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  1. 3D culture broadly regulates tumor cell hypoxia response and angiogenesis via pro-inflammatory pathways.

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    DelNero, Peter; Lane, Maureen; Verbridge, Scott S; Kwee, Brian; Kermani, Pouneh; Hempstead, Barbara; Stroock, Abraham; Fischbach, Claudia

    2015-07-01

    Oxygen status and tissue dimensionality are critical determinants of tumor angiogenesis, a hallmark of cancer and an enduring target for therapeutic intervention. However, it is unclear how these microenvironmental conditions interact to promote neovascularization, due in part to a lack of comprehensive, unbiased data sets describing tumor cell gene expression as a function of oxygen levels within three-dimensional (3D) culture. Here, we utilized alginate-based, oxygen-controlled 3D tumor models to study the interdependence of culture context and the hypoxia response. Microarray gene expression analysis of tumor cells cultured in 2D versus 3D under ambient or hypoxic conditions revealed striking interdependence between culture dimensionality and hypoxia response, which was mediated in part by pro-inflammatory signaling pathways. In particular, interleukin-8 (IL-8) emerged as a major player in the microenvironmental regulation of the hypoxia program. Notably, this interaction between dimensionality and oxygen status via IL-8 increased angiogenic sprouting in a 3D endothelial invasion assay. Taken together, our data suggest that pro-inflammatory pathways are critical regulators of tumor hypoxia response within 3D environments that ultimately impact tumor angiogenesis, potentially providing important therapeutic targets. Furthermore, these results highlight the importance of pathologically relevant tissue culture models to study the complex physical and chemical processes by which the cancer microenvironment mediates new vessel formation.

  2. Subcellular localization of PUMA regulates its pro-apoptotic activity in Burkitt's lymphoma B cells

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    Ambroise, Gorbatchev; Portier, Alain; Roders, Nathalie; Arnoult, Damien; Vazquez, Aimé

    2015-01-01

    The BH3-only protein PUMA (p53-upregulated modulator of apoptosis) is a major regulator of apoptosis. It belongs to the Bcl-2 family of proteins responsible for maintaining mitochondrial outer membrane integrity by controlling the intrinsic (mitochondrial) apoptotic pathway. We describe here a new pathway regulating PUMA activation through the control of its subcellular distribution. Surprisingly, neither PUMA upregulation in normal activated human B lymphocytes nor high levels of PUMA in Bur...

  3. TNF-α-induced up-regulation of pro-inflammatory cytokines is reduced by phosphatidylcholine in intestinal epithelial cells

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    Griffiths Gareth

    2009-07-01

    Full Text Available Abstract Background Phosphatidylcholine (PC is a major lipid of the gastrointestinal mucus layer. We recently showed that mucus from patients suffering from ulcerative colitis has low levels of PC. Clinical studies reveal that the therapeutic addition of PC to the colonic mucus using slow release preparations is beneficial. The positive role of PC in this disease is still unclear; however, we have recently shown that PC has an intrinsic anti-inflammatory property. It could be demonstrated that the exogenous application of PC inhibits membrane-dependent actin assembly and TNF-α-induced nuclear NF-κB activation. We investigate here in more detail the hypothesis that the exogenous application of PC has anti-inflammatory properties. Methods PC species with different fatty acid side chains were applied to differentiated and non-differentiated Caco-2 cells treated with TNF-α to induce a pro-inflammatory response. We analysed TNF-α-induced NF-κB-activation via the transient expression of a NF-κB-luciferase reporter system. Pro-inflammatory gene transcription was detected with the help of a quantitative real time (RT-PCR analysis. We assessed the binding of TNF-α to its receptor by FACS and analysed lipid rafts by isolating detergent resistant membranes (DRMs. Results The exogenous addition of all PC species tested significantly inhibited TNF-α-induced pro-inflammatory signalling. The expression levels of IL-8, ICAM-1, IP-10, MCP-1, TNF-α and MMP-1 were significantly reduced after PC pre-treatment for at least two hours. The effect was comparable to the inhibition of NF-kB by the NF-kB inhibitor SN 50 and was not due to a reduced binding of TNF-α to its receptor or a decreased surface expression of TNF-α receptors. PC was also effective when applied to the apical side of polarised Caco-2 cultures if cells were stimulated from the basolateral side. PC treatment changed the compartmentation of the TNF-α-receptors 1 and 2 to DRMs. Conclusion PC

  4. Constitutively active ErbB2 regulates cisplatin-induced cell death in breast cancer cells via pro- and antiapoptotic mechanisms

    DEFF Research Database (Denmark)

    Sigurðsson, Haraldur H; Olesen, Christina Wilkens; Dybboe, Rie;

    2015-01-01

    UNLABELLED: Despite the frequent expression of N-terminally truncated ErbB2 (ΔNErbB2/p95HER2) in breast cancer and its association with Herceptin resistance and poor prognosis, it remains poorly understood how ΔNErbB2 affects chemotherapy-induced cell death. Previously it was shown that ΔNErbB2...... upregulates acid extrusion from MCF-7 breast cancer cells and that inhibition of the Na(+)/H(+) exchanger (SLC9A1/NHE1) strongly sensitizes ΔNErbB2-expressing MCF-7 cells to cisplatin chemotherapy. The aim of this study was to identify the mechanism through which ΔNErbB2 regulates cisplatin-induced breast...... cisplatin exposure. IMPLICATIONS: This work reveals that ΔNErbB2 strongly affects several major pro- and antiapoptotic pathways and provides mechanistic insight into the role of NHE1 in chemotherapy resistance. These findings have relevance for defining therapy regimens in breast cancers with ΔNErbB2 and...

  5. Periostin down-regulation attenuates the pro-fibrogenic response of hepatic stellate cells induced by TGF-β1.

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    Hong, Li; Shejiao, Dai; Fenrong, Chen; Gang, Zhao; Lei, Dong

    2015-10-01

    Liver fibrosis is characterized by an exacerbated accumulation of deposition of the extracellular matrix (ECM), and the activation of hepatic stellate cells (HSC) plays a pivotal role in the development of liver fibrosis. Periostin has been shown to regulate cell adhesion, proliferation, migration and apoptosis; however, the involvement of periostin and its role in transforming growth factor (TGF)-β1-induced HSC activation remains unclear. We used RT-PCR and Western blot to evaluate the expression level of periostin in hepatic fibrosis tissues and HSCs, respectively. Cell proliferation was determined using the Cell Proliferation ELISA BrdU kit, cell cycle was analysed by flow cytometry. The expression of α-smooth muscle actin (α-SMA), collagen I, TGF-β1, p-Smad2 and p-Smad3 were determined by western blot. Our study found that periostin was up-regulated in liver fibrotic tissues and activated HSCs. In addition, siRNA-periostin suppressed TGF-β1-induced HSC proliferation. The HSC transfected with siRNA-periostin significantly inhibited TGF-β1-induced expression levels of α-SMA and collagen I. Furthermore, TGF-β1 stimulated the expression of periostin, and siRNA-periostin attenuated TGF-β1-induced Smad2/3 activation in HSCs. These results suggest that periostin may function as a novel regulator to modulate HSC activation, potentially by promoting the TGF-β1/Smad signalling pathway, and propose a strategy to target periostin for the treatment of liver fibrosis.

