic stem cells, low levels of intracellular reactive oxygen species (ROS(Low)) is known to help sustain stemness properties. In this report, we evaluated the hypothesis that ROS(Low) character conferred CIC properties in HNSCC. Sphere cultures define CIC in HNSCC cell populations (HN-CIC). We found that ROS(Low) cells in HN-CIC defined in this manner were more numerous than in parental HNSCC cells. Further, ROS(Low) cells frequently coexpressed CIC surface markers such as memGrp78 and Glut3. Exploiting flow cytometry to sort cells on the basis of their ROS level, we found that isolated ROS(Low) cells displayed relatively more CIC properties, including quiescence, chemoresistance, in vitro malignant properties, and tumorigenicity. Pharmacological depletion of ROS modulators in cisplatin-treated HN-CIC reduced CIC properties, enhancing cell differentiation and enhancing cisplatin-induced cell death. Overall, our work defined cell subpopulations in HNSCC on the basis of differential intracellular ROS levels, which associated with stemness and chemoresistance properties. On the basis of our findings, we suggest that strategies to promote intracellular ROS levels may heighten the efficacy of conventional chemotherapy used for HNSCC treatment.

  4. Selective release from cultured mammalian cells of heat-shock (stress) proteins that resemble glia-axon transfer proteins. (United States)

    Hightower, L E; Guidon, P T


    Cultured rat embryo cells were stimulated to rapidly release a small group of proteins that included several heat-shock proteins (hsp110, hsp71, hscp73) and nonmuscle actin. The extracellular proteins were analyzed by two-dimensional polyacrylamide gel electrophoresis. Heat-shocked cells released the same set of proteins as control cells with the addition of the stress-inducible hsp110 and hsp71. Release of these proteins was not blocked by either monensin or colchicine, inhibitors of the common secretory pathway. A small amount of the glucose-regulated protein grp78 was externalized by this pathway. The extracellular accumulation of these proteins was inhibited after they were synthesized in the presence of the lysine analogue aminoethyl cysteine. It is likely that the analogue-substituted proteins were misfolded and could not be released from cells, supporting our conclusion that a selective release mechanism is involved. Remarkably, actin and the squid heat-shock proteins homologous to rat hsp71 and hsp110 are also among a select group of proteins transferred from glial cells to the squid giant axon, where they have been implicated in neuronal stress responses (Tytell et al.: Brain Res., 363:161-164, 1986). Based in part on the similarities between these two sets of proteins, we hypothesized that these proteins were released from labile cortical regions of animal cells in response to perturbations of homeostasis in cells as evolutionarily distinct as cultured rat embryo cells and squid glial cells.

  5. Characterization of ionizing radiation-induced unfolded protein response in human vascular endothelial cells

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Eun Ju; Lee, Yoon Jin; Kang, Seong Man [Laboratory of Radiation Effect, Korea Institute of Radiological and Medical Sciences, Seoul (Korea, Republic of)


    Misfolded or unfolded proteins within the endoplasmic reticulum (ER stress), viral infection, or amino acid deprivation induce eukaryotic translation initiation factor 2α phosphorylation (eIF2α) in eukaryotic cells, repressing global protein synthesis coincident with preferential translation of activating transcription factor 4 (ATF4). ATF4 is a transcriptional activator of genes involved in amino acid metabolism, cellular redox homeostasis, and regulation of apoptosis. When the eIF2α/ATF4 pathway is initiated by ER stress, the pathway is referred toas the unfolded protein response (UPR). In addition to DNA, proteins may be initial and important targets of ionizing radiation (IR), and the damaged protein can trigger ER stress pathway. Recent investigations suggested that IR induces ER stress followed by UPR in various cell types including intestinal epithelial cells. We conducted this study to determine whether IR can activate UPR in human vascular endothelial cells. Our data have shown that IR increased PERK-dependent eIF2α phosphorylation accompanied by induction in ATF4 protein levels in human vascular endothelial cells without alterations in expressions of XBP-1s and GRP78. Based on these data, we suggest that IR selectively activates PERK branch of unfolded protein response in human vascular endothelial cells.

  6. Involvement of Endoplasmic Reticulum Stress in Capsaicin-Induced Apoptosis of Human Pancreatic Cancer Cells

    Directory of Open Access Journals (Sweden)

    Shengzhang Lin


    Full Text Available Capsaicin, main pungent ingredient of hot chilli peppers, has been shown to have anticarcinogenic effect on various cancer cells through multiple mechanisms. In this study, we investigated the apoptotic effect of capsaicin on human pancreatic cancer cells in both in vitro and in vivo systems, as well as the possible mechanisms involved. In vitro, treatment of both the pancreatic cancer cells (PANC-1 and SW1990 with capsaicin resulted in cells growth inhibition, G0/G1 phase arrest, and apoptosis in a dose-dependent manner. Knockdown of growth arrest- and DNA damage-inducible gene 153 (GADD153, a marker of the endoplasmic-reticulum-stress- (ERS- mediated apoptosis pathway, by specific siRNA attenuated capsaicin-induced apoptosis both in PANC-1 and SW1990 cells. Moreover, in vivo studies capsaicin effectively inhibited the growth and metabolism of pancreatic cancer and prolonged the survival time of pancreatic cancer xenograft tumor-induced mice. Furthermore, capsaicin increased the expression of some key ERS markers, including glucose-regulated protein 78 (GRP78, phosphoprotein kinase-like endoplasmic reticulum kinase (phosphoPERK, and phosphoeukaryotic initiation factor-2α (phospho-eIF2α, activating transcription factor 4 (ATF4 and GADD153 in tumor tissues. In conclusion, we for the first time provide important evidence to support the involvement of ERS in the induction of apoptosis in pancreatic cancer cells by capsaicin.

  7. A Taiwanese Propolis Derivative Induces Apoptosis through Inducing Endoplasmic Reticular Stress and Activating Transcription Factor-3 in Human Hepatoma Cells

    Directory of Open Access Journals (Sweden)

    Fat-Moon Suk


    Full Text Available Activating transcription factor-(ATF- 3, a stress-inducible transcription factor, is rapidly upregulated under various stress conditions and plays an important role in inducing cancer cell apoptosis. NBM-TP-007-GS-002 (GS-002 is a Taiwanese propolin G (PPG derivative. In this study, we examined the antitumor effects of GS-002 in human hepatoma Hep3B and HepG2 cells in vitro. First, we found that GS-002 significantly inhibited cell proliferation and induced cell apoptosis in dose-dependent manners. Several main apoptotic indicators were found in GS-002-treated cells, such as the cleaved forms of caspase-3, caspase-9, and poly(ADP-ribose polymerase (PARP. GS-002 also induced endoplasmic reticular (ER stress as evidenced by increases in ER stress-responsive proteins including glucose-regulated protein 78 (GRP78, growth arrest- and DNA damage-inducible gene 153 (GADD153, phosphorylated eukaryotic initiation factor 2α (eIF2α, phosphorylated protein endoplasmic-reticular-resident kinase (PERK, and ATF-3. The induction of ATF-3 expression was mediated by mitogen-activated protein kinase (MAPK signaling pathways in GS-002-treated cells. Furthermore, we found that GS-002 induced more cell apoptosis in ATF-3-overexpressing cells. These results suggest that the induction of apoptosis by the propolis derivative, GS-002, is partially mediated through ER stress and ATF-3-dependent pathways, and GS-002 has the potential for development as an antitumor drug.

  8. Valproate Attenuates Endoplasmic Reticulum Stress-Induced Apoptosis in SH-SY5Y Cells via the AKT/GSK3β Signaling Pathway (United States)

    Li, Zhengmao; Wu, Fenzan; Zhang, Xie; Chai, Yi; Chen, Daqing; Yang, Yuetao; Xu, Kebin; Yin, Jiayu; Li, Rui; Shi, Hongxue; Wang, Zhouguang; Li, Xiaokun; Xiao, Jian; Zhang, Hongyu


    Endoplasmic reticulum (ER) stress-induced apoptosis plays an important role in a range of neurological disorders, such as neurodegenerative diseases, spinal cord injury, and diabetic neuropathy. Valproate (VPA), a typical antiepileptic drug, is commonly used in the treatment of bipolar disorder and epilepsy. Recently, VPA has been reported to exert neurotrophic effects and promote neurite outgrowth, but its molecular mechanism is still unclear. In the present study, we investigated whether VPA inhibited ER stress and promoted neuroprotection and neuronal restoration in SH-SY5Y cells and in primary rat cortical neurons, respectively, upon exposure to thapsigargin (TG). In SH-SY5Y cells, cell viability was detected by the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) assay, and the expression of ER stress-related apoptotic proteins such as glucose‑regulated protein (GRP78), C/EBP homologous protein (CHOP), and cleaved caspase-12/-3 were analyzed with Western blot analyses and immunofluorescence assays. To explore the pathway involved in VPA-induced cell proliferation, we also examined p-AKT, GSK3β, p-JNK and MMP-9. Moreover, to detect the effect of VPA in primary cortical neurons, immunofluorescence staining of β-III tubulin and Anti-NeuN was analyzed in primary cultured neurons exposed to TG. Our results demonstrated that VPA administration improved cell viability in cells exposed to TG. In addition, VPA increased the levels of GRP78 and p-AKT and decreased the levels of ATF6, XBP-1, GSK3β, p-JNK and MMP-9. Furthermore, the levels of the ER stress-induced apoptosis response proteins CHOP, cleaved caspase-12 and cleaved caspase-3 were inhibited by VPA treatment. Meanwhile, VPA administration also increased the ratio of Bcl-2/Bax. Moreover, VPA can maintain neurite outgrowth of primary cortical neurons. Collectively, the neurotrophic effect of VPA is related to the inhibition of ER stress-induced apoptosis in SH-SY5Y cells and the

  9. Resveratrol-induced cytotoxicity in human Burkitt's lymphoma cells is coupled to the unfolded protein response

    Directory of Open Access Journals (Sweden)

    Yan Ying


    Full Text Available Abstract Background Resveratrol (RES, a natural phytoalexin found at high levels in grapes and red wine, has been shown to induce anti-proliferation and apoptosis of human cancer cell lines. However, the underlying molecular mechanisms are at present only partially understood. Method The effects of RES on activation of unfolded protein responses (UPR were evaluated using Western blotting, semi-quantitative and real-time RT-PCR. Cell death was evaluated using Annexin V/PI staining and subsequent FACS. Results Similar as tunicamycin, treatment with RES lead to the activation of all 3 branches of the UPR, with early splicing of XBP-1 indicative of IRE1 activation, phosphorylation of eIF2α consistent with ER resident kinase (PERK activation, activating transcription factor 6 (ATF6 splicing, and increase in expression levels of the downstream molecules GRP78/BiP, GRP94 and CHOP/GADD153 in human Burkitt's lymphoma Raji and Daudi cell lines. RES was shown to induce cell death, which could be attenuated by thwarting upregulation of CHOP. Conclusions Our data suggest that activation of the apoptotic arm of the UPR and its downstream effector CHOP/GADD153 is involved, at least in part, in RES-induced apoptosis in Burkitt's lymphoma cells.

  10. Antarctic skuas recognize individual humans. (United States)

    Lee, Won Young; Han, Yeong-Deok; Lee, Sang-Im; Jablonski, Piotr G; Jung, Jin-Woo; Kim, Jeong-Hoon


    Recent findings report that wild animals can recognize individual humans. To explain how the animals distinguish humans, two hypotheses are proposed. The high cognitive abilities hypothesis implies that pre-existing high intelligence enabled animals to acquire such abilities. The pre-exposure to stimuli hypothesis suggests that frequent encounters with humans promote the acquisition of discriminatory abilities in these species. Here, we examine individual human recognition abilities in a wild Antarctic species, the brown skua (Stercorarius antarcticus), which lives away from typical human settlements and was only recently exposed to humans due to activities at Antarctic stations. We found that, as nest visits were repeated, the skua parents responded at further distances and were more likely to attack the nest intruder. Also, we demonstrated that seven out of seven breeding pairs of skuas selectively responded to a human nest intruder with aggression and ignored a neutral human who had not previously approached the nest. The results indicate that Antarctic skuas, a species that typically inhabited in human-free areas, are able to recognize individual humans who disturbed their nests. Our findings generally support the high cognitive abilities hypothesis, but this ability can be acquired during a relatively short period in the life of an individual as a result of interactions between individual birds and humans.

  11. Recombinant Newcastle disease virus (rL-RVG) triggers autophagy and apoptosis in gastric carcinoma cells by inducing ER stress. (United States)

    Bu, Xuefeng; Zhao, Yinghai; Zhang, Zhijian; Wang, Mubin; Li, Mi; Yan, Yulan


    We have reported that the recombinant avirulent Newcastle disease virus (NDV) LaSota strain expressing the rabies virus glycoprotein (rL-RVG) could induce autophagy and apoptosis in gastric carcinoma cells. In the present study, we explored the upstream regulators, endoplasmic reticulum (ER) stress that induce autophagy and apoptosis and the relationships among them. For this purpose, SGC-7901 and HGC cells were infected with rL-RVG. NDV LaSota strain and phosphate-buffered saline (PBS) were treated as the control groups. Western blotting and immunofluorescence microscopy were used to detect the expression of the ER stress-related proteins glucose-regulated protein 78 (GRP78) and the transcription factor GADD153 (CHOP), among others. The expression of beclin-1 and the conversion of light chain (LC) 3-I were used to determine the occurrence of autophagy, and flow cytometry (FCM) and western blotting were used to examine apoptosis-related protein expression. Transmission electron microscopy was also performed to monitor the ultrastructure of the cells. Moreover, small interfering (si) RNA was used to knock down CHOP expression. rL-RVG treatment increased the expression of ER stress-related proteins, such as GRP78, CHOP, activating transcriptional factor 6 (ATF6), X-box-binding protein 1 (XBP-1), and phosphorylated eukaryotic initiation factor 2 (p-eIF2α), in a time- and concentration-dependent manner, and knockdown of CHOP reduced LC3-II conversion and beclin-1 expression. When ER stress was inhibited with 4-PBA, the expression of both autophagy-related proteins and apoptosis-related proteins markedly decreased. Interestingly, inhibition of autophagy with 3-methyladenine (3MA) decreased not only apoptosis-related protein expression but also ER stress-related protein expression. Moreover, we found that downregulation of the c-Jun N-terminal kinase (JNK) pathway by SP600125 reduced LC3-II conversion, beclin-1 expression and caspase-3 activation. Collectively, the

  12. Inhibition of homocysteine-induced endoplasmic reticulum stress and endothelial cell damage by l-serine and glycine. (United States)

    Sim, Woo-Cheol; Han, Inhoi; Lee, Wonseok; Choi, You-Jin; Lee, Kang-Yo; Kim, Dong Gwang; Jung, Seung-Hwan; Oh, Seon-Hee; Lee, Byung-Hoon


    Hyperhomocysteinemia is an independent risk factor for several cardiovascular diseases. The use of vitamins to modulate homocysteine metabolism substantially lowers the risk by reducing plasma homocysteine levels. In this study, we evaluated the effects of l-serine and related amino acids on homocysteine-induced endoplasmic reticulum (ER) stress and endothelial cell damage using EA.hy926 human endothelial cells. Homocysteine treatment decreased cell viability and increased apoptosis, which were reversed by cotreatment with l-serine. l-Serine inhibited homocysteine-induced ER stress as verified by decreased glucose-regulated protein 78kDa (GRP78) and C/EBP homologous protein (CHOP) expression as well as X-box binding protein 1 (xbp1) mRNA splicing. The effects of l-serine on homocysteine-induced ER stress are not attributed to intracellular homocysteine metabolism, but instead to decreased homocysteine uptake. Glycine exerted effects on homocysteine-induced ER stress, apoptosis, and cell viability that were comparable to those of l-serine. Although glycine did not affect homocysteine uptake or export, coincubation of homocysteine with glycine for 24h reduced the intracellular concentration of homocysteine. Taken together, l-serine and glycine cause homocysteine-induced endothelial cell damage by reducing the level of intracellular homocysteine. l-Serine acts by competitively inhibiting homocysteine uptake in the cells. However, the mechanism(s) by which glycine lowers homocysteine levels are unclear.

  13. Calcium Homeostasis and ER Stress in Control of Autophagy in Cancer Cells

    Directory of Open Access Journals (Sweden)

    Elżbieta Kania


    Full Text Available Autophagy is a basic catabolic process, serving as an internal engine during responses to various cellular stresses. As regards cancer, autophagy may play a tumor suppressive role by preserving cellular integrity during tumor development and by possible contribution to cell death. However, autophagy may also exert oncogenic effects by promoting tumor cell survival and preventing cell death, for example, upon anticancer treatment. The major factors influencing autophagy are Ca2+ homeostasis perturbation and starvation. Several Ca2+ channels like voltage-gated T- and L-type channels, IP3 receptors, or CRAC are involved in autophagy regulation. Glucose transporters, mainly from GLUT family, which are often upregulated in cancer, are also prominent targets for autophagy induction. Signals from both Ca2+ perturbations and glucose transport blockage might be integrated at UPR and ER stress activation. Molecular pathways such as IRE 1-JNK-Bcl-2, PERK-eIF2α-ATF4, or ATF6-XBP 1-ATG are related to autophagy induced through ER stress. Moreover ER molecular chaperones such as GRP78/BiP and transcription factors like CHOP participate in regulation of ER stress-mediated autophagy. Autophagy modulation might be promising in anticancer therapies; however, it is a context-dependent matter whether inhibition or activation of autophagy leads to tumor cell death.

  14. Tula hantavirus triggers pro-apoptotic signals of ER stress in Vero E6 cells. (United States)

    Li, Xiao-Dong; Lankinen, Hilkka; Putkuri, Niina; Vapalahti, Olli; Vaheri, Antti


    Tula virus is a member of the Hantavirus genus of the family Bunyaviridae. Viruses of this family have an unusual pattern of intracellular maturation at the ER-Golgi compartment. We recently found that Tula virus, similar to several other hantaviruses, is able to induce apoptosis in cultured cells [Li, X.D., Kukkonen, S., Vapalahti, O., Plyusnin, A., Lankinen, H., Vaheri, A., 2004. Tula hantavirus infection of Vero E6 cells induces apoptosis involving caspase 8 activation. J. Gen. Virol. 85, 3261-3268.]. However, the cellular mechanisms remain to be clarified. In this study, we demonstrate that the progressive replication of Tula virus in Vero E6 cells initiates several death programs that are intimately associated with ER stress: (1) early activation of ER-resident caspase-12; (2) phosphorylation of Jun NH2-terminal kinase (JNK) and its downstream target transcriptional factor, c-jun; (3) induction of the pro-apoptotic transcriptional factor, growth arrest- and DNA damage-inducible gene 153, or C/EBP homologous protein (Gadd153/chop); and (4) changes in the ER-membrane protein BAP31 implying cross-talk with the mitochondrial apoptosis pathway. Furthermore, we confirmed that a sustained ER stress was induced marked by an increased expression of an ER chaperone Grp78/BiP. Taken together, we have identified involvement of ER stress-mediated death program in Tula virus-infected Vero E6 cells which provides a new approach to understand the mechanisms in hantavirus-induced apoptosis.

  15. Characterization and Inducing Melanoma Cell Apoptosis Activity of Mannosylerythritol Lipids-A Produced from Pseudozyma aphidis.

    Directory of Open Access Journals (Sweden)

    Linlin Fan

    Full Text Available Mannosylerythritol lipids (MELs are natural glycolipid biosurfactants which have potential applications in the fields of food, cosmetic and medicine. In this study, MELs were produced from vegetable oil by Pseudozyma aphidis. Their structural data through LC/MS, GC/MS and NMR analysis revealed that MEL-A with two acetyls was the major compound and the identified homologs of MEL-A contained a length of C8 to C14 fatty acid chains. This glycolipid exhibited a surface tension of 27.69 mN/m at a critical micelle concentration (CMC, self-assembling into particles in the water solution. It was observed to induce cell growth-inhibition and apoptosis of B16 melanoma cells in a dose-dependent manner, as well as cause cell cycle arrest at the S phase. Further quantitative RT-PCR analysis and western blotting revealed an increasing tendency of both mRNA and protein expressions of Caspase-12, CHOP, GRP78 and Caspase-3, and a down-regulation of protein Bcl-2. Combined with the up regulation of signaling IRE1 and ATF6, it can be speculated that MEL-A-induced B16 melanoma cell apoptosis was associated with the endoplasmic reticulum stress (ERS.

  16. Genes encoding two Theileria parva antigens recognized by CD8+ T-cells exhibit sequence diversity in South Sudanese cattle populations but the majority of alleles are similar to the Muguga component of the live vaccine cocktail (United States)

    Pelle, Roger; Mwacharo, Joram M.; Njahira, Moses N.; Marcellino, Wani L.; Kiara, Henry; Malak, Agol K.; EL Hussein, Abdel Rahim M.; Bishop, Richard; Skilton, Robert A.


    East Coast fever (ECF), caused by Theileria parva infection, is a frequently fatal disease of cattle in eastern, central and southern Africa, and an emerging disease in South Sudan. Immunization using the infection and treatment method (ITM) is increasingly being used for control in countries affected by ECF, but not yet in South Sudan. It has been reported that CD8+ T-cell lymphocytes specific for parasitized cells play a central role in the immunity induced by ITM and a number of T. parva antigens recognized by parasite-specific CD8+ T-cells have been identified. In this study we determined the sequence diversity among two of these antigens, Tp1 and Tp2, which are under evaluation as candidates for inclusion in a sub-unit vaccine. T. parva samples (n = 81) obtained from cattle in four geographical regions of South Sudan were studied for sequence polymorphism in partial sequences of the Tp1 and Tp2 genes. Eight positions (1.97%) in Tp1 and 78 positions (15.48%) in Tp2 were shown to be polymorphic, giving rise to four and 14 antigen variants in Tp1 and Tp2, respectively. The overall nucleotide diversity in the Tp1 and Tp2 genes was π = 1.65% and π = 4.76%, respectively. The parasites were sampled from regions approximately 300 km apart, but there was limited evidence for genetic differentiation between populations. Analyses of the sequences revealed limited numbers of amino acid polymorphisms both overall and in residues within the mapped CD8+ T-cell epitopes. Although novel epitopes were identified in the samples from South Sudan, a large number of the samples harboured several epitopes in both antigens that were similar to those in the T. parva Muguga reference stock, which is a key component in the widely used live vaccine cocktail. PMID:28231338

  17. Noxa在胃腺癌细胞耐受内质网应激诱导凋亡中的作用%The role of Noxa in endoplasmic reticulum stress-induced apoptosis in gastric adenocarcinoma cell

    Institute of Scientific and Technical Information of China (English)

    王家钰; 张丽; 吴萍; 朱孝峰; 张旭东; 张林杰


    Objective To investigate the role and mechanism of Noxa in resistance of gastric adenocarcinoma cells to endoplasmic reticulum ( ER ) stress-induced apoptosis. Methods Apoptosis was measured by flow cytometry u-sing propidium iodide staining. The activation of Caspase-3 , Caspase-9 and protein levels of GRP78 , Noxa, Mcl-1 were detected by Western blot. The expression of Noxa mRNA was monitored by quantitative RT-PCR. Silencing of gene Mcl-1 , Noxa expression was accomplished by small interfering RNA ( siRNA ) techniques. Results Gastric adenocarcinoma cells were generally resistant to apoptosis induced by the ER-stress inducer tunicymicin( TM ). Exposure of TM resulted in less activation of Caspase-3 and Caspase-9 in gastric adenocarcinoma cells. The expression of Noxa and Mcl-1 were increased in protein level. Inhibiting Mcl-1 expression by siRNA in SGC-7901 cells increased the cell apoptosis. In contrast, inhibition of Noxa and Mcl-1 partially blocked tunicamycin-induced apoptosis in SGC-7901 cells. Conclusion Pro-apoptotic Bcl-2 family member Noxa is critical in gastric adenocarcinoma cells and the role of pro-apoptotic of Noxa is neutralized by the anti-apoptotic protein Mcl-1 , which is also increased by ER stress.%目的 研究胃腺癌细胞对内质网应激诱导细胞凋亡耐受中BH3-only家族蛋白Noxa的表达、作用以及相关机制.方法 衣霉素(TM)处理SGC-7901中分化胃腺癌细胞株及BGC-823低分化胃腺癌细胞株;采用PI单染法测定细胞凋亡率;Western blot法检测葡萄糖调节蛋白78(GRP78)、半胱天冬氨酸蛋白酶3(Caspase-3)、半胱天冬氨酸蛋白酶9(Caspase-9)、成人T细胞白血病诱导的PMA反应蛋白(Noxa)和髓样细胞白血病-1(Mcl-1)蛋白变化;小干扰RNA(siRNA)技术特异“沉默”Mcl-1和Noxa基因;实时荧光定量PCR检测Noxa mRNA水平变化.结果 TM处理胃腺癌细胞后,GRP78表达升高;胃腺癌细胞对TM诱导的内质网应激性凋亡耐受,Caspase-3和Caspase-9活

  18. Inhibition of autophagy enhances heat-induced apoptosis in human non-small cell lung cancer cells through ER stress pathways. (United States)

    Xie, Wen-Yue; Zhou, Xiang-Dong; Yang, Juan; Chen, Ling-Xiu; Ran, Dan-Hua


    The occurrence and mechanisms of autophagy induced by heat stress are not well known in lung cancer cells. Here, we have demonstrated that heat stress induces autophagy in A549 and NCI-H460 cells through morphological and biochemical analyses. The inhibition of autophagy by chloroquine, 3-methyladenine and Beclin 1 siRNA enhanced heat-induced apoptosis. Moreover, the combination of chloroquine and heat stress inhibited tumor growth and enhanced apoptosis in vivo experiments. In addition, heat-induced autophagy involved the ER stress pathway (PERK- or IRE1-dependent). Further, heat treatment led to the increased phosphorylation of AMPK and the decreased phosphorylation of mTOR in vitro and in vivo. Knockdown of GRP78 inhibited the AMPK-mTOR pathway, and the AMPK inhibitor compound C decreased heat-induced autophagy, suggesting that activation of ER stress was involved in autophagy induction and promotion of the AMPK-mTOR pathway. In conclusion, our data suggested that the heat treatment of lung cancer cells triggered protective autophagy, as mediated by ER stress. Thus, inhibition of autophagy can be a promising strategy to enhance hyperthermia in the treatment of lung cancer patients.

  19. The transition of mouse pluripotent stem cells from the naïve to the primed state requires Fas signaling through 3-O sulfated heparan sulfate structures recognized by the HS4C3 antibody

    Energy Technology Data Exchange (ETDEWEB)

    Hirano, Kazumi [Laboratory of Cell Biology, Department of Bioinformatics, Faculty of Engineering, Soka University, 1-236 Tangi-cho, Hachioji, Tokyo 192-8577 (Japan); Van Kuppevelt, Toin H. [Department of Biochemistry, Nijmegen Center for Molecular Life Sciences, Radboud University Nijmegen Medical Centre, 280 P.O. Box 9101, 6500 HB Nijmegen (Netherlands); Nishihara, Shoko, E-mail: [Laboratory of Cell Biology, Department of Bioinformatics, Faculty of Engineering, Soka University, 1-236 Tangi-cho, Hachioji, Tokyo 192-8577 (Japan)


    Highlights: ► Fas transcript increases during the transition from the naïve to the primed state. ► 3OST-5 transcript, the HS4C3 epitope synthesis gene, increases during the transition. ► Fas signaling regulates the transition from the naïve to the primed state. ► HS4C3-binding epitope regulates the transition from the naïve to the primed state. ► Fas signaling is regulated by the HS4C3 epitope during the transition. -- Abstract: The characteristics of pluripotent embryonic stem cells of human and mouse are different. The properties of human embryonic stem cells (hESCs) are similar to those of mouse epiblast stem cells (mEpiSCs), which are in a later developmental pluripotency state, the so-called “primed state” compared to mouse embryonic stem cells (mESCs) which are in a naïve state. As a result of the properties of the primed state, hESCs proliferate slowly, cannot survive as single cells, and can only be transfected with genes at low efficiency. Generating hESCs in the naïve state is necessary to overcome these problems and allow their application in regenerative medicine. Therefore, clarifying the mechanism of the transition between the naïve and primed states in pluripotent stem cells is important for the establishment of stable methods of generating naïve state hESCs. However, the signaling pathways which contribute to the transition between the naïve and primed states are still unclear. In this study, we carried out induction from mESCs to mEpiSC-like cells (mEpiSCLCs), and observed an increase in the activation of Fas signaling during the induction. The expression of Fgf5, an epiblast marker, was diminished by inhibition of Fas signaling using the caspase-8 and -3 blocking peptides, IETD and DEVD, respectively. Furthermore, during the induction, we observed increased expression of 3-O sulfated heparan sulfate (HS) structures synthesized by HS 3-O-sulfotransferase (3OST), which are recognized by the HS4C3 antibody (HS4C3-binding epitope

  20. Licochalcone A-Induced Human Bladder Cancer T24 Cells Apoptosis Triggered by Mitochondria Dysfunction and Endoplasmic Reticulum Stress

    Directory of Open Access Journals (Sweden)

    Xuan Yuan


    Full Text Available Licochalcone A (LCA, a licorice chalconoid, is considered to be a bioactive agent with chemopreventive potential. This study investigated the mechanisms involved in LCA-induced apoptosis in human bladder cancer T24 cells. LCA significantly inhibited cells proliferation, increased reactive oxygen species (ROS levels, and caused T24 cells apoptosis. Moreover, LCA induced mitochondrial dysfunction, caspase-3 activation, and poly-ADP-ribose polymerase (PARP cleavage, which displayed features of mitochondria-dependent apoptotic signals. Besides, exposure of T24 cells to LCA triggered endoplasmic reticulum (ER stress; as indicated by the enhancement in 78 kDa glucose-regulated protein (GRP 78, growth arrest and DNA damage-inducible gene 153/C/EBP homology protein (GADD153/CHOP expression, ER stress-dependent apoptosis is caused by the activation of ER-specific caspase-12. All the findings from our study suggest that LCA initiates mitochondrial ROS generation and induces oxidative stress that consequently causes T24 cell apoptosis via the mitochondria-dependent and the ER stress-triggered signaling pathways.

  1. Fisetin induces apoptosis and endoplasmic reticulum stress in human non-small cell lung cancer through inhibition of the MAPK signaling pathway. (United States)

    Kang, Kyoung Ah; Piao, Mei Jing; Madduma Hewage, Susara Ruwan Kumara; Ryu, Yea Seong; Oh, Min Chang; Kwon, Taeg Kyu; Chae, Sungwook; Hyun, Jin Won


    Fisetin (3,3',4',7-tetrahydroxyflavone), a dietary flavonoid compound, is currently being investigated for its anticancer effect in various cancer models, including lung cancer. Recent studies show that fisetin induces cell growth inhibition and apoptosis in the human non-small cell lung cancer line NCI-H460. In this study, we investigated whether fisetin can induce endoplasmic reticulum (ER) stress-mediated apoptosis in NCI-H460 cells. Fisetin induced mitochondrial reactive oxygen species (ROS) and characteristic signs of ER stress: ER staining; mitochondrial Ca(2+) overload; expression of ER stress-related proteins; glucose-regulated protein (GRP)-78, phosphorylation of protein kinase RNA (PKR)-like endoplasmic reticulum kinase (PERK) and phosphorylation of eukaryotic initiation factor-2 α subunit; cleavage of activating transcription factor-6; phosphorylation of inositol-requiring kinase-1 and splicing of X-box transcription factor-1; induction of C/EBP homologous protein and cleaved caspase-12. siRNA-mediated knockdown of CHOP and ATF-6 attenuated fisetin-induced apoptotic cell death. In addition, fisetin induced phosphorylation of ERK, JNK, and p38 MAPK. Moreover, silencing of the MAPK signaling pathway prevented apoptotic cell death. In summary, our results indicate that, in NCI-H460 cells, fisetin induces apoptosis and ER stress that is mediated by induction of the MAPK signaling pathway.

  2. Pinus densiflora leaf essential oil induces apoptosis via ROS generation and activation of caspases in YD-8 human oral cancer cells. (United States)

    Jo, Jeong-Rang; Park, Ju Sung; Park, Yu-Kyoung; Chae, Young Zoo; Lee, Gyu-Hee; Park, Gy-Young; Jang, Byeong-Churl


    The leaf of Pinus (P.) densiflora, a pine tree widely distributed in Asian countries, has been used as a traditional medicine. In the present study, we investigated the anticancer activity of essential oil, extracted by steam distillation, from the leaf of P. densiflora in YD-8 human oral squamous cell carcinoma (OSCC) cells. Treatment of YD-8 cells with P. densiflora leaf essential oil (PLEO) at 60 µg/ml for 8 h strongly inhibited proliferation and survival and induced apoptosis. Notably, treatment with PLEO led to generation of ROS, activation of caspase-9, PARP cleavage, down-regulation of Bcl-2, and phosphorylation of ERK-1/2 and JNK-1/2 in YD-8 cells. Treatment with PLEO, however, did not affect the expression of Bax, XIAP and GRP78. Importantly, pharmaco-logical inhibition studies demonstrated that treatment with vitamin E (an anti-oxidant) or z-VAD-fmk (a pan-caspase inhibitor), but not with PD98059 (an ERK-1/2 inhibitor) or SP600125 (a JNK-1/2 inhibitor), strongly suppressed PLEO-induced apoptosis in YD-8 cells and reduction of their survival. Vitamin E treatment further blocked activation of caspase-9 and Bcl-2 down-regulation induced by PLEO. Thus, these results demonstrate firstly that PLEO has anti-proliferative, anti-survival and pro-apoptotic effects on YD-8 cells and the effects are largely due to the ROS-dependent activation of caspases.

  3. Fenretinide induces ubiquitin-dependent proteasomal degradation of stearoyl-CoA desaturase in human retinal pigment epithelial cells. (United States)

    Samuel, William; Kutty, R Krishnan; Duncan, Todd; Vijayasarathy, Camasamudram; Kuo, Bryan C; Chapa, Krysten M; Redmond, T Michael


    Stearoyl-CoA desaturase (SCD, SCD1), an endoplasmic reticulum (ER) resident protein and a rate-limiting enzyme in monounsaturated fatty acid biosynthesis, regulates cellular functions by controlling the ratio of saturated to monounsaturated fatty acids. Increase in SCD expression is strongly implicated in the proliferation and survival of cancer cells, whereas its decrease is known to impair proliferation, induce apoptosis, and restore insulin sensitivity. We examined whether fenretinide, (N-(4-hydroxyphenyl)retinamide, 4HPR), which induces apoptosis in cancer cells and recently shown to improve insulin sensitivity, can modulate the expression of SCD. We observed that fenretinide decreased SCD protein and enzymatic activity in the ARPE-19 human retinal pigment epithelial cell line. Increased expression of BiP/GRP78, ATF4, and GADD153 implicated ER stress. Tunicamycin and thapsigargin, compounds known to induce ER stress, also decreased the SCD protein. This decrease was completely blocked by the proteasome inhibitor MG132. In addition, PYR41, an inhibitor of ubiquitin activating enzyme E1, blocked the fenretinide-mediated decrease in SCD. Immunoprecipitation analysis using anti-ubiquitin and anti-SCD antibodies and the blocking of SCD loss by PYR41 inhibition of ubiquitination further corroborate that fenretinide mediates the degradation of SCD in human RPE cells via the ubiquitin-proteasome dependent pathway. Therefore, the effect of fenretinide on SCD should be considered in its potential therapeutic role against cancer, type-2 diabetes, and retinal diseases.

  4. Recognizing child maltreatment in Bangladesh. (United States)

    Khan, N Z; Lynch, M A


    Concern is increasing in Bangladesh over child abuse, neglect, and exploitation. Children from all walks of life are being treated at the Child Development Center (CDC) Dhaka Shishu Hospital for neurodevelopmental problems resulting from abuse and neglect. Efforts to protect children from sexual harassment result in girls being isolated at home or married at an early age. Some young brides are eventually abandoned and forced into prostitution. Early marriage reflects the lack of acknowledgement of a period of adolescence and the belief that puberty is a marker of adulthood. Many girls aged 8-16 are employed as live-in domestic servants, and many suffer sexual as well as emotional abuse. Garment factories, on the other hand, offer girls an escape from extreme poverty, domestic service, and early marriage but are threatened by forces that condemn child labor. Rather than ending such opportunities, employers should be encouraged to provide employees with educational and welfare facilities. The CDC seeks to explore the extent and depth of the problem of child abuse while recognizing the special circumstances at work in Bangladesh. It is also necessary to raise awareness of these issues and of the discrepancies between the law and cultural practices. For example, the legal marriage age of 18 years for a woman and 21 years for a man is often ignored. Additional forms of abuse receiving the attention of women's organizations and human rights groups include the trafficking of children. A network of concerned organizations should be created to work against the child abuse, neglect, and exploitation that Bangladesh has pledged to overcome by signing the UN Convention on the Rights of the Child.

  5. Luteolin modulates 6-hydroxydopamine-induced transcriptional changes of stress response pathways in PC12 cells.

    Directory of Open Access Journals (Sweden)

    Ling-Wei Hu

    Full Text Available The neurotoxin 6-hydroxydopamine (6-OHDA, which causes transcriptional changes associated with oxidative and proteotoxic stress, has been widely used to generate an experimental model of Parkinson's disease. The food-derived compound luteolin has multi-target actions including antioxidant, anti-inflammatory and neurotrophic activities. The aim of this study is to investigate how luteolin affects 6-OHDA-mediated stress response pathways. The results showed that when PC12 cells were pre-treated with luteolin (20 µM 30 min prior to 6-OHDA (100 µM exposure, 6-OHDA-induced ROS overproduction, cytotoxicity, caspase-3 activation, and mRNA expression of BIM, TRB3 and GADD34 were significantly attenuated. Moreover, 6-OHDA-mediated cell cycle arrest and transcription of p53 target genes, p21, GADD45α and PUMA, were reduced by luteolin. Luteolin also significantly down-regulated 6-OHDA-mediated unfolded protein response (UPR, leading to decreases in phospho-eIF2α, ATF4, GRP78 and CHOP. In addition, luteolin attenuated 6-OHDA-induced Nrf2-mediated HO-1 and GCLC. Taken together, these results suggest that diminishing intracellular ROS formation and down-regulation of p53, UPR and Nrf2-ARE pathways may be involved in the neuroprotective effect of luteolin.

  6. Endoplasmic reticulum (ER Chaperones and Oxidoreductases: Critical Regulators of Tumor Cell Survival and Immunorecognition

    Directory of Open Access Journals (Sweden)

    Thomas eSimmen


    Full Text Available Endoplasmic reticulum (ER chaperones and oxidoreductases are abundant enzymes that mediate the production of fully folded secretory and transmembrane proteins. Resisting the Golgi and plasma membrane-directed bulk flow, ER chaperones and oxidoreductases enter retrograde trafficking whenever they are pulled outside of the ER. However, solid tumors are characterized by the increased production of reactive oxygen species (ROS, combined with reduced blood flow that leads to low oxygen supply and ER stress. Under these conditions, hypoxia and the unfolded protein response (UPR upregulate ER chaperones and oxidoreductases. When this occurs, ER oxidoreductases and chaperones become important regulators of tumor growth. However, under these conditions, these proteins not only promote the production of proteins, but also alter the properties of the plasma membrane and hence modulate tumor immune recognition. For instance, high levels of calreticulin serve as an eat-me signal on the surface of tumor cells. Conversely, both intracellular and surface BiP/GRP78 promotes tumor growth. Other ER folding assistants able to modulate the properties of tumor tissue include protein disulfide isomerase (PDI, Ero1α and GRP94. Understanding the roles and mechanisms of ER chaperones in regulating tumor cell functions and immunorecognition will lead to important insight for the development of novel cancer therapies.

  7. Cytotoxic T-lymphocyte clones, established by stimulation with the HLA-A2 binding p5365-73 wild type peptide loaded on dendritic cells In vitro, specifically recognize and lyse HLA-A2 tumour cells overexpressing the p53 protein

    DEFF Research Database (Denmark)

    Barfoed, Annette Malene; Petersen, T R; Kirkin, A F;


    to carry identical T-cell receptors. The CTL clone, 2D9, was shown to specifically lyse the HLA-A*0201+ squamous carcinoma cell line SCC9 and the breast cancer cell line MDA-MB-468. Our data demonstrate that human peripheral blood lymphocytes from normal healthy individuals comprise T cells capable...... of recognizing p53 derived wild type (self) peptides. Furthermore, the capacity of R9V specific T cell clones to exert HLA restricted cytotoxicity, argues that the R9V peptide is naturally presented on certain cancer cells. This supports the view that p53 derived wild type peptides might serve as candidate...... target antigens for the immunotherapeutic treatment of cancer....