  6. Lactobacilli regulate Staphylococcus aureus 161:2-induced pro-inflammatory T-cell responses in vitro.

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    Haileselassie, Yeneneh; Johansson, Maria A; Zimmer, Christine L; Björkander, Sophia; Petursdottir, Dagbjort H; Dicksved, Johan; Petersson, Mikael; Persson, Jan-Olov; Fernandez, Carmen; Roos, Stefan; Holmlund, Ulrika; Sverremark-Ekström, Eva

    2013-01-01

    There seems to be a correlation between early gut microbiota composition and postnatal immune development. Alteration in the microbial composition early in life has been associated with immune mediated diseases, such as autoimmunity and allergy. We have previously observed associations between the presence of lactobacilli and Staphylococcus (S.) aureus in the early-life gut microbiota, cytokine responses and allergy development in children. Consistent with the objective to understand how bacteria modulate the cytokine response of intestinal epithelial cell (IEC) lines and immune cells, we exposed IEC lines (HT29, SW480) to UV-killed bacteria and/or culture supernatants (-sn) from seven Lactobacillus strains and three S. aureus strains, while peripheral blood mononuclear cells (PBMC) and cord blood mononuclear cells (CBMC) from healthy donors were stimulated by bacteria-sn or with bacteria conditioned IEC-sn. Although the overall IEC response to bacterial exposure was characterized by limited sets of cytokine and chemokine production, S. aureus 161:2-sn induced an inflammatory response in the IEC, characterized by CXCL1/GROα and CXCL8/IL-8 production, partly in a MyD88-dependent manner. UV-killed bacteria did not induce a response in the IEC line, and a combination of both UV-killed bacteria and the bacteria-sn had no additive effect to that of the supernatant alone. In PBMC, most of the Lactobacillus-sn and S. aureus-sn strains were able to induce a wide array of cytokines, but only S. aureus-sn induced the T-cell associated cytokines IL-2, IL-17 and IFN-γ, independently of IEC-produced factors, and induced up regulation of CTLA-4 expression and IL-10 production by T-regulatory cells. Notably, S. aureus-sn-induced T-cell production of IFN- γ and IL-17 was down regulated by the simultaneous presence of any of the different Lactobacillus strains, while the IEC CXCL8/IL-8 response was unaltered. Thus these studies present a possible role for lactobacilli in

  7. Lactobacilli regulate Staphylococcus aureus 161:2-induced pro-inflammatory T-cell responses in vitro.

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    Yeneneh Haileselassie

    Full Text Available There seems to be a correlation between early gut microbiota composition and postnatal immune development. Alteration in the microbial composition early in life has been associated with immune mediated diseases, such as autoimmunity and allergy. We have previously observed associations between the presence of lactobacilli and Staphylococcus (S. aureus in the early-life gut microbiota, cytokine responses and allergy development in children. Consistent with the objective to understand how bacteria modulate the cytokine response of intestinal epithelial cell (IEC lines and immune cells, we exposed IEC lines (HT29, SW480 to UV-killed bacteria and/or culture supernatants (-sn from seven Lactobacillus strains and three S. aureus strains, while peripheral blood mononuclear cells (PBMC and cord blood mononuclear cells (CBMC from healthy donors were stimulated by bacteria-sn or with bacteria conditioned IEC-sn. Although the overall IEC response to bacterial exposure was characterized by limited sets of cytokine and chemokine production, S. aureus 161:2-sn induced an inflammatory response in the IEC, characterized by CXCL1/GROα and CXCL8/IL-8 production, partly in a MyD88-dependent manner. UV-killed bacteria did not induce a response in the IEC line, and a combination of both UV-killed bacteria and the bacteria-sn had no additive effect to that of the supernatant alone. In PBMC, most of the Lactobacillus-sn and S. aureus-sn strains were able to induce a wide array of cytokines, but only S. aureus-sn induced the T-cell associated cytokines IL-2, IL-17 and IFN-γ, independently of IEC-produced factors, and induced up regulation of CTLA-4 expression and IL-10 production by T-regulatory cells. Notably, S. aureus-sn-induced T-cell production of IFN- γ and IL-17 was down regulated by the simultaneous presence of any of the different Lactobacillus strains, while the IEC CXCL8/IL-8 response was unaltered. Thus these studies present a possible role for

  8. Regulation of growth hormone secretion by (pro)renin receptor

    OpenAIRE

    Tani, Yuji; Yamada, Shozo; Inoshita, Naoko; Hirata, Yukio; SHICHIRI, MASAYOSHI

    2015-01-01

    (Pro)renin receptor (PRR) has a single transmembrane domain that co-purifies with the vacuolar H+-ATPase (V-ATPase). In addition to its role in cellular acidification, V-ATPase has been implicated in membrane fusion and exocytosis via its Vo domain. Results from the present study show that PRR is expressed in pituitary adenoma cells and regulates growth hormone (GH) release via V-ATPase-induced cellular acidification. Positive PRR immunoreactivity was detected more often in surgically resecte...

  9. Hedgehog promotes neovascularization in pancreatic cancers by regulating Ang-1 and IGF-1 expression in bone-marrow derived pro-angiogenic cells.

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    Kazumasa Nakamura

    Full Text Available BACKGROUND: The hedgehog (Hh pathway has been implicated in the pathogenesis of cancer including pancreatic ductal adenocarcinoma (PDAC. Recent studies have suggested that the oncogenic function of Hh in PDAC involves signaling in the stromal cells rather than cell autonomous effects on the tumor cells. However, the origin and nature of the stromal cell type(s that are responsive to Hh signaling remained unknown. Since Hh signaling plays a crucial role during embryonic and postnatal vasculogenesis, we speculated that Hh ligand may act on tumor vasculature specifically focusing on bone marrow (BM-derived cells. METHODOLOGY/PRINCIPAL FINDINGS: Cyclopamine was utilized to inhibit the Hh pathway in human PDAC cell lines and their xenografts. BM transplants, co-culture systems of tumor cells and BM-derived pro-angiogenic cells (BMPCs were employed to assess the role of tumor-derived Hh in regulating the BM compartment and the contribution of BM-derived cells to angiogenesis in PDAC. Cyclopamine administration attenuated Hh signaling in the stroma rather than in the cancer cells as reflected by decreased expression of full length Gli2 protein and Gli1 mRNA specifically in the compartment. Cyclopamine inhibited the growth of PDAC xenografts in association with regression of the tumor vasculature and reduced homing of BM-derived cells to the tumor. Host-derived Ang-1 and IGF-1 mRNA levels were downregulated by cyclopamine in the tumor xenografts. In vitro co-culture and matrigel plug assays demonstrated that PDAC cell-derived Shh induced Ang-1 and IGF-1 production in BMPCs, resulting in their enhanced migration and capillary morphogenesis activity. CONCLUSIONS/SIGNIFICANCE: We identified the BMPCs as alternative stromal targets of Hh-ligand in PDAC suggesting that the tumor vasculature is an attractive therapeutic target of Hh blockade. Our data is consistent with the emerging concept that BM-derived cells make important contributions to epithelial

  10. Poliovirus protease 3C(pro) kills cells by apoptosis.

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    Barco, A; Feduchi, E; Carrasco, L

    2000-01-20

    The tetracycline-based Tet-Off expression system has been used to analyze the effects of poliovirus protease 3C(pro) on human cells. Stable HeLa cell clones that express this poliovirus protease under the control of an inducible, tightly regulated promoter were obtained. Tetracycline removal induces synthesis of 3C protease, followed by drastic morphological alterations and cellular death. Degradation of cellular DNA in nucleosomes and generation of apoptotic bodies are observed from the second day after 3C(pro) induction. The cleavage of poly(ADP-ribose) polymerase, an enzyme involved in DNA repair, occurs after induction of 3C(pro), indicating caspase activation by this poliovirus protease. The 3C(pro)-induced apoptosis is blocked by the caspase inhibitor z-VAD-fmk. Our findings suggest that the protease 3C is responsible for triggering apoptosis in poliovirus-infected cells by a mechanism that involves caspase activation.

  11. Lactate Regulates Metabolic and Pro-inflammatory Circuits in Control of T Cell Migration and Effector Functions.

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    Robert Haas

    2015-07-01

    Full Text Available Lactate has long been considered a "waste" by-product of cell metabolism, and it accumulates at sites of inflammation. Recent findings have identified lactate as an active metabolite in cell signalling, although its effects on immune cells during inflammation are largely unexplored. Here we ask whether lactate is responsible for T cells remaining entrapped in inflammatory sites, where they perpetuate the chronic inflammatory process. We show that lactate accumulates in the synovia of rheumatoid arthritis patients. Extracellular sodium lactate and lactic acid inhibit the motility of CD4+ and CD8+ T cells, respectively. This selective control of T cell motility is mediated via subtype-specific transporters (Slc5a12 and Slc16a1 that we find selectively expressed by CD4+ and CD8+ subsets, respectively. We further show both in vitro and in vivo that the sodium lactate-mediated inhibition of CD4+ T cell motility is due to an interference with glycolysis activated upon engagement of the chemokine receptor CXCR3 with the chemokine CXCL10. In contrast, we find the lactic acid effect on CD8+ T cell motility to be independent of glycolysis control. In CD4+ T helper cells, sodium lactate also induces a switch towards the Th17 subset that produces large amounts of the proinflammatory cytokine IL-17, whereas in CD8+ T cells, lactic acid causes the loss of their cytolytic function. We further show that the expression of lactate transporters correlates with the clinical T cell score in the synovia of rheumatoid arthritis patients. Finally, pharmacological or antibody-mediated blockade of subtype-specific lactate transporters on T cells results in their release from the inflammatory site in an in vivo model of peritonitis. By establishing a novel role of lactate in control of proinflammatory T cell motility and effector functions, our findings provide a potential molecular mechanism for T cell entrapment and functional changes in inflammatory sites that drive

  12. Anti-inflammatory drugs suppress proliferation and induce apoptosis through altering expressions of cell cycle regulators and pro-apoptotic factors in cultured human osteoblasts

    International Nuclear Information System (INIS)

    It has been reported that anti-inflammatory drugs (AIDs) inhibited bone repair in animal studies, and suppressed proliferation and induced cell death in rat osteoblast cultures. In this study, we further investigated the molecular mechanisms of AID effects on proliferation and cell death in human osteoblasts (hOBs). We examined the effects of dexamethasone (10-7 and 10-6 M), non-selective non-steroidal anti-inflammatory drugs (NSAIDs): indomethacin, ketorolac, piroxicam and diclofenac (10-5 and 10-4 M), and COX-2 inhibitor: celecoxib (10-6 and 10-5 M) on proliferation, cytotoxicity, cell death, and mRNA and protein levels of cell cycle and apoptosis-related regulators in hOBs. All the tested AIDs significantly inhibited proliferation and arrested cell cycle at G0/G1 phase in hOBs. Celecoxib and dexamethasone, but not non-selective NSAIDs, were found to have cytotoxic effects on hOB, and further demonstrated to induce apoptosis and necrosis (at higher concentration) in hOBs. We further found that indomethacin, celecoxib and dexamethasone increased the mRNA and protein expressions of p27kip1 and decreased those of cyclin D2 and p-cdk2 in hOBs. Bak expression was increased by celecoxib and dexamethasone, while Bcl-XL level was declined only by dexamethasone. Furthermore, the replenishment of PGE1, PGE2 or PGF2α did not reverse the effects of AIDs on proliferation and expressions of p27kip1 and cyclin D2 in hOBs. We conclude that the changes in expressions of regulators of cell cycle (p27kip1 and cyclin D2) and/or apoptosis (Bak and Bcl-XL) by AIDs may contribute to AIDs caused proliferation suppression and apoptosis in hOBs. This effect might not relate to the blockage of prostaglandin synthesis by AIDs

  13. The pro-adhesive and pro-survival effects of glucocorticoid in human ovarian cancer cells.

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    Yin, Lijuan; Fang, Fang; Song, Xinglei; Wang, Yan; Huang, Gaoxiang; Su, Jie; Hui, Ning; Lu, Jian

    2016-07-01

    Cell adhesion to extracellular matrix (ECM) is controlled by multiple signaling molecules and intracellular pathways, and is pivotal for survival and growth of cells from most solid tumors. Our previous works demonstrated that dexamethasone (DEX) significantly enhances cell adhesion and cell resistance to chemotherapeutics by increasing the levels of integrin β1, α4, and α5 in human ovarian cancer cells. However, it is unclear whether the components of ECM or other membrane molecules are also involved in the pro-adhesive effect of DEX in ovarian cancer cells. In this study, we demonstrated that the treatment of cells with DEX did not change the expression of collagens (I, III, and IV), laminin, CD44, and its principal ligand hyaluronan (HA), but significantly increased the levels of intracellular and secreted fibronectin (FN). Inhibiting the expression of FN with FN1 siRNA or blocking CD44, another FN receptor, with CD44 blocking antibody significantly attenuated the pro-adhesion of DEX, indicating that upregulation of FN mediates the pro-adhesive effect of DEX by its interaction with CD44 besides integrin β1. Moreover, DEX significantly enhanced cell resistance to the chemotherapeutic agent paclitaxel (PTX) by activating PI-3K-Akt pathway. Finally, we found that DEX also significantly upregulated the expression of MUC1, a transmembrane glycoprotein. Inhibiting the expression of MUC1 with MUC1 siRNA significantly attenuated the DEX-induced effects of pro-adhesion, Akt-activation, and pro-survival. In conclusion, these results provide new data that upregulation of FN and MUC1 by DEX contributes to DEX-induced pro-adhesion and protects ovarian cancer cells from chemotherapy. PMID:27151574

  14. Pro-B-cell-specific transcription and proapoptotic function of protein kinase Ceta.

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    Morrow, T A; Muljo, S A; Zhang, J; Hardwick, J M; Schlissel, M S

    1999-08-01

    Using a subtractive cloning scheme on cDNA prepared from primary pro-B and pre-B cells, we identified several genes whose products regulate apoptosis. We further characterized one of these genes, encoding protein kinase Ceta (PKCeta). PKCeta transcripts were readily detected in pro-B cells but were absent in pre-B cells. Although both a full-length and a truncated form of PKCeta were detectable in bone marrow pro-B cells, transition to the pre-B-cell stage was associated with increased relative levels of truncated PKCeta. We found that PKCeta is proteolyzed in apoptotic lymphocytes, generating a kinase-active fragment identical to the truncated form which is capable of inducing apoptosis when expressed in a pro-B cell line. Caspase-3 can generate an identical PKCeta cleavage product in vitro, and caspase inhibitors prevent the generation of this product during apoptosis in transfected cell lines. Inducible overexpression of either the full-length or truncated form of PKCeta results in cell cycle arrest at the G(1)/S transition. These results suggest that the expression and proteolytic activation of PKCeta play an important role in the regulation of cell division and cell death during early B-cell development. PMID:10409750

  15. ProSAAS-derived peptides are colocalized with neuropeptide Y and function as neuropeptides in the regulation of food intake.

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    Jonathan H Wardman

    Full Text Available ProSAAS is the precursor of a number of peptides that have been proposed to function as neuropeptides. Because proSAAS mRNA is highly expressed in the arcuate nucleus of the hypothalamus, we examined the cellular localization of several proSAAS-derived peptides in the mouse hypothalamus and found that they generally colocalized with neuropeptide Y (NPY, but not α-melanocyte stimulating hormone. However, unlike proNPY mRNA, which is upregulated by food deprivation in the mediobasal hypothalamus, neither proSAAS mRNA nor proSAAS-derived peptides were significantly altered by 1-2 days of food deprivation in wild-type mice. Furthermore, while proSAAS mRNA levels in the mediobasal hypothalamus were significantly lower in Cpe(fat/fat mice as compared to wild-type littermates, proNPY mRNA levels in the mediobasal hypothalamus and in other subregions of the hypothalamus were not significantly different between wild-type and Cpe(fat/fat mice. Intracerebroventricular injections of antibodies to two proSAAS-derived peptides (big LEN and PEN significantly reduced food intake in fasted mice, while injections of antibodies to two other proSAAS-derived peptides (little LEN and little SAAS did not. Whole-cell patch clamp recordings of parvocellular neurons in the hypothalamic paraventricular nucleus, a target of arcuate NPY projections, showed that big LEN produced a rapid and reversible inhibition of synaptic glutamate release that was spike independent and abolished by blocking postsynaptic G protein activity, suggesting the involvement of a postsynaptic G protein-coupled receptor and the release of a retrograde synaptic messenger. Taken together with previous studies, these findings support a role for proSAAS-derived peptides such as big LEN as neuropeptides regulating food intake.