  8. beta-catenin siRNA regulation of apoptosis- and angiogenesis-related gene expression in hepatocellular carcinoma cells: potential uses for gene therapy. (United States)

    Wang, Xin-Hong; Sun, Xun; Meng, Xiang-Wei; Lv, Zhi-Wu; Du, Ya-Ju; Zhu, Yan; Chen, Jing; Kong, De-Xia; Jin, Shi-Zhu


    The molecular mechanism responsible for hepatocellular carcinoma (HCC) development remains to be defined although a number of gene pathways have been shown to play an active role, such as Wnt/beta-catenin signaling. In this study, beta-catenin small interfering RNA (siRNA) was designed, synthesized, and transfected into HCC HepG2 cells. RT-PCR and western blot assays were performed to detect expression of altered genes and proteins, and the MTT assay was used to detect cell viability. Our data showed that beta-catenin mRNA and protein expression levels were effectively knocked down by beta-catenin siRNA and subsequently, tumor cell proliferation was significantly suppressed. Flow cytometry assay showed that tumor cells were arrested at the G0/G1 phase of the cell cycles. Molecularly, expression of Smad3, p-caspase-3, and Grp78 protein were upregulated after 72 h of beta-catenin siRNA transfection, whereas expression of TERT, caspase-3, XIAP, MMP-2, MMP-9, VEGF-A, VEGF-c, and bFGF protein were reduced. However, there was no change between the expression of STAT3 and the HSP27 protein following transfection. The results from the current study demonstrated the importance of the Wnt/beta-catenin signaling pathway in regulation of gene expression in HCC. Further studies are required to investigate the role of this pathway in HCC development and targeting of this pathway to control HCC.

  9. Protective effects of quercetin on dieldrin-induced endoplasmic reticulum stress and apoptosis in dopaminergic neuronal cells. (United States)

    Park, Euteum; Chun, Hong Sung


    Dieldrin, an organochlorine pesticide still used in several developing countries, has been proposed as a risk factor for Parkinson's disease. Quercetin is one of the potent bioactive flavonoids present in numerous plants. In this study, we investigated the protective effects of quercetin on neurotoxicity induced by dieldrin in cultured dopaminergic SN4741 cells. Our initial experiments showed that quercetin (10-40 μM) dose dependently prevented dieldrin (20 μM)-induced cytotoxicity in SN4741 cells. Pretreatment for 1 h with quercetin before dieldrin application could significantly suppress dieldrin-induced apoptotic characteristics, including nuclear condensation, DNA fragmentation, and caspase-3/7 activation. Results showed that dieldrin-induced markers of endoplasmic reticulum (ER) stress response such as chaperone GRP78, heme oxygenase-1, and phosphorylation of the α subunit of eukaryotic initiation factor 2. In addition, dieldrin reduced antiapoptotic Bcl-2 expression, but significantly elevated a proapoptotic transcription factor CHOP. Furthermore, RNA interference to CHOP almost completely repressed dieldrin-induced apoptotic cell death. Interestingly, quercetin prevented the changes in dieldrin-induced ER stress markers. These results suggest that quercetin may suppress the ER stress-CHOP pathway and dieldrin-induced apoptosis in dopaminergic neurons.

  10. 硫化氢对急性肺损伤大鼠肺泡上皮细胞内质网应激的调节%Regulatory effects of hydrogen sulfide on alveolar epithelial cell endoplasmic reticulum stress in rats with acute lung injury

    Institute of Scientific and Technical Information of China (English)

    刘志伟; 王海英; 关岚; 赵斌


    目的 探索内源性硫化氢对于油酸诱导的急性肺损伤大鼠肺泡上皮细胞内质网应激的调节作用.方法 雄性Sprague Dawley大鼠随机(随机数字法)被分入正常对照组、油酸组、油酸+硫氢化钠组及硫氢化钠对照组.每个组进一步分为2、4和6h3个时间点.对肺组织进行肺组织损伤半定量评分,检测肺组织湿、干质量比和匀浆硫化氢含量.通过免疫组化染色和蛋白印迹法检测内质网应激的标志蛋白(GRP78和elF2α)的表达.结果 肺损伤大鼠肺组织损伤半定量评分及湿、干质量比明显升高,而肺组织硫化氢含量,肺泡上皮细胞GRP78及eIF2α的表达明显降低.在硫氢化钠预处理的大鼠中,肺损伤半定量评分及肺组织湿、干质量比明显降低,而肺组织硫化氢含量,肺泡上皮细胞GRP78及eIF2α的表达明显升高.结论 内源性硫化氢可以通过促进肺泡上皮细胞内质网应激反应进而在肺损伤过程中起到保护作用.%Objective To study the regulatory effect of hydrogen sulfide (H2S) on endoplasmic reticulum stress in alveolar epithelial cells of rats with acute lung injury (ALI) induced by oleic acid (OA).Methods Seventy-two male Sprague Dawley (SD) rats were equally divided into control group (C group),oleic acid-induced ALI group (OA group),oleic acid-induced ALI with sodium hydrosulfide (NaHS) pretreatment group (OA + NaHS group) and sodium hydrosulfide treatment group (NaHS group).The model of acute lung injury was made by oleic acid intravenous injection in dose of 0.1 mL/kg.NaHS was injected intra-abdominally in dose of 1 ml/kg with concentration of 56 μmol/L 30 min before administration of oleic acid for pretreatment.In control groups,saline was used instead of oleic acid and NaHS in the equivalent volume.Six rats of each group were sacrificed at 2 h,4 h and 6 hours separately after modeling for observing the acute injury of lung tissue.Index of quantitative assessment of

  11. Recognizing, diagnosing, and treating rhabdomyolysis. (United States)

    Heard, Henry; Barker, James


    Rhabdomyolysis is an acute and potentially fatal syndrome characterized by striated muscle breakdown and subsequent release of muscle cell contents into the systemic circulation. The sudden release of large quantities of potassium, calcium, organic acids, and myoglobin into the bloodstream can cause renal tubal toxicity, cardiac dysrhythmias, and death. Complications can be managed and minimized if predicted and treated early, but patients may not have classic symptoms of rhabdomyolysis and may even be asymptomatic on presentation. This article reviews the pathophysiology, causes, diagnosis, and treatment of acute rhabdomyolysis.

  12. Saturated lipids decrease mitofusin 2 leading to endoplasmic reticulum stress activation and insulin resistance in hypothalamic cells. (United States)

    Diaz, Brenda; Fuentes-Mera, Lizeth; Tovar, Armando; Montiel, Teresa; Massieu, Lourdes; Martínez-Rodríguez, Herminia Guadalupe; Camacho, Alberto


    Endoplasmic reticulum (ER) and mitochondria dysfunction contribute to insulin resistance generation during obesity and diabetes. ER and mitochondria interact through Mitofusin 2 (MTF2), which anchors in the outer mitochondrial and ER membranes regulating energy metabolism. Ablation of MTF2 leads to ER stress activation and insulin resistance. Here we determine whether lipotoxic insult induced by saturated lipids decreases MTF2 expression leading to ER stress response in hypothalamus and its effects on insulin sensitivity using in vitro and in vivo models. We found that lipotoxic stimulation induced by palmitic acid, but not the monounsaturated palmitoleic acid, decreases MTF2 protein levels in hypothalamic mHypoA-CLU192 cells. Also, palmitic acid incubation activates ER stress response evidenced by increase in the protein levels of GRP78/BIP marker at later stage than MTF2 downregulation. Additionally, we found that MTF2 alterations induced by palmitic, but not palmitoleic, stimulation exacerbate insulin resistance in hypothalamic cells. Insulin resistance induced by palmitic acid is prevented by pre-incubation of the anti-inflammatory and the ER stress release reagents, sodium salicylate and 4 phenylbutirate, respectively. Finally, we demonstrated that lipotoxic insult induced by high fat feeding to mice decreases MTF2 proteins levels in arcuate nucleus of hypothalamus. Our data indicate that saturated lipids modulate MTF2 expression in hypothalamus coordinating the ER stress response and the susceptibility to insulin resistance.

  13. E platinum, a newly synthesized platinum compound, induces apoptosis through ROS-triggered ER stress in gastric carcinoma cells. (United States)

    Wang, Xiaoping; Guo, Qinglong; Tao, Lei; Zhao, Li; Chen, Yan; An, Teng; Chen, Zhen; Fu, Rong


    Gastric cancer (GC) is still one of the leading causes of death in cancer-related diseases. In this study, we aimed to investigate the antitumor effect of E Platinum, a newly platinum-based chemotherapeutic agent bearing the basic structure of Oxaliplatin, in a variety of gastric carcinoma cells and the underlying mechanisms. We demonstrated that E Platinum significantly induced apoptosis in gastric cancer cells via mitochondrial apoptotic pathway as a result of increased reactive oxygen species (ROS). We also found that E Platinum enhanced Ca(2+) flux out from the endoplasmic reticulum by increasing the protein expression of IP3R type 1 (IP3R1) and decreasing the expression of ERp44. Dysfunction of Ca(2+) homeostasis in endoplasmic reticulum (ER) leads to accumulation of unfolded proteins and ER stress. Mechanically, E Platinum increased ER stress associated protein expression such as GRP78, p-PERK, p-eIF2α, ATF4, and CHOP. However, knocking down CHOP reversed E Platinum-induced apoptosis by blocking mitochondrial apoptotic pathway. Furthermore, 10 mg/kg of E Platinum significantly suppressed BGC-823 tumor growth in vivo without toxicity, which correlated with induction of apoptosis and expression of ER stress related proteins in tumor tissues. Taken together, E Platinum inhibited tumor growth and induced apoptosis by ROS-mediated ER stress activation both in vitro and in vivo. Our study indicated that E Platinum may be a potential and effective treatment for gastric cancer in clinical. © 2016 Wiley Periodicals, Inc.

  14. The importance of recognizing postural pseudoanemia. (United States)

    Goldner, Fred; Jacob, Giris; Raj, Satish R; Robertson, David


    The determination of the packed red cell volume and the hemoglobin level has been paramount for monitoring anemia and blood loss for patients in the hospital setting. Recently, these variables have been studied during various control conditions including changes in posture. It has been found that the hematocrit changes markedly with alteration of body posture, in such a way that shifts of estimated blood volume of 1 pint can commonly be elicited by a simple change of posture from supine to upright or vice versa. Therefore, it is important to recognize that in addition to the numerous pathological conditions that may affect the value of the packed cell volume, certain physiological maneuvers may have an equal impact and may confound the accurate assessment of true pathological changes in these variables. Thus, changes in posture can lead to substantial changes in hematocrit, which may be attributed mistakenly to blood loss or acute anemia and may result in a cascade of unnecessary diagnostic costs. In reality, these changes represent postural pseudoanemia, a normal physiological response to a change in position from standing to lying.

  15. Chronic neuroprotective effects of low concentration lithium on SH-SY5Y cells: possible involvement of stress proteins and gene expression (United States)

    Nciri, Riadh; Bourogaa, Ezzeddine; Jbahi, Samira; Allagui, Mohamed Salah; Elfeki, Abdelfattah; Vincent, Christian; Croute, Françoise


    To investigate the molecular mechanism underlying the neuroprotective effect of lithium on cells, in this study, we exposed SH-SY5Y cells to 0.5 mmol/L lithium carbonate (Li2CO2) for 25–50 weeks and then detected the expression levels of some neurobiology related genes and post-translational modifications of stress proteins in SH-SY5Y cells. cDNA arrays showed that pyruvate kinase 2 (PKM2) and calmodulin 3 (CaM 3) expression levels were significantly down-regulated, phosphatase protein PP2A expression was lightly down-regulated, and casein kinase II (CK2), threonine/tyrosine phosphatase 7 (PYST2), and dopamine beta-hydroxylase (DBH) expression levels were significantly up-regulated. Besides, western blot analysis of stress proteins (HSP27, HSP70, GRP78 and GRP94) showed an over-expression of two proteins: a 105 kDa protein which is a hyper-phosphorylated isoform of GRP94, and a 108 kDa protein which is a phosphorylated tetramer of HSP27. These results suggest that the neuroprotective effects of lithium are likely related to gene expressions and post-translational modifications of proteins cited above. PMID:25206881

  16. CHIP, a carboxy terminus HSP-70 interacting protein, prevents cell death induced by endoplasmic reticulum stress in the central nervous system. (United States)

    Cabral Miranda, Felipe; Adão-Novaes, Juliana; Hauswirth, William W; Linden, Rafael; Petrs-Silva, Hilda; Chiarini, Luciana B


    Endoplasmic reticulum (ER) stress and protein misfolding are associated with various neurodegenerative diseases. ER stress activates unfolded protein response (UPR), an adaptative response. However, severe ER stress can induce cell death. Here we show that the E3 ubiquitin ligase and co-chaperone Carboxyl Terminus HSP70/90 Interacting Protein (CHIP) prevents neuron death in the hippocampus induced by severe ER stress. Organotypic hippocampal slice cultures (OHSCs) were exposed to Tunicamycin, a pharmacological ER stress inducer, to trigger cell death. Overexpression of CHIP was achieved with a recombinant adeno-associated viral vector (rAAV) and significantly diminished ER stress-induced cell death, as shown by analysis of propidium iodide (PI) uptake, condensed chromatin, TUNEL and cleaved caspase 3 in the CA1 region of OHSCs. In addition, overexpression of CHIP prevented upregulation of both CHOP and p53 both pro-apoptotic pathways induced by ER stress. We also detected an attenuation of eIF2a phosphorylation promoted by ER stress. However, CHIP did not prevent upregulation of BiP/GRP78 induced by UPR. These data indicate that overexpression of CHIP attenuates ER-stress death response while maintain ER stress adaptative response in the central nervous system. These results indicate a neuroprotective role for CHIP upon UPR signaling. CHIP emerge as a candidate for clinical intervention in neurodegenerative diseases associated with ER stress.

  17. Chronic neuroprotective effects of low concentration lithium on SH-SY5Y cells:possible involvement of stress proteins and gene expression

    Institute of Scientific and Technical Information of China (English)

    Riadh Nciri; Ezzeddine Bourogaa; Samira Jbahi; Mohamed Salah Allagui; Abdelfattah Elfeki; Christian Vincent; Franoise Croute


    To investigate the molecular mechanism underlying the neuroprotective effect of lithium on cells, in this study, we exposed SH-SY5Y cells to 0.5 mmol/L lithium carbonate (Li2CO2) for 25-50 weeks and then detected the expression levels of some neurobiology related genes and post-translational modifications of stress proteins in SH-SY5Y cells. cDNA arrays showed that pyruvate kinase 2 (PKM2) and calmodulin 3 (CaM 3) expression levels were signiifcantly down-regulated, phosphatase protein PP2A expression was lightly down-regulated, and casein kinase II (CK2), threonine/tyrosine phosphatase 7 (PYST2), and dopamine beta-hydroxylase (DBH) expression levels were signiifcantly up-regulated. Besides, western blot analysis of stress proteins (HSP27, HSP70, GRP78 and GRP94) showed an over-expression of two proteins:a 105 kDa protein which is a hyper-phosphorylated isoform of GRP94, and a 108 kDa protein which is a phosphorylated tetramer of HSP27. These results suggest that the neuroprotective effects of lithium are likely related to gene expressions and post-translational modiifcations of proteins cited above.

  18. 15,16-Dihydrotanshinone I, a Compound of Salvia miltiorrhiza Bunge, Induces Apoptosis through Inducing Endoplasmic Reticular Stress in Human Prostate Carcinoma Cells

    Directory of Open Access Journals (Sweden)

    Mao-Te Chuang


    Full Text Available 5,16-dihydrotanshinone I (DHTS is extracted from Salvia miltiorrhiza Bunge (tanshen root and was found to be the most effective compound of tanshen extracts against breast cancer cells in our previous studies. However, whether DHTS can induce apoptosis through an endoplasmic reticular (ER stress pathway was examined herein. In this study, we found that DHTS significantly inhibited the proliferation of human prostate DU145 carcinoma cells and induced apoptosis. DHTS was able to induce ER stress as evidenced by the upregulation of glucose regulation protein 78 (GRP78/Bip and CAAT/enhancer binding protein homologous protein/growth arrest- and DNA damage-inducible gene 153 (CHOP/GADD153, as well as increases in phosphorylated eukaryotic initiation factor 2α (eIF2α, c-jun N-terminal kinase (JNK, and X-box-binding protein 1 (XBP1 mRNA splicing forms. DHTS treatment also caused significant accumulation of polyubiquitinated proteins and hypoxia-inducible factor (HIF-1α, indicating that DHTS might be a proteasome inhibitor that is known to induce ER stress or enhance apoptosis caused by the classic ER stress-dependent mechanism. Moreover, DHTS-induced apoptosis was reversed by salubrinal, an ER stress inhibitor. Results suggest that DHTS can induce apoptosis of prostate carcinoma cells via induction of ER stress and/or inhibition of proteasome activity, and may have therapeutic potential for prostate cancer patients.

  19. Imipramine protects mouse hippocampus against tunicamycin-induced cell death. (United States)

    Ono, Yoko; Shimazawa, Masamitsu; Ishisaka, Mitsue; Oyagi, Atsushi; Tsuruma, Kazuhiro; Hara, Hideaki


    Endoplasmic reticulum (ER) stress is implicated in various diseases. Recently, some reports have suggested that the sigma-1 receptor may play a role in ER stress, and many antidepressants have a high affinity for the sigma-1 receptor. In the present study, we focused on imipramine, a widely used antidepressant, and investigated whether it might protect against the neuronal cell death induced by tunicamycin, an ER stress inducer. In mouse cultured hippocampal HT22 cells, imipramine inhibited cell death and caspase-3 activation induced by tunicamycin, although it did not alter the elevated expressions of 78 kDa glucose-regulated protein (GRP78) and C/EBP-homologous protein (CHOP). Interestingly, in such cells application of imipramine normalized the expression of the sigma-1 receptor, which was decreased by treatment with tunicamycin alone. Additionally, NE-100, a selective sigma-1 receptor antagonist, abolished the protective effect of imipramine against such tunicamycin-induced cell death. Imipramine inhibited the reduction of mitochondrial membrane potential induced by tunicamycin, and NE-100 blocked this modulating effect of imipramine. Furthermore, in anesthetized mice intracerebroventricular administration of tunicamycin decreased the number of neuronal cells in the hippocampus, particularly in the CA1 and dentate gyrus (DG) areas, and 7 days' imipramine treatment (10mg/kg/day; i.p.) significantly suppressed these reductions in CA1 and DG. These findings suggest that imipramine protects against ER stress-induced hippocampal neuronal cell death both in vitro and in vivo. Such protection may be partly due to the sigma-1 receptor.

  20. Dietary oxidized n-3 PUFA induce oxidative stress and inflammation: role of intestinal absorption of 4-HHE and reactivity in intestinal cells. (United States)

    Awada, Manar; Soulage, Christophe O; Meynier, Anne; Debard, Cyrille; Plaisancié, Pascale; Benoit, Bérengère; Picard, Grégory; Loizon, Emmanuelle; Chauvin, Marie-Agnès; Estienne, Monique; Peretti, Noël; Guichardant, Michel; Lagarde, Michel; Genot, Claude; Michalski, Marie-Caroline


    Dietary intake of long-chain n-3 PUFA is now widely advised for public health and in medical practice. However, PUFA are highly prone to oxidation, producing potentially deleterious 4-hydroxy-2-alkenals. Even so, the impact of consuming oxidized n-3 PUFA on metabolic oxidative stress and inflammation is poorly described. We therefore studied such effects and hypothesized the involvement of the intestinal absorption of 4-hydroxy-2-hexenal (4-HHE), an oxidized n-3 PUFA end-product. In vivo, four groups of mice were fed for 8 weeks high-fat diets containing moderately oxidized or unoxidized n-3 PUFA. Other mice were orally administered 4-HHE and euthanized postprandially versus baseline mice. In vitro, human intestinal Caco-2/TC7 cells were incubated with 4-hydroxy-2-alkenals. Oxidized diets increased 4-HHE plasma levels in mice (up to 5-fold, P intestine along with decreasing Paneth cell number (up to -19% in the duodenum). Both in vivo and in vitro, intestinal absorption of 4-HHE was associated with formation of 4-HHE-protein adducts and increased expression of glutathione peroxidase 2 (GPx2) and glucose-regulated protein 78 (GRP78). Consumption of oxidized n-3 PUFA results in 4-HHE accumulation in blood after its intestinal absorption and triggers oxidative stress and inflammation in the upper intestine.

  1. Fucoidan induces cancer cell apoptosis by modulating the endoplasmic reticulum stress cascades.

    Directory of Open Access Journals (Sweden)

    Shaohua Chen

    Full Text Available Cancer metastasis is the main cause leading to disease recurrence and high mortality in cancer patients. Therefore, inhibiting metastasis process or killing metastatic cancer cells by inducing apoptosis is of clinical importance in improving cancer patient survival. Previous studies revealed that fucoidan, a fucose-rich polysaccharide isolated from marine brown alga, is a promising natural product with significant anti-cancer activity. However, little is known about the role of endoplasmic reticulum (ER stress in fucoidan-induced cell apoptosis.We reported that fucoidan treatment inhibits cell growth and induces apoptosis in cancer cells. Fucoidan treatments resulted in down-regulation of the glucose regulated protein 78 (GRP78 in the metastatic MDA-MB-231 breast cancer cells, and of the ER protein 29 (ERp29 in the metastatic HCT116 colon cancer cells. However, fucoidan treatment promoted ER Ca2+-dependent calmodulin-dependent kinase II (CaMKII phosphorylation, Bcl-associated X protein (Bax and caspase 12 expression in MDA-MB-231 cells, but not in HCT116 cells. In both types of cancer cells, fucoidan activated the phosphorylation of eukaryotic initiation factor 2 alpha (p-eIF2α\\CCAAT/enhancer binding protein homologous protein (CHOP pro-apoptotic cascade and inhibited the phosphorylation of inositol-requiring kinase 1 (p-IRE-1\\X-box binding proteins 1 splicing (XBP-1s pro-survival cascade. Furthermore, CHOP knockdown prevented DNA damage and cell death induced by fucoidan.Fucoidan exerts its anti-tumor function by modulating ER stress cascades. Contribution of ER stress to the fucoidan-induced cell apoptosis augments our understanding of the molecular mechanisms underlying its anti-tumour activity and provides evidence for the therapeutic application of fucoidan in cancer.

  2. EGFR-independent autophagy induction with gefitinib and enhancement of its cytotoxic effect by targeting autophagy with clarithromycin in non-small cell lung cancer cells. (United States)

    Sugita, Shohei; Ito, Kentaro; Yamashiro, Yutaro; Moriya, Shota; Che, Xiao-Fang; Yokoyama, Tomohisa; Hiramoto, Masaki; Miyazawa, Keisuke


    Gefitinib (GEF), an inhibitor for EGFR tyrosine kinase, potently induces autophagy in non-small cell lung cancer (NSCLC) cell lines such as PC-9 cells expressing constitutively activated EGFR kinase by EGFR gene mutation as well as A549 and H226 cells with wild-type EGFR. Unexpectedly, GEF-induced autophagy was also observed in non-NSCLC cells such as murine embryonic fibroblasts (MEF) and leukemia cell lines K562 and HL-60 without EGFR expression. Knockout of EGFR gene in A549 cells by CRISPR/Cas9 system still exhibited autophagy induction after treatment with GEF, indicating that the autophagy induction by GEF is not mediated through inhibiting EGFR kinase activity. Combined treatment with GEF and clarithromycin (CAM), a macrolide antibiotic having the effect of inhibiting autophagy flux, enhances the cytotoxic effect in NSCLC cell lines, although treatment with CAM alone exhibits no cytotoxicity. GEF treatment induced up-regulation of endoplasmic reticulum (ER)-stress related genes such as CHOP/GADD153 and GRP78. Knockdown of CHOP in PC-9 cells and Chop-knockout MEF both exhibited less sensitivity to GEF than controls. Addition of CAM in culture medium resulted in further pronounced GEF-induced ER stress loading, while CAM alone exhibited no effect. These data suggest that GEF-induced autophagy functions as cytoprotective and indicates the potential therapeutic possibility of using CAM for GEF therapy. Furthermore, it is suggested that the intracellular signaling for autophagy initiation in response to GEF can be completely dissociated from EGFR, but unknown target molecule(s) of GEF for autophagy induction might exist.

  3. Potassium-3-beta-hydroxy-20-oxopregn-5-en-17-alpha-yl sulfate: a novel inhibitor of 78 kDa glucose-regulated protein

    Directory of Open Access Journals (Sweden)

    Mhaidat NM


    Full Text Available Nizar M Mhaidat,1,2 Qosay A Al-Balas,1 Karem H Alzoubi,1 Rowan F AlEjielat2 1Department of Clinical Pharmacy, Faculty of Pharmacy, Jordan University of Science and Technology, Irbid, 2Department of Clinical Pharmacy, Faculty of Pharmaceutical Sciences, Hashemite University, Zarqa, Jordan Background: Previous studies have shown the central role of 78 kDa glucose-regulated protein (GRP78 in colorectal cancer (CRC survival and chemoresistance. In the present study, we aimed to design a GRP78 inhibitor and test its potential to inhibit CRC cells growth. Materials and methods: Computer-aided drug design was used to establish novel compounds as potential inhibitors of GRP78. Discovery Studio 3.5 software was used to evaluate a series of designed compounds and assess their mode of binding to the active site of the protein. The cytotoxicity of the designed compounds was evaluated using the MTT assay and the propidium iodide method. The effect of the inhibitor on the expression of GRP78 was evaluated by immunoblotting. Results: Among the designed compounds, only potassium-3-beta-hydroxy-20-oxopregn-5-en-17-alpha-yl sulfate (PHOS has a potential to inhibit the growth of CRC cells. Inhibition of cellular growth was largely attributed to downregulation of GRP78 and induction of apoptotic cell death. Conclusion: These results introduce PHOS as a promising GRP78 inhibitor that could be used in future studies as a combination with chemotherapy in the treatment of CRC patients. Our ongoing studies aim to characterize PHOS safety profile as well as its mechanism of action. Keywords: UPR, apoptosis, GRP78, CRC, chemotherapy, computer-aided drug design (CADD

  4. Glucose-regulated protein 78 may play a crucial role in promoting the pulmonary microvascular remodeling in a rat model of hepatopulmonary syndrome (United States)

    Zhang, Huiying; Lv, Minli; Zhao, Zhongfu; Jia, Jiantao; Zhang, Lili; Xiao, Peng; Wang, Limin; Li, Chen; Ji, Jingquan; Tian, Xiaoxia; Li, Xujiong; Fan, Yimin; Lai, Lina; Liu, Yan; Li, Baohong; Zhang, Cuiying; Liu, Mingshe; Guo, Jianhong; Han, Dewu; Ji, Cheng


    Objective This study is to investigate the role of glucose-regulated protein 78 (GRP78) in the pulmonary microvascular remodeling during hepatopulmonary syndrome (HPS) development. Methods The rat models with liver cirrhosis and HPS were induced by multiple pathogenic factors for 4 to 8 wk. The concentrations of alanine transferase (ALT) and endotoxin in plasma were detected in the models, followed by the detection of GRP78 expression. RT-PCR, quantitative real-time PCR and Western blotting were employed to assess the mRNA and protein expression levels of vascular endothelial growth factor (VEGF), respectively. Immunohistochemistry staining was used to examine the expression of a specific vascular marker, factor VIII-related antigen (FVIII-RAg), and several cell proliferation- and apoptosis-related proteins, including CHOP/GADD153, caspase-12, Bcl-2 and nuclear factor (NF)-κB. Results The levels of endotoxin and ALT in plasma were gradually increased as the disease progressed, so did GRP78, which were in a positive correlation. The expression levels of VEGF (both mRNA and protein) and FVIII-RAg were significantly elevated in the HPS models, indicating active angiogenesis, which was also positively correlated with GRP78 expression. Furthermore, the expression levels of the pro-apoptotic proteins of CHOP/GADD153 and caspase-12 were dramatically decreased, while the anti-apoptotic proteins of Bcl-2 and NF-κB were significantly elevated, in the HPS models. There were also close correlations between these proteins and GRP78. Conclusions Over-expression of GRP78 in lungs may be the critical pathogenic factor for HPS. Through promoting cell proliferation and survival and inhibiting apoptosis, GRP78 may promote the pulmonary microvascular remodeling in HPS pathogenesis. Our results provide a potential therapeutic target for clinical prevention and treatment for HPS and related complications. PMID:24768185

  5. Tetrandrine Induces Apoptosis of Human Nasopharyngeal Carcinoma NPC-TW 076 Cells through Reactive Oxygen Species Accompanied by an Endoplasmic Reticulum Stress Signaling Pathway

    Directory of Open Access Journals (Sweden)

    Ya-Jing Lin


    Full Text Available Nasopharyngeal carcinoma (NPC is an epithelial malignancy of the head and neck and the incidence is higher in Southeast Asia. Tetrandrine (TET is a bisbenzylisoquinoline alkaloid, a natural product, and exhibits biological activities including action against many human cancer cell lines. However, the molecular mechanism of TET-induced cell apoptosis in human NPC cells is still unclear. In the present study, we investigated TET-induced apoptotic cell death and associated possible signal pathways on human nasopharyngeal carcinoma NPC-TW 076 cells in vitro. Phase contrast microscopy was used to examine cell morphology and DAPI staining was used to examine chromatin condensation. Flow cytometry assay was used to measure total viable cells, cell cycle and sub-G1 phase distribution, reactive oxygen species (ROS, Ca2+, and mitochondria membrane potential (ΔΨm in NPC-TW 076 cells. Results indicate that TET induced cell death through the cell morphological changes, caused G0/G1 phase arrest, increased ROS and Ca2+ production, and finally caused apoptotic cell death in NPC-TW 076 cells. There was no influence on the level of ΔΨm after TET treatment. Western blotting indicated that TET increased endoplasmic reticulum (ER stress associated protein expression such as GADD153, GRP78, ATF-6α and ATF-6 βwhich indicated that TET induced cell death through ER stress. ER stress is a potential target in cancer treatment, so the ability of TET to induce ER stress response and to activate programming cell death in NPC-TW 076 cells make this molecule become a promising anticancer agent.

  6. Tetrandrine Induces Apoptosis of Human Nasopharyngeal Carcinoma NPC-TW 076 Cells through Reactive Oxygen Species Accompanied by an Endoplasmic Reticulum Stress Signaling Pathway. (United States)

    Lin, Ya-Jing; Peng, Shu-Fen; Lin, Meng-Liang; Kuo, Chao-Lin; Lu, Kung-Wen; Liao, Ching-Lung; Ma, Yi-Shih; Chueh, Fu-Shin; Liu, Kuo-Ching; Yu, Fu-Shun; Chung, Jing-Gung


    Nasopharyngeal carcinoma (NPC) is an epithelial malignancy of the head and neck and the incidence is higher in Southeast Asia. Tetrandrine (TET) is a bisbenzylisoquinoline alkaloid, a natural product, and exhibits biological activities including action against many human cancer cell lines. However, the molecular mechanism of TET-induced cell apoptosis in human NPC cells is still unclear. In the present study, we investigated TET-induced apoptotic cell death and associated possible signal pathways on human nasopharyngeal carcinoma NPC-TW 076 cells in vitro. Phase contrast microscopy was used to examine cell morphology and DAPI staining was used to examine chromatin condensation. Flow cytometry assay was used to measure total viable cells, cell cycle and sub-G₁ phase distribution, reactive oxygen species (ROS), Ca(2+), and mitochondria membrane potential (ΔΨm) in NPC-TW 076 cells. Results indicate that TET induced cell death through the cell morphological changes, caused G₀/G₁ phase arrest, increased ROS and Ca(2+) production, and finally caused apoptotic cell death in NPC-TW 076 cells. There was no influence on the level of ΔΨm after TET treatment. Western blotting indicated that TET increased endoplasmic reticulum (ER) stress associated protein expression such as GADD153, GRP78, ATF-6α and ATF-6 βwhich indicated that TET induced cell death through ER stress. ER stress is a potential target in cancer treatment, so the ability of TET to induce ER stress response and to activate programming cell death in NPC-TW 076 cells make this molecule become a promising anticancer agent.

  7. Fisetin-induced apoptosis of human oral cancer SCC-4 cells through reactive oxygen species production, endoplasmic reticulum stress, caspase-, and mitochondria-dependent signaling pathways. (United States)

    Su, Chen-Hsuan; Kuo, Chao-Lin; Lu, Kung-Wen; Yu, Fu-Shun; Ma, Yi-Shih; Yang, Jiun-Long; Chu, Yung-Lin; Chueh, Fu-Shin; Liu, Kuo-Ching; Chung, Jing-Gung


    Oral cancer is one of the cancer-related diseases in human populations and its incidence rates are rising worldwide. Fisetin, a flavonoid from natural products, has been shown to exhibit anticancer activities in many human cancer cell lines but the molecular mechanism of fisetin-induced apoptosis in human oral cancer cells is still unclear; thus, in this study, we investigated fisetin-induced cell death and associated signal pathways on human oral cancer SCC-4 cells in vitro. We examined cell morphological changes, total viable cells, and cell cycle distribution by phase contrast microscopy and flow cytometry assays. Reactive oxygen species (ROS), Ca(2+) , mitochondria membrane potential (ΔΨm ), and caspase-8, -9, and -3 activities were also measured by flow cytometer. Results indicate that fisetin induced cell death through the cell morphological changes, caused G2/M phase arrest, induction of apoptosis, promoted ROS and Ca(2+) production, and decreased the level of ΔΨm and increased caspase-3, -8, and -9 activities in SCC-4 cells. DAPI staining and DNA gel electrophoresis were also used to confirm fisetin-induced cell apoptosis in SCC-4 cells. Western blotting also found out that Fisetin increased the proapoptotic proteins such as Bax and Bid and decreased the antiapoptotic proteins such as Bcl-2. Furthermore, results also showed that Fisetin increased the cytochrome c, AIF, and Endo G release from mitochondria in SCC-4 cells. We also used ATF-6α, ATF-6β, GADD153, and GRP78 which indicated that fisetin induced cell death through ER stress. Based on those observations, we suggest that fisetin induced cell apoptosis through ER stress, mitochondria-, and caspase-dependent pathways.

  8. Differential Influence of Anticancer Treatments and Angiogenesis on the Seric Titer of Autoantibody Used as Tumor and Metastasis Biomarker

    Directory of Open Access Journals (Sweden)

    Florence Defresne


    Full Text Available Early detection of tumor-specific autoantibodies (auto-Abs has the potential to be used for cancer screening and diagnosis. Whether auto-Ab may be useful to track metastatic progression or response to treatment is, however, largely unknown. To address these issues, the serological proteome was analyzed in an invasive but treatmentresponsive mouse tumor model. Among 40 serum-reactive proteins identified by multiplex analysis, we chose to focus on glucose-regulated protein 78 (GRP78, a chaperone protein involved in the endoplasmic reticulum stress response. We first validated GRP78 as a protein overexpressed and mislocalized in tumor cells. We then documented that an increase in GRP78 auto-Ab titer preceded the detection of a palpable tumor mass, correlated with metastatic progression, and was influenced by the onset of tumor neovascularization. We also found that chemotherapy and radiotherapy, both leading to inhibition of tumor growth, oppositely influenced the anti-GRP78 immune response. Whereas radiation increased the concentration of GRP78 auto-Ab by three-fold, the auto-Ab titer was reduced in response to bolus or metronomic administration of cyclophosphamide. Finally, we established a decrease in auto-Ab-producing B lymphocytes in response to chemotherapy and the overexpression of GRP78 together with a strong immunoglobulin response in irradiated tumors. In conclusion, we identified GRP78 auto-Ab as an early marker of tumor and metastatic progressions. However, the multiple influences of anticancer treatments on the humoral immune system calls for caution when exploiting such auto-Ab as markers of the tumor response.

  9. Gadolinium chloride elicits apoptosis in human osteosarcoma U-2 OS cells through extrinsic signaling, intrinsic pathway and endoplasmic reticulum stress. (United States)

    Tsai, Yuh-Feng; Huang, Ching-Wen; Chiang, Jo-Hua; Tsai, Fuu-Jen; Hsu, Yuan-Man; Lu, Chi-Cheng; Hsiao, Chen-Yu; Yang, Jai-Sing


    Gadolinium (Gd) compounds are important as magnetic resonance imaging (MRI) contrast agents, and are potential anticancer agents. However, no report has shown the effect of gadolinium chloride (GdCl3) on osteosarcoma in vitro. The present study investigated the apoptotic mechanism of GdCl3 on human osteosarcoma U-2 OS cells. Our results indicated that GdCl3 significantly reduced cell viability of U-2 OS cells in a concentration-dependent manner. GdCl3 led to apoptotic cell shrinkage and DNA fragmentation in U-2 OS cells as revealed by morphologic changes and TUNEL staining. Colorimetric assay analyses also showed that activities of caspase-3, caspase-8, caspase-9 and caspase-4 occurred in GdCl3-treated U-2 OS cells. Pretreatment of cells with pan-caspase inhibitor (Z-VAD-FMK) and specific inhibitors of caspase-3/-8/-9 significantly reduced cell death caused by GdCl3. The increase of cytoplasmic Ca2+ level, ROS production and the decrease of mitochondria membrane potential (ΔΨm) were observed by flow cytometric analysis in U-2 OS cells after GdCl3 exposure. Western blot analyses demonstrated that the levels of Fas, FasL, cytochrome c, Apaf-1, GADD153 and GRP78 were upregulated in GdCl3-treated U-2 OS cells. In conclusion, death receptor, mitochondria-dependent and endoplasmic reticulum (ER) stress pathways contribute to GdCl3-induced apoptosis in U-2 OS cells. GdCl3 might have potential to be used in treatment of osteosarcoma patients.

  10. Novel Role of ER Stress and Autophagy in Microcystin-LR Induced Apoptosis in Chinese Hamster Ovary Cells (United States)

    Zhang, Shenshen; Liu, Chuanrui; Li, Yang; Imam, Mustapha U.; Huang, Hui; Liu, Haohao; Xin, Yongjuan; Zhang, Huizhen


    Microcystin-LR (MC-LR) is a ubiquitous peptide that exhibits strong reproductive toxicity, although the mechanistic basis for such toxicity remains largely unknown. The present study was conducted to investigate the mechanisms underlying the adverse effects of exposure to MC-LR in Chinese hamster ovary (CHO) cells. The results showed that MC-LR inhibited the in vitro proliferation of CHO cells significantly, with an IC50 of 10 μM. Moreover, MC-LR-treated CHO cells revealed strong induction of cell cycle arrest and apoptosis. Additionally, exposure of CHO cells to MC-LR resulted in excess reactive oxygen species production and intracellular calcium release, with resultant endoplasmic reticulum stress (ERs). There was also extensive accumulation of autophagic vacuoles with the highest concentration of MC-LR used (10 μM). Furthermore, the expression of ERs (GRP78, ATF-6, PERK, IRE1, CHOP) and autophagy (Beclin1 and LC3II) proteins was increased, with concomitantly reduced expression of LC3I suggesting that ERs and autophagy were induced in CHO cells by MC-LR treatment. Conversely, pretreatment of CHO cells with 4-Phenyl butyric acid, the ERs inhibitor reduced the MC-LR-induced apoptotic cell death and cellular autophagy as evidenced by the reduced expression of Beclin1 and LC3II. Similarly, MC-LR treatment in combination with an autophagy inhibitor (3-methyladenine) increased apoptotic cell death compared with MC-LR alone, and induced ERs via upregulating ERs proteins. The overall results indicated that activation of ERs and autophagy are both associated with MC-LR-induced apoptosis in CHO cells. ERs may be a trigger of autophagy in this process. PMID:27877136

  11. Luteolin and Apigenin Attenuate 4-Hydroxy-2-Nonenal-Mediated Cell Death through Modulation of UPR, Nrf2-ARE and MAPK Pathways in PC12 Cells. (United States)

    Wu, Pei-Shan; Yen, Jui-Hung; Kou, Mei-Chun; Wu, Ming-Jiuan


    Luteolin and apigenin are dietary flavones and exhibit a broad spectrum of biological activities including antioxidant, anti-inflammatory, anti-cancer and neuroprotective effects. The lipid peroxidation product 4-hydroxy-2-nonenal (4-HNE) has been implicated as a causative agent in the development of neurodegenerative disorders. This study investigates the cytoprotective effects of luteolin and apigenin against 4-HNE-mediated cytotoxicity in neuronal-like catecholaminergic PC12 cells. Both flavones restored cell viability and repressed caspase-3 and PARP-1 activation in 4-HNE-treated cells. Luteolin also mitigated 4-HNE-mediated LC3 conversion and reactive oxygen species (ROS) production. Luteolin and apigenin up-regulated 4-HNE-mediated unfolded protein response (UPR), leading to an increase in endoplasmic reticulum chaperone GRP78 and decrease in the expression of UPR-targeted pro-apoptotic genes. They also induced the expression of Nrf2-targeted HO-1 and xCT in the absence of 4-HNE, but counteracted their expression in the presence of 4-HNE. Moreover, we found that JNK and p38 MAPK inhibitors significantly antagonized the increase in cell viability induced by luteolin and apigenin. Consistently, enhanced phosphorylation of JNK and p38 MAPK was observed in luteolin- and apigenin-treated cells. In conclusion, this result shows that luteolin and apigenin activate MAPK and Nrf2 signaling, which elicit adaptive cellular stress response pathways, restore 4-HNE-induced ER homeostasis and inhibit cytotoxicity. Luteolin exerts a stronger cytoprotective effect than apigenin possibly due to its higher MAPK, Nrf2 and UPR activation, and ROS scavenging activity.

  12. Recognizing textual entailment models and applications

    CERN Document Server

    Dagan, Ido; Sammons, Mark


    In the last few years, a number of NLP researchers have developed and participated in the task of Recognizing Textual Entailment (RTE). This task encapsulates Natural Language Understanding capabilities within a very simple interface: recognizing when the meaning of a text snippet is contained in the meaning of a second piece of text. This simple abstraction of an exceedingly complex problem has broad appeal partly because it can be conceived also as a component in other NLP applications, from Machine Translation to Semantic Search to Information Extraction. It also avoids commitment to any sp

  13. Paraphrase substitution for recognizing textual entailment

    NARCIS (Netherlands)

    Bosma, W.E.; Callison-Burch, C.; Nardi, A.; Peters, C.; Vicedo, J.L.


    We describe a method for recognizing textual entailment that uses the length of the longest common subsequence (LCS) between two texts as its decision criterion. Rather than requiring strict word matching in the common subsequences, we perform a flexible match using automatically generated paraphrase

  14. Paraphrase Substitution for Recognizing Textual Entailment

    NARCIS (Netherlands)

    Bosma, W.E.; Callison-Burch, C.; Peters, C.; Clough, P.; Gey, F.C.; Karlgren, J.; Magnini, B.; Oard, D.W.; de Rijke, M.; Stempfhuber, M.


    We describe a method for recognizing textual entailment that uses the length of the longest common subsequence (LCS) between two texts as its decision criterion. Rather than requiring strict word matching in the common subsequences, we perform a flexible match using automatically generated paraphras

  15. How should a speech recognizer work?

    NARCIS (Netherlands)

    Scharenborg, O.E.; Norris, D.G.; Bosch, L.F.M. ten; McQueen, J.M.


    Although researchers studying human speech recognition (HSR) and automatic speech recognition (ASR) share a common interest in how information processing systems (human or machine) recognize spoken language, there is little communication between the two disciplines. We suggest that this lack of comm

  16. Recognizing and Responding to Adolescent Depression. (United States)

    King, Stephen R.


    Depression is increasingly recognized as a problem affecting adolescents as well as adults. Adolescents are underserved with regard to treatment facilities. One solution is the comprehensive health care clinic providing a holistic approach to assessment and intervention. Policy recommendations, which include a role for the school system, are made.…

  17. Oxidized low density lipoprotein (LDL) affects hyaluronan synthesis in human aortic smooth muscle cells. (United States)

    Viola, Manuela; Bartolini, Barbara; Vigetti, Davide; Karousou, Evgenia; Moretto, Paola; Deleonibus, Sara; Sawamura, Tatsuya; Wight, Thomas N; Hascall, Vincent C; De Luca, Giancarlo; Passi, Alberto


    Thickening of the vessel in response to high low density lipoprotein(s) (LDL) levels is a hallmark of atherosclerosis, characterized by increased hyaluronan (HA) deposition in the neointima. Human native LDL trapped within the arterial wall undergoes modifications such as oxidation (oxLDL). The aim of our study is to elucidate the link between internalization of oxLDL and HA production in vitro, using human aortic smooth muscle cells. LDL were used at an effective protein concentration of 20-50 μg/ml, which allowed 80% cell viability. HA content in the medium of untreated cells was 28.9 ± 3.7 nmol HA-disaccharide/cell and increased after oxLDL treatment to 53.9 ± 5.6. OxLDL treatments doubled the transcripts of HA synthase HAS2 and HAS3. Accumulated HA stimulated migration of aortic smooth muscle cells and monocyte adhesiveness to extracellular matrix. The effects induced by oxLDL were inhibited by blocking LOX-1 scavenger receptor with a specific antibody (10 μg/ml). The cholesterol moiety of LDL has an important role in HA accumulation because cholesterol-free oxLDL failed to induce HA synthesis. Nevertheless, cholesterol-free oxLDL and unmodified cholesterol (20 μg/ml) induce only HAS3 transcription, whereas 22,oxysterol affects both HAS2 and HAS3. Moreover, HA deposition was associated with higher expression of endoplasmic reticulum stress markers (CHOP and GRP78). Our data suggest that HA synthesis can be induced in response to specific oxidized sterol-related species delivered through oxLDL.

  18. Autoreactivity to Glucose Regulated Protein 78 Links Emphysema and Osteoporosis in Smokers (United States)

    Zhang, Yingze; Xue, Jianmin; Fuhrman, Carl R.; Tan, Jiangning; Burger, Mathew; Kass, Daniel J.; Csizmadia, Eva; Otterbein, Leo; Chandra, Divay; Bhargava, Arpit; Pilewski, Joseph M.; Roodman, G. David; Sciurba, Frank C.; Duncan, Steven R.


    Rationale Emphysema and osteoporosis are epidemiologically associated diseases of cigarette smokers. The causal mechanism(s) linking these illnesses is unknown. We hypothesized autoimmune responses may be involved in both disorders. Objectives To discover an antigen-specific autoimmune response associated with both emphysema and osteoporosis among smokers. Methods Replicate nonbiased discovery assays indicated that autoimmunity to glucose regulated protein 78 (GRP78), an endoplasmic reticulum chaperone and cell surface signaling receptor, is present in many smokers. Subject assessments included spirometry, chest CT scans, dual x-ray absorptiometry, and immunoblots for anti-GRP78 IgG. Anti-GRP78 autoantibodies were isolated from patient plasma by affinity chromatography, leukocyte functions assessed by flow cytometry, and soluble metabolites and mediators measured by immunoassays. Measurements and Main Results Circulating anti-GRP78 IgG autoantibodies were detected in plasma specimens from 86 (32%) of the 265 smoking subjects. Anti-GRP78 autoantibodies were singularly prevalent among subjects with radiographic emphysema (OR 3.1, 95%CI 1.7–5.7, p = 0.003). Anti-GRP78 autoantibodies were also associated with osteoporosis (OR 4.7, 95%CI 1.7–13.3, p = 0.002), and increased circulating bone metabolites (p = 0.006). Among emphysematous subjects, GRP78 protein was an autoantigen of CD4 T-cells, stimulating lymphocyte proliferation (p = 0.0002) and IFN-gamma production (p = 0.03). Patient-derived anti-GRP78 autoantibodies had avidities for osteoclasts and macrophages, and increased macrophage NFkB phosphorylation (p = 0.005) and productions of IL-8, CCL-2, and MMP9 (p = 0.005, 0.007, 0.03, respectively). Conclusions Humoral and cellular GRP78 autoimmune responses in smokers have numerous biologically-relevant pro-inflammatory and other deleterious actions, and are associated with emphysema and osteoporosis. These findings may have relevance

  19. Autoreactivity to glucose regulated protein 78 links emphysema and osteoporosis in smokers.

    Directory of Open Access Journals (Sweden)

    Jessica Bon

    Full Text Available RATIONALE: Emphysema and osteoporosis are epidemiologically associated diseases of cigarette smokers. The causal mechanism(s linking these illnesses is unknown. We hypothesized autoimmune responses may be involved in both disorders. OBJECTIVES: To discover an antigen-specific autoimmune response associated with both emphysema and osteoporosis among smokers. METHODS: Replicate nonbiased discovery assays indicated that autoimmunity to glucose regulated protein 78 (GRP78, an endoplasmic reticulum chaperone and cell surface signaling receptor, is present in many smokers. Subject assessments included spirometry, chest CT scans, dual x-ray absorptiometry, and immunoblots for anti-GRP78 IgG. Anti-GRP78 autoantibodies were isolated from patient plasma by affinity chromatography, leukocyte functions assessed by flow cytometry, and soluble metabolites and mediators measured by immunoassays. MEASUREMENTS AND MAIN RESULTS: Circulating anti-GRP78 IgG autoantibodies were detected in plasma specimens from 86 (32% of the 265 smoking subjects. Anti-GRP78 autoantibodies were singularly prevalent among subjects with radiographic emphysema (OR 3.1, 95%CI 1.7-5.7, p = 0.003. Anti-GRP78 autoantibodies were also associated with osteoporosis (OR 4.7, 95%CI 1.7-13.3, p = 0.002, and increased circulating bone metabolites (p = 0.006. Among emphysematous subjects, GRP78 protein was an autoantigen of CD4 T-cells, stimulating lymphocyte proliferation (p = 0.0002 and IFN-gamma production (p = 0.03. Patient-derived anti-GRP78 autoantibodies had avidities for osteoclasts and macrophages, and increased macrophage NFkB phosphorylation (p = 0.005 and productions of IL-8, CCL-2, and MMP9 (p = 0.005, 0.007, 0.03, respectively. CONCLUSIONS: Humoral and cellular GRP78 autoimmune responses in smokers have numerous biologically-relevant pro-inflammatory and other deleterious actions, and are associated with emphysema and osteoporosis. These findings may have

  20. Apoptosis and necrosis induced by novel realgar quantum dots in human endometrial cancer cells via endoplasmic reticulum stress signaling pathway

    Directory of Open Access Journals (Sweden)

    Wang H


    Full Text Available Huan Wang,1–3 Zhengyun Liu,4 Ying Gou,3 Yu Qin,4 Yaze Xu,5 Jie Liu,4 Jin-Zhu Wu6 1Research Center for Medicine and Biology, 2Guizhou Provincial College-based Key Lab for Tumor Prevention and Treatment with Distinctive Medicines, 3Department of Microbiology, 4Key Lab for Basic Pharmacology of Ministry of Education, 5Pharmacy School, Zunyi Medical College, Zunyi, 6Department of Chemistry, School of Science, Harbin Institute of Technology, Harbin, People’s Republic of China Abstract: Realgar (AS4S4 has been used in traditional medicines for malignancy, but the poor water solubility is still a major hindrance to its clinical use. Realgar quantum dots (RQDs were therefore synthesized with improved water solubility and bioavailability. Human endometrial cancer JEC cells were exposed to various concentrations of RQDs to evaluate their anticancer effects and to explore mechanisms by the MTT assay, transmission electron microscopy (TEM, flow cytometry, real-time reverse transcriptase polymerase chain reaction (RT-PCR and Western blot analysis. Results revealed that the highest photoluminescence quantum yield of the prepared RQDs was up to approximately 70%, with the average size of 5.48 nm. RQDs induced antiproliferative activity against JEC cells in a concentration-dependent manner. In light microscopy and TEM examinations, RQDs induced vacuolization and endoplasmic reticulum (ER dilation in JEC cells in a concentration-dependent manner. ER stress by RQDs were further confirmed by increased expression of GADD153 and GRP78 at both mRNA and protein levels. ER stress further led to JEC cell apoptosis and necrosis, as evidenced by flow cytometry and mitochondrial membrane potential detection. Our findings demonstrated that the newly synthesized RQDs were effective against human endometrial cancer cells. The underlying mechanism appears to be, at least partly, due to ER stress leading to apoptotic cell death and necrosis. Keywords: realgar, quantum dots

  1. The invasion of tobacco mosaic virus RNA induces endoplasmic reticulum stress-related autophagy in HeLa cells. (United States)

    Li, Li; Wang, Li; Xiao, Ruijing; Zhu, Guoguo; Li, Yan; Liu, Changxuan; Yang, Ru; Tang, Zhiqing; Li, Jie; Huang, Wei; Chen, Lang; Zheng, Xiaoling; He, Yuling; Tan, Jinquan


    The ability of human cells to defend against viruses originating from distant species has long been ignored. Owing to the pressure of natural evolution and human exploration, some of these viruses may be able to invade human beings. If their 'fresh' host had no defences, the viruses could cause a serious pandemic, as seen with HIV, SARS (severe acute respiratory syndrome) and avian influenza virus that originated from chimpanzees, the common palm civet and birds, respectively. It is unknown whether the human immune system could tolerate invasion with a plant virus. To model such an alien virus invasion, we chose TMV (tobacco mosaic virus) and used human epithelial carcinoma cells (HeLa cells) as its 'fresh' host. We established a reliable system for transfecting TMV-RNA into HeLa cells and found that TMV-RNA triggered autophagy in HeLa cells as shown by the appearance of autophagic vacuoles, the conversion of LC3-I (light chain protein 3-I) to LC3-II, the up-regulated expression of Beclin1 and the accumulation of TMV protein on autophagosomal membranes. We observed suspected TMV virions in HeLa cells by TEM (transmission electron microscopy). Furthermore, we found that TMV-RNA was translated into CP (coat protein) in the ER (endoplasmic reticulum) and that TMV-positive RNA translocated from the cytoplasm to the nucleolus. Finally, we detected greatly increased expression of GRP78 (78 kDa glucose-regulated protein), a typical marker of ERS (ER stress) and found that the formation of autophagosomes was closely related to the expanded ER membrane. Taken together, our data indicate that HeLa cells used ERS and ERS-related autophagy to defend against TMV-RNA.

  2. Expression and Location of Glucose-regulated Protein 78 in Testis and Epididymis

    Directory of Open Access Journals (Sweden)

    W Wang


    Full Text Available Objective: To know the role of glucose-regulated protein 78 (GRP78/BiP/HSPA5 in spermatogenesis and its expression and location in the testis and epididymis. Methods: Immunohistochemistry and Western blot were used to detect GRP78 location and expression in the testis and epididymis. Results: Glucose-regulated protein 78 was observed in spermatocytes, round spermatids and interstitial cells of the testis and in principal cells of the epididymis. Glucose-regulated protein 78 was first detected in the rat testis at postnatal day 14. Thereafter, the protein level increased gradually with age and was maintained at a high and stable state after postnatal day 28. In the rat, GRP78 was expressed in the principal cells but not in clear cells of the epididymis. Conclusion: Glucose-regulated protein 78 participates in the process of spermatogenesis.

  3. Characterization of human mesenchymal stem cell secretome at early steps of adipocyte and osteoblast differentiation

    Directory of Open Access Journals (Sweden)

    Alessi Marie-Christine


    Full Text Available Abstract Background It is well established that adipose tissue plays a key role in energy storage and release but is also a secretory organ and a source of stem cells. Among different lineages, stem cells are able to differentiate into adipocytes and osteoblasts. As secreted proteins could regulate the balance between both lineages, we aimed at characterizing the secretome of human multipotent adipose-derived stem cell (hMADS at an early step of commitment to adipocytes and osteoblasts. Results A proteomic approach, using mono-dimensional electrophoresis and tandem mass spectrometry, allowed us to identify a total of 73 proteins at day 0 and day 3 of adipocyte and osteoblast differentiation. Analysis of identified proteins showed that 52 % corresponded to classical secreted proteins characterized by a signal peptide, that 37 % previously described in the extracellular compartment were devoid of signal peptide and that 11 % neither exhibited a signal peptide nor had been previously described extracellularly. These proteins were classified into 8 clusters according to their function. Quantitative analysis has been performed for 8 candidates: PAI-1, PEDF, BIGH3, PTX3, SPARC, ENO1, GRP78 and MMP2. Among them, PAI-1 was detected at day 0 and day 3 of osteoblast differentiation but never in adipocyte secretome. Furthermore we showed that PAI-1 mRNA was down-regulated in the bone of ovariectomized mice. Conclusion Given its regulation during the early events of hMADS cell differentiation and its status in ovariectomized mice, PAI-1 could play a role in the adipocyte/osteoblast balance and thus in bone diseases such as osteoporosis.

  4. Characterization of human mesenchymal stem cell secretome at early steps of adipocyte and osteoblast differentiation (United States)

    Chiellini, Chiara; Cochet, Olivia; Negroni, Luc; Samson, Michel; Poggi, Marjorie; Ailhaud, Gérard; Alessi, Marie-Christine; Dani, Christian; Amri, Ez-Zoubir


    Background It is well established that adipose tissue plays a key role in energy storage and release but is also a secretory organ and a source of stem cells. Among different lineages, stem cells are able to differentiate into adipocytes and osteoblasts. As secreted proteins could regulate the balance between both lineages, we aimed at characterizing the secretome of human multipotent adipose-derived stem cell (hMADS) at an early step of commitment to adipocytes and osteoblasts. Results A proteomic approach, using mono-dimensional electrophoresis and tandem mass spectrometry, allowed us to identify a total of 73 proteins at day 0 and day 3 of adipocyte and osteoblast differentiation. Analysis of identified proteins showed that 52 % corresponded to classical secreted proteins characterized by a signal peptide, that 37 % previously described in the extracellular compartment were devoid of signal peptide and that 11 % neither exhibited a signal peptide nor had been previously described extracellularly. These proteins were classified into 8 clusters according to their function. Quantitative analysis has been performed for 8 candidates: PAI-1, PEDF, BIGH3, PTX3, SPARC, ENO1, GRP78 and MMP2. Among them, PAI-1 was detected at day 0 and day 3 of osteoblast differentiation but never in adipocyte secretome. Furthermore we showed that PAI-1 mRNA was down-regulated in the bone of ovariectomized mice. Conclusion Given its regulation during the early events of hMADS cell differentiation and its status in ovariectomized mice, PAI-1 could play a role in the adipocyte/osteoblast balance and thus in bone diseases such as osteoporosis. PMID:18302751

  5. JA, a new type of polyunsaturated fatty acid isolated from Juglans mandshurica Maxim, limits the survival and induces apoptosis of heptocarcinoma cells. (United States)

    Gao, Xiu-Li; Lin, Hua; Zhao, Wei; Hou, Ya-Qin; Bao, Yong-Li; Song, Zhen-Bo; Sun, Lu-Guo; Tian, Shang-Yi; Liu, Biao; Li, Yu-Xin


    Juglans mandshurica Maxim (Juglandaceae) is a famous folk medicine for cancer treatment and some natural compounds isolated from it have been studied extensively. Previously we isolated a type of ω-9 polyunsaturated fatty acid (JA) from the bark of J. mandshurica, however little is known about its activity and the underlying mechanisms. In this study, we studied anti-tumor activity of JA on several human cancer cell lines. Results showed that JA is cytotoxic to HepG2, MDA-MB-231, SGC-7901, A549 and Huh7 cells at a concentration exerting minimal toxic effects on L02 cells. The selective toxicity of JA was better than other classical anti-cancer drugs. Further investigation indicated that JA could induce cell apoptosis, characterized by chromatin condensation, DNA fragmentation and activation of the apoptosis-associated proteins such as Caspase-3 and PARP-1. Moreover, we investigated the cellular apoptosis pathway involved in the apoptosis process in HepG2 cells. We found that proteins involved in mitochondrion (cleaved-Caspase-9, Apaf-1, HtrA2/Omi, Bax, and Mitochondrial Bax) and endocytoplasmic reticulum (XBP-1s, GRP78, cleaved-Caspase-7 and cleaved-Caspase-12) apoptotic pathways were up-regulated when cells were treated by JA. In addition, a morphological change in the mitochondrion was detected. Furthermore, we found that JA could inhibit DNA synthesis and induce G2/M cell cycle arrest. The expression of G2-to-M transition related proteins, such as CyclinB1 and phosphorylated-CDK1, were reduced. In contrast, the G2-to-M inhibitor p21 was increased in JA-treated cells. Overall, our results suggest that JA can induce mitochondrion- and endocytoplasmic reticulum-mediated apoptosis, and G2/M phase arrest in HepG2 cells, making it a promising therapeutic agent against hepatoma.

  6. Impairment of the ubiquitin-proteasome pathway by methyl N-(6-phenylsulfanyl-1H-benzimidazol-2-yl)carbamate leads to a potent cytotoxic effect in tumor cells: a novel antiproliferative agent with a potential therapeutic implication. (United States)

    Dogra, Nilambra; Mukhopadhyay, Tapas


    In recent years, there has been a great deal of interest in proteasome inhibitors as a novel class of anticancer drugs. We report that fenbendazole (FZ) (methyl N-(6-phenylsulfanyl-1H-benzimidazol-2-yl)carbamate) exhibits a potent growth-inhibitory activity against cancer cell lines but not normal cells. We show here, using fluorogenic substrates, that FZ treatment leads to the inhibition of proteasomal activity in the cells. Succinyl-Leu-Leu-Val-Tyr-methylcoumarinamide (MCA), benzyloxycarbonyl-Leu-Leu-Glu-7-amido-4-MCA, and t-butoxycarbonyl-Gln-Ala-Arg-7-amido-4-MCA fluorescent derivatives were used to assess chymotrypsin-like, post-glutamyl peptidyl-hydrolyzing, and trypsin-like protease activities, respectively. Non-small cell lung cancer cells transiently transfected with an expression plasmid encoding pd1EGFP and treated with FZ showed an accumulation of the green fluorescent protein in the cells due to an increase in its half-life. A number of apoptosis regulatory proteins that are normally degraded by the ubiquitin-proteasome pathway like cyclins, p53, and IκBα were found to be accumulated in FZ-treated cells. In addition, FZ induced distinct ER stress-associated genes like GRP78, GADD153, ATF3, IRE1α, and NOXA in these cells. Thus, treatment of human NSCLC cells with fenbendazole induced endoplasmic reticulum stress, reactive oxygen species production, decreased mitochondrial membrane potential, and cytochrome c release that eventually led to cancer cell death. This is the first report to demonstrate the inhibition of proteasome function and induction of endoplasmic reticulum stress/reactive oxygen species-dependent apoptosis in human lung cancer cell lines by fenbendazole, which may represent a new class of anticancer agents showing selective toxicity against cancer cells.

  7. Virulence Factors of Pseudomonas aeruginosa Induce Both the Unfolded Protein and Integrated Stress Responses in Airway Epithelial Cells.

    Directory of Open Access Journals (Sweden)

    Emily F A van 't Wout


    Full Text Available Pseudomonas aeruginosa infection can be disastrous in chronic lung diseases such as cystic fibrosis and chronic obstructive pulmonary disease. Its toxic effects are largely mediated by secreted virulence factors including pyocyanin, elastase and alkaline protease (AprA. Efficient functioning of the endoplasmic reticulum (ER is crucial for cell survival and appropriate immune responses, while an excess of unfolded proteins within the ER leads to "ER stress" and activation of the "unfolded protein response" (UPR. Bacterial infection and Toll-like receptor activation trigger the UPR most likely due to the increased demand for protein folding of inflammatory mediators. In this study, we show that cell-free conditioned medium of the PAO1 strain of P. aeruginosa, containing secreted virulence factors, induces ER stress in primary bronchial epithelial cells as evidenced by splicing of XBP1 mRNA and induction of CHOP, GRP78 and GADD34 expression. Most aspects of the ER stress response were dependent on TAK1 and p38 MAPK, except for the induction of GADD34 mRNA. Using various mutant strains and purified virulence factors, we identified pyocyanin and AprA as inducers of ER stress. However, the induction of GADD34 was mediated by an ER stress-independent integrated stress response (ISR which was at least partly dependent on the iron-sensing eIF2α kinase HRI. Our data strongly suggest that this increased GADD34 expression served to protect against Pseudomonas-induced, iron-sensitive cell cytotoxicity. In summary, virulence factors from P. aeruginosa induce ER stress in airway epithelial cells and also trigger the ISR to improve cell survival of the host.

  8. alpha-Linolenic acid protects renal cells against palmitic acid lipotoxicity via inhibition of endoplasmic reticulum stress. (United States)

    Katsoulieris, Elias; Mabley, Jon G; Samai, Mohamed; Green, Irene C; Chatterjee, Prabal K


    Unsaturated fatty acids may counteract the lipotoxicity associated with saturated fatty acids. Palmitic acid induced endoplasmic reticulum (ER) stress and caused apoptotic and necrotic cell death in the renal proximal tubular cell line, NRK-52E. We investigated whether alpha-linolenic acid, an unsaturated fatty acid, protected against ER stress and cell death induced by palmitic acid or by other non-nutrient ER stress generators. Incubation of NRK-52E cells for 24h with palmitic acid produced a significant increase in apoptosis and necrosis. Palmitic acid also increased levels of three indicators of ER stress - the phosphorylated form of the eukaryotic initiation factor 2alpha (eIF2alpha), C/EBP homologous protein (CHOP), and glucose regulated protein 78 (GRP78). alpha-Linolenic acid dramatically reduced cell death and levels of all three indicators of ER stress brought about by palmitic acid. Tunicamycin, which induces ER stress by glycosylation of proteins, produced similar effects to those obtained using palmitic acid; its effects were partially reversed by alpha-linolenic acid. Salubrinal (a phosphatase inhibitor) causes increased levels of the phosphorylated form of eIF2alpha - this effect was partially reversed by alpha-linolenic acid. Palmitoleate, a monosaturated fatty acid, had similar effects to those of alpha-linolenic acid. These results suggest that part of the mechanism of protection of the kidney by unsaturated fatty acids is through inhibition of ER stress, eIF2alpha phosphorylation and consequential reduction of CHOP protein expression and apoptotic renal cell death.

  9. Exogenous hepatitis B virus envelope proteins induce endoplasmic reticulum stress: involvement of cannabinoid axis in liver cancer cells (United States)

    Montalbano, Roberta; Honrath, Birgit; Wissniowski, Thaddeus Till; Elxnat, Moritz; Roth, Silvia; Ocker, Matthias; Quint, Karl; Churin, Yuri; Roederfeld, Martin; Schroeder, Dirk; Glebe, Dieter; Roeb, Elke; Fazio, Pietro Di


    HBV represents the most common chronic viral infection and major cause of hepatocellular carcinoma (HCC), although its exact role in liver tumorigenesis is unclear. Massive storage of the small (SHBs), middle (MHBs) and large surface (LHBs) HBV envelope proteins leads to cell stress and sustained inflammatory responses. Cannabinoid (CB) system is involved in the pathogenesis of liver diseases, stimulating acute and chronic inflammation, liver damage and fibrogenesis; it triggers endoplasmic reticulum (ER) stress response. The aim of our work was to investigate the activation of ER stress pathway after ectopic HBV envelope proteins expression, in liver cancer cells, and the role exerted by CB receptors. PCR, immunofluorescence and western blotting showed that exogenous LHBs and MHBs induce a clear ER stress response in Huh-7 cells expressing CB1 receptor. Up-regulation of the chaperone BiP/GRP78 (Binding Immunoglobulin Protein/Glucose-Regulated Protein 78) and of the transcription factor CHOP/GADD153 (C/EBP Homologous Protein/Growth Arrest and DNA Damage inducible gene 153), phosphorylation of PERK (PKR-like ER Kinase) and eIF2α (Eukaryotic Initiation Factor 2α) and splicing of XBP1 (X-box binding protein 1) was observed. CB1−/− HepG2 cells did not show any ER stress activation. Inhibition of CB1 receptor counteracted BiP expression in transfected Huh-7 and in HBV+ PLC/PRF/5 cells; whereas no effect was observed in HBV− HLF cells. These results suggest that HBV envelope proteins are able to induce the ER stress pathway. CB1 expression is directly correlated with ER stress function. Further investigations are needed to clarify the involvement of cannabinoid in HCC progression after HBV infection. PMID:26967385

  10. Recognizing frequency characteristics of gas sensor array

    Institute of Scientific and Technical Information of China (English)


    A novel method based on independent component analyzing (ICA) in frequency domain to distinguish the frequency characteristics of multi-sensor system is presented. The conditions of this type of ICA are considered and each step of resolving the problem is discussed. For a two gas sensor array, the frequency characteristics including amplitude-frequency and phase-frequency are recognized by this method, and cross-sensitivity between them is also eliminated. From the principle of similarity, the recognition m...

  11. Textual Entailment Recognizing by Theorem Proving Approach

    CERN Document Server

    Tatar, Doina


    In this paper we present two original methods for recognizing textual inference. First one is a modified resolution method such that some linguistic considerations are introduced in the unification of two atoms. The approach is possible due to the recent methods of transforming texts in logic formulas. Second one is based on semantic relations in text, as presented in WordNet. Some similarities between these two methods are remarked.

  12. Recognizing Action Units for Facial Expression Analysis. (United States)

    Tian, Ying-Li; Kanade, Takeo; Cohn, Jeffrey F


    Most automatic expression analysis systems attempt to recognize a small set of prototypic expressions, such as happiness, anger, surprise, and fear. Such prototypic expressions, however, occur rather infrequently. Human emotions and intentions are more often communicated by changes in one or a few discrete facial features. In this paper, we develop an Automatic Face Analysis (AFA) system to analyze facial expressions based on both permanent facial features (brows, eyes, mouth) and transient facial features (deepening of facial furrows) in a nearly frontal-view face image sequence. The AFA system recognizes fine-grained changes in facial expression into action units (AUs) of the Facial Action Coding System (FACS), instead of a few prototypic expressions. Multistate face and facial component models are proposed for tracking and modeling the various facial features, including lips, eyes, brows, cheeks, and furrows. During tracking, detailed parametric descriptions of the facial features are extracted. With these parameters as the inputs, a group of action units (neutral expression, six upper face AUs and 10 lower face AUs) are recognized whether they occur alone or in combinations. The system has achieved average recognition rates of 96.4 percent (95.4 percent if neutral expressions are excluded) for upper face AUs and 96.7 percent (95.6 percent with neutral expressions excluded) for lower face AUs. The generalizability of the system has been tested by using independent image databases collected and FACS-coded for ground-truth by different research teams.

  13. Picosecond pulsed electric fields induce apoptosis in HeLa cells via the endoplasmic reticulum stress and caspase-dependent signaling pathways. (United States)

    Chen, Wen-Juan; Xiong, Zheng-Ai; Zhang, Min; Yao, Chen-Guo; Zhao, Zhong-Yong; Hua, Yuan-Yuan; Zhou, Wei


    The non-invasive treatment of tumors with preserved fertility holds great promise. The application of pulsed electric field (PEF) is a new biomedical engineering technique for tumor therapy. Picosecond pulsed electric fields (psPEF) can be transferred to target deep tissue non-invasively and precisely; however, research of the biological effects of psPEF on cells is limited. Electric theory predicts that when the pulse duration decreases to nanoseconds and picoseconds, it will mainly affect organelles and lead to intracellular electromanipulations. Previous studies have shown that psPEF targets the mitochondria and induces apoptosis through a mitochondrial-mediated pathway in HeLa cells. The endoplasmic reticulum is also involved in the intrinsic pathways of apoptosis. In the present study, HeLa cells were exposed to psPEF to investigate the underlying mechanisms of apoptosis. MTT assay demonstrated that psPEF displayed strong growth inhibitory effects on HeLa cells. Treatment with psPEF led to marked cell apoptosis and cell cycle arrest at the G2/M phase. In addition, psPEF affected the phosphorylation levels of endoplasmic reticulum sensors and upregulated the expression of glucose-regulated protein 78 (GRP78), glucose-regulated protein 94 (GRP94) and CCAAT enhancer-binding protein (C/EBP) homologous protein (CHOP). These changes were accompanied by the elevation of intracellular Ca2+ concentrations. Furthermore, the activation of caspase-12, -9 and -3, led to the release of cytochrome c, as well as the upregulation of Bax and the downregulation of Bcl-2, as observed in the HeLa cells. Taken together, these data suggest that psPEF is an efficient apoptosis-inducing agent for HeLa cells, which exerts its effects, at least partially, via the endoplasmic reticulum stress and caspase-dependent signaling pathways.

  14. Proteomic Characterization of Annexin l (ANX1 and Heat Shock Protein 27 (HSP27 as Biomarkers for Invasive Hepatocellular Carcinoma Cells.

    Directory of Open Access Journals (Sweden)

    Ruo-Chiau Wang

    Full Text Available To search for reliable biomarkers and drug targets for management of hepatocellular carcinoma (HCC, we performed a global proteomic analysis of a pair of HCC cell lines with distinct differentiation statuses using 2-DE coupled with MALDI-TOF MS. In total, 106 and 55 proteins were successfully identified from the total cell lysate and the cytosolic, nuclear and membrane fractions in well-differentiated (HepG2 and poorly differentiated (SK-Hep-1 HCC clonal variants, respectively. Among these proteins, nine spots corresponding to proteins differentially expressed between HCC cell types were selected and confirmed by immunofluorescence staining and western blotting. Notably, Annexin 1 (ANX1, ANX-2, vimentin and stress-associated proteins, such as GRP78, HSP75, HSC-70, protein disulfide isomerase (PDI, and heat shock protein-27 (HSP27, were exclusively up-regulated in SK-Hep-1 cells. Elevated levels of ANX-4 and antioxidant/metabolic enzymes, such as MnSOD, peroxiredoxin, NADP-dependent isocitrate dehydrogenase, α-enolase and UDP-glucose dehydrogenase, were observed in HepG2 cells. We functionally demonstrated that ANX1 and HSP27 were abundantly overexpressed only in highly invasive types of HCC cells, such as Mahlavu and SK-Hep-1. Knockdown of ANX1 or HSP27 in HCC cells resulted in a severe reduction in cell migration. The in-vitro observations of ANX1 and HSP27 expressions in HCC sample was demonstrated by immunohistochemical stains performed on HCC tissue microarrays. Poorly differentiated HCC tended to have stronger ANX1 and HSP27 expressions than well-differentiated or moderately differentiated HCC. Collectively, our findings suggest that ANX1 and HSP27 are two novel biomarkers for predicting invasive HCC phenotypes and could serve as potential treatment targets.

  15. Safrole induces G0/G1 phase arrest via inhibition of cyclin E and provokes apoptosis through endoplasmic reticulum stress and mitochondrion-dependent pathways in human leukemia HL-60 cells. (United States)

    Yu, Chun-Shu; Huang, An-Cheng; Yang, Jai-Sing; Yu, Chien-Chih; Lin, Chin-Chung; Chung, Hsiung-Kwang; Huang, Yi-Ping; Chueh, Fu-Shin; Chung, Jing-Gung


    Safrole, a component of Piper betle inflorescence, is a carcinogen which has been demonstrated to induce apoptosis on human oral cancer HSC-3 cells in vitro and to inhibit HSC-3 cells in xenograft tumor cells in vivo. In our previous study, safrole promoted phagocytosis by macrophages and natural killer cell cytotoxicity in normal BALB/c mice. The cytotoxic effects of safrole on HL-60 cells were investigated by using flow cytometric analysis, comet assay, 4',6-diamidino-2-phenylindole (DAPI) staining, western blotting and confocal laser microscopy. The obtained results indicate that safrole induced a cytotoxic response through reducing the percentage of viable cells and induction of apoptosis in HL-60 cells in a dose-dependent manner. DAPI staining and comet assay also showed that safrole induced apoptosis (chromatin condensation) and DNA damage in HL-60 cells. The flow cytometric assay showed that safrole increased the production of reactive oxygen species (ROS) and Ca(2+) and reduced the mitochondrial membrane potential in HL-60 cells. Safrole enhanced the levels of the pro-apoptotic protein BAX, inhibited those of the anti-apoptotic protein BCL-2 and promoted the levels of apoptosis-inducing factor (AIF) and endonuclease G (Endo G) in HL-60 cells. Furthermore, safrole promoted the expression of glucose-regulated protein 78 (GRP78), growth arrest- and DNA damage-inducible gene 153 (GADD153) and of activating transcription factor 6α (ATF-6α). Based on these findings, we suggest that safrole-induced apoptosis in HL-60 cells is mediated through the ER stress and intrinsic signaling pathways.

  16. Endoplasmic reticulum stress mediates the anti-inflammatory effect of ethyl pyruvate in endothelial cells.

    Directory of Open Access Journals (Sweden)

    Ge Wang

    Full Text Available Ethyl pyruvate (EP is a simple aliphatic ester of the metabolic intermediate pyruvate that has been demonstrated to be a potent anti-inflammatory agent in a variety of in vivo and in vitro model systems. However, the protective effects and mechanisms underlying the actions of EP against endothelial cell (EC inflammatory injury are not fully understood. Previous studies have confirmed that endoplasmic reticulum stress (ERS plays an important role in regulating the pathological process of EC inflammation. In this study, our aim was to explore the effects of EP on tumor necrosis factor-α (TNF-α-induced inflammatory injury in human umbilical vein endothelial cells (HUVECs and to explore the role of ERS in this process. TNF-α treatment not only significantly increased the adhesion of monocytes to HUVECs and inflammatory cytokine (sICAM1, sE-selectin, MCP-1 and IL-8 production in cell culture supernatants but it also increased ICAM and MMP9 protein expression in HUVECs. TNF-α also effectively increased the ERS-related molecules in HUVECs (GRP78, ATF4, caspase12 and p-PERK. EP treatment effectively reversed the effects of the TNF-α-induced adhesion of monocytes on HUVECs, inflammatory cytokines and ERS-related molecules. Furthermore, thapsigargin (THA, an ERS inducer attenuated the protective effects of EP against TNF-α-induced inflammatory injury and ERS. The PERK siRNA treatment not only inhibited ERS-related molecules but also mimicked the protective effects of EP to decrease TNF-α-induced inflammatory injury. In summary, we have demonstrated for the first time that EP can effectively reduce vascular endothelial inflammation and that this effect at least in part depends on the attenuation of ERS.

  17. Melatonin Suppresses Autophagy Induced by Clinostat in Preosteoblast MC3T3-E1 Cells. (United States)

    Yoo, Yeong-Min; Han, Tae-Young; Kim, Han Sung


    Microgravity exposure can cause cardiovascular and immune disorders, muscle atrophy, osteoporosis, and loss of blood and plasma volume. A clinostat device is an effective ground-based tool for simulating microgravity. This study investigated how melatonin suppresses autophagy caused by simulated microgravity in preosteoblast MC3T3-E1 cells. In preosteoblast MC3T3-E1 cells, clinostat rotation induced a significant time-dependent increase in the levels of the autophagosomal marker microtubule-associated protein light chain (LC3), suggesting that autophagy is induced by clinostat rotation in these cells. Melatonin treatment (100, 200 nM) significantly attenuated the clinostat-induced increases in LC3 II protein, and immunofluorescence staining revealed decreased levels of both LC3 and lysosomal-associated membrane protein 2 (Lamp2), indicating a decrease in autophagosomes. The levels of phosphorylation of mammalian target of rapamycin (p-mTOR) (Ser2448), phosphorylation of extracellular signal-regulated kinase (p-ERK), and phosphorylation of serine-threonine protein kinase (p-Akt) (Ser473) were significantly reduced by clinostat rotation. However, their expression levels were significantly recovered by melatonin treatment. Also, expression of the Bcl-2, truncated Bid, Cu/Zn- superoxide dismutase (SOD), and Mn-SOD proteins were significantly increased by melatonin treatment, whereas levels of Bax and catalase were decreased. The endoplasmic reticulum (ER) stress marker GRP78/BiP, IRE1α, and p-PERK proteins were significantly reduced by melatonin treatment. Treatment with the competitive melatonin receptor antagonist luzindole blocked melatonin-induced decreases in LC3 II levels. These results demonstrate that melatonin suppresses clinostat-induced autophagy through increasing the phosphorylation of the ERK/Akt/mTOR proteins. Consequently, melatonin appears to be a potential therapeutic agent for regulating microgravity-related bone loss or osteoporosis.

  18. The multifaceted role of the embryonic gene Cripto-1 in cancer, stem cells and epithelial-mesenchymal transition. (United States)

    Klauzinska, Malgorzata; Castro, Nadia P; Rangel, Maria Cristina; Spike, Benjamin T; Gray, Peter C; Bertolette, Daniel; Cuttitta, Frank; Salomon, David


    Cripto-1 (CR-1)/Teratocarcinoma-derived growth factor1 (TDGF-1) is a cell surface glycosylphosphatidylinositol (GPI)-linked glycoprotein that can function either in cis (autocrine) or in trans (paracrine). The cell membrane cis form is found in lipid rafts and endosomes while the trans acting form lacking the GPI anchor is soluble. As a member of the epidermal growth factor (EGF)/Cripto-1-FRL-1-Cryptic (CFC) family, CR-1 functions as an obligatory co-receptor for the transforming growth factor-β (TGF-β) family members, Nodal and growth and differentiation factors 1 and 3 (GDF1/3) by activating Alk4/Alk7 signaling pathways that involve Smads 2, 3 and 4. In addition, CR-1 can activate non-Smad-dependent signaling elements such as PI3K, Akt and MAPK. Both of these pathways depend upon the 78kDa glucose regulated protein (GRP78). Finally, CR-1 can facilitate signaling through the canonical Wnt/β-catenin and Notch/Cbf-1 pathways by functioning as a chaperone protein for LRP5/6 and Notch, respectively. CR-1 is essential for early embryonic development and maintains embryonic stem cell pluripotentiality. CR-1 performs an essential role in the etiology and progression of several types of human tumors where it is expressed in a population of cancer stem cells (CSCs) and facilitates epithelial-mesenchymal transition (EMT). In this context, CR-1 can significantly enhance tumor cell migration, invasion and angiogenesis. Collectively, these facts suggest that CR-1 may be an attractive target in the diagnosis, prognosis and therapy of several types of human cancer.