  16. Silibinin attenuates ionizing radiation-induced pro-angiogenic response and EMT in prostate cancer cells

    International Nuclear Information System (INIS)

    Graphical abstract: Potential model showing mechanism of silibinin-mediated attenuation of IR-induced angiogenic phenotype and EMT in tumor cells. Silibinin counters radiation induced invasive and migratory phenotype of cancer cells by down-regulating mitogenic pathways activated by IR, leading to inhibition of molecules including VEGF, iNOS, MMPs and N-cadherin. Silibinin also reverses IR mediated E-cadherin down-regulation, inhibiting EMT in tumor cells. Silibinin also radiosensitizes endothelial cells, reduces capillary tube formation by targeting various pro-angiogenic molecules. Further, silibinin may inhibit autocrine and paracrine signaling between tumor and endothelial cells by decreasing the levels of VEGF and other signaling molecules activated in response to IR. - Highlights: • Silibinin radiosensitizes endothelial cells. • Silibinin targets ionization radiation (IR)-induced EMT in PCa cells. • Silibinin is in phase II clinical trial in PCa patients, hence clinically relevant. - Abstract: Radiotherapy of is well established and frequently utilized in prostate cancer (PCa) patients. However, recurrence following therapy and distant metastases are commonly encountered problems. Previous studies underline that, in addition to its therapeutic effects, ionizing radiation (IR) increases the vascularity and invasiveness of surviving radioresistant cancer cells. This invasive phenotype of radioresistant cells is an upshot of IR-induced pro-survival and mitogenic signaling in cancer as well as endothelial cells. Here, we demonstrate that a plant flavonoid, silibinin can radiosensitize endothelial cells by inhibiting expression of pro-angiogenic factors. Combining silibinin with IR not only strongly down-regulated endothelial cell proliferation, clonogenicity and tube formation ability rather it strongly (p < 0.001) reduced migratory and invasive properties of PCa cells which were otherwise marginally affected by IR treatment alone. Most of the pro

  17. Pro-apoptotic gene regulation in the Caribbean fruit fly, Anastrepha suspensa

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    Transcriptional activation of pro-apoptotic genes in response to cytotoxic stimuli is a conserved feature of the cell death pathway proposed for metazoans. However, understanding the extent of this conservation in insects, as well as other organisms, has been limited by the lack of known pro-apoptot...

  18. Chitosan drives anti-inflammatory macrophage polarisation and pro-inflammatory dendritic cell stimulation

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    MI Oliveira

    2012-07-01

    Full Text Available Macrophages and dendritic cells (DC share the same precursor and play key roles in immunity. Modulation of their behaviour to achieve an optimal host response towards an implanted device is still a challenge. Here we compare the differentiation process and polarisation of these related cell populations and show that they exhibit different responses to chitosan (Ch, with human monocyte-derived macrophages polarising towards an anti-inflammatory phenotype while their DC counterparts display pro-inflammatory features. Macrophages and DC, whose interactions with biomaterials are frequently analysed using fully differentiated cells, were cultured directly on Ch films, rather than exposed to the polymer after complete differentiation. Ch was the sole stimulating factor and activated both macrophages and DC, without leading to significant T cell proliferation. After 10 d on Ch, macrophages significantly down-regulated expression of pro-inflammatory markers, CD86 and MHCII. Production of pro-inflammatory cytokines, particularly TNF-α, decreased with time for cells cultured on Ch, while anti-inflammatory IL-10 and TGF-β1, significantly increased. Altogether, these results suggest an M2c polarisation. Also, macrophage matrix metalloproteinase activity was augmented and cell motility was stimulated by Ch. Conversely, DC significantly enhanced CD86 expression, reduced IL-10 secretion and increased TNF-α and IL-1β levels. Our findings indicate that cells with a common precursor may display different responses, when challenged by the same biomaterial. Moreover, they help to further comprehend macrophage/DC interactions with Ch and the balance between pro- and anti-inflammatory signals associated with implant biomaterials. We propose that an overall pro-inflammatory reaction may hide the expression of anti-inflammatory cytokines, likely relevant for tissue repair/regeneration.

  19. File list: ALL.Bld.10.AllAg.Pro-B_cells [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  20. File list: His.Bld.50.AllAg.Pro-B_cells [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  1. File list: ALL.Bld.20.AllAg.Pro-B_cells [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  2. Platelet adhesion and degranulation induce pro-survival and pro-angiogenic signalling in ovarian cancer cells.

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    Karl Egan

    Full Text Available Thrombosis is common in ovarian cancer. However, the interaction of platelets with ovarian cancer cells has not been critically examined. To address this, we investigated platelet interactions in a range of ovarian cancer cell lines with different metastatic potentials [HIO-80, 59M, SK-OV-3, A2780, A2780cis]. Platelets adhered to ovarian cancer cells with the most significant adhesion to the 59M cell line. Ovarian cancer cells induced platelet activation [P-selectin expression] in a dose dependent manner, with the most significant activation seen in response to the 59M cell line. The platelet antagonists [cangrelor, MRS2179, and apyrase] inhibited 59M cell induced activation suggesting a P2Y12 and P2Y1 receptor mediated mechanism of platelet activation dependent on the release of ADP by 59M cells. A2780 and 59M cells potentiated PAR-1, PAR-4, and TxA2 receptor mediated platelet activation, but had no effect on ADP, epinephrine, or collagen induced activation. Analysis of gene expression changes in ovarian cancer cells following treatment with washed platelets or platelet releasate showed a subtle but valid upregulation of anti-apoptotic, anti-autophagy pro-angiogenic, pro-cell cycle and metabolic genes. Thus, ovarian cancer cells with different metastatic potential adhere and activate platelets differentially while both platelets and platelet releasate mediate pro-survival and pro-angiogenic signals in ovarian cancer cells.

  3. Molecular mechanisms of differentiation of murine pro-inflammatory gamma-delta T cell subsets

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    Bruno eSilva-Santos

    2013-12-01

    Full Text Available Gamma-delta (gd T cells are unconventional innate-like lymphocytes that actively participate in protective immunity against tumors and infectious organisms including bacteria, viruses and parasites. However, gd T cells are also involved in the development of inflammatory and autoimmune diseases. gd T cells are functionally characterized by very rapid production of pro-inflammatory cytokines, while also impacting on (slower but long-lasting adaptive immune responses. This makes it crucial to understand the molecular mechanisms that regulate  T cell effector functions. Although they share many similarities with ab T cells, our knowledge of the molecular pathways that control effector functions in gd T cells still lags significantly behind. In this review, we focus on the segregation of interferon-gamma versus interleukin-17 production in murine thymic-derived gd T cell subsets defined by CD27 and CCR6 expression levels. We summarize the most recent studies that disclose the specific epigenetic and transcriptional mechanisms that govern the stability or plasticity of discrete pro-inflammatory gd T cell subsets, whose manipulation may be valuable for regulating (autoimmune responses.