  19. The multifaceted role of the embryonic gene Cripto-1 in cancer, stem cells and epithelial-mesenchymal transition (United States)

    Klauzinska, Malgorzata; Castro, Nadia P.; Rangel, Maria Cristina; Spike, Benjamin T.; Gray, Peter C.; Bertolette, Daniel; Cuttitta, Frank; Salomon, David


    Cripto-1 (CR-1)/Teratocarcinoma-derived growth factor1 (TDGF-1) is a cell surface glycosylphosphatidylinositol (GPI)-linked glycoprotein that can function either in cis (autocrine) or in trans (paracrine). The cell membrane cis form is found in lipid rafts and endosomes while the trans acting form lacking the GPI anchor is soluble. As a member of the epidermal growth factor (EGF)/Cripto-1-FRL-1-Cryptic (CFC) family, CR-1 functions as an obligatory co-receptor for the transforming growth factor-β (TGF-β) family members, Nodal and growth and differentiation factors 1 and 3 (GDF1/3) by activating Alk4/Alk7 signaling pathways that involve Smads 2, 3 and 4. In addition, CR-1 can activate non-Smad-dependent signaling elements such as PI3K, Akt and MAPK. Both of these pathways depend upon the 78 kDa glucose regulated protein (GRP78). Finally, CR-1 can facilitate signaling through the canonical Wnt/β-catenin and Notch/Cbf-1 pathways by functioning as a chaperone protein for LRP5/6 and Notch, respectively. CR-1 is essential for early embryonic development and maintains embryonic stem cell pluripotentiality. CR-1 performs an essential role in the etiology and progression of several types of human tumors where it is expressed in a population of cancer stem cells (CSCs) and facilitates epithelial-mesenchymal transition (EMT). In this context, CR-1 can significantly enhance tumor cell migration, invasion and angiogenesis. Collectively, these facts suggest that CR-1 may be an attractive target in the diagnosis, prognosis and therapy of several types of human cancer. PMID:25153355

  20. Di(2-ethylhexyl) phthalate-induced apoptosis in rat INS-1 cells is dependent on activation of endoplasmic reticulum stress and suppression of antioxidant protection. (United States)

    Sun, Xia; Lin, Yi; Huang, Qiansheng; Shi, Junpeng; Qiu, Ling; Kang, Mei; Chen, Yajie; Fang, Chao; Ye, Ting; Dong, Sijun


    Di(2-ethylhexyl) phthalate (DEHP) is used as plasticizer and is ubiquitously found in the environment. Exposure to DEHP has been linked to an increased incidence of type 2 diabetes. Pancreatic β-cell dysfunction is a hallmark of type 2 diabetes; however, it is unknown whether DEHP exposure contributes to this risk. Here, we aimed to investigate the cytotoxic effects of DEHP on INS-1 cells and to further explore the related underlying mechanisms. INS-1 cells were exposed to 0, 5, 25, 125 or 625 μM DEHP for 24 hrs. Cell viability, glucose-stimulated insulin secretion, reactive oxygen species (ROS) generation, cellular antioxidant response, Ca(2+) homoeostasis and the levels of genes and proteins involved in endoplasmic reticulum (ER) stress were measured. The results showed that DEHP decreased insulin secretion and content and induced apoptosis in INS-1 cells in a dose-dependent manner. Furthermore, ROS generation was increased and Nrf2-dependent antioxidant defence protection was dysregulated in INS-1 cells after DEHP exposure. Most importantly, DEHP effectively depleted ER Ca(2+) and triggered the ER stress response as demonstrated by the elevated transcription and translation of the ER chaperone GRP78 and GRP94, the increased phosphorylation of protein kinase R-like endoplasmic reticulum kinase (PERK) and its downstream substrate eukaryotic translation initiation factor 2α (eIF2α), as well as the increased levels of activating transcription factor 4 (ATF4) and C/EBP homologous protein (CHOP). Taken together, DEHP exerted toxic effects on INS-1 cells by inducing apoptosis, which is dependent on the activation of the PERK-ATF4-CHOP ER stress signalling pathway and the suppression of Nrf2-dependent antioxidant protection.

  1. Recognizing Patterns In Log-Polar Coordinates (United States)

    Weiman, Carl F. R.


    Log-Hough transform is basis of improved method for recognition of patterns - particularly, straight lines - in noisy images. Takes advantage of rotational and scale invariance of mapping from Cartesian to log-polar coordinates, and offers economy of representation and computation. Unification of iconic and Hough domains simplifies computations in recognition and eliminates erroneous quantization of slopes attributable to finite spacing of Cartesian coordinate grid of classical Hough transform. Equally efficient recognizing curves. Log-Hough transform more amenable to massively parallel computing architectures than traditional Cartesian Hough transform. "In-place" nature makes it possible to apply local pixel-neighborhood processing.

  2. Recognizing frequency characteristics of gas sensor array

    Institute of Scientific and Technical Information of China (English)


    A novel method based on independent component analyzing (ICA) in frequency domain to distinguish the frequency characteristics of multi-sensor system is presented. The conditions of this type of ICA are considered and each step of resolving the problem is discussed. For a two gas sensor array, the frequency characteristics including amplitude-frequency and phase-frequency are recognized by this method, and cross-sensitivity between them is also eliminated. From the principle of similarity, the recognition mean square error is no more than 0.085.

  3. Prodigiosin activates endoplasmic reticulum stress cell death pathway in human breast carcinoma cell lines

    Energy Technology Data Exchange (ETDEWEB)

    Pan, Mu-Yun [Institute of Biomedical Sciences, National Chung Hsing University, Taichung, Taiwan (China); Shen, Yuh-Chiang [Institute of Biomedical Sciences, National Chung Hsing University, Taichung, Taiwan (China); National Research Institute of Chinese Medicine, Taipei, Taiwan (China); Lu, Chien-Hsing [Department of Obstetrics and Gynecology, Taichung Veterans General Hospital, Taichung, Taiwan (China); Department of Obstetrics and Gynecology, National Yang-Ming University School of Medicine, Taipei, Taiwan (China); Yang, Shu-Yi [Institute of Biomedical Sciences, National Chung Hsing University, Taichung, Taiwan (China); Ho, Tsing-Fen [Department of Medical Laboratory Science and Biotechnology, Central Taiwan University of Science and Technology, Taichung, Taiwan (China); Peng, Yu-Ta [Institute of Biomedical Sciences, National Chung Hsing University, Taichung, Taiwan (China); Chang, Chia-Che, E-mail: [Institute of Biomedical Sciences, National Chung Hsing University, Taichung, Taiwan (China); Agricultural Biotechnology Center, National Chung Hsing University, Taichung, Taiwan (China); Graduate Institute of Basic Medical Science, China Medical University, Taichung, Taiwan (China)


    Prodigiosin is a bacterial tripyrrole pigment with potent cytotoxicity against diverse human cancer cell lines. Endoplasmic reticulum (ER) stress is initiated by accumulation of unfolded or misfolded proteins in the ER lumen and may induce cell death when irremediable. In this study, the role of ER stress in prodigiosin-induced cytotoxicity was elucidated for the first time. Comparable to the ER stress inducer thapsigargin, prodigiosin up-regulated signature ER stress markers GRP78 and CHOP in addition to activating the IRE1, PERK and ATF6 branches of the unfolded protein response (UPR) in multiple human breast carcinoma cell lines, confirming prodigiosin as an ER stress inducer. Prodigiosin transcriptionally up-regulated CHOP, as evidenced by its promoting effect on the CHOP promoter activity. Of note, knockdown of CHOP effectively lowered prodigiosin's capacity to evoke PARP cleavage, reduce cell viability and suppress colony formation, highlighting an essential role of CHOP in prodigiosin-induced cytotoxic ER stress response. In addition, prodigiosin down-regulated BCL2 in a CHOP-dependent manner. Importantly, restoration of BCL2 expression blocked prodigiosin-induced PARP cleavage and greatly enhanced the survival of prodigiosin-treated cells, suggesting that CHOP-dependent BCL2 suppression mediates prodigiosin-elicited cell death. Moreover, pharmacological inhibition of JNK by SP600125 or dominant-negative blockade of PERK-mediated eIF2α phosphorylation impaired prodigiosin-induced CHOP up-regulation and PARP cleavage. Collectively, these results identified ER stress-mediated cell death as a mode-of-action of prodigiosin's tumoricidal effect. Mechanistically, prodigiosin engages the IRE1–JNK and PERK–eIF2α branches of the UPR signaling to up-regulate CHOP, which in turn mediates BCL2 suppression to induce cell death. Highlights: ► Prodigiosin is a bacterial tripyrrole pigment with potent anticancer effect. ► Prodigiosin is herein identified

  4. Identification and Charaterization of Genes Encoding Melanoma Antigens Recognized by Tumor-infiltrating Lymphocytes

    Institute of Scientific and Technical Information of China (English)

    Rong-FuWang; StevenA.Rosenberg


    Adoptive transfer of tumor infiltrating lymphocytes (TILs) into the autologous patient with melanoma resulted in the objective regression of tumor, suggesting that these TILs recognize tumor rejection antigens on the tumor cells.

  5. Peptide-Based Vaccinology: Experimental and Computational Approaches to Target Hypervariable Viruses through the Fine Characterization of Protective Epitopes Recognized by Monoclonal Antibodies and the Identification of T-Cell-Activating Peptides

    Directory of Open Access Journals (Sweden)

    Matteo Castelli


    Full Text Available Defining immunogenic domains of viral proteins capable of eliciting a protective immune response is crucial in the development of novel epitope-based prophylactic strategies. This is particularly important for the selective targeting of conserved regions shared among hypervariable viruses. Studying postinfection and postimmunization sera, as well as cloning and characterization of monoclonal antibodies (mAbs, still represents the best approach to identify protective epitopes. In particular, a protective mAb directed against conserved regions can play a key role in immunogen design and in human therapy as well. Experimental approaches aiming to characterize protective mAb epitopes or to identify T-cell-activating peptides are often burdened by technical limitations and can require long time to be correctly addressed. Thus, in the last decade many epitope predictive algorithms have been developed. These algorithms are continually evolving, and their use to address the empirical research is widely increasing. Here, we review several strategies based on experimental techniques alone or addressed by in silico analysis that are frequently used to predict immunogens to be included in novel epitope-based vaccine approaches. We will list the main strategies aiming to design a new vaccine preparation conferring the protection of a neutralizing mAb combined with an effective cell-mediated response.

  6. Ants recognize foes and not friends. (United States)

    Guerrieri, Fernando J; Nehring, Volker; Jørgensen, Charlotte G; Nielsen, John; Galizia, C Giovanni; d'Ettorre, Patrizia


    Discriminating among individuals and rejecting non-group members is essential for the evolution and stability of animal societies. Ants are good models for studying recognition mechanisms, because they are typically very efficient in discriminating 'friends' (nest-mates) from 'foes' (non-nest-mates). Recognition in ants involves multicomponent cues encoded in cuticular hydrocarbon profiles. Here, we tested whether workers of the carpenter ant Camponotus herculeanus use the presence and/or absence of cuticular hydrocarbons to discriminate between nest-mates and non-nest-mates. We supplemented the cuticular profile with synthetic hydrocarbons mixed to liquid food and then assessed behavioural responses using two different bioassays. Our results show that (i) the presence, but not the absence, of an additional hydrocarbon elicited aggression and that (ii) among the three classes of hydrocarbons tested (unbranched, mono-methylated and dimethylated alkanes; for mono-methylated alkanes, we present a new synthetic pathway), only the dimethylated alkane was effective in eliciting aggression. Our results suggest that carpenter ants use a fundamentally different mechanism for nest-mate recognition than previously thought. They do not specifically recognize nest-mates, but rather recognize and reject non-nest-mates bearing odour cues that are novel to their own colony cuticular hydrocarbon profile. This begs for a reappraisal of the mechanisms underlying recognition systems in social insects.

  7. Stereoscopic Offset Makes Objects Easier to Recognize.

    Directory of Open Access Journals (Sweden)

    Baptiste Caziot

    Full Text Available Binocular vision is obviously useful for depth perception, but it might also enhance other components of visual processing, such as image segmentation. We used naturalistic images to determine whether giving an object a stereoscopic offset of 15-120 arcmin of crossed disparity relative to its background would make the object easier to recognize in briefly presented (33-133 ms, temporally masked displays. Disparity had a beneficial effect across a wide range of disparities and display durations. Most of this benefit occurred whether or not the stereoscopic contour agreed with the object's luminance contour. We attribute this benefit to an orienting of spatial attention that selected the object and its local background for enhanced 2D pattern processing. At longer display durations, contour agreement provided an additional benefit, and a separate experiment using random-dot stimuli confirmed that stereoscopic contours plausibly contributed to recognition at the longer display durations in our experiment. We conclude that in real-world situations binocular vision confers an advantage not only for depth perception, but also for recognizing objects from their luminance patterns and bounding contours.

  8. Stereoscopic Offset Makes Objects Easier to Recognize. (United States)

    Caziot, Baptiste; Backus, Benjamin T


    Binocular vision is obviously useful for depth perception, but it might also enhance other components of visual processing, such as image segmentation. We used naturalistic images to determine whether giving an object a stereoscopic offset of 15-120 arcmin of crossed disparity relative to its background would make the object easier to recognize in briefly presented (33-133 ms), temporally masked displays. Disparity had a beneficial effect across a wide range of disparities and display durations. Most of this benefit occurred whether or not the stereoscopic contour agreed with the object's luminance contour. We attribute this benefit to an orienting of spatial attention that selected the object and its local background for enhanced 2D pattern processing. At longer display durations, contour agreement provided an additional benefit, and a separate experiment using random-dot stimuli confirmed that stereoscopic contours plausibly contributed to recognition at the longer display durations in our experiment. We conclude that in real-world situations binocular vision confers an advantage not only for depth perception, but also for recognizing objects from their luminance patterns and bounding contours.

  9. Melatonin inhibits tunicamycin-induced endoplasmic reticulum stress and insulin resistance in skeletal muscle cells. (United States)

    Quan, Xiaojuan; Wang, Juyan; Liang, Chunlian; Zheng, Huadong; Zhang, Lin


    The prevalence of type 2 diabetes mellitus (T2D) is increasing worldwide. Melatonin possesses various beneficial metabolic actions, decreased levels of which may accelerate T2D. Endoplasmic reticulum stress (ERS) has been linked to insulin resistance in multiple tissues, but the role of melatonin on ERS and insulin resistance in skeletal muscle has not yet been investigated. In this study, the results showed that tunicamycin decreased insulin-stimulated Akt phosphorylation, but promoted the phosphorylation of protein kinase R-like ER protein kinase (PERK) time-dependently in C2C12 cells. Consistently, ERS gene markers, including binding immunoglobulin protein (BIP)/glucose regulated protein 78 (GRP78) expression and the splicing of X box binding protein 1 (XBP-1), were activated by tunicamycin time-dependently. Interestingly, melatonin pretreatment reversed the elevated PERK phosphorylation, as well as the activation of Bip expression and XBP-1 splicing, and prevented the inhibitory effect of tunicamycin on Akt phosphorylation. In addition, the insulin-provoked glucose transport was reduced by tunicamycin, and then promoted by melatonin pretreatment. A strong phosphorylation of inositol-requiring enzyme 1 (IRE-1), c-JUN NH2-terminal kinase (JNK), and insulin receptor substrate 1 (IRS-1) serine, and simultaneously, a dramatic decrease of IRS-1 tyrosine phosphorylation were observed in the presence of tunicamycin, leading to a blockade of insulin signaling, which was reversed by melatonin pretreatment. Furthermore, luzindole pretreatment acted inversely with melatonin action on glucose uptake and insulin signaling. Therefore, these results demonstrated that melatonin pretreatment inhibited the activated role of tunicamycin on ERS and insulin resistance through melatonin receptor-mediated IRE-1/JNK/IRS-1 insulin signaling in skeletal muscle cells.

  10. Effects of Apigenin on Cisplatin sensitivity of esophageal squamous cell carcinoma TE-1 cells and its mechanisms%芹菜素对食管鳞状细胞癌TE-1细胞顺铂敏感性的影响及机制

    Institute of Scientific and Technical Information of China (English)

    朱鸿武; 谢子英; 李炳玲; 雷昌贤; 周梅花; 孙大勇; 赵亚刚


    目的 探讨芹菜素对食管鳞状细胞癌TE-1细胞顺铂敏感性的影响及机制. 方法 通过CCK-8实验检测芹菜素对TE-1细胞的抑制作用, 再通过CCK-8实验和平板克隆实验检测TE-1细胞在芹菜素作用下对顺铂敏感性的改变, 最后通过Western blot和Hoechst 33258核染色等实验检测芹菜素导致TE-1细胞对顺铂敏感性发生变化的具体机制. 结果 芹菜素显著地抑制了食管鳞状细胞癌TE-1细胞的生长,且这一作用具有浓度依赖性;CCK-8实验和平板克隆实验结果显示联合芹菜素可增强TE-1细胞对顺铂的敏感性,且这一作用具有浓度依赖性; 芹菜素可以增加TE-1细胞在顺铂作用下的凋亡, 减少内源性GRP78蛋白的表达. 结论 芹菜素可以增加TE-1细胞对顺铂的敏感性,可能成为一种新的食管鳞状细胞癌化疗辅助用药.%Objective To investigate the effects of Apigenin on Cisplatin sensitivity of esophageal squamous cell carci-noma TE-1 cells and its mechanisms. Methods Firstly, inhibition effect of Apigenin on TE-1 cells was tested using CCK-8 assay. Secondly, while combined with Apigenin, Cisplatin sensitivity change of TE-1 cells was detected by CCK-8 assay and colony formation assay. Finally, Western blot and Hoechst 33258 nuclear staining assays were used to investigate the mechanisms of Apigenin-induced Cisplatin sensitivity changes of TE-1 cells. Results Firstly, it demonstrated that Apigenin induced cytotoxic effect on esophageal squamous cell carcinoma TE-1 cells in a dose-de-pendent manner. Secondly, CCK-8 assay and colony formation assay also indicated that the Cisplatin sensitivity of TE-1 cells was increased by Apigenin in a dose-dependent manner. In addition, treatment of TE-1 cells with Apigenin displayed significant Cisplatin-induced apoptosis and reducted of endogenous GRP78 expression. Conclusion Apigenin can increase Cisplatin sensitivity of TE-1 cells and it is a promising adjuvant drug in the

  11. Kaempferol Inhibits Endoplasmic Reticulum Stress-Associated Mucus Hypersecretion in Airway Epithelial Cells And Ovalbumin-Sensitized Mice. (United States)

    Park, Sin-Hye; Gong, Ju-Hyun; Choi, Yean-Jung; Kang, Min-Kyung; Kim, Yun-Ho; Kang, Young-Hee


    Mucus hypersecretion is an important pathological feature of chronic airway diseases, such as asthma and pulmonary diseases. MUC5AC is a major component of the mucus matrix forming family of mucins in the airways. The initiation of endoplasmic reticulum (ER)-mediated stress responses contributes to the pathogenesis of airway diseases. The present study investigated that ER stress was responsible for airway mucus production and this effect was blocked by the flavonoid kaempferol. Oral administration of ≥10 mg/kg kaempferol suppressed mucus secretion and goblet cell hyperplasia observed in the bronchial airway and lung of BALB/c mice sensitized with ovalbumin (OVA). TGF-β and tunicamycin promoted MUC5AC induction after 72 h in human bronchial airway epithelial BEAS-2B cells, which was dampened by 20 μM kaempferol. Kaempferol inhibited tunicamycin-induced ER stress of airway epithelial cells through disturbing the activation of the ER transmembrane sensor ATF6 and IRE1α. Additionally, this compound demoted the induction of ER chaperones such as GRP78 and HSP70 and the splicing of XBP-1 mRNA by tunicamycin. The in vivo study further revealed that kaempferol attenuated the induction of XBP-1 and IRE1α in epithelial tissues of OVA-challenged mice. TGF-β and tunicamycin induced TRAF2 with JNK activation and such induction was deterred by kaempferol. The inhibition of JNK activation encumbered the XBP-1 mRNA splicing and MUC5AC induction by tunicamycin and TGF-β. These results demonstrate that kaempferol alleviated asthmatic mucus hypersecretion through blocking bronchial epithelial ER stress via the inhibition of IRE1α-TRAF2-JNK activation. Therefore, kaempferol may be a potential therapeutic agent targeting mucus hypersecretion-associated pulmonary diseases.

  12. The SGK1 Inhibitor SI113 Induces Autophagy, Apoptosis and Endoplasmic Reticulum Stress in Endometrial Cancer Cells. (United States)

    Conza, Domenico; Mirra, Paola; Calì, Gaetano; Tortora, Teresa; Insabato, Luigi; Fiory, Francesca; Schenone, Silvia; Amato, Rosario; Beguinot, Francesco; Perrotti, Nicola; Ulianich, Luca


    Endometrial cancer is often characterized by PI3K/AKT pathway deregulation. Recently it has been suggested that SGK1, a serine/threonine protein kinase that shares structural and functional similarities with the AKT family, might play a role in cancer, since its expression and/or activity has been found to be deregulated in different human tumors. However, the role of SGK1 in endometrial cancer has been poorly investigated. Here we show that SGK1 expression is increased in tissue specimens from neoplastic endometrium. The SGK1 inhibitor SI113 induced a significant reduction of endometrial cancer cells viability, measured by the (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay. This effect was associated to the increase of autophagy, as revealed by the increase of the markers LC3B-II and beclin I, detected by both immunofluorescence and western blot analysis. SI113 treatment caused also apoptosis of endometrial cancer cells, evidenced by the cleavage of the apoptotic markers PARP and Caspase-9. Intriguingly, these effects were associated to the induction of endoplasmic reticulum stress markers GRP78 and CHOP evaluated by both Real-Time RT-PCR and Western Blot analysis. Increased expression of SGK1 in endometrial cancer tissues suggest a role for SGK1 in this type of cancer, as reported for other malignancies. Moreover, the efficacy of SI113 in affecting endometrial cancer cells viability, possibly via endoplasmic reticulum stress activation, identifies SGK1 as an attractive molecular target for new tailored therapeutic intervention for the treatment of endometrial cancer. This article is protected by copyright. All rights reserved.

  13. Low intracellular ATP levels exacerbate carcinogen-induced inflammatory stress response and inhibit in vitro tubulogenesis in human brain endothelial cells

    Directory of Open Access Journals (Sweden)

    Elizabeth Tahanian


    Full Text Available Elizabeth Tahanian, Sabrina Peiro, Borhane AnnabiLaboratoire d'Oncologie Moléculaire, Centre de Recherche BioMED, Département de Chimie, Université du Québec à Montréal, Montréal, Québec, CanadaAbstract: Solid tumor development requires angiogenesis and is correlated to the expression of inflammatory markers through cellular metabolic and energetic adaptation. While high glycolysis rates enable the cancer cell compartment to generate adenosine triphosphate (ATP, very little is known about the impact of low intracellular ATP concentrations within the vascular endothelial cell compartment, which is responsible for tumor angiogenesis. Here, we investigated the effect of 2-deoxy-D-glucose (2-DG, a glucose analog that inhibits glycolysis through intracellular ATP depletion, on human brain microvascular endothelial cell (HBMEC angiogenic properties. While preformed capillaries remained unaffected, we found that in vitro tubulogenesis was dose-dependently decreased by 2-DG and that this correlated with reduced intracellular ATP levels. Procarcinogenic signaling was induced with phorbol 12-myristate 13-acetate (PMA and found to trigger the proinflammatory marker cyclooxygenase-2 (COX-2 and endoplasmic reticulum (ER stress marker GRP78 expression, whose inductions were potentiated when PMA was combined with 2-DG treatment. Inversely, PMA-induced matrix-metalloproteinase-9 (MMP-9 gene expression and protein secretion were abrogated in the presence of 2-DG, and this can be partially explained by reduced nuclear factor-κB signaling. Collectively, we provide evidence for an intracellular ATP requirement in order for tubulogenesis to occur, and we link increases in ER stress to inflammation. A better understanding of the metabolic adaptations of the vascular endothelial cells that mediate tumor vascularization will help the development of new drugs and therapies.Keywords: endoplasmic reticulum stress, MMP-9, COX-2, 2-deoxy-D-glucose, endothelial

  14. Recognizing Charles Bonnet syndrome in the elderly. (United States)

    Walsh, Christina M; Hilas, Olga


    Charles Bonnet syndrome (CBS) is an under-recognized and commonly misdiagnosed condition characterized by the presence of visual hallucinations that psychologically normal people acknowledge as being unreal. It is commonly associated with ocular pathology and usually observed in elderly individuals with visual impairment. The exact etiology of CBS is unknown; however, the presentation of hallucinations is believed to be a result of functional deterioration of the central and peripheral nervous systems. Eradication of hallucinations and recurrent episodes has been seen with the use of neuroleptic and anticonvulsant agents. Correction of underlying ocular disorders and low-vision rehabilitation may also help in the resolution of visions. Careful patient assessment is necessary to appropriately diagnose CBS and determine the best approach to management.

  15. Improved AAG based recognization of machining feature

    Institute of Scientific and Technical Information of China (English)


    The lost information caused by feature interaction is restored by using auxiliary faces(AF)and virtual links(VL).The delta volume of the interacted features represented by concave attachable connected graph (CACG)can be decomposed into several isolated features represented by complete concave adjacency graph (CCAG).We can recognize the features sketchy type by using CCAG as a hint; the exact type of the feature can be attained by deleting the auxiliary faces from the isolated feature.United machining feature(UMF)is used to represent the features that can be machined in the same machining process.It is important to the rationalizing of the process plans and reduce the time costing in machining.An example is given to demonstrate the effectiveness of this method.

  16. Recognizing asthma mimics and asthma complications. (United States)

    Amundson, Dennis; Seda, Gilbert; Daheshia, Massoud


    Asthma is a chronic inflammatory disorder of the airways characterized by airflow obstruction, bronchial hyperreactivity, and underlying inflammation. Two common reasons asthmatics fail standard therapy are incorrect diagnosis and failure to recognize underlying contributing factors. A correct diagnosis of asthma is of great importance to military practitioners since misdiagnosis or uncontrolled asthma affects an individual's operational readiness or determines whether one can receive a medical waiver to enlist in military service. This article presents four cases of patients with dyspnea that have conditions which mimic asthma or complicate asthma management: vocal cord dysfunction misdiagnosed as asthma, respiratory bronchiolitis interstitial lung disease mistaken as asthma, difficult-to-control asthma because of bronchiectasis and allergic bronchopulmonary aspergillosis, and difficult and fatal asthma. Asthma is contrasted to other respiratory disorders, and an outlined approach to asthma diagnosis and management is presented using the Global Initiative for Asthma guidelines.

  17. Recognizing depression in palliative care patients. (United States)

    Noorani, Nazneen Hyder; Montagnini, Marcos


    Clinically significant depression is a common psychiatric disorder in patients with advanced and terminal diseases. Depression is often unrecognized and untreated and it causes major suffering to patients and families. Having adequate knowledge and skills to properly recognize depression in patients with advanced illnesses is essential for providing comprehensive end-of-life care. The objective of this paper is to review the key elements of the assessment of depression in palliative care patients. We also discuss the challenges of making the diagnosis, review the risk factors associated with depression and describe the features of the most common assessment tools that have been studied in this population. Finally, we highlight how to differentiate depression from normal grief, as the overlap between these conditions imposes a diagnostic challenge.

  18. 46 CFR 160.077-9 - Recognized laboratory. (United States)


    ... 46 Shipping 6 2010-10-01 2010-10-01 false Recognized laboratory. 160.077-9 Section 160.077-9... Recognized laboratory. (a) A manufacturer seeking Coast Guard approval of a product under this subpart shall... to a recognized independent laboratory. The following laboratories are recognized under §...

  19. 46 CFR 160.049-8 - Recognized laboratory. (United States)


    ... 46 Shipping 6 2010-10-01 2010-10-01 false Recognized laboratory. 160.049-8 Section 160.049-8... Recognized laboratory. (a) A manufacturer seeking Coast Guard approval of a product under this subpart shall... to a recognized independent laboratory. The following laboratories are recognized under §...

  20. 46 CFR 162.039-5 - Recognized laboratory. (United States)


    ... 46 Shipping 6 2010-10-01 2010-10-01 false Recognized laboratory. 162.039-5 Section 162.039-5... Recognized laboratory. (a) A recognized laboratory is one which is regularly engaged in the examination... motorboats. The following laboratories are recognized, and the semiportable fire extinguishers bearing...

  1. 46 CFR 160.048-8 - Recognized laboratory. (United States)


    ... 46 Shipping 6 2010-10-01 2010-10-01 false Recognized laboratory. 160.048-8 Section 160.048-8... Recognized laboratory. (a) A manufacturer seeking Coast Guard approval of a product under this subpart shall... to a recognized independent laboratory. The following laboratories are recognized under §...

  2. Activation of the Akt-NF-kappaB pathway by subtilase cytotoxin through the ATF6 branch of the unfolded protein response. (United States)

    Yamazaki, Hiroaki; Hiramatsu, Nobuhiko; Hayakawa, Kunihiro; Tagawa, Yasuhiro; Okamura, Maro; Ogata, Ryouji; Huang, Tao; Nakajima, Shotaro; Yao, Jian; Paton, Adrienne W; Paton, James C; Kitamura, Masanori


    Shiga toxin has the potential to induce expression of inflammation-associated genes, although the underlying mechanisms are not well understood. We examined the effects of subtilase cytotoxin (SubAB), an AB(5) toxin produced by some Shiga toxigenic Escherichia coli, on the activation of NF-kappaB. SubAB is known to be a protease which selectively degrades GRP78/Bip. Treatment of NRK-52E cells with SubAB caused rapid cleavage of GRP78. Following the degradation of GRP78, transient activation of NF-kappaB was observed with a peak at 6-12 h; the activation subsided within 24 h despite the continuous absence of intact GRP78. The activation of NF-kappaB was preceded by transient phosphorylation of Akt. Treatment of the cells with a selective inhibitor of Akt1/2 or an inhibitor of PI3K attenuated SubAB-induced NF-kappaB activation, suggesting that activation of Akt is an event upstream of NF-kappaB. Degradation of GRP78 caused the unfolded protein response (UPR), and inducers of the UPR mimicked the stimulatory effects of SubAB on Akt and NF-kappaB. SubAB triggered the three major branches of the UPR including the IRE1-XBP1, PERK, and ATF6 pathways. Dominant-negative inhibition of IRE1alpha, XBP1, or PERK did not attenuate activation of NF-kappaB by SubAB. In contrast, genetic and pharmacological inhibition of ATF6 significantly suppressed SubAB-triggered Akt phosphorylation and NF-kappaB activation. These results suggested that loss of GRP78 by SubAB leads to transient phosphorylation of Akt and consequent activation of NF-kappaB through the ATF6 branch of the UPR.

  3. Arsenite-induced autophagy is associated with proteotoxicity in human lymphoblastoid cells

    Energy Technology Data Exchange (ETDEWEB)

    Bolt, Alicia M.; Zhao, Fei; Pacheco, Samantha; Klimecki, Walter T., E-mail:


    Epidemiological studies of arsenic-exposed populations have provided evidence that arsenic exposure in humans is associated with immunosuppression. Previously, we have reported that arsenite-induced toxicity is associated with the induction of autophagy in human lymphoblastoid cell lines (LCL). Autophagy is a cellular process that functions in the degradation of damaged cellular components, including protein aggregates formed by misfolded or damaged proteins. Accumulation of misfolded or damaged proteins in the endoplasmic reticulum (ER) lumen causes ER stress and activates the unfolded protein response (UPR). In an effort to investigate the mechanism of autophagy induction by arsenite in the LCL model, we examined the potential contribution of ER stress and activation of the UPR. LCL exposed to sodium arsenite for 8-days induced expression of UPR-activated genes, including CHOP and GRP78, at the RNA and the protein level. Evidence for activation of the three arms of the UPR was observed. The arsenite-induced activation of the UPR was associated with an accumulation of protein aggregates containing p62 and LC3, proteins with established roles in the sequestration and autophagic clearance of protein aggregates. Taken together, these data provide evidence that arsenite-induced autophagy is associated with the generation of ER stress, activation of the UPR, and formation of protein aggregates that may be targeted to the lysosome for degradation. -- Highlights: ► Arsenite induces endoplasmic reticulum stress and the unfolded protein response. ► Arsenite induces the formation of protein aggregates that contain p62 and LC3-II. ► Time-course data suggests that arsenite-induced autophagy precedes ER stress.

  4. Targeting bortezomib-induced aggresome formation using vinorelbine enhances the cytotoxic effect along with ER stress loading in breast cancer cell lines (United States)

    Miyahara, Kana; Kazama, Hiromi; Kokuba, Hiroko; Komatsu, Seiichiro; Hirota, Ayako; Takemura, Jun; Hirasawa, Kazuhiro; Moriya, Shota; Abe, Akihisa; Hiramoto, Masaki; Ishikawa, Takashi; Miyazawa, Keisuke


    The ubiquitin-proteasome and autophagy-lysosome pathways are two major self-digestive systems for cellular proteins. Ubiquitinated misfolded proteins are degraded mostly by proteasome. However, when ubiquitinated proteins accumulate beyond the capacity of proteasome clearance, they are transported to the microtubule-organizing center (MTOC) along the microtubules to form aggresomes, and subsequently some of them are degraded by the autophagy-lysosome system. We previously reported that macrolide antibiotics such as azithromycin and clarithromycin block autophagy flux, and that concomitant treatment with the proteasome inhibitor bortezomib (BZ) and macrolide enhances endoplasmic reticulum (ER) stress-mediated apoptosis in breast cancer cells. As ubiquitinated proteins are concentrated at the aggresome upon proteasome failure, we focused on the microtubule as the scaffold of this transport pathway for aggresome formation. Treatment of metastatic breast cancer cell lines (e.g., MDA-MB-231 cells) with BZ resulted in induction of aggresomes, which immunocytochemistry detected as a distinctive eyeball-shaped vimentin-positive inclusion body that formed in a perinuclear lesion, and that electron microscopy detected as a sphere of fibrous structure with some dense amorphous deposit. Vinorelbine (VNR), which inhibits microtubule polymerization, more effectively suppressed BZ-induced aggresome formation than paclitaxel (PTX), which stabilizes microtubules. Combined treatment using BZ and VNR, but not PTX, enhanced the cytotoxic effect and apoptosis induction along with pronounced ER stress loading such as upregulation of GRP78 and CHOP/GADD153. The addition of azithromycin to block autophagy flux in the BZ plus VNR-containing cell culture further enhanced the cytotoxicity. These data suggest that suppression of BZ-induced aggresome formation using an inhibitory drug such as VNR for microtubule polymerization is a novel strategy for meta-static breast cancer therapy. PMID

  5. Brucella suis Vaccine Strain 2 Induces Endoplasmic Reticulum Stress that Affects Intracellular Replication in Goat Trophoblast Cells In vitro. (United States)

    Wang, Xiangguo; Lin, Pengfei; Li, Yang; Xiang, Caixia; Yin, Yanlong; Chen, Zhi; Du, Yue; Zhou, Dong; Jin, Yaping; Wang, Aihua


    Brucella has been reported to impair placental trophoblasts, a cellular target where Brucella efficiently replicates in association with the endoplasmic reticulum (ER), and ultimately trigger abortion in pregnant animals. However, the precise effects of Brucella on trophoblast cells remain unclear. Here, we describe the infection and replication of Brucella suis vaccine strain 2 (B.suis.S2) in goat trophoblast cells (GTCs) and the cellular and molecular responses induced in vitro. Our studies demonstrated that B.suis.S2 was able to infect and proliferate to high titers, hamper the proliferation of GTCs and induce apoptosis due to ER stress. Tunicamycin (Tm), a pharmacological chaperone that strongly mounts ER stress-induced apoptosis, inhibited B.suis.S2 replication in GTCs. In addition, 4 phenyl butyric acid (4-PBA), a pharmacological chaperone that alleviates ER stress-induced apoptosis, significantly enhanced B.suis.S2 replication in GTCs. The Unfolded Protein Response (UPR) chaperone molecule GRP78 also promoted B.suis.S2 proliferation in GTCs by inhibiting ER stress-induced apoptosis. We also discovered that the IRE1 pathway, but not the PERK or ATF6 pathway, was activated in the process. However, decreasing the expression of phosphoIRE1α and IRE1α proteins with Irestatin 9389 (IRE1 antagonist) in GTCs did not affect the proliferation of B.suis.S2. Although GTC implantation was not affected upon B.suis.S2 infection, progesterone secretion was suppressed, and prolactin and estrogen secretion increased; these effects were accompanied by changes in the expression of genes encoding key steroidogenic enzymes. This study systematically explored the mechanisms of abortion in Brucella infection from the viewpoint of pathogen invasion, ER stress and reproductive endocrinology. Our findings may provide new insight for understanding the mechanisms involved in goat abortions caused by Brucella infection.

  6. Brucella suis vaccine strain 2 induces endoplasmic reticulum stress that affects intracellular replication in goat trophoblast cells in vitro

    Directory of Open Access Journals (Sweden)

    Xiangguo eWang


    Full Text Available Brucella has been reported to impair placental trophoblasts, a cellular target where Brucella efficiently replicates in association with the endoplasmic reticulum (ER, and ultimately trigger abortion in pregnant animals. However, the precise effects of Brucella on trophoblast cells remain unclear. Here, we describe the infection and replication of Brucella suis vaccine strain 2 (B.suis.S2 in goat trophoblast cells (GTCs and the cellular and molecular responses induced in vitro. Our studies demonstrated that B.suis.S2 was able to infect and proliferate to high titers, hamper the proliferation of GTCs and induce apoptosis due to ER stress. Tunicamycin (Tm, a pharmacological chaperone that strongly mounts ER stress-induced apoptosis, inhibited B.suis.S2 replication in GTCs. In addition, 4 phenyl butyric acid (4-PBA, a pharmacological chaperone that alleviates ER stress-induced apoptosis, significantly enhanced B.suis.S2 replication in GTCs. The Unfolded Protein Response (UPR chaperone molecule GRP78 also promoted B.suis.S2 proliferation in GTCs by inhibiting ER stress-induced apoptosis. We also discovered that the IRE1 pathway, but not the PERK or ATF6 pathway, was activated in the process. However, decreasing the expression of phosphoIRE1α and IRE1α proteins with Irestatin 9389 (IRE1 antagonist in GTCs did not affect the proliferation of B.suis.S2. Although GTC implantation was not affected upon B.suis.S2 infection, progesterone secretion was suppressed, and prolactin and estrogen secretion increased; these effects were accompanied by changes in the expression of genes encoding key steroidogenic enzymes. This study systematically explored the mechanisms of abortion in Brucella infection from the viewpoint of pathogen invasion, ER stress and reproductive endocrinology. Our findings may provide new insight for understanding the mechanisms involved in goat abortions caused by Brucella infection.

  7. Inhibition of neutral sphingomyelinase decreases elevated levels of inducible nitric oxide synthase and apoptotic cell death in ocular hypertensive rats

    Energy Technology Data Exchange (ETDEWEB)

    Aslan, Mutay, E-mail: [Department of Medical Biochemistry, Akdeniz University Faculty of Medicine, Antalya (Turkey); Basaranlar, Goksun [Department of Biophysics, Akdeniz University Faculty of Medicine, Antalya (Turkey); Unal, Mustafa [Department of Ophthalmology, Akdeniz University Faculty of Medicine, Antalya (Turkey); Ciftcioglu, Akif [Department of Pathology, Akdeniz University Faculty of Medicine, Antalya (Turkey); Derin, Narin [Department of Biophysics, Akdeniz University Faculty of Medicine, Antalya (Turkey); Mutus, Bulent [Department of Chemistry and Biochemistry, University of Windsor, Windsor, Ontario (Canada)


    Endoplasmic reticulum (ER) stress and excessive nitric oxide production via induction of inducible nitric oxide synthase (NOS2) have been implicated in the pathogenesis of neuronal retinal cell death in ocular hypertension. Neutral sphingomyelinase (N-SMase)/ceramide pathway can regulate NOS2 expression, hence this study determined the role of selective neutral sphingomyelinase (N-SMase) inhibition on retinal NOS2 levels, ER stress, apoptosis and visual evoked potentials (VEPs) in a rat model of elevated intraocular pressure (EIOP). NOS2 expression and retinal protein nitration were significantly greater in EIOP and significantly decreased with N-SMase inhibition. A significant increase was observed in retinal ER stress markers pPERK, CHOP and GRP78 in EIOP, which were not significantly altered by N-SMase inhibition. Retinal TUNEL staining showed increased apoptosis in all EIOP groups; however N-SMase inhibition significantly decreased the percent of apoptotic cells in EIOP. Caspase-3, -8 and -9 activities were significantly increased in EIOP and returned to baseline levels following N-SMase inhibition. Latencies of all VEP components were significantly prolonged in EIOP and shortened following N-SMase inhibition. Data confirm the role of nitrative injury in EIOP and highlight the protective effect of N-SMase inhibition in EIOP via down-regulation of NOS2 levels and nitrative stress. - Highlights: • Inhibition of N-SMase decreases NOS2 levels in ocular hypertension. • Inhibition of N-SMase decreases protein nitration in ocular hypertension. • Inhibition of N-SMase decreases caspase activation in ocular hypertension. • Inhibition of N-SMase decreases apoptosis in ocular hypertension.

  8. Recognizing disguised faces: human and machine evaluation.

    Directory of Open Access Journals (Sweden)

    Tejas Indulal Dhamecha

    Full Text Available Face verification, though an easy task for humans, is a long-standing open research area. This is largely due to the challenging covariates, such as disguise and aging, which make it very hard to accurately verify the identity of a person. This paper investigates human and machine performance for recognizing/verifying disguised faces. Performance is also evaluated under familiarity and match/mismatch with the ethnicity of observers. The findings of this study are used to develop an automated algorithm to verify the faces presented under disguise variations. We use automatically localized feature descriptors which can identify disguised face patches and account for this information to achieve improved matching accuracy. The performance of the proposed algorithm is evaluated on the IIIT-Delhi Disguise database that contains images pertaining to 75 subjects with different kinds of disguise variations. The experiments suggest that the proposed algorithm can outperform a popular commercial system and evaluates them against humans in matching disguised face images.

  9. Development of NATO's recognized environmental picture (United States)

    Teufert, John F.; Trabelsi, Mourad


    An important element for the fielding of a viable, effective NATO Response Force (NRF) is access to meteorological, oceanographic, geospatial data (GEOMETOC) and imagery. Currently, the available GEOMETOC information suffers from being very fragmented. NATO defines the Recognised Environmental Picture as controlled information base for GEOMETOC data. The NATO REP proposes an architecture that is both flexible and open. The focus lies on enabling a network-centric approach. The key into achieving this is relying on using open, well recognized standards that apply to both the data exchange protocols and the data formats. Communication and information exchange based on open standards enables system interoperability. Diverse systems, each with unique, specialized contributions to an increased understanding of the battlespace, can now cooperate to a manageable information sphere. By clearly defining responsibilities in the generation of information, a reduction in data transfer overhead is achieved . REP identifies three main stages in the dissemination of GEOMETOC data. These are Collection, Fusion (and Analysis) and Publication. A REP architecture has been successfully deployed during the NATO Coalition Warrior Interoperability Demonstration (CWID) in Lillehammer, Norway during June 2005. CWID is an annual event to validate and improve the interoperability of NATO and national Consultation and command, control, communications, computers, intelligence, surveillance, and reconnaissance (C4ISR) systems. With a test case success rate of 84%, it was able to provide relevant GEOMETOC support to the main NRF component headquarters. In 2006, the REP architecture will be deployed and validated during the NATO NRF Steadfast live exercises.