  4. Protein stability regulators screening assay (Pro-SRSA): protein degradation meets the CRISPR-Cas9 library.

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    Wu, Yuanzhong; Kang, Tiebang

    2016-01-01

    The regulation of protein stability is a fundamental issue for biophysical processes, but there has not previously been a convenient and unbiased method of identifying regulators of protein stability. However, as reported in the article entitled "A genome-scale CRISPR-Cas9 screening method for protein stability reveals novel regulators of Cdc25A," recently published in Cell Discovery, our team developed a protein stability regulators screening assay (Pro-SRSA) by combining the whole-genome clustered regularly interspaced short palindromic repeats Cas9 (CRISPR-Cas9) library with a dual-fluorescence-based protein stability reporter and high-throughput sequencing to screen for regulators of protein stability. Based on our findings, we are confident that this efficient and unbiased screening method at the genome scale will be used by researchers worldwide to identify regulators of protein stability. PMID:27357860

  5. Treg cells expressing the co-inhibitory molecule TIGIT selectively inhibit pro-inflammatory Th1 and Th17 cell responses

    OpenAIRE

    Joller, Nicole; Lozano, Ester; Burkett, Patrick R.; Patel, Bonny; Xiao, Sheng; Zhu, Chen; Xia, Junrong; Tan, Tze G.; Sefik, Esen; Yajnik, Vijay; Sharpe, Arlene H.; Quintana, Francisco J.; Mathis, Diane; Benoist, Christophe; Hafler, David A.

    2014-01-01

    Foxp3+ T regulatory (Treg) cells regulate immune responses and maintain self-tolerance. Recent work shows that Treg cells are comprised of many subpopulations with specialized regulatory functions. Here we identified Foxp3+ T cells expressing the co-inhibitory molecule TIGIT as a distinct Treg cell subset that specifically suppresses pro-inflammatory T helper 1 (Th1) and Th17 cell, but not Th2 cell responses. Transcriptional profiling characterized TIGIT+ Treg cells as an activated Treg subse...

  6. Regulation of intestinal immune responses through TLR activation: implications for pro- and prebiotics

    Directory of Open Access Journals (Sweden)

    Sander eDe Kivit

    2014-02-01

    Full Text Available The intestinal mucosa is constantly facing a high load of antigens including bacterial antigens derived from the microbiota and food. Despite this, the immune cells present in the gastrointestinal tract do not initiate a pro-inflammatory immune response. Toll-like receptors (TLRs are pattern recognition receptors expressed by various cells in the gastrointestinal tract, including intestinal epithelial cells (IEC and resident immune cells in the lamina propria. Many diseases, including chronic intestinal inflammation (e.g. inflammatory bowel disease, irritable bowel syndrome (IBS, allergic gastroenteritis (e.g. eosinophilic gastroenteritis and allergic IBS and infections are nowadays associated with a deregulated microbiota. The microbiota may directly interact with TLR. In addition, differences in intestinal TLR expression in health and disease may suggest that TLR play an essential role in disease pathogenesis and may be novel targets for therapy. TLR signaling in the gut is involved in either maintaining intestinal homeostasis or the induction of an inflammatory response. This mini review provides an overview of the current knowledge regarding the contribution of intestinal epithelial TLR signaling in both tolerance induction or promoting intestinal inflammation, with a focus on food allergy. We will also highlight a potential role of the microbiota in regulating gut immune responses, especially through TLR activation.

  7. Regulation of Intestinal Immune Responses through TLR Activation: Implications for Pro- and Prebiotics.

    Science.gov (United States)

    de Kivit, Sander; Tobin, Mary C; Forsyth, Christopher B; Keshavarzian, Ali; Landay, Alan L

    2014-01-01

    The intestinal mucosa is constantly facing a high load of antigens including bacterial antigens derived from the microbiota and food. Despite this, the immune cells present in the gastrointestinal tract do not initiate a pro-inflammatory immune response. Toll-like receptors (TLRs) are pattern recognition receptors expressed by various cells in the gastrointestinal tract, including intestinal epithelial cells (IEC) and resident immune cells in the lamina propria. Many diseases, including chronic intestinal inflammation (e.g., inflammatory bowel disease), irritable bowel syndrome (IBS), allergic gastroenteritis (e.g., eosinophilic gastroenteritis and allergic IBS), and infections are nowadays associated with a deregulated microbiota. The microbiota may directly interact with TLR. In addition, differences in intestinal TLR expression in health and disease may suggest that TLRs play an essential role in disease pathogenesis and may be novel targets for therapy. TLR signaling in the gut is involved in either maintaining intestinal homeostasis or the induction of an inflammatory response. This mini review provides an overview of the current knowledge regarding the contribution of intestinal epithelial TLR signaling in both tolerance induction or promoting intestinal inflammation, with a focus on food allergy. We will also highlight a potential role of the microbiota in regulating gut immune responses, especially through TLR activation. PMID:24600450

  8. Macrophage pro-inflammatory response to Francisella novicida infection is regulated by SHIP.

    Directory of Open Access Journals (Sweden)

    Kishore V L Parsa

    2006-07-01

    Full Text Available Francisella tularensis, a Gram-negative facultative intracellular pathogen infecting principally macrophages and monocytes, is the etiological agent of tularemia. Macrophage responses to F. tularensis infection include the production of pro-inflammatory cytokines such as interleukin (IL-12, which is critical for immunity against infection. Molecular mechanisms regulating production of these inflammatory mediators are poorly understood. Herein we report that the SH2 domain-containing inositol phosphatase (SHIP is phosphorylated upon infection of primary murine macrophages with the genetically related F. novicida, and negatively regulates F. novicida-induced cytokine production. Analyses of the molecular details revealed that in addition to activating the MAP kinases, F. novicida infection also activated the phosphatidylinositol 3-kinase (PI3K/Akt pathway in these cells. Interestingly, SHIP-deficient macrophages displayed enhanced Akt activation upon F. novicida infection, suggesting elevated PI3K-dependent activation pathways in absence of SHIP. Inhibition of PI3K/Akt resulted in suppression of F. novicida-induced cytokine production through the inhibition of NFkappaB. Consistently, macrophages lacking SHIP displayed enhanced NFkappaB-driven gene transcription, whereas overexpression of SHIP led to decreased NFkappaB activation. Thus, we propose that SHIP negatively regulates F. novicida-induced inflammatory cytokine response by antagonizing the PI3K/Akt pathway and suppressing NFkappaB-mediated gene transcription. A detailed analysis of phosphoinositide signaling may provide valuable clues for better understanding the pathogenesis of tularemia.

  9. Caspase-1-like regulation of the proPO-system and role of ppA and caspase-1-like cleaved peptides from proPO in innate immunity.

    Science.gov (United States)

    Jearaphunt, Miti; Noonin, Chadanat; Jiravanichpaisal, Pikul; Nakamura, Seiko; Tassanakajon, Anchalee; Söderhäll, Irene; Söderhäll, Kenneth

    2014-04-01

    Invertebrates rely on innate immunity to respond to the entry of foreign microorganisms. One of the important innate immune responses in arthropods is the activation of prophenoloxidase (proPO) by a proteolytic cascade finalized by the proPO-activating enzyme (ppA), which leads to melanization and the elimination of pathogens. Proteolytic cascades play a crucial role in innate immune reactions because they can be triggered more quickly than immune responses that require altered gene expression. Caspases are intracellular proteases involved in tightly regulated limited proteolysis of downstream processes and are also involved in inflammatory responses to infections for example by activation of interleukin 1ß. Here we show for the first time a link between caspase cleavage of proPO and release of this protein and the biological function of these fragments in response to bacterial infection in crayfish. Different fragments from the cleavage of proPO were studied to determine their roles in bacterial clearance and antimicrobial activity. These fragments include proPO-ppA, the N-terminal part of proPO cleaved by ppA, and proPO-casp1 and proPO-casp2, the fragments from the N-terminus after cleavage by caspase-1. The recombinant proteins corresponding to all three of these peptide fragments exhibited bacterial clearance activity in vivo, and proPO-ppA had antimicrobial activity, as evidenced by a drastic decrease in the number of Escherichia coli in vitro. The bacteria incubated with the proPO-ppA fragment were agglutinated and their cell morphology was altered. Our findings show an evolutionary conserved role for caspase cleavage in inflammation, and for the first time show a link between caspase induced inflammation and melanization. Further we give a more detailed understanding of how the proPO system is regulated in time and place and a role for the peptide generated by activation of proPO as well as for the peptides resulting from Caspase 1 proteolysis.