  10. Perspective: Recognizing and rewarding clinical scholarship. (United States)

    Grigsby, R Kevin; Thorndyke, Luanne


    Faculty members in medical schools and academic medical centers are in a constant process of generating new knowledge. The cornerstone of academia--and academic medicine--is scholarship. Traditionally, tenure and/or academic promotion in the professorial ranks is awarded to those who meet institutional criteria in the missions of research, teaching, and service, including patient care. In the academic review process, priority is often placed on a record of demonstrated, consistent success in traditional laboratory research, also known as the scholarship of discovery. More recently, there has been greater recognition of other forms of scholarship: education, application, and integration. These forms of scholarship, although less recognized, also result in the generation of new knowledge. In an attempt to understand the breadth and scope of clinical scholarship, the authors searched the extant literature in academic medicine for a definition of clinical scholarship and expanded the search to disciplines outside of medicine. They found that succinct, discrete definitions of clinical scholarship have been published in other disciplines, but not in academic medicine. After reviewing definitions of clinical scholarship from other disciplines, adapting definitions of educational scholarship in academic medicine, and including qualities unique to clinical scholarship, the authors developed a framework for understanding clinical scholarship in academic medicine as a means for opening a dialogue within the academic medical community. This dialogue hopefully will lead to formulating a succinct, discrete definition of clinical scholarship that will allow greater recognition and reward for clinical scholars in the promotion and tenure process.

  11. Dogs recognize dog and human emotions. (United States)

    Albuquerque, Natalia; Guo, Kun; Wilkinson, Anna; Savalli, Carine; Otta, Emma; Mills, Daniel


    The perception of emotional expressions allows animals to evaluate the social intentions and motivations of each other. This usually takes place within species; however, in the case of domestic dogs, it might be advantageous to recognize the emotions of humans as well as other dogs. In this sense, the combination of visual and auditory cues to categorize others' emotions facilitates the information processing and indicates high-level cognitive representations. Using a cross-modal preferential looking paradigm, we presented dogs with either human or dog faces with different emotional valences (happy/playful versus angry/aggressive) paired with a single vocalization from the same individual with either a positive or negative valence or Brownian noise. Dogs looked significantly longer at the face whose expression was congruent to the valence of vocalization, for both conspecifics and heterospecifics, an ability previously known only in humans. These results demonstrate that dogs can extract and integrate bimodal sensory emotional information, and discriminate between positive and negative emotions from both humans and dogs.

  12. Sternocostoclavicular Hyperostosis: An Ill-Recognized Disease (United States)

    Roed, Bolette; Kristensen, Tatiana; Thorsen, Søren; Poulsen Bloch, Klaus; Afzelius, Pia


    Sternocostoclavicular hyperostosis (SCCH) is an ill-recognized, rarely diagnosed disease. Today, SCCH is widely considered part of the synovitis, acne, pustulosis, hyperostosis and osteitis (SAPHO) syndrome. SCCH develops over years with intermittent attacks of pain, swelling, and reddening of the sternocostoclavicular region. The disease causes progressive hyperostosis, fusion of the sternocostoclavicular joints, and soft tissue ossification. SCCH is chronic, non-malignant, and occurs predominantly bilaterally in middle-aged women. The incidence of the disease is unknown. We present a case of isolated SCCH, where chest radiographs showed a clear development of bilateral disease over the course of more than a decade. Whole-body bone scintigraphy was performed and was suggestive of SCCH. The diagnosis was established as late as 14 years from the onset of symptoms. During this period, the patient underwent several inconclusive examinations, resulting in a delay of diagnosis and in prolonged and aggravated symptoms. With this case report, we want to draw attention to SCCH and the importance of early diagnosis of the disease. PMID:27527220

  13. Salvianolic acid A attenuates TNF-α- and D-GalN-induced ER stress-mediated and mitochondrial-dependent apoptosis by modulating Bax/Bcl-2 ratio and calcium release in hepatocyte LO2 cells. (United States)

    Yan, Xiaojing; Jiang, Zequn; Bi, Lei; Yang, Ye; Chen, Weiping


    Salvianolic acid (Sal A) is a water-soluble compound extracted from Radix Salvia miltiorrhiza (danshen), which has been widely used to treat acute hepatitis and hepatic damage in traditional Chinese medicine. The aim of the present study was to delineate the antiapoptotic signaling pathways involved in Sal A's hepato-protective action in hepatocyte LO2 cells and to further elucidate the mechanism by which Sal A elicits the antiapoptotic effects on hepatocytes. Here, the study showed that Sal A had antiapoptotic effects on the TNF-α/D-GalN-treated LO2 cells. Moreover, Western blotting demonstrated that the levels of p-eIF2α, ATF4, GRP78, CHOP and caspase-4 were markedly decreased in Sal A group. Additionally, the decrease of the cell mitochondrial membrane permeability and increase of ΔΨm were detected in Sal A-treated cells by high-content screening (HCS) analysis. And the levels of cleaved-caspase-9, cleaved-caspase-3, apoptosis-inducing factor (AIF), Apaf-1, and Cytc (cyto) were downregulated, while Cytc (mito) was upregulated by Sal A via Western blotting. Furthermore, the decreased levels of Bax/Bcl-2 ratio and calcium release were measured in Sal A-treated cells. In summary, Sal A attenuates TNF-α- and D-GalN-induced both ER stress and mitochondrial-dependent apoptosis by suppression of Bax/Bcl-2 ratio and prevention of calcium release, which support the notion that Sal A could be developed into a novel hepatic protectant.

  14. O-Glycosylation of NnTreg Lymphocytes Recognized by the Amaranthus leucocarpus Lectin

    Directory of Open Access Journals (Sweden)

    María C. Jiménez-Martínez


    Full Text Available O-glycosidically-linked glycans have been involved in development, maturation, homing, and immune regulation in T cells. Previous reports indicate that Amaranthus leucocarpus lectin (ALL, specific for glycans containing galactose-N-acetylgalactosamine and N-acetylgalactosamine, recognizes human naïve CD27+CD25+CD4+ T cells. Our aim was to evaluate the phenotype of CD4+ T cells recognized by ALL in peripheral blood mononuclear cells obtained from healthy volunteers. CD4+ T cells were isolated by negative selection using magnetic beads-labeled monoclonal antibodies; the expression of T regulatory cell phenotypic markers was assessed on ALL-recognized cells. In addition, IL-4, IL-10, IFN-γ, and TGF-β intracellular production in ALL+ cells was also evaluated. The analyses of phenotypic markers and intracellular cytokines were performed through flow cytometry. ALL-recognized CD4+ T cells were mainly CD45RA+, CCR7+ cells. Although 52±10% CD25+Foxp3+ cells were positive to ALL, only 34±4% of ALL+ cells corresponded to CD25+Foxp3− cells. Intracellular cytokines in freshly obtained ALL+CD4+ T cells exhibited 8% of IL-4, 15% of IL-10, 2% of IFN-γ, and 15% of TGF-β, whereas ALL−CD4+ T cells depicted 1% of IL-4, 2% of IL-10, <1% of IFN-γ, and 6% of TGF-β. Our results show that galactose-N-acetylgalactosamine and N-galactosamine-bearing CD4+ T cells expressed phenotypic markers of NnTreg cells.

  15. Can a CNN recognize Catalan diet? (United States)

    Herruzo, P.; Bolaños, M.; Radeva, P.


    Nowadays, we can find several diseases related to the unhealthy diet habits of the population, such as diabetes, obesity, anemia, bulimia and anorexia. In many cases, these diseases are related to the food consumption of people. Mediterranean diet is scientifically known as a healthy diet that helps to prevent many metabolic diseases. In particular, our work focuses on the recognition of Mediterranean food and dishes. The development of this methodology would allow to analise the daily habits of users with wearable cameras, within the topic of lifelogging. By using automatic mechanisms we could build an objective tool for the analysis of the patient's behavior, allowing specialists to discover unhealthy food patterns and understand the user's lifestyle. With the aim to automatically recognize a complete diet, we introduce a challenging multi-labeled dataset related to Mediter-ranean diet called FoodCAT. The first type of label provided consists of 115 food classes with an average of 400 images per dish, and the second one consists of 12 food categories with an average of 3800 pictures per class. This dataset will serve as a basis for the development of automatic diet recognition. In this context, deep learning and more specifically, Convolutional Neural Networks (CNNs), currently are state-of-the-art methods for automatic food recognition. In our work, we compare several architectures for image classification, with the purpose of diet recognition. Applying the best model for recognising food categories, we achieve a top-1 accuracy of 72.29%, and top-5 of 97.07%. In a complete diet recognition of dishes from Mediterranean diet, enlarged with the Food-101 dataset for international dishes recognition, we achieve a top-1 accuracy of 68.07%, and top-5 of 89.53%, for a total of 115+101 food classes.

  16. The SARS coronavirus spike glycoprotein is selectively recognized by lung surfactant protein D and activates macrophages

    DEFF Research Database (Denmark)

    Leth-Larsen, Rikke; Zhong, Fei; Chow, Vincent T K;


    Da glycosylated protein. It was not secreted in the presence of tunicamycin and was detected as a 130 kDa protein in the cell lysate. The purified S-protein bound to Vero but not 293T cells and was itself recognized by lung surfactant protein D (SP-D), a collectin found in the lung alveoli. The binding required...

  17. Expression of hereditary hemochromatosis C282Y HFE protein in HEK293 cells activates specific endoplasmic reticulum stress responses

    Directory of Open Access Journals (Sweden)

    Norris Suzanne


    Full Text Available Abstract Background Hereditary Hemochromatosis (HH is a genetic disease associated with iron overload, in which individuals homozygous for the mutant C282Y HFE associated allele are at risk for the development of a range of disorders particularly liver disease. Conformational diseases are a class of disorders associated with the expression of misfolded protein. HFE C282Y is a mutant protein that does not fold correctly and consequently is retained in the Endoplasmic Reticulum (ER. In this context, we sought to identify ER stress signals associated with mutant C282Y HFE protein expression, which may have a role in the molecular pathogenesis of HH. Results Vector constructs of Wild type HFE and Mutant C282Y HFE were made and transfected into HEK293 cell lines. We have shown that expression of C282Y HFE protein triggers both an unfolded protein response (UPR, as revealed by the increased GRP78, ATF6 and CHOP expression, and an ER overload response (EOR, as indicated by NF-κB activation. Furthermore, C282Y HFE protein induced apoptotic responses associated with activation of ER stress. Inhibition studies demonstrated that tauroursodeoxycholic acid, an endogenous bile acid, downregulates these events. Finally, we found that the co-existence of both C282Y HFE and Z alpha 1-antitrypsin protein (the protein associated with the liver disease of Z alpha 1-antitrypsin deficiency expression on ER stress responses acted as potential disease modifiers with respect to each other. Conclusion Our novel observations suggest that both the ER overload response (EOR and the unfolded protein response (UPR are activated by mutant C282Y HFE protein.

  18. 46 CFR 90.10-35 - Recognized classification society. (United States)


    ... 46 Shipping 4 2010-10-01 2010-10-01 false Recognized classification society. 90.10-35 Section 90... classification society. The term recognized classification society means the American Bureau of Shipping or other classification society recognized by the Commandant....

  19. 46 CFR 42.05-60 - Recognized classification society. (United States)


    ... 46 Shipping 2 2010-10-01 2010-10-01 false Recognized classification society. 42.05-60 Section 42... society. The term recognized classification society means the American Bureau of Shipping or other classification society recognized by the Commandant, as provided in 46 U.S.C. 5107, and who also may be...

  20. 46 CFR 160.060-9 - Recognized laboratory. (United States)


    ... 46 Shipping 6 2010-10-01 2010-10-01 false Recognized laboratory. 160.060-9 Section 160.060-9..., Adult and Child § 160.060-9 Recognized laboratory. (a) A manufacturer seeking Coast Guard approval of a... shall apply for approval directly to a recognized independent laboratory. The following laboratories...

  1. 46 CFR 160.052-9 - Recognized laboratory. (United States)


    ... 46 Shipping 6 2010-10-01 2010-10-01 false Recognized laboratory. 160.052-9 Section 160.052-9..., Adult and Child § 160.052-9 Recognized laboratory. (a) A manufacturer seeking Coast Guard approval of a... shall apply for approval directly to a recognized independent laboratory. The following laboratories...

  2. 46 CFR 160.047-7 - Recognized laboratory. (United States)


    ... 46 Shipping 6 2010-10-01 2010-10-01 false Recognized laboratory. 160.047-7 Section 160.047-7... and Child § 160.047-7 Recognized laboratory. (a) A manufacturer seeking Coast Guard approval of a... shall apply for approval directly to a recognized independent laboratory. The following laboratories...

  3. 46 CFR 164.012-12 - Recognized laboratory. (United States)


    ... 46 Shipping 6 2010-10-01 2010-10-01 false Recognized laboratory. 164.012-12 Section 164.012-12...: SPECIFICATIONS AND APPROVAL MATERIALS Interior Finishes for Merchant Vessels § 164.012-12 Recognized laboratory. A recognized laboratory is one which is operated as a nonprofit public service and is...

  4. 46 CFR 164.019-17 - Recognized laboratory. (United States)


    ... 46 Shipping 6 2010-10-01 2010-10-01 false Recognized laboratory. 164.019-17 Section 164.019-17...: SPECIFICATIONS AND APPROVAL MATERIALS Personal Flotation Device Components § 164.019-17 Recognized laboratory. (a) General. A laboratory may be designated as a recognized laboratory under this subpart if it is—...

  5. 46 CFR 160.064-7 - Recognized laboratory. (United States)


    ... 46 Shipping 6 2010-10-01 2010-10-01 false Recognized laboratory. 160.064-7 Section 160.064-7...: SPECIFICATIONS AND APPROVAL LIFESAVING EQUIPMENT Marine Buoyant Devices § 160.064-7 Recognized laboratory. (a) A... laboratory. The following laboratories are recognized under § 159.010-7 of this part, to perform testing...

  6. 原癌基因c-jun在毒胡萝卜素内酯诱导小鼠胚胎成纤维细胞凋亡中的作用%The role of c-jun in thapsigargin-induced mouse fibroblast cell apoptosis

    Institute of Scientific and Technical Information of China (English)

    张洁; 周方舟; 曹新冉; 任宏生; 楚玉峰; 王爱红; 董波; 赵鹏


    Objective To examine the impact of c-jun on thapsigargin-induced fibroblast cell apoptosis and address the mecha-nisms.Methods The thapsigargin was used to treat c-jun-/-mouse fibroblast cells,wild-type mouse fibroblasts and reconstitution of c-jun expression in c-jun-/-cells(c-jun Re).Cell viability was evaluated by trypan blue dye exclusion.Apoptosis was detected by fluorescence ac-tivated cell sorting(FACS)and DNA staining.c-jun,caspase-12,Grp78,Gadd153 and α-tubulin were analyzed by immunoblotting.Confocal images of cells loaded with the ER-Tracker and visualized by two-photon microscopy.Results Wild type(c-jun+/+),c-jun-/-and c-jun Re cells exhibited dramatic differences in sensitivity to TG.The c-jun-/-cells were the most sensitive,while c-jun Re cells were relatively resistant,to TG-induced cell death(P<0.05).TG treatment led to the activation of caspase-12,as indicated by the cleavage of procaspase-12, in c-jun-/-cells in a time-dependent manner.In contrast,caspase-12 activation was significantly attenuated in wild type fibroblast cells and abrogated in c-jun Re cells in response to TG treatment(P<0.05).c-jun-/-cells exhibited a large degree of apoptosis after treatment with TG,while c-jun Re cells demonstrated relative resistance to TG-induced apoptosis.c-jun-/-mouse fibroblast cells were more sensitive to TG-induced cell death compared to wild-type mouse fibroblasts,while reconstitution of c-jun expression in c-jun-/-cells(c-Jun Re)enhanced re-sistance to TG-induced cell death(P<0.05).The expression levels of ER chaperones Grp78 and Gadd153 induced by TG were lower in c-jun Re than those in c-jun-/-cells.ER-tracker found different patterns in the ER structure between c-jun-/-and c-jun Re cells.Conclusions Thapsigargin could cause mouse fibroblast cells apoptosis.c-jun gene expression changes of apoptosis degree,this change is mediated by endoplasmic reticulum stress.c-jun gene expression plays a protective role in thapsigargin-induced fibroblast cell

  7. Inhibition of glucose regulated protein 78 in sensitizing adriamycin-induced apoptosis of human breast cancer cells%抑制葡萄糖调节蛋白78表达对阿霉素诱导人乳腺癌SK-BR-3细胞凋亡的增敏作用

    Institute of Scientific and Technical Information of China (English)

    刘宏莉; 龚萍; 刘浩; 张旭东; 蒋志文


    目的:探讨特异性下调葡萄糖调节蛋白78(GRP78)对阿霉素(adriamycin,ADR)诱导人乳腺癌SK-BR-3细胞凋亡的影响,以期为乳腺癌化疗提供新的靶点.方法:培养人乳腺癌SK-BR-3细胞,用ADR(1 mg/L)处理人乳腺癌SK-BR-3细胞0、6、12、24、36 h,Western blot检测GRP78的表达;ADR处理SK-BR-3细胞48 h后,用溴化丙啶染色测定细胞凋亡率;用小分子干扰RNA siRNA预处理SK-BR-3细胞,再予ADR同上处理,检测细胞凋亡率,比较siRNA作用前后细胞凋亡率的变化.结果:ADR可诱导内质网应激,上调GRP78的表达,SK-BR-3细胞对ADR诱导的细胞凋亡率<30%;siRNA可明显阻断GRP78的表达,显著增加ADR诱导的细胞凋亡作用,凋亡率达到(62.30±0.88)%(P<0.01).结论:抑制GRP78的表达可增强人乳腺癌SK-BR-3细胞对ADR经内质网应激途径诱导细胞凋亡的敏感性,促进肿瘤细胞的凋亡,增强其抗肿瘤作用.

  8. Triptolide induced cell death through apoptosis and autophagy in murine leukemia WEHI-3 cells in vitro and promoting immune responses in WEHI-3 generated leukemia mice in vivo. (United States)

    Chan, Shih-Feng; Chen, Ya-Yin; Lin, Jen-Jyh; Liao, Ching-Lung; Ko, Yang-Ching; Tang, Nou-Ying; Kuo, Chao-Lin; Liu, Kuo-Ching; Chung, Jing-Gung


    Triptolide, a traditional Chinese medicine, obtained from Tripterygium wilfordii Hook F, has anti-inflammatory, antiproliferative, and proapoptotic properties. We investigated the potential efficacy of triptolide on murine leukemia by measuring the triptolide-induced cytotoxicity in murine leukemia WEHI-3 cells in vitro. Results indicated that triptolide induced cell morphological changes and induced cytotoxic effects through G0/G1 phase arrest, induction of apoptosis. Flow cytometric assays showed that triptolide increased the production of reactive oxygen species, Ca(2+) release and mitochondrial membrane potential (ΔΨm ), and activations of caspase-8, -9, and -3. Triptolide increased protein levels of Fas, Fas-L, Bax, cytochrome c, caspase-9, Endo G, Apaf-1, PARP, caspase-3 but reduced levels of AIF, ATF6α, ATF6β, and GRP78 in WEHI-3 cells. Triptolide stimulated autophagy based on an increase in acidic vacuoles, monodansylcadaverine staining for LC-3 expression and increased protein levels of ATG 5, ATG 7, and ATG 12. The in vitro data suggest that the cytotoxic effects of triptolide may involve cross-talk between cross-interaction of apoptosis and autophagy. Normal BALB/c mice were i.p. injected with WEHI-3 cells to generate leukemia and were oral treatment with triptolide at 0, 0.02, and 0.2 mg/kg for 3 weeks then animals were weighted and blood, liver, spleen samples were collected. Results indicated that triptolide did not significantly affect the weights of animal body, spleen and liver of leukemia mice, however, triptolide significant increased the cell populations of T cells (CD3), B cells (CD19), monocytes (CD11b), and macrophage (Mac-3). Furthermore, triptolide increased the phagocytosis of macrophage from peripheral blood mononuclear cells (PBMC) but not effects from peritoneum. Triptolide promoted T and B cell proliferation at 0.02 and 0.2 mg/kg treatment when cells were pretreated with Con A and LPS stimulation, respectively; however, triptolide

  9. Proteomic mapping of stimulus-specific signaling pathways involved in THP-1 cells exposed to Porphyromonas gingivalis or its purified components. (United States)

    Saba, Julian A; McComb, Mark E; Potts, Donna L; Costello, Catherine E; Amar, Salomon


    Periodontitis is an inflammatory disease initiated by host-parasite interactions which contributes to connective tissue destruction and alveolar bone resorption. Porphyromonas gingivalis (P.g.), a black-pigmented Gram-negative anaerobic bacterium, is a major pathogen in the development and progression of periodontitis. To characterize the role that P. gingivalis and its cell surface components play in disease processes, we investigated the differential expression of proteins induced by live P.g., P.g. LPS, and P.g. FimA, using two-dimensional gel electrophoresis in combination with mass spectrometry. We have tested whether, at the level of protein expression, unique signaling pathways are differentially induced by the bacterial components P.g. LPS and P.g. FimA, as compared to live P.g. We found that P.g. LPS stimulation of THP-1 up-regulated the expression of a set of proteins compared to control: deoxyribonuclease, actin, carbonic anhydrase 2, alpha enolase, adenylyl cyclase-associated protein (CAP1), protein disulfide isomerase (PDI), glucose regulated protein (grp78), and 70-kDa heat shock protein (HSP70), whereas FimA treatment did not result in statistically significant changes to protein levels versus the control. Live P.g. stimulation resulted in 12 differentially expressed proteins: CAP1, tubulin beta-2 chain, ATP synthase beta chain, tubulin alpha-6 chain, PDI, vimentin, 60-kDa heat shock protein, and nucleolin were found to be up-regulated, while carbonic anhydrase II, beta-actin, and HSP70 were down-regulated relative to control. These differential changes by the bacteria and its components are interpreted as preferential signal pathway activation in host immune/inflammatory responses to P.g. infection.

  10. Human peripheral blood monocytes display surface antigens recognized by monoclonal antinuclear antibodies

    Energy Technology Data Exchange (ETDEWEB)

    Holers, V.M.; Kotzin, B.L.


    The authors used monoclonal anti-nuclear autoantibodies and indirect immunofluorescence to examine normal human peripheral blood mononuclear leukocytes for the presence of cell surface nuclear antigens. Only one monoclonal anti-histone antibody (MH-2) was found to bind to freshly isolated PBL, staining approximately 10% of large cells. However, after cells were placed into culture for 16-24 h, a high percentage (up to 60%) of large-sized cells were recognized by an anti-DNA (BWD-1) and several different antihistone monoclonal antibodies (BWH-1, MH-1, and MH-2). These antibodies recognize separate antigenic determinants on chromatin and histones extracted from chromatin. The histone antigen-positive cells were viable, and the monoclonal antibodies could be shown to be binding to the cell surface and not to the nucleus. Using monoclonal antibodies specific for monocytes and T cells, and complement-mediated cytotoxicity, the cells bearing histone antigens were shown to be primarily monocytes. The appearance of histone and DNA antigen-positive cells was nearly completely inhibited by the addition of low concentrations of cycloheximide at initiation of the cultures. In contrast, little effect on the percentage of positive cells was detected if cells were exposed to high doses of gamma irradiation before culture. These data further support the existence of cell surface nuclear antigens on selected cell subsets, which may provide insight into the immunopathogenesis of systemic lupus erythematosus and related autoimmune diseases.

  11. Endoplasmic reticulum stress is chronically activated in chronic pancreatitis. (United States)

    Sah, Raghuwansh P; Garg, Sushil K; Dixit, Ajay K; Dudeja, Vikas; Dawra, Rajinder K; Saluja, Ashok K


    The pathogenesis of chronic pancreatitis (CP) is poorly understood. Endoplasmic reticulum (ER) stress has now been recognized as a pathogenic event in many chronic diseases. However, ER stress has not been studied in CP, although pancreatic acinar cells seem to be especially vulnerable to ER dysfunction because of their dependence on high ER volume and functionality. Here, we aim to investigate ER stress in CP, study its pathogenesis in relation to trypsinogen activation (widely regarded as the key event of pancreatitis), and explore its mechanism, time course, and downstream consequences during pancreatic injury. CP was induced in mice by repeated episodes of acute pancreatitis (AP) based on caerulein hyperstimulation. ER stress leads to activation of unfolded protein response components that were measured in CP and AP. We show sustained up-regulation of unfolded protein response components ATF4, CHOP, GRP78, and XBP1 in CP. Overexpression of GRP78 and ATF4 in human CP confirmed the experimental findings. We used novel trypsinogen-7 knock-out mice (T(-/-)), which lack intra-acinar trypsinogen activation, to clarify the relationship of ER stress to intra-acinar trypsinogen activation in pancreatic injury. Comparable activation of ER stress was seen in wild type and T(-/-) mice. Induction of ER stress occurred through pathologic calcium signaling very early in the course of pancreatic injury. Our results establish that ER stress is chronically activated in CP and is induced early in pancreatic injury through pathologic calcium signaling independent of trypsinogen activation. ER stress may be an important pathogenic mechanism in pancreatitis that needs to be explored in future studies.

  12. When Do Infants Begin Recognizing Familiar Words in Sentences? (United States)

    DePaolis, Rory A.; Vihman, Marilyn M.; Keren-Portnoy, Tamar


    Previous studies have shown that by 11 but not by 10 months infants recognize words that have become familiar from everyday life independently of the experimental setting. This study explored the ability of 10-, 11-, and 12- month-old infants to recognize familiar words in sentential context, without experimental training. The headturn preference…

  13. Recognizing Strokes in Tennis Videos Using Hidden Markov Models

    NARCIS (Netherlands)

    Petkovic, M.; Jonker, W.; Zivkovic, Z.


    This paper addresses content-based video retrieval with an emphasis on recognizing events in tennis game videos. In particular, we aim at recognizing different classes of tennis strokes using automatic learning capability of Hidden Markov Models. Driven by our domain knowledge, a robust player segme

  14. The repertoire of glycosphingolipids recognized by Vibrio cholerae.

    Directory of Open Access Journals (Sweden)

    John Benktander

    Full Text Available The binding of cholera toxin to the ganglioside GM1 as the initial step in the process leading to diarrhea is nowadays textbook knowledge. In contrast, the knowledge about the mechanisms for attachment of Vibrio cholerae bacterial cells to the intestinal epithelium is limited. In order to clarify this issue, a large number of glycosphingolipid mixtures were screened for binding of El Tor V. cholerae. Several specific interactions with minor complex non-acid glycosphingolipids were thereby detected. After isolation of binding-active glycosphingolipids, characterization by mass spectrometry and proton NMR, and comparative binding studies, three distinct glycosphingolipid binding patterns were defined. Firstly, V. cholerae bound to complex lacto/neolacto glycosphingolipids with the GlcNAcβ3Galβ4GlcNAc sequence as the minimal binding epitope. Secondly, glycosphingolipids with a terminal Galα3Galα3Gal moiety were recognized, and the third specificity was the binding to lactosylceramide and related compounds. V. cholerae binding to lacto/neolacto glycosphingolipids, and to the other classes of binding-active compounds, remained after deletion of the chitin binding protein GbpA. Thus, the binding of V. cholerae to chitin and to lacto/neolacto containing glycosphingolipids represents two separate binding specificities.

  15. Effects of the Combination of the Main Active Components of Astragalus and Panax notoginseng on Inflammation and Apoptosis of Nerve Cell after Cerebral Ischemia-Reperfusion. (United States)

    Huang, Xiao-Ping; Ding, Huang; Lu, Jin-Dong; Tang, Ying-Hong; Deng, Bing-Xiang; Deng, Chang-Qing


    Astragalus and Panax notoginseng are commonly used to treat cardio-cerebrovascular diseases in China and are often combined together to promote curative effect. We speculate that the enhancement of the combination on anticerebral ischemia injury may come from the main active components. The purpose of this work was to probe the effects and mechanisms of Astragaloside IV (the active component of Astragalus) combined with Ginsenoside Rg1, Ginsenoside Rb1, and Notoginsenoside R1 (the active components of P. notoginseng) to antagonize ischemia/reperfusion (I/R) injury via inflammation and apoptosis. C57BL/6 mice were randomly divided into sham, model, Astragaloside IV, Ginsenoside Rg1, Ginsenoside Rb1, Notoginsenoside R1, four active components combination, and Edaravone groups. After administration for 3 days, bilateral common carotid arteries (CCA) were occluded with artery clip for 20[Formula: see text]min followed by reperfusion for 24[Formula: see text]h. Our results showed that the survival rate of nerve cell in hippocampal CA1 decreased while the apoptotic rate increased, and the level of caspase-3 protein in brain tissues was elevated, the expressions of TNF-a, IL-1, and ICAM-1 mRNA as well as phosphorylated nuclear factor kappa B (NF-κB) inhibitor protein α (p-IκBa) in brain tissues were up-regulated, and the nuclear translocation rate of NF-κB was raised. Additionally, the protein expressions of phosphorylated tyrosine kinase 1 (p-JAK1), phosphorylated signal transducer and activator of transcription-1 (p-STAT1), glucose regulated protein 78 (GRP78), caspase-12, and phosphorylated c-Jun N-terminal kinases 1/2 (p-JNK1/2) in brain tissues were also significantly strengthened after I/R for 24 h. All drugs could increase neurocyte survival rate in hippocampal CA1, decrease the apoptotic rate, and inhibit caspase-3 protein expression, in contrast, the effects of four active components combination were better than those of active components alone. In addition

  16. Regulation of AKT phosphorylation at Ser473 and Thr308 by endoplasmic reticulum stress modulates substrate specificity in a severity dependent manner.

    Directory of Open Access Journals (Sweden)

    Hong Wa Yung

    Full Text Available Endoplasmic reticulum (ER stress is a common factor in the pathophysiology of diverse human diseases that are characterised by contrasting cellular behaviours, from proliferation in cancer to apoptosis in neurodegenerative disorders. Coincidently, dysregulation of AKT/PKB activity, which is the central regulator of cell growth, proliferation and survival, is often associated with the same diseases. Here, we demonstrate that ER stress modulates AKT substrate specificity in a severity-dependent manner, as shown by phospho-specific antibodies against known AKT targets. ER stress also reduces both total and phosphorylated AKT in a severity-dependent manner, without affecting activity of the upstream kinase PDK1. Normalisation to total AKT revealed that under ER stress phosphorylation of Thr308 is suppressed while that of Ser473 is increased. ER stress induces GRP78, and siRNA-mediated knock-down of GRP78 enhances phosphorylation at Ser473 by 3.6 fold, but not at Thr308. Substrate specificity is again altered. An in-situ proximity ligation assay revealed a physical interaction between GRP78 and AKT at the plasma membrane of cells following induction of ER stress. Staining was weak in cells with normal nuclear morphology but stronger in those displaying rounded, condensed nuclei. Co-immunoprecipitation of GRP78 and P-AKT(Ser473 confirmed the immuno-complex consists of non-phosphorylated AKT (Ser473 and Thr308. The interaction is likely specific as AKT did not bind to all molecular chaperones, and GRP78 did not bind to p70 S6 kinase. These findings provide one mechanistic explanation for how ER stress contributes to human pathologies demonstrating contrasting cell fates via modulation of AKT signalling.

  17. Involvement of caspase-12-dependent apoptotic pathway in ionic radiocontrast urografin-induced renal tubular cell injury

    Energy Technology Data Exchange (ETDEWEB)

    Wu, Cheng Tien [Institute of Toxicology, College of Medicine, National Taiwan University, Taipei, Taiwan (China); Weng, Te I. [Department of Forensic Medicine, College of Medicine, National Taiwan University, Taipei, Taiwan (China); Chen, Li Ping [Department of Dentistry, Chang Gang Memorial Hospital, Chang Gang University, Taoyuan, Taiwan (China); Chiang, Chih Kang [Department of Integrated Diagnostics and Therapeutics, National Taiwan University Hospital and National Taiwan University College of Medicine, Taipei, Taiwan (China); Department of Internal Medicine, National Taiwan University Hospital and National Taiwan University College of Medicine, Taipei, Taiwan (China); Liu, Shing Hwa, E-mail: [Institute of Toxicology, College of Medicine, National Taiwan University, Taipei, Taiwan (China); Department of Urology, National Taiwan University Hospital, Taipei, Taiwan (China)


    Contrast medium (CM) induces a direct toxic effect on renal tubular cells. This toxic effect subjects in the disorder of CM-induced nephropathy. Our previous work has demonstrated that CM shows to activate the endoplasmic reticulum (ER)-related adaptive unfolding protein response (UPR) activators. Glucose-regulated protein 78 (GRP78)/eukaryotic initiation factor 2α (eIF2α)-related pathways play a protective role during the urografin (an ionic CM)-induced renal tubular injury. However, the involvement of ER stress-related apoptotic signals in the urografin-induced renal tubular cell injury remains unclear. Here, we examined by the in vivo and in vitro experiments to explore whether ER stress-regulated pro-apoptotic activators participate in urografin-induced renal injury. Urografin induced renal tubular dilation, tubular cells detachment, and necrosis in the kidneys of rats. The tubular apoptosis, ER stress-related pro-apoptotic transcriptional factors, and kidney injury marker-1 (kim-1) were also conspicuously up-regulated in urografin-treated rats. Furthermore, treatment of normal rat kidney (NRK)-52E tubular cells with urografin augmented the expressions of activating transcription factor-6 (ATF-6), C/EBP homologous protein (CHOP), Bax, caspase-12, JNK, and inositol-requiring enzyme (IRE) 1 signals. Urografin-induced renal tubular cell apoptosis was not reversed by the inhibitors of ATF-6, JNK signals or CHOP siRNA transfection, but it could be partially reversed by the inhibitor of caspase-12. Taken together, the present results and our previous findings suggest that exposure of CM/urografin activates the ER stress-regulated survival- and apoptosis-related signaling pathways in renal tubular cells. Caspase-12-dependent apoptotic pathway may be partially involved in the urografin-induced nephropathy. -- Highlights: ► Ionic contrast medium-urografin induces renal tubular cell apoptosis. ► Urografin induces the ER stress-regulated survival and apoptosis

  18. Recognizing Advanced Heart Failure and Knowing Your Options (United States)

    ... Disease Venous Thromboembolism Aortic Aneurysm More Recognizing Advanced Heart Failure and Knowing Your Options Updated:Mar 25,2016 ... need in the future. Treatment Options for Advanced Heart Failure Major Interventions Open-heart surgery: For patients with ...

  19. A novel approach in recognizing magnetic material with simplified algorithm

    KAUST Repository

    Talukdar, Abdul Hafiz Ibne


    In this article a cost-effective and simple system (circuit and algorithm) which allows recognizing different kinds of films by their magneto-field conductive properties is demonstrated. The studied signals are generated by a proposed circuit. This signal was further analyzed (recognized) in frequency domain creating the Fourier frequency spectrum which is easily used to detect the response of magnetic sample. The novel algorithm in detecting magnetic field is presented here with both simulation and experimental results. © 2011 IEEE.

  20. The PERK-eIF2α signaling pathway is involved in TCDD-induced ER stress in PC12 cells. (United States)

    Duan, Zhiqing; Zhao, Jianya; Fan, Xikang; Tang, Cuiying; Liang, Lingwei; Nie, Xiaoke; Liu, Jiao; Wu, Qiyun; Xu, Guangfei


    Studies have shown that 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) induces apoptotic cell death in neuronal cells. However, whether this is the result of endoplasmic reticulum (ER) stress-mediated apoptosis remains unknown. In this study, we determined whether ER stress plays a role in the TCDD-induced apoptosis of pheochromocytoma (PC12) cells and primary neurons. PC12 cells were exposed to different TCDD concentrations (1, 10, 100, 200, or 500nM) for varying lengths of time (1, 3, 6, 12, or 24h). TCDD concentrations much higher than 10nM (100, 200, or 500nM) markedly increased glucose-regulated protein (GRP78) and C/EBP homologous protein (CHOP) levels, which are hallmarks of ER stress. We also evaluated the effects of TCDD on ER morphology in PC12 cells and primary neurons that were treated with different TCDD concentrations (1, 10, 50, or 200nM) for 24h. Ultrastructural ER alterations were observed with transmission electron microscopy in PC12 cells and primary neurons treated with high concentrations of TCDD. Furthermore, TCDD-induced ER stress significantly promoted the activation of the PKR-like ER kinase (PERK), a sensor for the unfolded protein response (UPR), and its downstream target eukaryotic translation initiation factor 2 α (eIF2α); in contrast, TCDD did not appear to affect inositol-requiring enzyme 1 (IRE1) and activating transcription factor 6 (ATF6), two other UPR sensors. Importantly, TCDD significantly inhibited eIF2α phosphorylation and triggered apoptosis in PC12 cells after 6-24h of treatment. Salubrinal, which activates the PERK-eIF2α pathway, significantly enhanced eIF2α phosphorylation in PC12 cells and attenuated the TCDD-induced cell death. In contrast, knocking down eIF2α using small interfering RNA markedly enhanced TCDD-induced cell death. Together, these results indicate that the PERK-eIF2α pathway plays an important role in TCDD-induced ER stress and apoptosis in PC12 cells.

  1. Recognizing the intensity of strength training exercises with wearable sensors. (United States)

    Pernek, Igor; Kurillo, Gregorij; Stiglic, Gregor; Bajcsy, Ruzena


    In this paper we propose a system based on a network of wearable accelerometers and an off-the-shelf smartphone to recognize the intensity of stationary activities, such as strength training exercises. The system uses a hierarchical algorithm, consisting of two layers of Support Vector Machines (SVMs), to first recognize the type of exercise being performed, followed by recognition of exercise intensity. The first layer uses a single SVM to recognize the type of the performed exercise. Based on the recognized type a corresponding intensity prediction SVM is selected on the second layer, specializing in intensity prediction for the recognized type of exercise. We evaluate the system for a set of upper-body exercises using different weight loads. Additionally, we compare the most important features for exercise and intensity recognition tasks and investigate how different sliding window combinations, sensor configurations and number of training subjects impact the algorithm performance. We perform all of the experiments for two different types of features to evaluate the feasibility of implementation on resource constrained hardware. The results show the algorithm is able to recognize exercise types with approximately 85% accuracy and 6% intensity prediction error. Furthermore, due to similar performance using different types of features, the algorithm offers potential for implementation on resource constrained hardware.

  2. Increased expression of 78 kD glucose-regulated protein promotes cardiomyocyte apoptosis in a rat model of liver cirrhosis (United States)

    Zhang, Lili; Zhang, Huiying; Lv, Minli; Jia, Jiantao; Fan, Yimin; Tian, Xiaoxia; Li, Xujiong; Li, Baohong; Ji, Jingquan; Wang, Limin; Zhao, Zhongfu; Han, Dewu; Ji, Cheng


    Aims: This study was to investigate the role and underlying mechanism of 78 kD glucose-regulated protein (GRP78) in cardiomyocyte apoptosis in a rat model of liver cirrhosis. Methods: A rat model of liver cirrhosis was established with multiple pathogenic factors. A total of 42 male SD rats were randomly divided into the liver cirrhosis group and control group. Cardiac structure analysis was performed to assess alterations in cardiac structure. Cardiomyocytes apoptosis was detected by TdT-mediated dUTP nick end labeling method. Expression of GRP78, CCAAT/enhancer-binding protein homologous protein (CHOP), caspase-12, nuclear factor kappa-light-chain-enhancer of activated B cells p65 subunit (NF-κB p65) and B cell lymphoma-2 (Bcl-2) was detected by immunohistochemical staining. Results: The ratios of left ventricular wall thickness to heart weight and heart weight to body weight were significantly increased with the progression of liver cirrhosis (P < 0.05). Apoptosis index of cardiomyocytes was significantly increased with the progression of liver cirrhosis (P < 0.05). The expression levels of GRP78, CHOP and caspase-12 were significantly increased in the progression of liver cirrhosis (P < 0.05). The expression levels of NF-κB p65 and Bcl-2 were highest in the 4-wk liver cirrhosis, and they were decreased in the 6-wk and 8-wk in the progression of liver cirrhosis. GRP78 expression levels were positively correlated with apoptosis index, CHOP and caspase-12 expression levels (P < 0.05). CHOP expression levels were negatively correlated with NF-κB p65 and Bcl-2 expression levels (P < 0.05). Conclusion: Increased expression of GRP78 promotes cardiomyocyte apoptosis in rats with cirrhotic cardiomyopathy. PMID:26464674

  3. Research of recognizing intelligence based on commanding decision-making

    Institute of Scientific and Technical Information of China (English)

    Liu Jingxue; Fei Qi


    In commanding decision-making, the commander usually needs to know a lot of situations(intelligence) on the adversary. Because of the military intelligence with opposability, it is inevitable that intelligence personnel take some deceptive information released by the rival as intelligence data in the process of intelligence gathering. Since the failure of intelligence is likely to lead to a serious aftereffect, the recognition of intelligence is a very important problem. An elementary research on recognizing military intelligence and puts forward a systematic processing method are made. First, the types and characteristics of military intelligence are briefly discussed, a research thought of recognizing military intelligence by means of recognizing military hypotheses are presented. Next, the reasoning mode and framework for recognizing military hypotheses are presented from the angle of psychology of intelligence analysis and non-monotonic reasoning. Then, a model for recognizing military hypothesis is built on the basis of fuzzy judgement information given by intelligence analysts. A calculative example shows that the model has the characteristics of simple calculation and good maneuverability. Last, the methods that selecting the most likely hypothesis from the survival hypotheses via final recognition are discussed.

  4. Detection of serum autoantibodies to melanocyte and correlation between melanoma antigen recognized by T-cells and vitiligo in children%白癜风患儿血清中抗黑素细胞抗体检测及黑素瘤抗原-1与该病的关系

    Institute of Scientific and Technical Information of China (English)

    陈谨萍; 李海翩; 金盛华; 张金桃; 李军


    Objective To detect the serum levels of melanocyte antibodies and explore the relation between melanoma antigen recognized by T-cells (MART-1) and vitiligo in children. Methods The serum samples were collected from children with vitiligo to test the autoantibodies, and divided into low- and high-titer group according to the test results. Melanocytes were incubated with the serum samples, and the changes of melanocyte surface antigen were evaluated using specific MART-1 antibody. Results The serum melanocyte antibody levels in children with vitiligo were significantly higher than those in normal subjects. The expression level of melanocyte surface antigen MART-1 increased obviously after incubation of the melanocyte with high antibody titer serum samples, and MART-1 was found to specifically bind to specific MART-1 antibody. Conclusion Melanocytes MART-1 may correlate to the autoimmune mechanism in children with vitiligo.%目的 了解儿童白癜风患者血清中抗黑素细胞抗体情况及探讨黑素瘤抗原-1(MART-1)与儿童白癜风的关系.方法 采集患儿血清.检测自身抗体,根据抗体滴度高低分成两组,与正常人黑素细胞进行孵育,利用MART-1特异性抗体检测孵育后黑素细胞表面抗原MART-1的变化.结果 白癜风患儿血清抗黑素细胞抗体滴度升高.与高滴度抗体患儿血清孵育后的MART-1表达明显增加,并能与MART-1特异性抗体丰H结合.结论 部分儿童白癜风是免疫相关的,MART-1与儿童白癜风可能相关联.

  5. Recognizing Variable Environments The Theory of Cognitive Prism

    CERN Document Server

    Dong, Tiansi


    Normal adults do not have any difficulty in recognizing their homes. But can artificial systems do in the same way as humans? This book collects interdisciplinary evidences and presents an answer from the perspective of computing, namely, the theory of cognitive prism. To recognize an environment, an intelligent system only needs to classify objects, structures them based on the connection relation (not through measuring!), subjectively orders the objects, and compares with the target environment, whose knowledge is similarly structured. The intelligent system works, therefore, like a prism: when a beam of light (a scene) reaches (is perceived) to an optical prism (by an intelligent system), some light (objects) is reflected (are neglected), those passed through (the recognized objects) are distorted (are ordered differently). So comes the term 'cognitive prism'! Two fundamental propositions used in the theory can be informally stated as follow: an orientation relation is a kind of distance comparison relatio...

  6. Hand posture recognizer based on separator wavelet networks (United States)

    Bouchrika, Tahani; Jemai, Olfa; Zaied, Mourad; Ben Amar, Chokri


    This paper presents a novel hand posture recognizer based on separator wavelet networks (SWNs). Aiming at creating a robust and rapid hand posture recognizer, we have contributed by proposing a new training algorithm for the wavelet network classifier based on fast wavelet transform (FWN). So, the contribution resides in reducing the number of WNs modeling training data. To make that, inspiring from the adaboost feature selection method, we thought to create SWNs (n-1 WNs for n classes) instead of modeling each training sample by its wavelet network (WN). By proposing the new training algorithm, the recognition phase will be positively influenced. It will be more rapid thanks to the reduction of the number of comparisons between test images WNs and training WNs. Comparisons with other works, employing universal hand posture datasets are presented and discussed. Obtained results have shown that the new hand posture recognizer is comparable to previously established ones.

  7. You look familiar: how Malaysian Chinese recognize faces.

    Directory of Open Access Journals (Sweden)

    Chrystalle B Y Tan

    Full Text Available East Asian and white Western observers employ different eye movement strategies for a variety of visual processing tasks, including face processing. Recent eye tracking studies on face recognition found that East Asians tend to integrate information holistically by focusing on the nose while white Westerners perceive faces featurally by moving between the eyes and mouth. The current study examines the eye movement strategy that Malaysian Chinese participants employ when recognizing East Asian, white Western, and African faces. Rather than adopting the Eastern or Western fixation pattern, Malaysian Chinese participants use a mixed strategy by focusing on the eyes and nose more than the mouth. The combination of Eastern and Western strategies proved advantageous in participants' ability to recognize East Asian and white Western faces, suggesting that individuals learn to use fixation patterns that are optimized for recognizing the faces with which they are more familiar.

  8. Apoptosis of HeLa cells induced by a new targeting photosensitizer-based PDT via a mitochondrial pathway and ER stress

    Directory of Open Access Journals (Sweden)

    Li D


    Full Text Available Donghong Li,1 Lei Li,2 Pengxi Li,1 Yi Li,3 Xiangyun Chen1 1State Key Laboratory of Trauma, Burn and Combined Injury, The Second Department of Research Institute of Surgery, 2The First Department of Research Institute of Surgery, 3Cancer Center, Daping Hospital, Third Military Medical University, Chongqing, People’s Republic of China Abstract: Photodynamic therapy (PDT is emerging as a viable treatment for many cancers. To decrease the cutaneous photosensitivity induced by PDT, many attempts have been made to search for a targeting photosensitizer; however, few reports describe the molecular mechanism of PDT mediated by this type of targeting photosensitizer. The present study aimed to investigate the molecular mechanism of PDT induced by a new targeting photosensitizer (PS I, reported previously by us, on HeLa cells. Apoptosis is the primary mode of HeLa cell death in our system, and apoptosis occurs in a manner dependent on concentration, irradiation dose, and drug–light intervals. After endocytosis mediated by the folate receptor, PS I was primarily localized to the mitochondria and the endoplasmic reticulum (ER of HeLa cells. PS I PDT resulted in rapid increases in intracellular reactive oxygen species (ROS production and Ca2+ concentration, both of which reached a peak nearly simultaneously at 15 minutes, followed by the loss of mitochondrial membrane potential at 30 minutes, release of cytochrome c from mitochondria into the cytoplasm, downregulation of Bcl-2 expression, and upregulation of Bax expression. Meanwhile, activation of caspase-3, -9, and -12, as well as induction of C/EBP homologous protein (CHOP and glucose-regulated protein (GRP78, in HeLa cells after PS I PDT was also detected. These results suggest that apoptosis of HeLa cells induced by PS I PDT is not only triggered by ROS but is also regulated by Ca2+ overload. Mitochondria and the ER serve as the subcellular targets of PS I PDT, the effective activation of which

  9. Overexpression of glycosylated proteins in cervical cancer recognized by the Machaerocereus eruca agglutinin Overexpression of glycosylated proteins in cervical cancer recognized by the Machaerocereus eruca agglutinin

    Directory of Open Access Journals (Sweden)

    Carlos Solórzano


    Full Text Available In cervical cancer, glycosylation has been suggested as being involved in both its carcinogenesis and
    invasive capacity. In this work, we analyzed mucin type O-glycosylation in biopsies of invasive cervical cancer in
    FIGO stage II B through histochemistry using lectins specific for O-glycosidically linked glycans. Our results
    reveal that the lectin Machaerocereus eruca (MeA, specific for Gal in a Fuca1,2 (GalNAca1,3 Galb1,4 showed
    increased recognition of tumoral cells and tumoral stroma tissue compared to other lectins with similar specificity;
    healthy cervical tissue was negative for MeA. Trypsin treatment of recognized tissues abolished MeA’s recognition;
    moreover, interaction of MeA was inhibited with oligosaccharides from mucin. As demonstrated by
    Western blot of 2-D electrophoresis, MeA recognized ten glycoproteins in the range from 122 to 42 kDa in
    cervical cancer lysates. The LC-ESI-MS/MS analysis of the MeAs’ recognized peptides revealed that the latter
    matched mainly with the amino acid sequences of lamin A/C, vimentin, elongation factor 2, keratin 1, and beta
    actin. Our results suggest that MeA recognizes a complex of over-expressed O-glycosidically-linked proteins that
    play a relevant role in cervical cancer’s invasive capacity. O-glycosylation participates in the disassembly of intercellular
    junctions favoring cancer progression.In cervical cancer, glycosylation has been suggested as being involved in both its carcinogenesis and
    invasive capacity. In this work, we analyzed mucin type O-glycosylation in biopsies of invasive cervical cancer in
    FIGO stage II B through histochemistry using lectins specific for O-glycosidically linked glycans. Our results
    reveal that the lectin Machaerocereus eruca (MeA, specific for Gal in a Fuca1,2 (GalNAca1,3 Galb1,4 showed
    increased recognition of tumoral cells and tumoral stroma tissue compared to other lectins

  10. Overexpression of glycosylated proteins in cervical cancer recognized by the Machaerocereus eruca agglutinin. (United States)

    Solórzano, Carlos; Angel Mayoral, Miguel; de los Angeles Carlos, María; Berumen, Jaime; Guevara, Jorge; Raúl Chávez, Francisco; Mendoza-Hernández, Guillermo; Agundis, Concepción; Zenteno, Edgar


    In cervical cancer, glycosylation has been suggested as being involved in both its carcinogenesis and invasive capacity. In this work, we analyzed mucin type O-glycosylation in biopsies of invasive cervical cancer in FIGO stage II B through histochemistry using lectins specific for O-glycosidically linked glycans. Our results reveal that the lectin Machaerocereus eruca (MeA, specific for Gal in a Fucα1,2 (GalNAcα1,3) Galβ1,4) showed increased recognition of tumoral cells and tumoral stroma tissue compared to other lectins with similar specificity; healthy cervical tissue was negative for MeA. Trypsin treatment of recognized tissues abolished MeA's recognition;moreover, interaction of MeA was inhibited with oligosaccharides from mucin. As demonstrated by Western blot of 2-D electrophoresis, MeA recognized ten glycoproteins in the range from 122 to 42 kDa in cervical cancer lysates. The LC-ESI-MS/MS analysis of the MeAs' recognized peptides revealed that the latter matched mainly with the amino acid sequences of lamin A/C, vimentin, elongation factor 2, keratin 1, and beta actin. Our results suggest that MeA recognizes a complex of over-expressed O-glycosidically-linked proteins that play a relevant role in cervical cancer's invasive capacity. O-glycosylation participates in the disassembly of intercellular junctions favoring cancer progression.

  11. Recognize the Signs: Reading Young Adult Literature to Address Bullying (United States)

    Pytash, Kristine E.; Morgan, Denise N.; Batchelor, Katherine E.


    This article summarizes preservice teachers' experiences in a book club that read young adult literature focused on issues related to bullying. Preservice teachers learned to recognize various incidents of bullying in the books. They also began to consider how they might handle incidents of bullying in their future classrooms. (Contains 2 figures.)

  12. Recognizing Multi-user Activities using Body Sensor Networks

    DEFF Research Database (Denmark)

    Gu, Tao; Wang, Liang; Chen, Hanhua


    The advances of wireless networking and sensor technology open up an interesting opportunity to infer human activities in a smart home environment. Existing work in this paradigm focuses mainly on recognizing activities of a single user. In this work, we address the fundamental problem of recogni...

  13. Recognizing Job Health Hazards. Module SH-08. Safety and Health. (United States)

    Center for Occupational Research and Development, Inc., Waco, TX.

    This student module on recognizing job health hazards is one of 50 modules concerned with job safety and health. This module presents the four general categories of environmental conditions or stresses: chemical, physical, biological, and ergonomic. Following the introduction, 14 objectives (each keyed to a page in the text) the student is…

  14. Young children's ability to recognize advertisements in web page designs. (United States)

    Ali, Moondore; Blades, Mark; Oates, Caroline; Blumberg, Fran


    Identifying what is, and what is not an advertisement is the first step in realizing that an advertisement is a marketing message. Children can distinguish television advertisements from programmes by about 5 years of age. Although previous researchers have investigated television advertising, little attention has been given to advertisements in other media, even though other media, especially the Internet, have become important channels of marketing to children. We showed children printed copies of invented web pages that included advertisements, half of which had price information, and asked the children to point to whatever they thought was an advertisement. In two experiments we tested a total of 401 children, aged 6, 8, 10 and 12 years of age, from the United Kingdom and Indonesia. Six-year-olds recognized a quarter of the advertisements, 8-year-olds recognized half the advertisements, and the 10- and 12-year-olds recognized about three-quarters. Only the 10- and 12-year-olds were more likely to identify an advertisement when it included a price. We contrast our findings with previous results about the identification of television advertising, and discuss why children were poorer at recognizing web page advertisements. The performance of the children has implications for theories about how children develop an understanding of advertising.

  15. Islamic context influences how emotion is recognized from the eyes

    Directory of Open Access Journals (Sweden)

    Mariska eKret


    Full Text Available Previous research has shown a negative bias in the perception of whole facial expressions from out-group members. Whether or not emotion recognition from the eyes is already sensitive to contextual information is presently a matter of debate. In three experiments we tested whether emotions can be recognized from the upper part of the face only when just the eyes are visible and whether this recognition is affected by context cues, such as various Islamic headdresses versus a cap or a scarf. Our results indicate that fear is still well recognized from a briefly flashed (100ms image of the eyes in the context of a burqa leaving the eyes visible for less than 20%. Moreover, the type of headdress influences how emotions are recognized. In a group of participants from non-Islamic background, fear was recognized better from women wearing a niqāb than from women wearing a cap and a shawl, whereas the opposite pattern was observed for happy and sad expressions. Anxiety levels and/or explicit negative associations with the Islam as measured via questionnaires did not mediate the effects. Consistent with the face literature, we conclude that the recognition of emotions from the eyes is also influenced by context.

  16. Recognizing Risk-of-Failure in Communication Design Projects (United States)

    Yee, Joyce; Lievesley, Matthew; Taylor, Louise


    The pace of commercial graphic design practice presents very few opportunities to conduct user research after a project's launch. This makes the design team's ability to anticipate and address risks during the design development phase even more important, recognized in the astute observation from Tim Brown, CEO of leading international design…

  17. Development of monoclonal antibodies that recognize Treponema pallidum.


    Saunders, J M; Folds, J D


    We developed a panel of monoclonal antibodies to Treponema pallidum (Nichols) antigens, some of which recognize treponemal antigens on T. pallidum (Nichols), T. pallidum strain 14, and Treponema phagedenis biotype Reiter. The antibodies were detected by either an enzyme-linked immunosorbent assay or a radioimmunoassay.

  18. Recognizing and Fostering Creativity in Technological Design Education (United States)

    Cropley, David; Cropley, Arthur


    The importance of creativity in technological design education is now clearly recognized, both in everyday understanding and also in formal curriculum guidelines. Design offers special opportunities for creativity because of the "openness" of problems (ill-defined problems, the existence of a variety of pathways to the solution, the absence of…

  19. 46 CFR 188.10-59 - Recognized classification society. (United States)


    ... 46 Shipping 7 2010-10-01 2010-10-01 false Recognized classification society. 188.10-59 Section 188.10-59 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY (CONTINUED) OCEANOGRAPHIC RESEARCH... classification society. This term means the American Bureau of Shipping or other classification...

  20. Open Badges: Novel Means to Motivate, Scaffold and Recognize Learning (United States)

    Jovanovic, Jelena; Devedzic, Vladan


    This report is centered on the emerging concept and technology of Open Badges (OBs) that are offering novel means and practices of motivating, scaffolding, recognizing, and credentialing learning. OBs are closely associated with values such as openness and learners' agency, participatory learning and peer-learning communities. This report points…

  1. Recognizing Family Dynamics in the Treatment of Chronic Fatigue Syndrome (United States)

    Sperry, Len


    Chronic fatigue syndrome (CFS) is an increasingly common chronic medical condition that affects not only patients but also their families. Because family dynamics, particularly the family life cycle, can and does influence the disease process, those providing counseling to CFS patients and their families would do well to recognize these dynamics.…

  2. The synthesis and molecular recognization of the polyamine transporter of hydrazine-modified diamine conjugates

    Institute of Scientific and Technical Information of China (English)

    Jun Jun Zhou; Hao Huang; Song Qiang Xie; Yu Xia Wang; Jin Zhao; Chao Jie Wang


    A series of four novel hydrazine-modified diamine conjugates (7a-b, 8a-b) were synthesized and evaluated for cytotoxicityagainst Melanoma B 16, α-difluoromethylornithine (DFMO)-treated B 16, spermidine (SPD)-treated B 16, Mouse leukemia L 1210and Hela cell lines. Both the DFMO-B 16 and SPD-B 16 experiments indicated that conjugates 7a-b and 8a-b could recognize thepolyamine transporter (PAT) and enter the cells in part or in whole via PAT, although they were not as efficient as the reference, 9-anthracenemethyl homospermidine (1).

  3. Peripheral blood lymphocytes from low-grade squamous intraepithelial lesions patients recognize vaccine antigens in the presence of activated dendritic cells, and produced high levels of CD8 + IFNγ + T cells and low levels of IL-2 when induced to proliferate

    Directory of Open Access Journals (Sweden)

    Hernández-Montes Jorge


    Full Text Available Abstract Background Most infections with human papillomavirus (HPV are resolved without clinical intervention, but a minority evolves into chronic lesions of distinct grades, including cervical-uterine cancer. It is known that in most cases the immune system mediates elimination of HPV infection. However, the mechanism of immune evasion leading to HPV persistence and development of early cervical lesions is not fully understood. The aim of the present work was to evaluate the potential of peripheral blood leukocytes (PBL from low-grade squamous intraepithelial lesions (LSIL patients to be activated ex-vivo by vaccine antigens, the participation of cytotoxic lymphocytes and regulatory T cells, and to determine the secretion of Th1 and Th2 cytokines mediated by stimulation of T cell receptors. Results We found that PBL from LSIL patients showed a significantly lower proliferation rate to vaccine antigens as compared to that of healthy donors, even though there was not a difference in the presence of antibodies to those antigens in sera from both groups. We did not find differences in either the frequency of CD4 + CD25 + FoxP3+ in PBL, or the levels of IL-4, IL-5 and IL-10 in plasma or conditioned media from PBL incubated with TcR agonists in vitro, between the two groups. However, we detected a lower production of IL-2 and a higher proportion of CD8 + IFNγ + cells in PBL from LSIL patients as compared with PBL from normal donors. We also observed that PBL from patients infected by HPV-16 and −18 were not able to proliferate in the presence of soluble HPV antigens added to the culture; however, a high level of proliferation was attained when these antigens were presented by activated dendritic cells. Conclusions Our results suggest that the immunodeficiency reported in LSIL patients could be due to the inability of specific cytotoxic T lymphocytes that for some unknown reason are present but unable to mount a response when

  4. Murine leukemia RL male 1 and sarcoma Meth A antigens recognized by cytotoxic T lymphocytes (CTL). (United States)

    Uenaka, Akiko; Nakayama, Eiichi


    Peptide elution and expression cloning methods have been used to identify T cell-recognized antigens for which no molecular information is available. We identified a unique tumor antigen peptide pRL1a, IPGLPLSL that is recognized by CTL on BALB/c RL male 1 leukemia by peptide elution. The sequence of the peptide corresponded to the normally untranslated 5' region of akt. Cytotoxicity was generated in BALB/c spleen cells by in vivo and in vitro sensitization with pRL1a peptide in the form of multiple antigen peptide (MAP), but not the original form. pRL1a MAP immunization had a significant growth-inhibitory effect. pRL1a MAP was mostly internalized into the endosomal compartment of antigen-presenting cells, leaked to the cytosol, and degraded, and the pRL1a peptide produced was presented through the MHC class I pathway. In vivo depletion of CD4 T cells from tumor-inoculated BALB/c mice caused RL male 1 regression. Overexpression of the RLakt molecule seemed to induce CD4 immunoregulatory cells, which resulted in progressive RL male 1 growth in BALB/c mice. In vivo administration of anti-CD25 mAb (PC61) caused regression of RL male 1, suggesting that CD4(+) CD25(+) immunoregulatory cells were involved in the tumor growth. Recently, we improved the sensitivity and the efficacy of T cell antigen cloning from cDNA expression libraries by using large- and small-scale ELISPOT assays. Using the IFN-gamma ELISPOT method, we obtained a cDNA clone S35 of 937 bp recognized by AT-1 CTL on BALB/c Meth A sarcoma. S35 was a part of the retinoic acid-regulated nuclear matrix-associated protein (ramp). AT-1 CTL recognized the peptide LGAEAIFRL, which was derived from a newly created open reading frame due to the exon 14 extension.

  5. Platelets recognize brain-specific glycolipid structures, respond to neurovascular damage and promote neuroinflammation.

    Directory of Open Access Journals (Sweden)

    Ilya Sotnikov

    Full Text Available Platelets respond to vascular damage and contribute to inflammation, but their role in the neurodegenerative diseases is unknown. We found that the systemic administration of brain lipid rafts induced a massive platelet activation and degranulation resulting in a life-threatening anaphylactic-like response in mice. Platelets were engaged by the sialated glycosphingolipids (gangliosides integrated in the rigid structures of astroglial and neuronal lipid rafts. The brain-abundant gangliosides GT1b and GQ1b were specifically recognized by the platelets and this recognition involved multiple receptors with P-selectin (CD62P playing the central role. During the neuroinflammation, platelets accumulated in the central nervous system parenchyma, acquired an activated phenotype and secreted proinflammatory factors, thereby triggering immune response cascades. This study determines a new role of platelets which directly recognize a neuronal damage and communicate with the cells of the immune system in the pathogenesis of neurodegenerative diseases.

  6. Analysis of Gait Pattern to Recognize the Human Activities

    Directory of Open Access Journals (Sweden)

    Jay Prakash Gupta


    Full Text Available Human activity recognition based on the computer vision is the process of labelling image sequences with action labels. Accurate systems for this problem are applied in areas such as visual surveillance, human computer interaction and video retrieval. The challenges are due to variations in motion, recording settings and gait differences. Here we propose an approach to recognize the human activities through gait. Activity recognition through Gait is the process of identifying an activity by the manner in which they walk. The identification of human activities in a video, such as a person is walking, running, jumping, jogging etc are important activities in video surveillance. We contribute the use of Model based approach for activity recognition with the help of movement of legs only. Experimental results suggest that our method are able to recognize the human activities with a good accuracy rate and robust to shadows present in the videos.

  7. Federally-Recognized Tribes of the Columbia-Snake Basin.

    Energy Technology Data Exchange (ETDEWEB)

    United States. Bonneville Power Administration


    This is an omnibus publication about the federally-recognized Indian tribes of the Columbia-Snake river basin, as presented by themselves. It showcases several figurative and literal snapshots of each tribe, bits and pieces of each tribe`s story. Each individual tribe or tribal confederation either submitted its own section to this publication, or developed its own section with the assistance of the writer-editor. A federally-recognized tribe is an individual Indian group, or confederation of Indian groups, officially acknowledged by the US government for purposes of legislation, consultation and benefits. This publication is designed to be used both as a resource and as an introduction to the tribes. Taken together, the sections present a rich picture of regional indian culture and history, as told by the tribes.

  8. Forms can be recognized from dynamic occlusion alone. (United States)

    Stappers, P J


    Direct and indirect theories of perception differ on whether form perception depends on higher order invariants or on features in the retinal image. The present paper describes a demonstration that an object can be recognized through a higher order pattern (dynamic occlusion) without any of the object's features being displayed. Stimuli consist of computer stimulations of black wireframe objects moving in front of, and occluding, a random layout of point lights on a black background. In this way, no single videoframe of the stimuli displays any of the object's features, and motion of the amodal object in front of the light points is necessary for the form to become visible. The forms can also be recognized when isoluminous colours are used for background and point lights. Finally, it is noted that, if the observer can actively control the motion of the object, e.g., by moving a computer mouse, recognition is enhanced as in Gibson's (1962) experiment on active touch.

  9. Slice&Dice: Recognizing Food Preparation Activities Using Embedded Accelerometers (United States)

    Pham, Cuong; Olivier, Patrick

    Within the context of an endeavor to provide situated support for people with cognitive impairments in the kitchen, we developed and evaluated classifiers for recognizing 11 actions involved in food preparation. Data was collected from 20 lay subjects using four specially designed kitchen utensils incorporating embedded 3-axis accelerometers. Subjects were asked to prepare a mixed salad in our laboratory-based instrumented kitchen environment. Video of each subject's food preparation activities were independently annotated by three different coders. Several classifiers were trained and tested using these features. With an overall accuracy of 82.9% our investigation demonstrated that a broad set of food preparation actions can be reliably recognized using sensors embedded in kitchen utensils.

  10. Recognizing young children with high potential: U-STARS∼PLUS. (United States)

    Coleman, Mary Ruth


    Hands-on science is the ideal platform for observing young children's ability to solve problems, think deeply, and use their creative ingenuity to explore the world around them. Science is naturally interesting and offers authentic reasons to read for information and use math skills to collect, compile, and analyze data. This chapter will share one approach to nurturing and recognizing young children with high-potential: U-STARS∼PLUS (Using Science, Talents, and Abilities to Recognize Students∼Promoting Learning for Underrepresented Students). Each of the five components (high-end learning environments; teacher's observations of potential; engaging science activities; partnerships with parents; and capacity building for system change) will be explained. Concrete examples will be given for each area showing how it works and why it is important. Special attention will be paid to the needs of educationally vulnerable gifted children who remain underserved: racially, ethnically, and linguistically different; economically disadvantaged, and children who are twice exceptional (2e).

  11. Recognizing shipbuilding parts using artificial neural networks and Fourier descriptors


    Sanders, David


    A pattern recognition system is described for recognizing shipbuilding parts using artificial neural networks and Fourier descriptors. The system uses shape contour information that is invariant of size, translation, and rotation. Fourier descriptors provide information, and the neural networks make decisions about the shapes. A brief review of the current state of the art is included, and results from testing show that the system distinguished between various shapes and proved to be a valid ...

  12. Organizing to learn: recognizing and cultivating learning communities. (United States)

    Doty, Elizabeth A


    One of the most effective ways to learn is in a community of people with a shared purpose. Therefore, by recognizing and cultivating the learning communities that arise within most organizational structures, we are actually organizing ourselves to learn. Using stories to inspire the reader to reflect and apply these concepts, the author explores learning communities in a variety of fields, including examples that involve entire organizations, single workgroups, and those that cross organizational boundaries.

  13. Recognizing Age-Separated Face Images: Humans and Machines


    Daksha Yadav; Richa Singh; Mayank Vatsa; Afzel Noore


    Humans utilize facial appearance, gender, expression, aging pattern, and other ancillary information to recognize individuals. It is interesting to observe how humans perceive facial age. Analyzing these properties can help in understanding the phenomenon of facial aging and incorporating the findings can help in designing effective algorithms. Such a study has two components - facial age estimation and age-separated face recognition. Age estimation involves predicting the age of an individua...

  14. Capsular Weakness around Breast Implant: A Non-Recognized Complication



    Capsular contraction is a frequent complication following breast augmentation. On the other hand, capsular weakness, a not widely recognized complication, may occur around the implant. A weak capsule allows the migration of the prosthesis to the lateral region of the thoracic region or inferiorly, towards the abdomen, due to gravitational forces. The cause of capsular weakness remains unresolved. Implant malposition, with lateral or downward displacement, breast asymmetry, improper contour, w...

  15. Smart responsive microcapsules capable of recognizing heavy metal ions. (United States)

    Pi, Shuo-Wei; Ju, Xiao-Jie; Wu, Han-Guang; Xie, Rui; Chu, Liang-Yin


    Smart responsive microcapsules capable of recognizing heavy metal ions are successfully prepared with oil-in-water-in-oil double emulsions as templates for polymerization in this study. The microcapsules are featured with thin poly(N-isopropylacrylamide-co-benzo-18-crown-6-acrylamide) (P(NIPAM-co-BCAm)) membranes, and they can selectively recognize special heavy metal ions such as barium(II) or lead(II) ions very well due to the "host-guest" complexation between the BCAm receptors and barium(II) or lead(II) ions. The stable BCAm/Ba(2+) or BCAm/Pb(2+) complexes in the P(NIPAM-co-BCAm) membrane cause a positive shift of the volume phase transition temperature of the crosslinked P(NIPAM-co-BCAm) hydrogel to a higher temperature, and the repulsion among the charged BCAm/Ba(2+) or BCAm/Pb(2+) complexes and the osmotic pressure within the P(NIPAM-co-BCAm) membranes result in the swelling of microcapsules. Induced by recognizing barium(II) or lead(II) ions, the prepared microcapsules with P(NIPAM-co-BCAm) membranes exhibit isothermal and significant swelling not only in outer and inner diameters but also in the membrane thickness. The proposed microcapsules in this study are highly attractive for developing smart sensors and/or carriers for detection and/or elimination of heavy metal ions.

  16. Giardia mitosomal protein import machinery differentially recognizes mitochondrial targeting signals. (United States)

    Nyindodo-Ogari, Lilian; Schwartzbach, Steven D; Estraño, Carlos E


    Giardia lamblia mitosomes are believed to be vestigial mitochondria which lack a genome. Similar to higher eukaryotes, mitosomal proteins possess either N-terminal or internal mitosomal targeting sequences. To date, some components of the higher eukaryote archetypal mitochondrial protein import apparatus have been identified and characterized in Giardia mitosomes; therefore, it is expected that mitochondrial signals will be recognized by the mitosomal protein import system. To further determine the level of conservation of the Giardia mitosome protein import apparatus, we expressed mitochondrial proteins from higher eukaryotes in Giardia. These recombinant proteins include Tom20 and Tom22; two components of the mitochondrial protein import machinery. Our results indicate that N-terminal mitochondrial targeting sequence is recognized by the mitosomal protein import machinery; however, interestingly the internal mitochondrial targeting sequences of higher eukaryotes are not recognized by the mitosome. Our results indicate that Giardia mitosome protein transport machinery shows differential recognition of higher eukaryotic mitochondria transfer signals, suggesting a divergence of the transport system in G. lamblia. Therefore, our data support the hypothesis that the protein import machinery in Giardia lamblia mitosome is an incomplete vestigial derivative of mitochondria components.

  17. Comprehensive Context Recognizer Based on Multimodal Sensors in a Smartphone

    Directory of Open Access Journals (Sweden)

    Sungyoung Lee


    Full Text Available Recent developments in smartphones have increased the processing capabilities and equipped these devices with a number of built-in multimodal sensors, including accelerometers, gyroscopes, GPS interfaces, Wi-Fi access, and proximity sensors. Despite the fact that numerous studies have investigated the development of user-context aware applications using smartphones, these applications are currently only able to recognize simple contexts using a single type of sensor. Therefore, in this work, we introduce a comprehensive approach for context aware applications that utilizes the multimodal sensors in smartphones. The proposed system is not only able to recognize different kinds of contexts with high accuracy, but it is also able to optimize the power consumption since power-hungry sensors can be activated or deactivated at appropriate times. Additionally, the system is able to recognize activities wherever the smartphone is on a human’s body, even when the user is using the phone to make a phone call, manipulate applications, play games, or listen to music. Furthermore, we also present a novel feature selection algorithm for the accelerometer classification module. The proposed feature selection algorithm helps select good features and eliminates bad features, thereby improving the overall accuracy of the accelerometer classifier. Experimental results show that the proposed system can classify eight activities with an accuracy of 92.43%.

  18. Recognizing surgeon's actions during suture operations from video sequences (United States)

    Li, Ye; Ohya, Jun; Chiba, Toshio; Xu, Rong; Yamashita, Hiromasa


    Because of the shortage of nurses in the world, the realization of a robotic nurse that can support surgeries autonomously is very important. More specifically, the robotic nurse should be able to autonomously recognize different situations of surgeries so that the robotic nurse can pass necessary surgical tools to the medical doctors in a timely manner. This paper proposes and explores methods that can classify suture and tying actions during suture operations from the video sequence that observes the surgery scene that includes the surgeon's hands. First, the proposed method uses skin pixel detection and foreground extraction to detect the hand area. Then, interest points are randomly chosen from the hand area so that their 3D SIFT descriptors are computed. A word vocabulary is built by applying hierarchical K-means to these descriptors, and the words' frequency histogram, which corresponds to the feature space, is computed. Finally, to classify the actions, either SVM (Support Vector Machine), Nearest Neighbor rule (NN) for the feature space or a method that combines "sliding window" with NN is performed. We collect 53 suture videos and 53 tying videos to build the training set and to test the proposed method experimentally. It turns out that the NN gives higher than 90% accuracies, which are better recognition than SVM. Negative actions, which are different from either suture or tying action, are recognized with quite good accuracies, while "Sliding window" did not show significant improvements for suture and tying and cannot recognize negative actions.

  19. Anomalous expression of heat shock protein 27 and glucose regulated protein 78 contributed to cardiac allograft vasculopathy%热休克蛋白27和糖调节蛋白78分布异常对心脏移植后血管病变的影响

    Institute of Scientific and Technical Information of China (English)

    雷虹; 尹海辉; 章庆春; 严中亚


    目的 观察移植后心脏组织中热休克蛋白27(HSP27)和糖调节蛋白78(GRP78)的组织分布,探讨移植后心脏血管病变(CAV)的发病机制.方法 建立大鼠同基因(n=8)和异基因(n=8)心脏移植的Ono模型,采用免疫组织化学法[链霉菌抗生物素蛋白-过氧化物酶(SP)法]验证移植后心脏组织中HSP27和GRP78的组织分布.结果 同基因植组,HSP27和GRP78在心脏组织内均匀分布;异基因移植组,HSP27和GRP78在环血管周围心肌组织中高表达(2.24±0.36比1.04±0.10,P<0.01;1.51±0.12比0.85±0.30,P<0.05),而在增生的冠状动脉组织内几乎不表达(2.24±0.36比0.25±0.08,P<0.01;1.51±0.12比0.16±0.10,P<0.01).结论 HSP27和GRP78在异基因移植后心脏组织中分布不均,导致冠状动脉组织无力对抗免疫损伤,诱发血管平滑肌细胞过度增殖可能是CAV发生、发展的重要原因.%Objective To observe the protein expression of heat shock protein 27 (HSP27) and glucose regulated protein 78 (GRP78) in rats transplanted hearts,and provide some clues to elucidate the pathogenesis of cardiac allograft vasculopathy (CAV).Methods The hearts were transplanted from Lewis to Sprague-Dawely rats as allograft (n =8) and from Lewis to Lewis rats as isograft (n =8) based on Ono' s model.The protein expression of HSP27 and GRP78 was detected by using immunohistochemistry [streptavid-inperoxidase (SP) method].Results The expression of HSP27 and GRP78 was stained uniformly in cardiac isografts,but that was anomalous in cardiac allografts,which showed higher expression on cardiac muscle (2.24 ± 0.36 vs.1.04 ± 0.10,P < 0.01 ; 1.51 ± 0.12 vs.0.85 ± 0.30,P < 0.05) and lower expression on blood vessels in biopsies with CAV (2.24 ±0.36 vs.0.25 ±0.08,P <0.01 ; 1.51 ±0.12 vs.0.16 ± 0.10,P < 0.01).Conclusion The less expression of HSP27 and GRP78 on blood vessels decreased the defense to immunologic injury,which stimulated the over-proliferation of vascular

  20. Cells

    Directory of Open Access Journals (Sweden)

    Zhao-Hui Jin


    Full Text Available As cancer stem cells (CSCs are postulated to play critical roles in cancer development, including metastasis and recurrence, CSC imaging would provide valuable information for cancer treatment and lead to CSC-targeted therapy. To assess the possibility of in vivo CSC targeting, we conducted basic studies on radioimmunotargeting of cancer cells positive for CD133, a CSC marker recognized in various cancers. Antibodies against CD133 were labeled with 125I, and their in vitro cell binding properties were tested. Using the same isotype IgG as a control, in vivo biodistribution of the labeled antibody retaining immunoreactivity was examined in mice bearing an HCT116 xenograft in which a population of the cancer cells expressed CD133. Intratumoral distribution of the labeled antibody was examined and compared to the CD133 expression pattern. The 125I-labeled anti-CD133 antibody showed a modest but significantly higher accumulation in the HCT116 xenograft compared to the control IgG. The intratumoral distribution of the labeled antibody mostly overlapped with the CD133 expression, whereas the control IgG was found in the area close to the necrotic tumor center. Our results indicate that noninvasive in vivo targeting of CSCs could be possible with radiolabeled antibodies against cell membrane markers.

  1. Male tawny dragons use throat patterns to recognize rivals (United States)

    Osborne, Louise; Umbers, Kate D. L.; Backwell, Patricia R. Y.; Keogh, J. Scott


    The ability to distinguish between familiar and unfamiliar conspecifics is important for many animals, especially territorial species since it allows them to avoid unnecessary interactions with individuals that pose little threat. There are very few studies, however, that identify the proximate cues that facilitate such recognition in visual systems. Here, we show that in tawny dragons ( Ctenophorus decresii), males can recognize familiar and unfamiliar conspecific males based on morphological features alone, without the aid of chemical or behavioural cues. We further show that it is the colour pattern of the throat patches (gular) that facilitates this recognition.

  2. Recognizing and Treating Malaria in U.S. Residents

    Centers for Disease Control (CDC) Podcasts


    This podcast is an overview of the Clinician Outreach and Communication Activity (COCA) Call: It's a Small World After All: Dengue and Malaria in U.S. Residents - Recognizing and Treating These Mosquito-borne Diseases. CDC's David Townes discusses clinical presentation, transmission, prevention strategies, new treatments, and malaria resources available to health care providers.  Created: 6/9/2010 by Division of Parasitic Diseases and Malaria, Center for Global Health and Emergency Communication System (ECS)/Joint Information Center (JIC); Office of Public Health Preparedness and Response (OPHPR).   Date Released: 6/15/2010.

  3. [Idiopathic environmental intolerance: 2 disabling entities to recognize]. (United States)

    Bensefa-Colas, Lynda; Dupas, Dominique


    Idiopathic environmental intolerance is characterized by a variety of non-specific symptoms involving several organs within the same individual, and attributed to the exposure to chemical odors (multiple chemical sensitivities) or to the exposure to electromagnetic fields (electromagnetic hypersensitivity). Symptoms occur following an exposure to agents generally regarded as harmless due to the low levels of exposure, and they do not answer to any definition of organic diseases. The lack of established etiology renders treatment difficult. It is important for practitioner to recognize such disorders and assess the social and professional impact so as to improve patients' quality of life.

  4. New efficient algorithm for recognizing handwritten Hindi digits (United States)

    El-Sonbaty, Yasser; Ismail, Mohammed A.; Karoui, Kamal


    In this paper a new algorithm for recognizing handwritten Hindi digits is proposed. The proposed algorithm is based on using the topological characteristics combined with statistical properties of the given digits in order to extract a set of features that can be used in the process of digit classification. 10,000 handwritten digits are used in the experimental results. 1100 digits are used for training and another 5500 unseen digits are used for testing. The recognition rate has reached 97.56%, a substitution rate of 1.822%, and a rejection rate of 0.618%.

  5. Antibodies recognizing both IgM isotypes in Atlantic salmon

    DEFF Research Database (Denmark)

    Hedfors, Ida Aagård; Bakke, Hege; Skjødt, Karsten


    these molecules. The present study aimed at identifying tools to separate IgM positive (IgM(+)) B cells from IgM negative (IgM(-)) non-B cell populations using flow cytometry. Several monoclonal antibodies (mAbs), and one polyclonal antibody (pAb) to both rainbow trout (Oncorhynchus mykiss) and Atlantic salmon...... defined, mostly due to the lack of appropriate working tools like antibodies and functional assays. Membrane bound molecules like immunoglobulins (Ig) serve as cell surface markers for specific cell subsets and the identification of cells relies upon the production of specific antibodies towards...... of IgM(+) cells in the respective tissues in salmon. To our surprise, this seemingly simple task did not reveal similar staining patterns for all antibodies as expected, but rather large differences in the number of positively stained cells were discovered. In short, positively stained cells by each...

  6. Monoclonal Antibodies Recognizing HIV-1 gp41 Could Inhibit Env-Mediated Syncytium Formation

    Institute of Scientific and Technical Information of China (English)

    ZHANG Geng; CHEN Yinghua


    Some monoclonal antibodies (mAbs) could inhibit infection by HIV-1. In this study, four mAbs against HIV-1 gp41 were prepared in mice. All four mAbs could bind to the recombinant soluble gp41 and recognize the native envelope glycoprotein gp160 expressed on the HIV-Env+ CHO-WT cell in flow cytometry analysis. Interestingly, the results show that all four mAbs purified by affinity chromatography could inhibit HIV-1 Env-mediated membrane fusion (syncytium formation) by 40%-60% at 10 μg/mL, which implies potential inhibitory activities against HIV-1.