  10. Caspase-1-like regulation of the proPO-system and role of ppA and caspase-1-like cleaved peptides from proPO in innate immunity.

    Directory of Open Access Journals (Sweden)

    Miti Jearaphunt

    2014-04-01

    Full Text Available Invertebrates rely on innate immunity to respond to the entry of foreign microorganisms. One of the important innate immune responses in arthropods is the activation of prophenoloxidase (proPO by a proteolytic cascade finalized by the proPO-activating enzyme (ppA, which leads to melanization and the elimination of pathogens. Proteolytic cascades play a crucial role in innate immune reactions because they can be triggered more quickly than immune responses that require altered gene expression. Caspases are intracellular proteases involved in tightly regulated limited proteolysis of downstream processes and are also involved in inflammatory responses to infections for example by activation of interleukin 1ß. Here we show for the first time a link between caspase cleavage of proPO and release of this protein and the biological function of these fragments in response to bacterial infection in crayfish. Different fragments from the cleavage of proPO were studied to determine their roles in bacterial clearance and antimicrobial activity. These fragments include proPO-ppA, the N-terminal part of proPO cleaved by ppA, and proPO-casp1 and proPO-casp2, the fragments from the N-terminus after cleavage by caspase-1. The recombinant proteins corresponding to all three of these peptide fragments exhibited bacterial clearance activity in vivo, and proPO-ppA had antimicrobial activity, as evidenced by a drastic decrease in the number of Escherichia coli in vitro. The bacteria incubated with the proPO-ppA fragment were agglutinated and their cell morphology was altered. Our findings show an evolutionary conserved role for caspase cleavage in inflammation, and for the first time show a link between caspase induced inflammation and melanization. Further we give a more detailed understanding of how the proPO system is regulated in time and place and a role for the peptide generated by activation of proPO as well as for the peptides resulting from Caspase 1

  11. Regulation of Intestinal Immune Responses through TLR Activation: Implications for Pro- and Prebiotics

    OpenAIRE

    de Kivit, Sander; Tobin, Mary C.; Forsyth, Christopher B.; Keshavarzian, Ali; Landay, Alan L.

    2014-01-01

    The intestinal mucosa is constantly facing a high load of antigens including bacterial antigens derived from the microbiota and food. Despite this, the immune cells present in the gastrointestinal tract do not initiate a pro-inflammatory immune response. Toll-like receptors (TLRs) are pattern recognition receptors expressed by various cells in the gastrointestinal tract, including intestinal epithelial cells (IEC) and resident immune cells in the lamina propria. Many diseases, including chron...

  12. Differential regulation of pro- and antiapoptotic proteins in fish adipocytes during hypoxic conditions.

    Science.gov (United States)

    Ekambaram, Padmini; Parasuraman, Parimala; Jayachandran, Tharani

    2016-06-01

    Worldwide, the frequencies and magnitudes of hypoxic events in estuarine waters have increased considerably over the past two decades. Fish populations are suitable indicators for the assessment of quality of aquatic ecosystems and often comprise a variety of adaptation systems by triggering oxidants, antioxidants and hypoxia-responsive signaling proteins. Signaling pathway may lead to cell survival or cell death which is fine-tuned by both positive and negative factors, which includes hypoxia-inducible factor-1α (HIF1α), heat-shock protein-70 (HSP70), phospho-c-Jun N-terminal kinase 1/2 (p-JNK1/2) and apoptosis signal-regulating kinase-1 (ASK1). In the present study, we attempt to determine stress-mediated signaling changes and molecular mechanism behind the cell survival by comparing adipocytes of fish from field hypoxic condition and laboratory-induced hypoxic condition (in vitro hypoxia). Comparison of field and laboratory studies in fish adipocytes showed differential expression of HIF1α, HSP70, p-JNK1/2 and ASK1 with altered oxidants and antioxidants. Further, the results also suggest that in vitro hypoxic conditions mimic field hypoxic conditions. Trends of hypoxia response were same in in vitro hypoxia of control adipocytes as in Ennore estuary, and hypoxia response was more pronounced in the test adipocytes under in vitro hypoxic condition. Results of the present work suggest that hypoxia is the major crusade of water pollutants affecting fish by differential regulation of pro- and antiapoptotic proteins probably through HSP70. This may play a vital role by providing cytoprotection in pollutant-induced stressed fish adipocytes substantiated by the in vitro hypoxic studies. PMID:26744268

  13. dlk1/FA1 regulates the function of human bone marrow mesenchymal stem cells by modulating gene expression of pro-inflammatory cytokines and immune response-related factors

    DEFF Research Database (Denmark)

    Abdallah, Basem M.; Boissy, Patrice; Tan, Qihua;

    2007-01-01

    HG-U133A microarrays. In response to Dlk1 expression, 128 genes were significantly up-regulated (with >2-fold; p inflammatory cytokines, in addition to factors involved in the complement system......MSC are associated with Dlk1-induced cytokine expression. Furthermore, Dlk1 promoted B cell proliferation, synergized the immune response effects of the bacterial endotoxin lipopolysaccharide on hMSC, and led to marked transactivation of the NF-kappaB. Our data suggest a new role for Dlk1 in regulating the multiple...

  14. Globular adiponectin induces a pro-inflammatory response in human astrocytic cells

    Energy Technology Data Exchange (ETDEWEB)

    Wan, Zhongxiao; Mah, Dorrian; Simtchouk, Svetlana [School of Health and Exercise Sciences, University of British Columbia Okanagan, Kelowna, BC (Canada); Klegeris, Andis [Department of Biology, University of British Columbia Okanagan, Kelowna, BC (Canada); Little, Jonathan P., E-mail: jonathan.little@ubc.ca [School of Health and Exercise Sciences, University of British Columbia Okanagan, Kelowna, BC (Canada)

    2014-03-28

    Highlights: • Adiponectin receptors are expressed in human astrocytes. • Globular adiponectin induces secretion of IL-6 and MCP-1 from cultured astrocytes. • Adiponectin may play a pro-inflammatory role in astrocytes. - Abstract: Neuroinflammation, mediated in part by activated brain astrocytes, plays a critical role in the development of neurodegenerative disorders, including Alzheimer’s disease (AD). Adiponectin is the most abundant adipokine secreted from adipose tissue and has been reported to exert both anti- and pro-inflammatory effects in peripheral tissues; however, the effects of adiponectin on astrocytes remain unknown. Shifts in peripheral concentrations of adipokines, including adiponectin, could contribute to the observed link between midlife adiposity and increased AD risk. The aim of the present study was to characterize the effects of globular adiponectin (gAd) on pro-inflammatory cytokine mRNA expression and secretion in human U373 MG astrocytic cells and to explore the potential involvement of nuclear factor (NF)-κB, p38 mitogen-activated protein kinase (MAPK), extracellular signal-regulated kinase (ERK)1/2, c-Jun N-terminal kinase (JNK) and phosphatidylinositide 3-kinases (PI3 K) signaling pathways in these processes. We demonstrated expression of adiponectin receptor 1 (adipoR1) and adipoR2 in U373 MG cells and primary human astrocytes. gAd induced secretion of interleukin (IL)-6 and monocyte chemoattractant protein (MCP)-1, and gene expression of IL-6, MCP-1, IL-1β and IL-8 in U373 MG cells. Using specific inhibitors, we found that NF-κB, p38MAPK and ERK1/2 pathways are involved in gAd-induced induction of cytokines with ERK1/2 contributing the most. These findings provide evidence that gAd may induce a pro-inflammatory phenotype in human astrocytes.