  7. Recognizing and predicting movement effects: identifying critical movement features. (United States)

    Cañal-Bruland, Rouwen; Williams, A Mark


    It is not clear whether the critical features used to discriminate movements are identical to those involved in predicting the same movement's effects and consequently, whether the mechanisms underlying recognition and anticipation differ. We examined whether people rely on different kinematic information when required to recognize differences in the movement pattern in comparison to when they have to anticipate the outcome of these same movements. Naïve participants were presented with paired presentations of point-light animated tennis shots that ended at racket-ball contact. We instructed them either to judge whether the movements observed were the same or different or to predict shot direction (left vs. right). In addition, we locally manipulated the kinematics of point-light figures in an effort to identify the critical features used when making recognition and anticipation judgments. It appears that observers rely on different sources of information when required to recognize movement differences compared to when they need to anticipate the outcome of the same observed movements. Findings are discussed with reference to recent ideas focusing on the role of perceptual and motor resonance in perceptual judgments.

  8. Patient Selection in Plastic Surgery: Recognizing Body Dysmorphic Disorder

    Directory of Open Access Journals (Sweden)

    Cihan Sahin


    Full Text Available Plastic surgery is a branch of medicine that provides significant improvements to the people with positive changes. But first of all, this branch has a characteristic which requires analysing patients' psychological situation very carefully. Plastic surgeons are often confronted by patients with mental disorders seeking aesthetic surgery. It is imperative for surgeons to recognize possible underlying psychiatric illnesses. Common psychiatric conditions seen in cosmetic surgery patients include body dysmorphic disorder (BDD, narcissistic personality disorder and histrionic personality disorders. BDD is of particular importance to plastic surgeons. Because outrageous dissatisfaction with one's appearance may conceal psychopathologic traits that are not always easily recognizable, and which, if neglected, may result in serious iatrogenic and medicolegal consequences, we hope that this paper will help plastic surgeons in ultimately preventing patient and surgeon dissatisfaction within the population of patients with psychiatric disorders, and should recognize the diagnostic features of body dysmorphic disorder and screen psychologically unstable patients who may never be satisfied with surgery. [Arch Clin Exp Surg 2013; 2(2.000: 109-115

  9. Novel Moment Features Extraction for Recognizing Handwritten Arabic Letters

    Directory of Open Access Journals (Sweden)

    Gheith Abandah


    Full Text Available Problem statement: Offline recognition of handwritten Arabic text awaits accurate recognition solutions. Most of the Arabic letters have secondary components that are important in recognizing these letters. However these components have large writing variations. We targeted enhancing the feature extraction stage in recognizing handwritten Arabic text. Approach: In this study, we proposed a novel feature extraction approach of handwritten Arabic letters. Pre-segmented letters were first partitioned into main body and secondary components. Then moment features were extracted from the whole letter as well as from the main body and the secondary components. Using multi-objective genetic algorithm, efficient feature subsets were selected. Finally, various feature subsets were evaluated according to their classification error using an SVM classifier. Results: The proposed approach improved the classification error in all cases studied. For example, the improvements of 20-feature subsets of normalized central moments and Zernike moments were 15 and 10%, respectively. Conclusion/Recommendations: Extracting and selecting statistical features from handwritten Arabic letters, their main bodies and their secondary components provided feature subsets that give higher recognition accuracies compared to the subsets of the whole letters alone.

  10. MR Imaging Findings in Children with First Recognized Seizure (United States)

    Kalnin, Andrew J.; Fastenau, Philip S.; deGrauw, Ton J.; Musick, Beverly S.; Perkins, Susan M.; Johnson, Cynthia S.; Mathews, Vincent P.; Egelhoff, John C.; Dunn, David W.; Austin, Joan K.


    This study characterized structural abnormalities associated with onset of seizures in children using MRI and a standardized classification system in a large prospective cohort. A total of 281 children aged 6 to 14 years completed an MRI within six months of their first recognized seizure. Most MRI examinations were performed with a standardized, dedicated seizure protocol, and all were scored using a standard scoring system. At least one MRI abnormality was identified in 87 of the 281 (31%) of children with first recognized seizure. Two or more abnormalities were identified in 34 (12%). The most common abnormalities were ventricular enlargement (51%), leukomalacia/gliosis (23%), gray matter lesions such as heterotopias and cortical dysplasia (12%), volume loss (12%), various other white matter lesions (9%), and encephalomalacia (6%). Abnormalities defined as significant, or potentially related to seizures, occurred in 40 (14%). Temporal lobe and hippocampal abnormalities were detected at a higher frequency than in previous studies (13/87). Use of MRI and a standardized reliable and valid scoring system demonstrated a higher rate of abnormal findings than previous investigations, including findings that might have been considered incidental in the past. Practice parameters may need to be revised to expand the definition of significant abnormalities and to support wider use of MRI in children with newly diagnosed seizures. PMID:19027586

  11. Recognizing induced emotions of happiness and sadness from dance movement.

    Directory of Open Access Journals (Sweden)

    Edith Van Dyck

    Full Text Available Recent research revealed that emotional content can be successfully decoded from human dance movement. Most previous studies made use of videos of actors or dancers portraying emotions through choreography. The current study applies emotion induction techniques and free movement in order to examine the recognition of emotional content from dance. Observers (N = 30 watched a set of silent videos showing depersonalized avatars of dancers moving to an emotionally neutral musical stimulus after emotions of either sadness or happiness had been induced. Each of the video clips consisted of two dance performances which were presented side-by-side and were played simultaneously; one of a dancer in the happy condition and one of the same individual in the sad condition. After every film clip, the observers were asked to make forced-choices concerning the emotional state of the dancer. Results revealed that observers were able to identify the emotional state of the dancers with a high degree of accuracy. Moreover, emotions were more often recognized for female dancers than for their male counterparts. In addition, the results of eye tracking measurements unveiled that observers primarily focus on movements of the chest when decoding emotional information from dance movement. The findings of our study show that not merely portrayed emotions, but also induced emotions can be successfully recognized from free dance movement.

  12. On learning to recognize spectrally reduced speech: II. Individual differences (United States)

    Chiu, Peter; Eng, Michelle; Strange, Bethany; Yampolsky, Sasha; Waters, Gloria


    When patients with cochlear implants attempt speech recognition, or when listeners with normal hearing try to recognize spectrally reduced speech, performance may suffer because of additional demands on cognitive resources imposed by sensory degradation. Some previous studies have reported a significant correlation between digit span and recognition of spectrally reduced speech, but others have not [Eisenberg et al., J. Acoust. Soc. Am. 107, 2704-2710 (2000)]. In this study, we administered 3 separate measures of working memory capacity (i.e., digit span, alphabet span, and sentence span) and 1 measure of reading ability (American National Adult Reading Test, ANART) to normal-hearing listeners who participated in our previous studies [Chiu et al., J. Acoust. Soc. Am. 109, 2501 (2001) and Fishbeck and Chiu, J. Acoust. Soc. Am. 109, 2504 (2001)]. Preliminary data show that the listener's ability to recognize spectrally reduced speech shows the strongest relationship with ANART scores and the weakest relationship with digit span. Implications of the current findings on theories of speech recognition and working memory will be discussed. [Work supported by URC, Univ. of Cincinnati.

  13. Recognizing Bharatnatyam Mudra Using Principles of Gesture Recognition

    Directory of Open Access Journals (Sweden)

    Shweta Mozarkar


    Full Text Available A primary goal of gesture recognition research is to create a system which can identify specific human gestures and use them to convey information for the device control. Gesture Recognition is interpreting human gestures via mathematical algorithms. Indian classical Dance uses the expressive gestures called Mudra as a supporting visual mode of communication with the audience. These mudras are expressive meaningful (static or dynamic positions of body parts. This project attempts to recognize the mudra sequence using Image-processing and Pattern Recognition techniques and link the result to understand the corresponding expressions of the Indian classical dance via interpretation of few static Bharatnatyam Mudras. Here, a novel approach of computer aided recognition of Bharatnatyam Mudras is proposed using the saliency technique which uses the hypercomplex representation (i.e., quaternion Fourier Transform of the image, to highlight the object from background and in order to get the salient features of the static double hand mudra image. K Nearest Neighbor algorithm is used for classification. The entry giving the minimum difference for all the mudra features is the match for the given input image. Finally emotional description for the recognized mudra image is displayed.

  14. Recognizing age-separated face images: humans and machines. (United States)

    Yadav, Daksha; Singh, Richa; Vatsa, Mayank; Noore, Afzel


    Humans utilize facial appearance, gender, expression, aging pattern, and other ancillary information to recognize individuals. It is interesting to observe how humans perceive facial age. Analyzing these properties can help in understanding the phenomenon of facial aging and incorporating the findings can help in designing effective algorithms. Such a study has two components--facial age estimation and age-separated face recognition. Age estimation involves predicting the age of an individual given his/her facial image. On the other hand, age-separated face recognition consists of recognizing an individual given his/her age-separated images. In this research, we investigate which facial cues are utilized by humans for estimating the age of people belonging to various age groups along with analyzing the effect of one's gender, age, and ethnicity on age estimation skills. We also analyze how various facial regions such as binocular and mouth regions influence age estimation and recognition capabilities. Finally, we propose an age-invariant face recognition algorithm that incorporates the knowledge learned from these observations. Key observations of our research are: (1) the age group of newborns and toddlers is easiest to estimate, (2) gender and ethnicity do not affect the judgment of age group estimation, (3) face as a global feature, is essential to achieve good performance in age-separated face recognition, and (4) the proposed algorithm yields improved recognition performance compared to existing algorithms and also outperforms a commercial system in the young image as probe scenario.

  15. Recognizing age-separated face images: humans and machines.

    Directory of Open Access Journals (Sweden)

    Daksha Yadav

    Full Text Available Humans utilize facial appearance, gender, expression, aging pattern, and other ancillary information to recognize individuals. It is interesting to observe how humans perceive facial age. Analyzing these properties can help in understanding the phenomenon of facial aging and incorporating the findings can help in designing effective algorithms. Such a study has two components--facial age estimation and age-separated face recognition. Age estimation involves predicting the age of an individual given his/her facial image. On the other hand, age-separated face recognition consists of recognizing an individual given his/her age-separated images. In this research, we investigate which facial cues are utilized by humans for estimating the age of people belonging to various age groups along with analyzing the effect of one's gender, age, and ethnicity on age estimation skills. We also analyze how various facial regions such as binocular and mouth regions influence age estimation and recognition capabilities. Finally, we propose an age-invariant face recognition algorithm that incorporates the knowledge learned from these observations. Key observations of our research are: (1 the age group of newborns and toddlers is easiest to estimate, (2 gender and ethnicity do not affect the judgment of age group estimation, (3 face as a global feature, is essential to achieve good performance in age-separated face recognition, and (4 the proposed algorithm yields improved recognition performance compared to existing algorithms and also outperforms a commercial system in the young image as probe scenario.


    Directory of Open Access Journals (Sweden)

    Selvanayaki K.S


    Full Text Available Nowadays, automatic detection of text from the vehicles is an important problem in many applications. Text information present in an image can be easily understood by both human and computer. It has wide applications such as license plate reading, sign detection, identification of destination places, mobile text recognition and so on. This problem is challenging due to complex backgrounds, the non-uniform illuminations, variations of text font, size and line orientation. Once the text is identified, it can be analyzed, recognized and interpreted. Hence, there is a need for a better algorithm for detection and localization of text from vehicles. A method is proposed for detecting text from vehicles. The method makes use of features such as Histogram of oriented Gradients (HOG and Local Binary Pattern (LBP. These features are stored which can be further used for feature matching at the time of classification. After the text region is being detected, it can be further subjected to character segmentation and recognition thereby identifying the destination places. The ability to recognize text area from the vehicles, especially buses has obvious applications like traffic management in the bus stands. The obtained results are verified and performance parameters like speed, precision and recall are determined.

  17. Antibodies recognizing different domains of the polymeric immunoglobulin receptor. (United States)

    Solari, R; Kühn, L; Kraehenbuhl, J P


    The receptor responsible for the transepithelial transport of IgA dimer antibodies is a transmembrane glycoprotein known as membrane secretory component (SCm). During transport, the membrane anchoring domain is cleaved and the ectoplasmic domain of the receptor (SCs) remains tightly bound to the IgA dimer in exosecretions. We have produced monoclonal antibodies with distinct specificities against both cytoplasmic and ectoplasmic epitopes of rabbit SCm. One antibody (anti-SC303) reacted both with SCm and free SCs but not with SCs bound to IgA dimer (SIgA). Therefore, it recognized an epitope close to the IgA dimer binding site. The other monoclonal antibody (anti-SC166), which was unable to react with SCs, bound to the 15-kDa cytoplasmic extension of the membrane-spanning domain of the receptor. A polyclonal antibody (GaR-SC), raised in a goat against rabbit milk SCs, reacted with a subpopulation of SCs not recognized by the anti-SC303 monoclonal antibody and in addition also reacted with covalently bound sIgA. The three antibodies cross-reacted with rat SCm. We demonstrate the ability of the anti-SC166 monoclonal antibody to immunoadsorb subcellular organelles as a result of the cytoplasmic orientation of its epitope. Our data indicate that there are functional differences between the high- and low-molecular-weight families of SC in terms of IgA dimer binding.

  18. Recognizing articulated objects and object articulation in SAR images (United States)

    Bhanu, Bir; Jones, Grinnell, III; Ahn, Joon S.


    The focus of this paper is recognizing articulated objects and the pose of the articulated parts in SAR images. Using SAR scattering center locations as features, the invariance with articulation (i.e. turret rotation for the T72, T80 and M1a tanks, missile erect vs. down for the SCUD launcher) is shown as a function of object azimuth. Similar data is shown for configuration differences in the MSTAR (Public) Targets. The UCR model-based recognition engine (which uses non- articulated models to recognize articulated, occluded and non-standard configuration objects) is described and target identification performance results are given as confusion matrices and ROC curves for six inch and one foot resolution XPATCH images and the one foot resolution MSTAR data. Separate body and turret models are developed that are independent of the relative positions between the body and the turret. These models are used with a subsequent matching technique to refine the pose of the body and determine the pose of the turret. An expression of the probability that a random match will occur is derived and this function is used to set thresholds to minimize the probability of a random match for the recognition system. Results for identification, body pose and turret pose are presented as a function of percent occlusion for articulated XPATCH data and results are given for identification and body pose for articulated MSTAR data.

  19. Recognizing articulated objects using a region-based invariant transform. (United States)

    Weiss, Isaac; Ray, Manjit


    In this paper, we present a new method for representing and recognizing objects, based on invariants of the object's regions. We apply the method to articulated objects in low-resolution, noisy range images. Articulated objects such as a back-hoe can have many degrees of freedom, in addition to the unknown variables of viewpoint. Recognizing such an object in an image can involve a search in a high-dimensional space that involves all these unknown variables. Here, we use invariance to reduce this search space to a manageable size. The low resolution of our range images makes it hard to use common features such as edges to find invariants. We have thus developed a new "featureless" method that does not depend on feature detection. Instead of local features, we deal with whole regions of the object. We define a "transform" that converts the image into an invariant representation on a grid, based on invariant descriptors of entire regions centered around the grid points. We use these region-based invariants for indexing and recognition. While the focus here is on articulation, the method can be easily applied to other problems such as the occlusion of fixed objects.

  20. Endoplasmic reticulum stress is involved in podocyte apoptosis induced by saturated fatty acid palmitate

    Institute of Scientific and Technical Information of China (English)

    TAO Jian-ling; WEN Yu-bing; SHI Bing-yang; ZHANG Hong; RUAN Xiong-zhong; LI Hang; LI Xue-mei; DONG Wen-ji; LI Xue-wang


    Background Podocyte apoptosis is recently indicated as an early phenomenon of diabetic nephropathy.Pancreatic β-cells exposed to saturated free fatty acid palmitate undergo irreversible endoplasmic reticulum (ER) stress and consequent apoptosis,contributing to the onset of diabetes.We hypothesized that palmitate could induce podocyte apoptosis via ER stress,which initiates or aggravates proteinuria in diabetic nephropathy.Methods Podocyte apoptosis was detected by 4',6-diamidio-2-phenylindole (DAPI) stained apoptotic cell count and Annexin V-PI stain.The expressions of ER molecule chaperone glucose-regulated protein 78 (GRP78),indicators of ER-associated apoptosis C/EBP homologous protein (CHOP),and Bcl-2 were assayed by Western blotting and real-time PCR.GRP78 and synaptopodin were co-localized by immunofluorescence stain.Results Palmitate significantly increased the percentage of cultured apoptotic murine podocytes time-dependently when loading 0.75 mmol/L (10 hours,13 hours,and 15 hours compared with 0 hour,P <0.001) and dose-dependently when loading palmitate ranging from 0.25 to 1.00 mmol/L for 15 hours (compared to control,P <0.001).Palmitate time-dependently and dose-dependently increased the protein expression of GRP78 and CHOP,and decreased that of Bcl-2.Palmitate loading ranging from 0.5 to 1.0 mmol/L for 12 hours significantly increased mRNA of GRP78 and CHOP,and decreased that of Bcl-2 compared to control (P <0.001),with the maximum concentration being 0.75 mmol/L.Palmitate 0.5 mmol/L loading for 3 hours,8 hours,and 12 hours significantly increased mRNA of GRP78 and CHOP,and decreased that of Bcl-2 compared to 0 hour (P <0.001),with the maximum effect at 3 hours.Confocal microscopy demonstrated that GRP78 expression was significantly increased when exposed to 0.5 mmol/L of palmitate for 8 hours compared to control.Conclusion Palmitate could induce podocyte apoptosis via ER stress,suggesting podocyte apoptosis and consequent proteinuria caused

  1. Review of melanoma antigens recognized by monoclonal antibodies (MAbs). Their functional significance and applications in diagnosis and treatment of melanoma. (United States)

    Hersey, P


    The introduction of monoclonal antibody techniques has led to a rapid advance in information concerning antigenic structures in melanoma cell membranes. These have been classified according to the extent of their expression on cells of other tissues, but it is evident that a more precise classification based on their biochemical nature is possible. Several monoclonal antibodies appear to define antigens restricted to melanoma cells and fetal tissues. Many antibodies recognize antigens shared with gliomas and nevi, whereas other groups can be defined which recognize antigens on melanocytes or other carcinomas. One of the commonly detected antigens was shown to be a high molecular weight (MW) proteoglycan which may be involved in reactions with other cells and the intercellular matrix. A second antigen was shown to be a ganglioside which may have receptor functions in cells. A third was shown to be a glycoprotein with iron transport functions. The latter antigen and the large MW proteoglycan have been a focus of attention for in vivo targeting studies in treatment and diagnosis. The ganglioside, large MW proteoglycan and a melanocarcinoma antigen may be detected in the circulation of patients and are being evaluated for monitoring of disease activity in patients with melanoma. Several monoclonals may be of value in histological evaluation of melanoma, e.g. diagnosis of preneoplastic lesions, metastatic lesions of unknown origin and identification of cell structures related to metastatic behaviour in the host. Further studies should help to define cellular structures recognized by the immune system in humans.

  2. Cytotoxicity of CdTe quantum dots in human umbilical vein endothelial cells: the involvement of cellular uptake and induction of pro-apoptotic endoplasmic reticulum stress. (United States)

    Yan, Ming; Zhang, Yun; Qin, Haiyan; Liu, Kezhou; Guo, Miao; Ge, Yakun; Xu, Mingen; Sun, Yonghong; Zheng, Xiaoxiang


    Cadmium telluride quantum dots (CdTe QDs) have been proposed to induce oxidative stress, which plays a crucial role in CdTe QDs-mediated mitochondrial-dependent apoptosis in human umbilical vein endothelial cells (HUVECs). However, the direct interactions of CdTe QDs with HUVECs and their potential impairment of other organelles like endoplasmic reticulum (ER) in HUVECs are poorly understood. In this study, we reported that the negatively charged CdTe QDs (-21.63±0.91 mV), with good dispersity and fluorescence stability, were rapidly internalized via endocytosis by HUVECs, as the notable internalization could be inhibited up to 95.52% by energy depletion (NaN3/deoxyglucose or low temperature). The endocytosis inhibitors (methyl-β-cyclodextrin, genistein, sucrose, chlorpromazine, and colchicine) dramatically decreased the uptake of CdTe QDs by HUVECs, suggesting that both caveolae/raft- and clathrin-mediated endocytosis were involved in the endothelial uptake of CdTe QDs. Using immunocytochemistry, a striking overlap of the internalized CdTe QDs and ER marker was observed, which indicates that QDs may be transported to ER. The CdTe QDs also caused remarkable ER stress responses in HUVECs, confirmed by significant dilatation of ER cisternae, upregulation of ER stress markers GRP78/GRP94, and activation of protein kinase RNA-like ER kinase-eIF2α-activating transcription factor 4 pathway (including phosphorylation of both protein kinase RNA-like ER kinase and eIF2α and elevated level of activating transcription factor 4). CdTe QDs further promoted an increased C/EBP homologous protein expression, phosphorylation of c-JUN NH2-terminal kinase, and cleavage of ER-resident caspase-4, while the specific inhibitor (SP600125, Z-LEVD-fmk, or salubrinal) significantly attenuated QDs-triggered apoptosis, indicating that all three ER stress-mediated apoptosis pathways were activated and the direct participation of ER in the CdTe QDs-caused apoptotic cell death in HUVECs. Our

  3. ATF6 as a Transcription Activator of the Endoplasmic Reticulum Stress Element: Thapsigargin Stress-Induced Changes and Synergistic Interactions with NF-Y and YY1


    Li, Mingqing; Baumeister, Peter; Roy, Binayak; Phan, Trevor; Foti, Dolly; Luo, Shengzhan; Lee, Amy S.


    ATF6, a member of the leucine zipper protein family, can constitutively induce the promoter of glucose-regulated protein (grp) genes through activation of the endoplasmic reticulum (ER) stress element (ERSE). To understand the mechanism of grp78 induction by ATF6 in cells subjected to ER calcium depletion stress mediated by thapsigargin (Tg) treatment, we discovered that ATF6 itself undergoes Tg stress-induced changes. In nonstressed cells, ATF6, which contains a putative short transmembrane ...

  4. A Novel Handwritten Letter Recognizer Using Enhanced Evolutionary Neural Network (United States)

    Mahmoudi, Fariborz; Mirzashaeri, Mohsen; Shahamatnia, Ehsan; Faridnia, Saed

    This paper introduces a novel design for handwritten letter recognition by employing a hybrid back-propagation neural network with an enhanced evolutionary algorithm. Feeding the neural network consists of a new approach which is invariant to translation, rotation, and scaling of input letters. Evolutionary algorithm is used for the global search of the search space and the back-propagation algorithm is used for the local search. The results have been computed by implementing this approach for recognizing 26 English capital letters in the handwritings of different people. The computational results show that the neural network reaches very satisfying results with relatively scarce input data and a promising performance improvement in convergence of the hybrid evolutionary back-propagation algorithms is exhibited.

  5. An NC algorithm for recognizing tree adjoining languages

    Energy Technology Data Exchange (ETDEWEB)

    Pali, M.A. (New Jersey Institute of Technology, Newark, NJ (United States)); Shende, S.M. (Univ. of Nebraska, Lincoln, NE (United States))


    A parallel algorithm is presented for recognizing the class of languages generated by tree adjoining grammars, a tree rewriting system which has applications in natural language processing. This class of languages is known to properly include all context-free languages; for example, the noncontext-free sets [l brace]a'b'c[r brace] and [l brace]ww[r brace] are in this class. It is shown that the recognition problem for tree adjoining languages can be solved by a concurrent read, concurrent write parallel random-assess machine (CRCW PRAM) in O(log n) time using polynomially many processors. Thus, the class of tree adjoining languages is in AC[sup 1] and hence in NC. This extends a previous result for context-free languages.

  6. Combined pulmonary fibrosis and emphysema: an increasingly recognized condition

    Directory of Open Access Journals (Sweden)

    Olívia Meira Dias


    Full Text Available Combined pulmonary fibrosis and emphysema (CPFE has been increasingly recognized in the literature. Patients with CPFE are usually heavy smokers or former smokers with concomitant lower lobe fibrosis and upper lobe emphysema on chest HRCT scans. They commonly present with severe breathlessness and low DLCO, despite spirometry showing relatively preserved lung volumes. Moderate to severe pulmonary arterial hypertension is common in such patients, who are also at an increased risk of developing lung cancer. Unfortunately, there is currently no effective treatment for CPFE. In this review, we discuss the current knowledge of the pathogenesis, clinical characteristics, and prognostic factors of CPFE. Given that most of the published data on CPFE are based on retrospective analysis, more studies are needed in order to address the role of emphysema and its subtypes; the progression of fibrosis/emphysema and its correlation with inflammation; treatment options; and prognosis.

  7. Serendipitous learning: Recognizing and fostering the potential of microblogging

    Directory of Open Access Journals (Sweden)

    Ilona Buchem


    Full Text Available This paper introduces the concept of serendipitous learning in the context of microblogging and discusses the potential of unplanned and unexpected discoveries for learning. Serendipitous learning as a subset of incidental learning refers to learning through gaining new insights, discovering unrevealed aspects and recognizing seemingly unrelated connections. This type of learning can occur by chance and as a by-product of other activities like information browsing through social status updates in microblogs. It is argued that engaging in microblogging in an open social network of users frequently generating new information enhances the possibilities of serendipitous discovery. The paper discusses possible factors facilitating serendipitous learning and concludes with recommendations for future research.

  8. Recognizing and managing the immunologic reactions in leprosy. (United States)

    Kamath, Sonia; Vaccaro, Seth A; Rea, Thomas H; Ochoa, Maria T


    Immunologic reactions are an important aspect of leprosy that significantly impacts the course of the disease and the associated disability. Reversal reaction (type 1), erythema nodosum leprosum (type 2), and Lucio phenomenon are the 3 leprosy reactions, and they are most commonly seen in patients with the lepromatous and borderline categories of the disease. Because these forms of leprosy are the most common types seen in the United States, it is particularly important for physicians to be able to recognize and treat them. The reactions may occur before, during, or after treatment with multidrug therapy. Reversal reactions are the most common cause of nerve damage in leprosy, and erythema nodosum leprosum may also lead to neuritis. Although there have not been enough studies to confirm the most effective management regimens, treatment of reversal reaction and Lucio phenomenon with prednisone and of erythema nodosum leprosum with thalidomide and/or prednisone may help improve symptoms and prevent further disability.

  9. In vivo amelioration of endogenous antitumor autoantibodies via low-dose P4N through the LTA4H/activin A/BAFF pathway. (United States)

    Lin, Yu-Ling; Tsai, Nu-Man; Hsieh, Cheng-Hao; Ho, Shu-Yi; Chang, Jung; Wu, Hsin-Yi; Hsu, Ming-Hua; Chang, Chia-Ching; Liao, Kuang-Wen; Jackson, Tiffany L B; Mold, David E; Huang, Ru Chih C


    Cancer progression is associated with the development of antitumor autoantibodies in patients' sera. Although passive treatment with antitumor antibodies has exhibited remarkable therapeutic efficacy, inhibitory effects on tumor progression by endogenous antitumor autoantibodies (EAAs) have been limited. In this study, we show that P4N, a derivative of the plant lignan nordihydroguaiaretic acid (NDGA), enhanced the production of EAAs and inhibited tumor growth at low noncytotoxic concentrations via its immunoregulatory activity. Intratumoral injection of P4N improved the quantity and quality of EAAs, and passive transfer of P4N-induced EAAs dramatically suppressed lung metastasis formation and prolonged the survival of mice inoculated with metastatic CT26 tumor cells. P4N-induced EAAs specifically recognized two surface antigens, 78-kDa glucose-regulated protein (GRP78) and F1F0 ATP synthase, on the plasma membrane of cancer cells. Additionally, P4N treatment led to B-cell proliferation, differentiation to plasma cells, and high titers of autoantibody production. By serial induction of autocrine and paracrine signals in monocytes, P4N increased B-cell proliferation and antibody production via the leukotriene A4 hydrolase (LTA4H)/activin A/B-cell activating factor (BAFF) pathway. This mechanism provides a useful platform for studying and seeking a novel immunomodulator that can be applied in targeting therapy by improving the quantity and quality of the EAAs.

  10. Predefined GPGRAFY-Epitope-Specific Monoclonal Antibodies with Different Activities for Recognizing Native HIV-1 gp120

    Institute of Scientific and Technical Information of China (English)

    蓝灿辉; 田海军; 陈应华


    A seven-amino acid epitope GPGRAFY at the tip of the V3 loop in HIV-1 gp120 is the principal neutralizing epitope,and a subset of anti-V3 antibodies specific for this epitope shows a broad range of neutralizing activity.GPGRAFY-epitope-specific neutralizing antibodies were produced using predefined GPGRAFY-epitope-specific peptides instead of a natural or recombinant gp120 bearing this epitope.All six monoclonal antibodies (mAbs) could recognize the GPGRAFY-epitope on peptides and two of the antibodies,9D8 and 2D7,could recognize recombinant gp120 in enzymelinked immunosorkentassy (ELISA) assays.In the flow cytometry analysis,the mAbs 9D8 and 2D7 could bind to HIV-Env+ CHO-WT cells and the specific bindings could be inhibited by the GPGRAFY-epitope peptide,which suggests that these two mAbs could recognize the native envelope protein gp120 expressed on the cell membrane.However,in syncytium assays,none of the mAbs was capable of inhibiting HIV-Env-mediated cell membrane fusion.The different activities for recognizing native HIV-1 gp120 might be associated with different antibody affinities against the epitopes.The development of conformational mimics of the neutralization epitope in the gp120 V3 loop could elicit neutralizing mAbs with high affinity.

  11. The ability of IgY to recognize surface proteins of Streptococcus mutans

    Directory of Open Access Journals (Sweden)

    Basri A. Gani


    Full Text Available Background: Streptococcus mutans are gram positive bacteria classified into viridians group, and have a role in pathogenesis of dental caries. It’s adhesion to the tooth surface is mediated by cell surface proteins, which interact with specific receptor located in tooth pellicle. Glucan binding protein, Glukosyltransferase, and antigen I/II are basic proteins of S. mutans, which have a role in initiating the interaction. A previous study showed that chicken’s IgY can interfere the interaction. Purpose: The objective of this study was to assess the ability of IgY in recognizing the surface molecule of Streptococcus mutans expressed by various serotypes (c, d, e, f and a strain derived from IPB, Bogor. Method: Western blot was used as a method to determine such capability. Result: The result showed that IgY has a potency to recognize antigen I/II, but not the other proteins on the cell surface of all bacteria tested. Conclusion: The ability of IgY to bind the surface protein, antigen I/II, indicates that this avian antibody could be used as a candidate for anti-adhesion in preventing dental caries.

  12. Autoantibodies from patients with primary biliary cirrhosis recognize a region within the nucleoplasmic domain of inner nuclear membrane protein LBR. (United States)

    Lin, F; Noyer, C M; Ye, Q; Courvalin, J C; Worman, H J


    Autoantibodies from rare patients with primary biliary cirrhosis (PBC) recognize LBR, or lamin B receptor, an integral membrane protein of the inner nuclear membrane. Human LBR has a nucleoplasmic, amino-terminal domain of 208 amino acids followed by a carboxyl-terminal domain with eight putative transmembrane segments. Autoantibodies against LBR from four patients with PBC recognized the nucleoplasmic, amino-terminal domain but not the carboxyl-terminal domain. Immunoblotting of smaller fusion proteins demonstrated that these autoantibodies recognized a conformational epitope(s) contained within the stretch of amino acids from 1 to 60. These results, combined with those of previous studies, show that autoepitopes of nuclear membrane proteins are located within their nucleocytoplasmic domains and that autoantibodies from patients with PBC predominantly react with one domain of a protein antigen. This work also provides further characterization of anti-LBR antibodies that have found utility as reagents in cell biology research.

  13. Characterization of CTL Recognized Epitopes on Human Breast Tumors (United States)


    25. Christinck, E.R., Luscher , M.A., Barber, B.H., and Williams, D.B. (1991) Peptide binding to class I MHC on living cells and quantitation of...ER, Luscher MA, Barber BH, and Williams DB. Peptide binding to class I MHC on living cells and quantitation of complexes required for CTL lysis

  14. Identification of the epitopes on HCV core protein recognized by HLA-A2 restricted cytotoxic T lymphocytes

    Institute of Scientific and Technical Information of China (English)

    Hong-Chao Zhou; De-Zhong Xu; Xue-Ping Wang; Jing-Xia Zhang; Ying-Huang; Yong-Ping Yan; Yong Zhu; Bo-Quan Jin


    AIM To identify hepatitis C virus (HCV) core protein epitopes recognized by HLA-A2 restricted cytotoxic T lymphocyte (CTL). METHODS Utilizing the method of computer prediction followed by a 4 h 51 Cr-release assay confirmation. RESULTS The results showed that peripheral blood mononuclear cells (PBMC) obtained from two HLA-A2 positive donors who were infected with HCV could lyse autologous target cells labeled with peptide "ALAHGVFAL (core TS0-158)".The rates of specific lysis of the cells from the two donors were 37.5% and 15.8%,respectively. Blocking of the CTL response with anti-CD4 mAb caused no significant decrease of the specific lysis.But blocking of CTL response with anti-CD8 mAb could abolish the Iysis. CONCLUSION The peptide (core 150 - 158 ) is the candidate epitope recognized by HLA-A2 restricted CTL.

  15. The human immune system recognizes neopeptides derived from mitochondrial DNA deletions. (United States)

    Duvvuri, Bhargavi; Duvvuri, Venkata R; Wang, Chao; Chen, Lina; Wagar, Lisa E; Jamnik, Veronica; Wu, Jianhong; Yeung, Rae S M; Grigull, Jörg; Watts, Tania H; Wu, Gillian E


    Mutations in mitochondrial (mt) DNA accumulate with age and can result in the generation of neopeptides. Immune surveillance of such neopeptides may allow suboptimal mitochondria to be eliminated, thereby avoiding mt-related diseases, but may also contribute to autoimmunity in susceptible individuals. To date, the direct recognition of neo-mtpeptides by the adaptive immune system has not been demonstrated. In this study we used bioinformatics approaches to predict MHC binding of neopeptides identified from known deletions in mtDNA. Six such peptides were confirmed experimentally to bind to HLA-A*02. Pre-existing human CD4(+) and CD8(+) T cells from healthy donors were shown to recognize and respond to these neopeptides. One remarkably promiscuous immunodominant peptide (P9) could be presented by diverse MHC molecules to CD4(+) and/or CD8(+) T cells from 75% of the healthy donors tested. The common soil microbe, Bacillus pumilus, encodes a 9-mer that differs by one amino acid from P9. Similarly, the ATP synthase F0 subunit 6 from normal human mitochondria encodes a 9-mer with a single amino acid difference from P9 with 89% homology to P9. T cells expanded from human PBMCs using the B. pumilus or self-mt peptide bound to P9/HLA-A2 tetramers, arguing for cross-reactivity between T cells with specificity for self and foreign homologs of the altered mt peptide. These findings provide proof of principal that the immune system can recognize peptides arising from spontaneous somatic mutations and that such responses might be primed by foreign peptides and/or be cross-reactive with self.

  16. A deep convolutional neural network for recognizing foods (United States)

    Jahani Heravi, Elnaz; Habibi Aghdam, Hamed; Puig, Domenec


    Controlling the food intake is an efficient way that each person can undertake to tackle the obesity problem in countries worldwide. This is achievable by developing a smartphone application that is able to recognize foods and compute their calories. State-of-art methods are chiefly based on hand-crafted feature extraction methods such as HOG and Gabor. Recent advances in large-scale object recognition datasets such as ImageNet have revealed that deep Convolutional Neural Networks (CNN) possess more representation power than the hand-crafted features. The main challenge with CNNs is to find the appropriate architecture for each problem. In this paper, we propose a deep CNN which consists of 769; 988 parameters. Our experiments show that the proposed CNN outperforms the state-of-art methods and improves the best result of traditional methods 17%. Moreover, using an ensemble of two CNNs that have been trained two different times, we are able to improve the classification performance 21:5%.

  17. Feature Engineering for Recognizing Adverse Drug Reactions from Twitter Posts

    Directory of Open Access Journals (Sweden)

    Hong-Jie Dai


    Full Text Available Social media platforms are emerging digital communication channels that provide an easy way for common people to share their health and medication experiences online. With more people discussing their health information online publicly, social media platforms present a rich source of information for exploring adverse drug reactions (ADRs. ADRs are major public health problems that result in deaths and hospitalizations of millions of people. Unfortunately, not all ADRs are identified before a drug is made available in the market. In this study, an ADR event monitoring system is developed which can recognize ADR mentions from a tweet and classify its assertion. We explored several entity recognition features, feature conjunctions, and feature selection and analyzed their characteristics and impacts on the recognition of ADRs, which have never been studied previously. The results demonstrate that the entity recognition performance for ADR can achieve an F-score of 0.562 on the PSB Social Media Mining shared task dataset, which outperforms the partial-matching-based method by 0.122. After feature selection, the F-score can be further improved by 0.026. This novel technique of text mining utilizing shared online social media data will open an array of opportunities for researchers to explore various health related issues.

  18. Structural basis of Zika virus helicase in recognizing its substrates

    Directory of Open Access Journals (Sweden)

    Hongliang Tian


    Full Text Available Abstract The recent explosive outbreak of Zika virus (ZIKV infection has been reported in South and Central America and the Caribbean. Neonatal microcephaly associated with ZIKV infection has already caused a public health emergency of international concern. No specific vaccines or drugs are currently available to treat ZIKV infection. The ZIKV helicase, which plays a pivotal role in viral RNA replication, is an attractive target for therapy. We determined the crystal structures of ZIKV helicase-ATP-Mn2+ and ZIKV helicase-RNA. This is the first structure of any flavivirus helicase bound to ATP. Comparisons with related flavivirus helicases have shown that although the critical P-loop in the active site has variable conformations among different species, it adopts an identical mode to recognize ATP/Mn2+. The structure of ZIKV helicase-RNA has revealed that upon RNA binding, rotations of the motor domains can cause significant conformational changes. Strikingly, although ZIKV and dengue virus (DENV apo-helicases share conserved residues for RNA binding, their different manners of motor domain rotations result in distinct individual modes for RNA recognition. It suggests that flavivirus helicases could have evolved a conserved engine to convert chemical energy from nucleoside triphosphate to mechanical energy for RNA unwinding, but different motor domain rotations result in variable RNA recognition modes to adapt to individual viral replication.

  19. Recognizing Presentations of Pemphigoid Gestationis: A Case Study

    Directory of Open Access Journals (Sweden)

    Sadie Henry


    Full Text Available Introduction. Pemphigoid gestationis (PG is an autoimmune blistering disease that occurs in approximately 1 in 50,000 pregnancies. Failing to recognize PG may lead to inadequate maternal treatment and possible neonatal complications. Case Report. At 18 weeks of gestation, a 36-year-old otherwise healthy Caucasian G4P1 presented with pruritic papules on her anterior thighs, initially treated with topical steroids. At 31 weeks of gestation, she was switched to oral steroids after her rash and pruritus worsened. The patient had an uncomplicated SVD of a healthy female infant at 37 weeks of gestation and was immediately tapered off steroid treatment, resulting in a severe postpartum flare of her disease. Discussion. This case was similar to reported cases of pruritic urticarial papules followed by blisters; however, this patient had palm, sole, and mucous membrane involvement, which is rare. Biopsy for direct immunofluorescence or ELISA is the preferred test for diagnosis. Previous case reports describe severe postdelivery flares that require higher steroid doses. Obstetrical providers need to be familiar with this disease although it is rare, as this condition can be easily confused with other dermatoses of pregnancy. Adequate treatment is imperative for the physical and psychological well-being of the mother and infant.

  20. Recognizing and interpreting gestures on a mobile robot

    Energy Technology Data Exchange (ETDEWEB)

    Kortenkamp, D.; Huber, E.; Bonasso, R.P. [Metrica, Inc., NASA Johnson Space Center, Houston, TX (United States)


    Gesture recognition is an important skill for robots that work closely with humans. Gestures help to clarify spoken commands and are a compact means of relaying geometric information. We have developed a real-time, three-dimensional gesture recognition system that resides on-board a mobile robot. Using a coarse three-dimensional model of a human to guide stereo measurements of body parts, the system is capable of recognizing six distinct gestures made by an unadorned human in an unaltered environment. An active vision approach focuses the vision system`s attention on small, moving areas of space to allow for frame rate processing even when the person and/or the robot are moving. This paper describes the gesture recognition system, including the coarse model and the active vision approach. This paper also describes how the gesture recognition system is integrated with an intelligent control architecture to allow for complex gesture interpretation and complex robot action. Results from experiments with an actual mobile robot are given.

  1. Recognizing sights, smells, and sounds with gnostic fields.

    Directory of Open Access Journals (Sweden)

    Christopher Kanan

    Full Text Available Mammals rely on vision, audition, and olfaction to remotely sense stimuli in their environment. Determining how the mammalian brain uses this sensory information to recognize objects has been one of the major goals of psychology and neuroscience. Likewise, researchers in computer vision, machine audition, and machine olfaction have endeavored to discover good algorithms for stimulus classification. Almost 50 years ago, the neuroscientist Jerzy Konorski proposed a theoretical model in his final monograph in which competing sets of "gnostic" neurons sitting atop sensory processing hierarchies enabled stimuli to be robustly categorized, despite variations in their presentation. Much of what Konorski hypothesized has been remarkably accurate, and neurons with gnostic-like properties have been discovered in visual, aural, and olfactory brain regions. Surprisingly, there have not been any attempts to directly transform his theoretical model into a computational one. Here, I describe the first computational implementation of Konorski's theory. The model is not domain specific, and it surpasses the best machine learning algorithms on challenging image, music, and olfactory classification tasks, while also being simpler. My results suggest that criticisms of exemplar-based models of object recognition as being computationally intractable due to limited neural resources are unfounded.

  2. Recognizing genes and other components of genomic structure

    Energy Technology Data Exchange (ETDEWEB)

    Burks, C. (Los Alamos National Lab., NM (USA)); Myers, E. (Arizona Univ., Tucson, AZ (USA). Dept. of Computer Science); Stormo, G.D. (Colorado Univ., Boulder, CO (USA). Dept. of Molecular, Cellular and Developmental Biology)


    The Aspen Center for Physics (ACP) sponsored a three-week workshop, with 26 scientists participating, from 28 May to 15 June, 1990. The workshop, entitled Recognizing Genes and Other Components of Genomic Structure, focussed on discussion of current needs and future strategies for developing the ability to identify and predict the presence of complex functional units on sequenced, but otherwise uncharacterized, genomic DNA. We addressed the need for computationally-based, automatic tools for synthesizing available data about individual consensus sequences and local compositional patterns into the composite objects (e.g., genes) that are -- as composite entities -- the true object of interest when scanning DNA sequences. The workshop was structured to promote sustained informal contact and exchange of expertise between molecular biologists, computer scientists, and mathematicians. No participant stayed for less than one week, and most attended for two or three weeks. Computers, software, and databases were available for use as electronic blackboards'' and as the basis for collaborative exploration of ideas being discussed and developed at the workshop. 23 refs., 2 tabs.

  3. Allelic barley MLA immune receptors recognize sequence-unrelated avirulence effectors of the powdery mildew pathogen. (United States)

    Lu, Xunli; Kracher, Barbara; Saur, Isabel M L; Bauer, Saskia; Ellwood, Simon R; Wise, Roger; Yaeno, Takashi; Maekawa, Takaki; Schulze-Lefert, Paul


    Disease-resistance genes encoding intracellular nucleotide-binding domain and leucine-rich repeat proteins (NLRs) are key components of the plant innate immune system and typically detect the presence of isolate-specific avirulence (AVR) effectors from pathogens. NLR genes define the fastest-evolving gene family of flowering plants and are often arranged in gene clusters containing multiple paralogs, contributing to copy number and allele-specific NLR variation within a host species. Barley mildew resistance locus a (Mla) has been subject to extensive functional diversification, resulting in allelic resistance specificities each recognizing a cognate, but largely unidentified, AVRa gene of the powdery mildew fungus, Blumeria graminis f. sp. hordei (Bgh). We applied a transcriptome-wide association study among 17 Bgh isolates containing different AVRa genes and identified AVRa1 and AVRa13, encoding candidate-secreted effectors recognized by Mla1 and Mla13 alleles, respectively. Transient expression of the effector genes in barley leaves or protoplasts was sufficient to trigger Mla1 or Mla13 allele-specific cell death, a hallmark of NLR receptor-mediated immunity. AVRa1 and AVRa13 are phylogenetically unrelated, demonstrating that certain allelic MLA receptors evolved to recognize sequence-unrelated effectors. They are ancient effectors because corresponding loci are present in wheat powdery mildew. AVRA1 recognition by barley MLA1 is retained in transgenic Arabidopsis, indicating that AVRA1 directly binds MLA1 or that its recognition involves an evolutionarily conserved host target of AVRA1 Furthermore, analysis of transcriptome-wide sequence variation among the Bgh isolates provides evidence for Bgh population structure that is partially linked to geographic isolation.

  4. Aspects of abuse: recognizing and responding to child maltreatment. (United States)

    Jackson, Allison M; Kissoon, Natalie; Greene, Christian


    Child maltreatment is a public health problem and toxic stress impacting at least 1 in 8 children by the age of 18 years. Maltreatment can take the form of physical and sexual abuse, neglect, and emotional maltreatment. While some children may experience only one form of maltreatment, others may survive multiple forms, and in some cases particularly complex forms of maltreatment such as torture and medical child abuse. When considering maltreatment, providers should be adept at obtaining a thorough history not only from the parent but when appropriate also from the patient. The most common form of child maltreatment is neglect, which encompasses nutritional and medical neglect, as well as other forms such as physical and emotional neglect. Talking with caregivers about stressors and barriers to care may give insight into the etiology for neglect and is an opportunity for the provider to offer or refer for needed assistance. Familiarity with injury patterns and distribution in the context of developmental milestones and injury mechanisms is critical to the recognition of physical abuse. While most anogenital exam results of child victims of sexual abuse are normal, knowing the normal variations for the female genitalia, and thereby recognizing abnormal findings, is important not only forensically but also more importantly for patient care. Pattern recognition does not only apply to specific injuries or constellation of injuries but also applies to patterns of behavior. Harmful patterns of behavior include psychological maltreatment and medical child abuse, both of which cause significant harm to patients. As health professionals serving children and families, pediatric providers are in a unique position to identify suspected maltreatment and intervene through the health care system in order to manage the physical and psychological consequences of maltreatment and to promote the safety and well-being of children and youth by making referrals to child protective

  5. Novel monoclonal antibodies recognizing different subsets of lymphocytes from the common marmoset (Callithrix jacchus). (United States)

    Ito, Ryoji; Maekawa, Shin-ichiro; Kawai, Kenji; Suemizu, Hiroshi; Suzuki, Shuzo; Ishii, Hajime; Tanioka, Yoshikuni; Satake, Masanobu; Yagita, Hideo; Habu, Sonoko; Ito, Mamoru


    Callithrix jacchus, the common marmoset, is a small new world primate that is considered effective as an experimental animal model for various human diseases. In this study, we generated monoclonal antibodies (mAbs) against common marmoset lymphocytes for immunological studies on the common marmoset. We established five hybridoma clones, 6C9, 10D7, 6F10, 7A4 and 5A1, producing anti-marmoset mAbs against cell surface antigens on marmoset T and/or B lymphocytes. We confirmed that 6C9 and 10D7 antibodies recognized CD45 antigen, and 6F10 antibody recognized CD8 antigen by flow cytometry using marmoset cDNA transfectants. We also tested them for application of immunoprecipitation, Western blot analysis and immunohistochemistry. We found that immunohistochemistry using marmoset spleen sections could be applied with all established mAbs but immunoprecipitation and the Western blot analysis could be applied with 6F10 and 10D7 antibodies but not with the other three mAbs. These results show that these monoclonal antibodies are useful for advancing immunological research on the common marmoset.

  6. The D-6 mouse monoclonal antibody recognizes the CD74 cytoplasmic tail. (United States)

    Genève, Laetitia; Gauthier, Catherine; Thibodeau, Jacques


    The invariant chain (Ii; CD74) is a multifunctional protein of the immune system and a major player in the presentation of exogenous antigens to T cells. In the endoplasmic reticulum (ER), Ii assists the folding and trafficking of MHC class II molecules. In the present study, we characterized the recently commercialized D-6 monoclonal antibody (MAb) made against a polypeptide spanning the entire sequence of the p33 isoform of human Ii. Using transgenic mice expressing the human p35 isoform, we showed by flow cytometry that D-6 only slightly cross-reacts with mouse Ii in permeabilized splenocytes. Analysis of the human B lymphoblastoid cell line LG2 revealed that D-6 recognizes Ii only upon membrane permeabilization. Variants of Ii bearing specific mutations or deletions were transfected in human cells to map the D-6 epitope. Our results showed that this MAb binds to the N-terminal cytoplasmic domain of Ii and that the epitope was destroyed upon mutagenesis of the two leucine-based endosomal targeting motifs. Thus, D-6 cannot be used for rapid flow cytometric assessment of CD74 cell surface expression and would be ineffective as a drug conjugate for the treatment of hematological malignancies.

  7. 76 FR 59247 - Environmental Impact Considerations, Food Additives, and Generally Recognized As Safe Substances... (United States)


    ... Environmental Impact Considerations, Food Additives, and Generally Recognized As Safe Substances; Technical... considerations, food additives, and generally recognized as safe (GRAS) substances to correct minor errors in the... affecting certain regulations regarding environmental impact considerations (part 25), food additives...

  8. Lifting as We Climb: Recognizing Intersectional Gender Violence in Law

    Directory of Open Access Journals (Sweden)

    Shreya Atrey


    Full Text Available This paper interrogates the meaning of lifting all women as we climb the ladder of gender equality and justice by recognizing that gender violence affects women differently. This is because violence against women is perpetrated not only on the basis of their gender or sex but also other identities of race, religion, caste, region, age, disability, nationality, sexual orientation etc. With reference to CEDAW jurisprudence and examples from India, I seek to explain this understanding with the help of a normative framework of ‘intersectional integrity’. The framework insists on considering claimants as a whole by tracing unique and shared patterns of gender violence when it is also based on other identities such as race, religion, caste, region, age, disability, nationality, and sexual orientation. I argue that applying the framework allows us to diagnose and address the nature of violence suffered on multiple identities, in a clear and comprehensive way. Este artículo cuestiona el sentido de levantar a todas las mujeres a medida que se asciende la escalera de la igualdad de género y la justicia, reconociendo que la violencia de género afecta a las mujeres de manera diferente. Esto se debe a que la violencia contra las mujeres se comete no sólo sobre la base de su género o sexo, sino también por su raza, religión, casta, región, edad, discapacidad, nacionalidad, orientación sexual, etc. Se pretende explicar esta afirmación con la ayuda de un marco normativo de “integridad interseccional”, a través de referencias a la jurisprudencia del CEDAW y ejemplos de la India. El marco insiste en considerar a las demandantes en su conjunto, trazando patrones únicos y compartidos de violencia de género cuando se basa también en otras identidades como raza, religión, casta, región, edad, discapacidad, nacionalidad, orientación sexual. Se sostiene que la aplicación del marco permite diagnosticar y abordar la naturaleza de la violencia

  9. Guanxinkang Decoction Exerts Its Antiatherosclerotic Effect Partly through Inhibiting the Endoplasmic Reticulum Stress

    Directory of Open Access Journals (Sweden)

    Hao Wang


    Full Text Available Purpose. To investigate the antiatherosclerotic effect of Guanxinkang (GXK decoction on the apoptosis, mitochondrial membrane potential (MMP, and endoplasmic reticulum stress (ERS of human umbilical vein endothelial cells (HUVEC pretreated with homocysteinemia (HCY. Materials and Methods. HUVEC were randomly divided into 5 groups: (1 blank control group (control, (2 model control group (model, (3 GXK low dose group, (4 GXK medium dose group, and (5 GXK high dose group. For the three GXK groups, HCY was given to reach the concentration of 3.0 mmol/L after HUVEC had been incubated with rabbit serum containing GXK for two hours. At 3, 6, 12, and 24 h after HCY had been incubated with the cells, the HUVEC were collected for test of the apoptosis rate, MMP, and GRP78 protein (reflecting ERS. Results. In the model control group, the apoptosis rate and GRP 78 protein expression of HUVEC significantly increased (P<0.05, while MMP significantly decreased (P<0.05 compared with the blank control group. After GXK treatment of medium and high doses, the apoptosis rate and the GRP 78 protein expression significantly (P<0.05 decreased, while MMP significantly increased (P<0.05 in a time-dependent manner compared with the model control group. Conclusion. GXK can antagonize the injury of HUVEC caused by HCY and the antagonism effect increases with the concentration and treatment duration of GXK, with the possible mechanism of GXK antagonism being through inhibiting ERS caused by HCY.

  10. Region-specific vulnerability to endoplasmic reticulum stress-induced neuronal death in rat brain after status epilepticus

    Indian Academy of Sciences (India)

    Jing Chen; Hu Guo; Guo Zheng; Zhong-Nan Shi


    We sought to clarify the involvement and the intra-cerebral distribution variability of C/EBP homologous protein (CHOP), a representative molecule related to endoplasmic reticulum (ER) stress-induced cell death signalling pathways, in neuronal death resulting from status epilepticus in rats. The expression patterns of CHOP and glucose-regulated protein (GRP) 78, a good marker of ER stress, were assessed by Western blotting, real-time PCR, Hoechst and immunohistochemistry in the hippocampus, cortex and striatum on a status epilepticus (SE) model. Double-fluorescent staining of CHOP and the terminal deoxynucleotidyl transferase-mediated DNA nick-end labelling (TUNEL) method were performed to clarify the involvement of CHOP in cell death. SE resulted in a time-dependent increase in the expression of GRP78 and CHOP. The expression of GRP78 protein was increased at 3, 6 and 12 h after SE and no brain region variability was found. The expression of CHOP protein was also increased, reached its peak at 24 h and remained high at 48 h. CHOP protein expression, however, showed brain region variability with highest expression noted in the hippocampus followed by the striatum, and lowest in the cortex. The up-regulation of CHOP occurring at the transcriptional level was demonstrated by real-time PCR. Double fluorescence showed that CHOP expression strongly correlated with neurons undergoing apoptosis. The results indicated that SE compromises the function of the ER and that the hippocampus is more vulnerable than the cortex and the striatum.

  11. Propofol Prevents Renal Ischemia-Reperfusion Injury via Inhibiting the Oxidative Stress Pathways

    Directory of Open Access Journals (Sweden)

    Yingjie Li


    Full Text Available Background/Aims: Renal ischemia/reperfusion injury (IRI is a risk for acute renal failure and delayed graft function in renal transplantation and cardiac surgery. The purpose of this study is to determine whether propofol could attenuate renal IRI and explore related mechanism. Methods: Male rat right kidney was removed, left kidney was subjected to IRI. Propofol was intravenously injected into rats before ischemia. The kidney morphology and renal function were analyzed. The expression of Bax, Bcl-2, caspase-3, cl-caspase-3, GRP78, CHOP and caspase-12 were detected by Western blot analysis. Results: IR rats with propofol pretreatment had better renal function and less tubular apoptosis than untreated IR rats. Propofol pretreated IR rats had lower Bax/Bcl-2 ratio and less cleaved caspase-3. The protein expression levels of GRP78, CHOP and caspase-12 decreased significantly in propofol pretreated IR rats. In vitro cell model showed that propofol significantly increased the viability of NRK-52E cells that were subjected to hypoxia/reoxygenation (H/R in a dose-dependent manner. The effect of propofol on the expression regulation of Bax, Bcl-2, caspase-3, GRP78, CHOP was consistent in both in vitro and in vivo models. Conclusion: Experimental results suggest that propofol prevents renal IRI via inhibiting oxidative stress.

  12. The galactose-recognizing system of rat peritoneal macrophages; identification and characterization of the receptor molecule. (United States)

    Kelm, S; Schauer, R


    Resident rat peritoneal macrophages express a galactose-recognizing system, which mediates binding and uptake of cells and glycoproteins exposing terminal galactose residues. Here we describe the identification, isolation, and characterization of the corresponding receptor molecule. Using photoaffinity labelling of adherent peritoneal macrophages with the 4-azido-6-125I-salicylic acid derivative of anti-freeze glycoprotein 8 followed by SDS-PAGE and autoradiography, we identified the receptor of these cells as a protein with an apparent molecular mass of 42 kDa. Furthermore, cell surface receptors were radioiodinated by an affinity-supported labelling technique using the conjugate of asialoorosomucoid and lactoperoxidase, followed by extraction and isolation by affinity chromatography. Finally, the native receptor was isolated and analysed. To estimate its binding activity in solutions, a suitable binding assay was developed, using the precipitation of receptor-ligand complex with polyethylene glycol to separate bound from unbound 125I-asialoorosomucoid, which was used as ligand. It is shown that the isolated receptor binds to galactose-exposing particles and distinguishes between sialidase-treated and -untreated erythrocytes, similar to peritoneal macrophages. The binding characteristics of the membrane-bound and the solubilized receptor are described in the following paper of Lee et al.

  13. Circulating microparticles carry oxidation-specific epitopes and are recognized by natural IgM antibodies. (United States)

    Tsiantoulas, Dimitrios; Perkmann, Thomas; Afonyushkin, Taras; Mangold, Andreas; Prohaska, Thomas A; Papac-Milicevic, Nikolina; Millischer, Vincent; Bartel, Caroline; Hörkkö, Sohvi; Boulanger, Chantal M; Tsimikas, Sotirios; Fischer, Michael B; Witztum, Joseph L; Lang, Irene M; Binder, Christoph J


    Oxidation-specific epitopes (OSEs) present on apoptotic cells and oxidized low density lipoprotein (OxLDL) represent danger-associated molecular patterns that are recognized by different arcs of innate immunity, including natural IgM antibodies. Here, we investigated whether circulating microparticles (MPs), which are small membrane vesicles released by apoptotic or activated cells, are physiological carriers of OSEs. OSEs on circulating MPs isolated from healthy donors and patients with ST-segment elevation myocardial infarction (STE-MI) were characterized by flow cytometry using a panel of OSE-specific monoclonal antibodies. We found that a subset of MPs carry OSEs on their surface, predominantly malondialdehyde (MDA) epitopes. Consistent with this, a majority of IgM antibodies bound on the surface of circulating MPs were found to have specificity for MDA-modified LDL. Moreover, we show that MPs can stimulate THP-1 (human acute monocytic leukemia cell line) and human primary monocytes to produce interleukin 8, which can be inhibited by a monoclonal IgM with specificity for MDA epitopes. Finally, we show that MDA(+) MPs are elevated at the culprit lesion site of patients with STE-MI. Our results identify a subset of OSE(+) MPs that are bound by OxLDL-specific IgM. These findings demonstrate a novel mechanism by which anti-OxLDL IgM antibodies could mediate protective functions in CVD.

  14. Distinct oligoclonal band antibodies in multiple sclerosis recognize ubiquitous self-proteins. (United States)

    Brändle, Simone M; Obermeier, Birgit; Senel, Makbule; Bruder, Jessica; Mentele, Reinhard; Khademi, Mohsen; Olsson, Tomas; Tumani, Hayrettin; Kristoferitsch, Wolfgang; Lottspeich, Friedrich; Wekerle, Hartmut; Hohlfeld, Reinhard; Dornmair, Klaus


    Oligoclonal Ig bands (OCBs) of the cerebrospinal fluid are a hallmark of multiple sclerosis (MS), a disabling inflammatory disease of the central nervous system (CNS). OCBs are locally produced by clonally expanded antigen-experienced B cells and therefore are believed to hold an important clue to the pathogenesis. However, their target antigens have remained unknown, mainly because it was thus far not possible to isolate distinct OCBs against a background of polyclonal antibodies. To overcome this obstacle, we copurified disulfide-linked Ig heavy and light chains from distinct OCBs for concurrent analysis by mass spectrometry and aligned patient-specific peptides to corresponding transcriptome databases. This method revealed the full-length sequences of matching chains from distinct OCBs, allowing for antigen searches using recombinant OCB antibodies. As validation, we demonstrate that an OCB antibody from a patient with an infectious CNS disorder, neuroborreliosis, recognized a Borrelia protein. Next, we produced six recombinant antibodies from four MS patients and identified three different autoantigens. All of them are conformational epitopes of ubiquitous intracellular proteins not specific to brain tissue. Our findings indicate that the B-cell response in MS is heterogeneous and partly directed against intracellular autoantigens released during tissue destruction. In addition to helping elucidate the role of B cells in MS, our approach allows the identification of target antigens of OCB antibodies in other neuroinflammatory diseases and the production of therapeutic antibodies in infectious CNS diseases.

  15. Recognize and classify pneumoconiosis; Pneumokoniosen erkennen und klassifizieren

    Energy Technology Data Exchange (ETDEWEB)

    Hering, K.G.; Hofmann-Preiss, K. [Klinikum Westfalen, Knappschaftskrankenhaus, Dortmund (Germany)


    In the year 2012, out of the 10 most frequently recognized occupational diseases 6 were forms of pneumoconiosis. With respect to healthcare and economic aspects, silicosis and asbestos-associated diseases are of foremost importance. The latter are to be found everywhere and are not restricted to large industrial areas. Radiology has a central role in the diagnosis and evaluation of occupational lung disorders. In cases of known exposure mainly to asbestos and quartz, the diagnosis of pneumoconiosis, with few exceptions will be established primarily by the radiological findings. As these disorders are asymptomatic for a long time they are quite often detected as incidental findings in examinations for other reasons. Therefore, radiologists have to be familiar with the pattern of findings of the most frequent forms of pneumoconiosis and the differential diagnoses. For reasons of equal treatment of the insured a quality-based, standardized performance, documentation and evaluation of radiological examinations is required in preventive procedures and evaluations. Above all, a standardized low-dose protocol has to be used in computed tomography (CT) examinations, although individualized concerning the dose, in order to keep radiation exposure as low as possible for the patient. The International Labour Office (ILO) classification for the coding of chest X-rays and the international classification of occupational and environmental respiratory diseases (ICOERD) classification used since 2004 for CT examinations meet the requirements of the insured and the occupational insurance associations as a means of reproducible and comparable data for decision-making. (orig.) [German] Im Jahr 2012 waren 6 der 10 am haeufigsten anerkannten Berufskrankheiten Pneumokoniosen. Gesundheitspolitisch und oekonomisch stehen dabei die Silikose und asbestassoziierte Erkrankungen im Vordergrund. Insbesondere Letztere treten ubiquitaer auf und sind nicht an grosse Industriestandorte gebunden

  16. Selenoprotein S/SEPS1 modifies endoplasmic reticulum stress in Z variant alpha1-antitrypsin deficiency.

    LENUS (Irish Health Repository)

    Kelly, Emer


    Z alpha(1)-antitrypsin (ZAAT) deficiency is a disease associated with emphysematous lung disease and also with liver disease. The liver disease of AAT deficiency is associated with endoplasmic reticulum (ER) stress. SEPS1 is a selenoprotein that, through a chaperone activity, decreases ER stress. To determine the effect of SEPS1 on ER stress in ZAAT deficiency, we measured activity of the grp78 promoter and levels of active ATF6 as markers of the unfolded protein response in HepG2 cells transfected with the mutant form of AAT, a ZAAT transgene. We evaluated levels of NFkappaB activity as a marker of the ER overload response. To determine the effect of selenium supplementation on the function of SEPS1, we investigated glutathione peroxidase activity, grp78 promoter activity, and NFkappaB activity in the presence or absence of selenium. SEPS1 reduced levels of active ATF6. Overexpression of SEPS1 also inhibited grp78 promoter and NFkappaB activity, and this effect was enhanced in the presence of selenium supplementation. This finding demonstrates a role for SEPS1 in ZAAT deficiency and suggests a possible therapeutic potential for selenium supplementation.

  17. Flavivirus-cross-reactive, HLA-DR15-restricted epitope on NS3 recognized by human CD4+ CD8- cytotoxic T lymphocyte clones. (United States)

    Kurane, I; Okamoto, Y; Dai, L C; Zeng, L L; Brinton, M A; Ennis, F A


    The role of flavivirus-cross-reactive T lymphocytes in recovery from and pathogenesis of flavivirus infections is not known. In the present paper, we have defined a flavivirus-cross-reactive epitope recognized by two CD4+ CD8- cytotoxic T lymphocyte (CTL) clones, JK4 and JK43. The T cell clones were established from the peripheral blood T lymphocytes of a dengue-4-immune donor, using a limiting-dilution method with dengue-4 antigen. These two T cell clones were cross-reactive for dengue virus types 1, 2, 3 and 4, yellow fever virus and West Nile virus, and recognized NS3 protein. The smallest synthetic peptide recognized by these T cell clones was an identical 9 amino acid peptide which contains amino acids 146 to 154 (VIGLYGNGV) of dengue-4 NS3. HLA-DR15 was the restriction allele for recognition of this epitope by JK4 and JK43. JK4 and JK43 both used T cell receptor V alpha 8, but JK4 used V beta 8 and JK43 used V beta 2. This result indicates that this epitope is recognized by two flavivirus-cross-reactive CD4+ T cell clones which originated from different T cells in vivo.

  18. Gastric siderosis: An under-recognized and rare clinical entity

    Directory of Open Access Journals (Sweden)

    Jiten P Kothadia


    Full Text Available The increased deposition of iron in gastric mucosa is known as gastric siderosis. It is believed that the only regulated step of the iron metabolism cycle occurs during absorption in the small intestine. Once this system becomes overwhelmed due to either local or widespread iron levels, then iron can be absorbed very quickly by a passive concentration-dependent mechanism. This excess iron is initially stored in the liver but later can be found in the pancreas, heart and joints. Excess iron is not expected to deposit in the gastric mucosa. This gastric deposition has been found in association with hemochromatosis, oral iron medications, alcohol abuse, blood transfusions, hepatic cirrhosis and spontaneous portacaval shunt with esophageal varices. The precise mechanism of this iron deposition in gastric epithelial and stromal cells is still not well understood; thus, identification of iron in gastric mucosa raises many questions. On histology, the pattern of deposition is variable, and recognition of the pattern is often useful to choose the appropriate workup for the patient and to diagnose and possibly treat the cause of iron overload. In this article, we have described a well-referenced review of this rare clinical entity with different histological patterns, diagnostic tests and the clinical significance of the different patterns of iron deposition.

  19. Recognizing Immunoglobulin G4-Related Overlap Syndromes in Patients with Pancreatic and Hepatobiliary Diseases

    Directory of Open Access Journals (Sweden)

    Aldo J Montano-Loza


    Full Text Available The first description of autoimmune pancreatitis and elevated serum immunoglobulin-G4 (IgG4 in 2001 heralded further reports of several related autoimmune diseases with raised IgG4 levels. It is now recognized that a spectrum of overlap syndromes associated with increased IgG4 and biopsy evidence of IgG4-producing plasma cells, which has now been convincingly linked with cholangitis, autoimmune hepatitis, Sjögren’s syndrome, nephritis and retroperitoneal fibrosis. Collectively, this disease cluster is referred to as IgG4-related systemic disease. The importance of making the correct diagnosis is underscored by the management of individuals with IgG4-related systemic disease. In the first instance, patients generally have a dramatic response to immunosuppressive therapy, whereas patients with other forms of cholangitis and pancreatitis do not. Also, surgical management of pancreatic malignancy can be avoided once the correct diagnosis of IgG4-related disease has been made. In the present review, an overview of the current information regarding the role of IgG4 and IgG4-positive cells affecting the biliary system, pancreas and liver is provided.

  20. The natural antibody repertoire of sharks and humans recognizes the potential universe of antigens. (United States)

    Adelman, Miranda K; Schluter, Samuel F; Marchalonis, John J


    In ancestral sharks, a rapid emergence in the evolution of the immune system occurred, giving jawed-vertebrates the necessary components for the combinatorial immune response (CIR). To compare the natural antibody (NAb) repertoires of the most divergent vertebrates with the capacity to produce antibodies, we isolated NAbs to the same set of antigens by affinity chromatography from two species of Carcharhine sharks and from human polyclonal IgG and IgM antibody preparations. The activities of the affinity-purified anti-T-cell receptor (anti-TCR) NAbs were compared with those of monoclonal anti-TCR NAbs that were generated from a systemic lupus erythematosus patient. We report that sharks and humans, representing the evolutionary extremes of vertebrate species sharing the CIR, have NAbs to human TCRs, Igs, the human senescent cell antigen, and to numerous retroviral antigens, indicating that essential features of the combinatorial repertoire and the capacity to recognize the potential universe of antigens is shared among all jawed-vertebrates.

  1. 内质网应激PERK途径在高糖诱导血管平滑肌细胞向成骨样细胞转化中的作用%PERK pathway in transformation of smooth muscle cells to osteoblast-like cells induced by high glucose

    Institute of Scientific and Technical Information of China (English)

    申红远; 白静; 汤喆; 梁伟; 马清华; 刘秀华; 王禹


    目的:研究高糖刺激SD大鼠胸主动脉血管平滑肌细胞(VSMCs)向成骨样细胞转分化中内质网应激蛋白激酶R样内质网激酶(PERK)途径的作用。方法原代培养VSMCs,分为正常对照组(5mmol/L D-葡萄糖),甘露醇组(5mmol/L D-葡萄糖+25mmol/L甘露醇),高糖组(30mmol/L D-葡萄糖),高糖+PERK-小干扰RNA(siRNA)转染组(30mmol/L D-葡萄糖+PERK-siRNA转染),高糖+RNA干扰阴性对照组(30mmol/L D-葡萄糖+乱序RNA转染)。分别作用48和72h。逆转录-聚合酶链反应及Western blot分别从信使RNA(mRNA)和蛋白质水平检测不同时间点内质网应激相关分子GRP78、PERK、p-PERK、eIF2α、p-eIF2α、ATF4的改变,以及成骨样细胞标志性分子核心转录因子(Cbfα-1)、骨钙素以及平滑肌细胞标志性分子α-SMA的改变情况。应用碱性磷酸酶(ALP)活性检测试剂盒检测ALP的活性。结果与正常对照组和甘露醇组比较,在mRNA水平,高糖组和高糖+RNA干扰阴性对照组PERK和eIF2α表达升高(P<0.05),PERK-siRNA转染后其表达水平均降低(P<0.05),在蛋白质水平,PERK、p-PERK、eIF2α、p-eIF2α表达升高(P<0.05),PERK-siRNA干预后其表达均降低(P<0.05)。高糖刺激VSMCs后ALP活性升高(P<0.05),PERK-siRNA转染后其表达均降低(P<0.05)。结论内质网应激PERK途径在高糖诱导VSMCs由收缩表型向成骨样细胞表型转化中发挥作用,抑制PERK途径可以部分阻止这一转化过程。%Objective To investigate the role of endoplasmic reticulum stress protein kinase R-like endoplasmic reticulum kinase (PERK) pathway in the transdifferentiation of SD rat aortic vascular smooth muscle cells (VSMCs) to osteoblast-like cells by high glucose. Methods The primary cultured VSMCs from rat’s aortic segments were divided into 5 groups, including normal control group (5mmol/L D-glucose), mannitol group (5mmol/L D

  2. Doublecortin Is Excluded from Growing Microtubule Ends and Recognizes the GDP-Microtubule Lattice. (United States)

    Ettinger, Andreas; van Haren, Jeffrey; Ribeiro, Susana A; Wittmann, Torsten


    Many microtubule (MT) functions are mediated by a diverse class of proteins (+TIPs) at growing MT plus ends that control intracellular MT interactions and dynamics and depend on end-binding proteins (EBs) [1]. Cryoelectron microscopy has recently identified the EB binding site as the interface of four tubulin dimers that undergoes a conformational change in response to β-tubulin GTP hydrolysis [2, 3]. Doublecortin (DCX), a MT-associated protein (MAP) required for neuronal migration during cortical development [4, 5], binds to the same site as EBs [6], and recent in vitro studies proposed DCX localization to growing MT ends independent of EBs [7]. Because this conflicts with observations in neurons [8, 9] and the molecular function of DCX is not well understood, we revisited intracellular DCX dynamics at low expression levels. Here, we report that DCX is not a +TIP in cells but, on the contrary, is excluded from the EB1 domain. In addition, we find that DCX-MT interactions are highly sensitive to MT geometry. In cells, DCX binding was greatly reduced at MT segments with high local curvature. Remarkably, this geometry-dependent binding to MTs was completely reversed in the presence of taxanes, which reconciles incompatible observations in cells [9] and in vitro [10]. We propose a model explaining DCX specificity for different MT geometries based on structural changes induced by GTP hydrolysis that decreases the spacing between adjacent tubulin dimers [11]. Our data are consistent with a unique mode of MT interaction in which DCX specifically recognizes this compacted GDP-like MT lattice.

  3. Bullous pemphigoid and linear IgA dermatosis sera recognize a similar 120-kDa keratinocyte collagenous glycoprotein with antigenic cross-reactivity to BP180

    NARCIS (Netherlands)

    Pas, HH; Kloosterhuis, GJ; Heeres, K; vanderMeer, JB; Jonkman, MF


    Circulating IgG from a large subset of bullous pein; phigoid (SP) patients reacted on immunoblot with a 120-kDa protein in conditioned keratinocyte culture medium and in keratinocyte cell extracts, A protein with a similar molecular weight was recognized by circulating IgA from a subset of patients

  4. Sticholysin II: a pore-forming toxin as a probe to recognize sphingomyelin in artificial and cellular membranes. (United States)

    Garcia, Paloma Sanchez; Chieppa, Gabriele; Desideri, Alessandro; Cannata, Stefano; Romano, Elena; Luly, Paolo; Rufini, Stefano


    Sphingomyelin is a major component of membrane rafts, and also is a precursor of many bioactive molecules. The sphingomyelin plays important biological roles and alterations of its metabolism are the basis of some genetic disorders such as the Niemann Pick disease. A complete understanding of its biological role is frustrated by the lack of efficient tools for its recognition in the cell. Sticholysin II (StnII) is a 20 kDa protein from the sea-anemone Stichodactyla helianthus which shows a cytotoxic activity by forming oligomeric aqueous pores in the cell plasma membrane. A recent NMR analysis indicates that the sticholysin II binds specifically to sphingomyelin by two domains that recognize respectively the hydrophilic (i.e. phosphorylcholine) and the hydrophobic (i.e. ceramide) moieties of the molecule. Aim of our research has been to verify the possible employ of an antibody against the StnII to investigate the localization and the dynamics of sphingomyelin in cell membranes. For this purpose, we developed a monoclonal antibody (named A10) against the toxin and we tested its ability to bind StnII after binding to sphingomyelin. A10 antibody is able to recognize the sticholysin II both in its native form and after SDS treatment, being the protein still suitable for many analytic techniques such as ELISA, western blotting and immunofluorescence. The high affinity of the toxin for the sphingomyelin in cell membranes has been demonstrated by microscopic immuno-localization and western blot analysis; both methods confirmed that sphingomyelin is the molecular acceptor for StnII also in cell membranes. Finally, we studied the specificity of the toxin for sphingomyelin by a cell membrane-double labelling method, using cholera toxin, specific for the ganglioside GM1, and sticholysin II. The results obtained show that there is no cross-reactivity between the two toxins, confirming that sticholysin II is able to discriminate among membrane domains with sphingomyelin with

  5. Oxidation-specific epitopes are danger-associated molecular patterns recognized by pattern recognition receptors of innate immunity

    DEFF Research Database (Denmark)

    Miller, Yury I; Choi, Soo-Ho; Wiesner, Philipp


    are a major target of innate immunity, recognized by a variety of "pattern recognition receptors" (PRRs). By analogy with microbial "pathogen-associated molecular patterns" (PAMPs), we postulate that host-derived, oxidation-specific epitopes can be considered to represent "danger (or damage......)-associated molecular patterns" (DAMPs). We also argue that oxidation-specific epitopes present on apoptotic cells and their cellular debris provided the primary evolutionary pressure for the selection of such PRRs. Furthermore, because many PAMPs on microbes share molecular identity and/or mimicry with oxidation...

  6. 抑制MEK对内质网应激诱导乳腺癌细胞凋亡的增敏作用%Inhibition of MEK sensitizes human breast carcinoma cells to endoplasmic reticulum pathway's apoptosis

    Institute of Scientific and Technical Information of China (English)

    杨芬; 刘浩; 张旭东; 蒋志文


    目的 探讨抑制MEK/ERK信号通路对人乳腺癌细胞内质网(endoplasmic reticulum, ER)应激途径细胞凋亡的影响,以期为乳腺癌化疗提供新的靶点.方法 不同浓度(0、1.5、3、6、9、12 μmol*L-1)衣霉素(tunicamycin, TM)处理乳腺癌细胞SK-BR-3,48 h后溴化丙啶(propidium iodide,PI)染色检测细胞凋亡率;TM(3 μmol*L-1)处理SK-BR-3细胞不同时间(0、6、12、24、36 h),Western blot检测葡萄糖调节蛋白78(glucose-regulated protein 78, GRP78)、ERK1/2、pERK1/2的表达;MEK抑制剂U0126(20 μmol*L-1)预处理1 h后再给予TM(3 μmol*L-1)同上处理,检测上述指标,比较U0126作用前后上述指标的变化.结果 SK-BR-3细胞对TM诱导的细胞凋亡率<20%,且TM上调GRP78的表达;TM没有诱导ERK1/2的进一步激活;U0126明显增加TM诱导的细胞凋亡率(78%),同时下调GRP78的表达和阻断TM对GRP78的上调作用.结论 MEK/ERK信号通路的抑制增强人乳腺癌细胞SK-BR-3对ER应激途径细胞凋亡的敏感性,抑制非折叠蛋白反应(unfolded protein response, UPR)的诱导.

  7. 21 CFR 170.30 - Eligibility for classification as generally recognized as safe (GRAS). (United States)


    ... 21 Food and Drugs 3 2010-04-01 2009-04-01 true Eligibility for classification as generally recognized as safe (GRAS). 170.30 Section 170.30 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF... Safety § 170.30 Eligibility for classification as generally recognized as safe (GRAS). (a)...

  8. A Safe Education for All: Recognizing and Stemming Harassment in Music Classes and Ensembles (United States)

    Carter, Bruce Allen


    This article addresses the pervasiveness of harassment in schools in the United States and presents ways to recognize and stem bullying in music classrooms. Music educators are in a unique position to recognize atypical behaviors in their students. Music educators who teach middle and high school ensembles often retain the same students in their…

  9. Recognizing Multi-user Activities using Wearable Sensors in a Smart Home

    DEFF Research Database (Denmark)

    Wang, Liang; Gu, Tao; Tao, Xianping


    The advances of wearable sensors and wireless networks oer many opportunities to recognize human activities from sensor readings in pervasive computing. Existing work so far focuses mainly on recognizing activities of a single user in a home environment. However, there are typically multiple inha...

  10. Image segmentation and feature extraction for recognizing strokes in tennis game videos

    NARCIS (Netherlands)

    Zivkovic, Z.; Heijden, van der F.; Petkovic, M.; Jonker, W.; Langendijk, R.L.; Heijnsdijk, J.W.J.; Pimentel, A.D.; Wilkinson, M.H.F.


    This paper addresses the problem of recognizing human actions from video. Particularly, the case of recognizing events in tennis game videos is analyzed. Driven by our domain knowledge, a robust player segmentation algorithm is developed real video data. Further, we introduce a number of novel feat

  11. Automated facial coding software outperforms people in recognizing neutral faces as neutral from standardized datasets

    NARCIS (Netherlands)

    Lewinski, P.


    Little is known about people’s accuracy of recognizing neutral faces as neutral. In this paper, I demonstrate the importance of knowing how well people recognize neutral faces. I contrasted human recognition scores of 100 typical, neutral front-up facial images with scores of an arguably objective j

  12. 29 CFR 779.368 - Printing and engraving establishments not recognized as retail. (United States)


    ... retail. 779.368 Section 779.368 Labor Regulations Relating to Labor (Continued) WAGE AND HOUR DIVISION... FAIR LABOR STANDARDS ACT AS APPLIED TO RETAILERS OF GOODS OR SERVICES Exemptions for Certain Retail or... recognized as retail. (a) An establishment which is engaged in printing and engraving is not recognized as...

  13. 78 FR 7460 - Stakeholder Meeting on the Nationally Recognized Testing Laboratory Program (United States)


    ... Laboratory Program AGENCY: Occupational Safety and Health Administration (OSHA), Labor. ACTION: Notice of... concerning Nationally Recognized Testing Laboratory (NRTL) Program policies. The meeting will focus on the... standards require that a Nationally Recognized Testing Laboratory (NRTL) approve thirty-eight (38) types...

  14. Mining Emerging Patterns for Recognizing Activities of Multiple Users in Pervasive Computing

    DEFF Research Database (Denmark)

    Gu, Tao; Wu, Zhanqing; Wang, Liang;


    Understanding and recognizing human activities from sensor readings is an important task in pervasive computing. Existing work on activity recognition mainly focuses on recognizing activities for a single user in a smart home environment. However, in real life, there are often multiple inhabitants...... activity models, and propose an Emerging Pattern based Multi-user Activity Recognizer (epMAR) to recognize both single-user and multiuser activities. We conduct our empirical studies by collecting real-world activity traces done by two volunteers over a period of two weeks in a smart home environment...... sensor readings in a home environment, and propose a novel pattern mining approach to recognize both single-user and multi-user activities in a unified solution. We exploit Emerging Pattern – a type of knowledge pattern that describes significant changes between classes of data – for constructing our...

  15. Recognition of voice commands using adaptation of foreign language speech recognizer via selection of phonetic transcriptions (United States)

    Maskeliunas, Rytis; Rudzionis, Vytautas


    In recent years various commercial speech recognizers have become available. These recognizers provide the possibility to develop applications incorporating various speech recognition techniques easily and quickly. All of these commercial recognizers are typically targeted to widely spoken languages having large market potential; however, it may be possible to adapt available commercial recognizers for use in environments where less widely spoken languages are used. Since most commercial recognition engines are closed systems the single avenue for the adaptation is to try set ways for the selection of proper phonetic transcription methods between the two languages. This paper deals with the methods to find the phonetic transcriptions for Lithuanian voice commands to be recognized using English speech engines. The experimental evaluation showed that it is possible to find phonetic transcriptions that will enable the recognition of Lithuanian voice commands with recognition accuracy of over 90%.

  16. Recognizing Hypothermia

    Centers for Disease Control (CDC) Podcasts


    Hypothermia is a serious medical condition that strikes during very cold weather or when people are chilled from rain, sweat, or cold water.  Created: 11/1/2007 by Emergency Communications System.   Date Released: 12/13/2007.

  17. Recognizing Symptoms (United States)

    ... bowel habits. Learn More About Pain in IBS Symptoms Won’t Stop Everyone suffers from bowel changes ... They can be constant or keep coming back. Symptoms Change Some or all of IBS symptoms can ...

  18. Adaptive Resonance Theory: how a brain learns to consciously attend, learn, and recognize a