  15. JNK and macroautophagy activation by bortezomib has a pro-survival effect in primary effusion lymphoma cells.

    Directory of Open Access Journals (Sweden)

    Marisa Granato

    Full Text Available Understanding the mechanisms of autophagy induction and its role during chemotherapeutic treatments is of fundamental importance in order to manipulate it to improve the outcome of chemotherapy. In particular whether the bortezomib-induced autophagy plays a pro-survival or pro-death role is still controversial. In this study we investigated if bortezomib induced endoplasmic reticulum (ER stress and activated autophagy in Primary Effusion Lymphoma (PEL cells and how they influenced cell survival. We found that bortezomib induced up-regulation of the pro-survival and pro-death ER stress molecules BIP and CHOP and activated c-Jun NH2-terminal kinase (JNK, resulting in Bcl-2 phosphorylation and induction of autophagy. JNK and autophagy activation played a pro-survival role in this setting, thus their inhibition increased the bortezomib cytotoxic effect and PARP cleavage in PEL cells. Based on our results we suggest that the combination of bortezomib with JNK or autophagy inhibitors could be exploited to improve the outcome of therapy of this aggressive B cell lymphoma.

  16. Slit2 is decreased after spontaneous labour in myometrium and regulates pro-labour mediators.

    Science.gov (United States)

    Lim, Ratana; Liong, Stella; Barker, Gillian; Lappas, Martha

    2014-12-01

    Preterm birth, a global healthcare problem, is commonly associated with inflammation. As Slit2 plays an emerging role in inflammation, the purpose of this study was to determine the effect of Slit2 on labour mediators in human gestational tissues. Slit2 mRNA and protein expression were assessed using qRT-PCR and immunohistochemistry in foetal membranes and myometrium obtained before and after labour. Slit2 silencing was achieved using siRNA in primary myometrial cells. Pro-inflammatory and pro-labour mediators were evaluated by qRT-PCR, ELISA and gelatin zymography. Slit2 mRNA and protein expression were found to be significantly lower in myometrium after labour onset. There was no effect of term or preterm labour on Slit2 expression in foetal membranes. Slit2 mRNA expression was decreased in myometrium treated with LPS and IL-1β. Slit2 siRNA in myometrial cells increased IL-1β-induced pro-inflammatory cytokine gene expression and release (IL-6 and IL-8), COX-2 expression and prostaglandin PGE2 and PGF2α release, and MMP-9 gene expression and pro MMP-9 release. There was no effect of Slit2 siRNA on IL-1β-induced NF-κB transcriptional activity. Our results demonstrate that Slit2 is decreased in human myometrium after labour and our knock-down studies describe an anti-inflammatory effect of Slit2 in myometrial cells.

  17. Transcriptional regulation of cardiac genes balance pro- and anti-hypertrophic mechanisms in hypertrophic cardiomyopathy

    Directory of Open Access Journals (Sweden)

    Nina Gennebäck

    2012-06-01

    Full Text Available Hypertrophic cardiomyopathy (HCM is characterized by unexplained left ventricular hypertrophy. HCM is often hereditary, but our knowledge of the mechanisms leading from mutation to phenotype is incomplete. The transcriptional expression patterns in the myocar - dium of HCM patients may contribute to understanding the mechanisms that drive and stabilize the hypertrophy. Cardiac myectomies/biopsies from 8 patients with hypertrophic obstructive cardiomyopathy (HOCM and 5 controls were studied with whole genome Illumina microarray gene expression (detecting 18 189 mRNA. When comparing HOCM myocardium to controls, there was significant transcriptional down-regulation of the MYH6, EGR1, APOB and FOS genes, and significant transcriptional up-regulation of the ACE2, JAK2, NPPA (ANP, APOA1 and HDAC5 genes. The transcriptional regulation revealed both pro- and anti-hypertrophic mechanisms. The pro-hypertrophic response was explained by the transcriptional down-regulation of MYH6, indicating that the switch to the fetal gene program is maintained, and the transcriptional up-regulation of JAK2 in the JAK-STAT pathway. The anti-hypertrophic response was seen as a transcriptional down-regulation of the immediate early genes (IEGs, FOS and EGR1, and a transcriptional up-regulation of ACE2 and HDAC5. This can be interpreted as a transcriptional endogenous protection system in the heart of the HOCM patients, neither growing nor suppressing the already hypertrophic myocardium.

  18. CCAAT/enhancer binding protein β (C/EBPβ isoforms as transcriptional regulators of the pro-invasive CDH3/P-cadherin gene in human breast cancer cells.

    Directory of Open Access Journals (Sweden)

    André Albergaria

    Full Text Available P-cadherin is a cell-cell adhesion molecule codified by the CDH3 gene, which expression is highly associated with undifferentiated cells in normal adult epithelial tissues, as well as with poorly differentiated carcinomas. In breast cancer, P-cadherin is frequently overexpressed in high-grade tumours and is a well-established indicator of aggressive tumour behaviour and poor patient prognosis. However, till now, the mechanisms controlling CDH3 gene activation have been poorly explored. Since we recently described the existence of several CCAAT/Enhancer Binding Protein β (C/EBPβ transcription factor binding sites at the CDH3 promoter, the aim of this study was to assess if the distinct C/EBPβ isoforms were directly involved in the transcriptional activation of the CDH3 gene in breast cancer cells. DNA-protein interactions, mutation analysis and luciferase reporter assay studies have been performed. We demonstrated that C/EBPβ is co-expressed with P-cadherin in breast cancer cells and all the three isoforms function as transcriptional regulators of the CDH3 gene, directly interacting with specific regions of its promoter. Interestingly, this transcriptional activation was only reflected at the P-cadherin protein level concerning the LIP isoform. Taken together, our data show that CDH3 is a newly defined transcriptional target gene of C/EBPβ isoforms in breast cancer, and we also identified the binding sites that are relevant for this activation.

  19. The effect of pro-inflammatory cytokines on immunophenotype, differentiation capacity and immunomodulatory functions of human mesenchymal stem cells.

    Science.gov (United States)

    Pourgholaminejad, Arash; Aghdami, Nasser; Baharvand, Hossein; Moazzeni, Seyed Mohammad

    2016-09-01

    Mesenchymal stem cells (MSCs), as cells with potential clinical utilities, have demonstrated preferential incorporation into inflammation sites. Immunophenotype and immunomodulatory functions of MSCs could alter by inflamed-microenvironments due to the local pro-inflammatory cytokine milieu. A major cellular mediator with specific function in promoting inflammation and pathogenicity of autoimmunity are IL-17-producing T helper 17 (Th17) cells that polarize in inflamed sites in the presence of pro-inflammatory cytokines such as Interleukin-1β (IL-1β), IL-6 and IL-23. Since MSCs are promising candidate for cell-based therapeutic strategies in inflammatory and autoimmune diseases, Th17 cell polarizing factors may alter MSCs phenotype and function. In this study, human bone-marrow-derived MSCs (BM-MSC) and adipose tissue-derived MSCs (AD-MSC) were cultured with or without IL-1β, IL-6 and IL-23 as pro-inflammatory cytokines. The surface markers and their differentiation capacity were measured in cytokine-untreated and cytokine-treated MSCs. MSCs-mediated immunomodulation was analyzed by their regulatory effects on mixed lymphocyte reaction (MLR) and the level of IL-10, TGF-β, IL-4, IFN-γ and TNF-α production as immunomodulatory cytokines. Pro-inflammatory cytokines showed no effect on MSCs morphology, immunophenotype and co-stimulatory molecules except up-regulation of CD45. Adipogenic and osteogenic differentiation capacity increased in CD45+ MSCs. Moreover, cytokine-treated MSCs preserved the suppressive ability of allogeneic T cell proliferation and produced higher level of TGF-β and lower level of IL-4. We concluded pro-inflammatory cytokines up-regulate the efficacy of MSCs in cell-based therapy of degenerative, inflammatory and autoimmune disorders. PMID:27288632

  20. NCAM regulates cell motility

    DEFF Research Database (Denmark)

    Prag, Søren; Lepekhin, Eugene A; Kolkova, Kateryna;

    2002-01-01

    Cell migration is required during development of the nervous system. The regulatory mechanisms for this process, however, are poorly elucidated. We show here that expression of or exposure to the neural cell adhesion molecule (NCAM) strongly affected the motile behaviour of glioma cells...... independently of homophilic NCAM interactions. Expression of the transmembrane 140 kDa isoform of NCAM (NCAM-140) caused a significant reduction in cellular motility, probably through interference with factors regulating cellular attachment, as NCAM-140-expressing cells exhibited a decreased attachment...... to a fibronectin substratum compared with NCAM-negative cells. Ectopic expression of the cytoplasmic part of NCAM-140 also inhibited cell motility, presumably via the non-receptor tyrosine kinase p59(fyn) with which NCAM-140 interacts. Furthermore, we showed that the extracellular part of NCAM acted as a paracrine...

  1. Pro-inflammatory cytokines downregulate Hsp27 and cause apoptosis of human retinal capillary endothelial cells

    Science.gov (United States)

    Nahomi, Rooban B.; Palmer, Allison; Roth, Katelyn E.; Fort, Patrice E.; Nagaraj, Ram H.

    2013-01-01

    The formation of acellular capillaries in the retina, a hallmark feature of diabetic retinopathy, is caused by apoptosis of endothelial cells and pericytes. The biochemical mechanism of such apoptosis remains unclear. Small heat shock proteins play an important role in the regulation of apoptosis. In the diabetic retina, pro-inflammatory cytokines are upregulated. In this study, we investigated the effects of pro-inflammatory cytokines on small heat shock protein 27 (Hsp27) in human retinal endothelial cells (HREC). In HREC cultured in the presence of cytokine mixtures (CM), a significant downregulation of Hsp27 at the protein and mRNA level occurred, with no effect on HSF-1, the transcription factor for Hsp27. The presence of high glucose (25 mM) amplified the effects of cytokines on Hsp27. CM activated indoleamine 2,3-dioxygenase (IDO) and enhanced the production of kynurenine and ROS. An inhibitor of IDO, 1-methyl tryptophan (MT), inhibited the effects of CM on Hsp27. CM also upregulated NOS2 and, consequently, nitric oxide (NO). A NOS inhibitor, L-NAME, and a ROS scavenger blocked the CM-mediated Hsp27 downregulation. While a NO donor in the culture medium did not decrease the Hsp27 content, a peroxynitrite donor and exogenous peroxynitrite did. The cytokines and high glucose-induced apoptosis of HREC were inhibited by MT and L-NAME. Downregulation of Hsp27 by a siRNA treatment promoted apoptosis in HREC. Together, these data suggest that pro-inflammatory cytokines induce the formation of ROS and NO, which, through the formation of peroxynitrite, reduce the Hsp27 content and bring about apoptosis of retinal capillary endothelial cells. PMID:24252613

  2. ProBDNF inhibits collective migration and chemotaxis of rat Schwann cells.

    Science.gov (United States)

    Ding, You-Quan; Li, Xuan-Yang; Xia, Guan-Nan; Ren, Hong-Yi; Zhou, Xin-Fu; Su, Bing-Yin; Qi, Jian-Guo

    2016-10-01

    Schwann cell migration, including collective migration and chemotaxis, is essential for the formation of coordinate interactions between Schwann cells and axons during peripheral nerve development and regeneration. Moreover, limited migration of Schwann cells imposed a serious obstacle on Schwann cell-astrocytes intermingling and spinal cord repair after Schwann cell transplantation into injured spinal cords. Recent studies have shown that mature brain-derived neurotrophic factor, a member of the neurotrophin family, inhibits Schwann cell migration. The precursor form of brain-derived neurotrophic factor, proBDNF, was expressed in the developing or degenerating peripheral nerves and the injured spinal cords. Since "the yin and yang of neurotrophin action" has been established as a common sense, proBDNF would be expected to promote Schwann cell migration. However, we found, in the present study, that exogenous proBDNF also inhibited in vitro collective migration and chemotaxis of RSC 96 cells, a spontaneously immortalized rat Schwann cell line. Moreover, proBDNF suppressed adhesion and spreading of those cells. At molecular level, proBDNF inhibits F-actin polymerization and focal adhesion dynamics in cultured RSC 96 cells. Therefore, our results suggested a special case against the classical opinion of "the yin and yang of neurotrophin action" and implied that proBDNF might modulate peripheral nerve development or regeneration and spinal cord repair through perturbing native or transplanted Schwann cell migration.

  3. E2F1-Mediated Induction of NFYB Attenuates Apoptosis via Joint Regulation of a Pro-Survival Transcriptional Program.

    Directory of Open Access Journals (Sweden)

    Xiaolei Jiang

    Full Text Available The E2F1 transcription factor regulates cell proliferation and apoptosis through the control of a considerable variety of target genes. Previous work has detailed the role of other transcription factors in mediating the specificity of E2F function. Here we identify the NF-YB transcription factor as a novel direct E2F1 target. Genome-wide expression analysis of the effects of NFYB knockdown on E2F1-mediated transcription identified a large group of genes that are co-regulated by E2F1 and NFYB. We also provide evidence that knockdown of NFYB enhances E2F1-induced apoptosis, suggesting a pro-survival function of the NFYB/E2F1 joint transcriptional program. Bioinformatic analysis suggests that deregulation of these NFY-dependent E2F1 target genes might play a role in sarcomagenesis as well as drug resistance.

  4. An anti-inflammatory oligopeptide produced by Entamoeba histolytica down-regulates the expression of pro-inflammatory chemokines.

    Science.gov (United States)

    Utrera-Barillas, Dolores; Velazquez, Juan R; Enciso, Antonio; Cruz, Samira Muñoz; Rico, Guadalupe; Curiel-Quesada, Everardo; Teran, Luis M; Kretschmer, Roberto R

    2003-10-01

    Axenically grown Entamoeba histolytica produces a pentapeptide (Met-Gln-Cys-Asn-Ser) with anti-inflammatory properties that, among others, inhibits the in vitro and in vivo locomotion of human monocytes, sparing polymorphonuclear leucocytes from this effect [hence the name originally given. Monocyte Locomotion Inhibitory Factor (MLIF)]. A synthetic construct of this peptide displays the same effects as the native material. We now added MLIF to resting and PMA-stimulated cells of a human monocyte cell line and measured the effect upon mRNA and protein expression of pro-inflammatory chemokines (RANTES, IP-10, MIP-1alpha, MIP-1beta, MCP-1, IL-8, I-309 and lymphotactin) and the shared CC receptor repertoire. The constitutive expression of these chemokines and the CC receptors was unaffected, whereas induced expression of MIP-1alpha, MIP-1beta, and I-309, and that of the CCR1 receptor--all involved in monocyte chemotaxis--was significantly inhibited by MLIF. This suggests that the inhibition of monocyte functions by MLIF may not only be exerted directly on these cells, but also--and perhaps foremost--through a conglomerate down-regulation of endogenous pro-inflammatory chemokines.

  5. Retrotransposon derepression leads to activation of the unfolded protein response and apoptosis in pro-B cells.

    Science.gov (United States)

    Pasquarella, Alessandra; Ebert, Anja; Pereira de Almeida, Gustavo; Hinterberger, Maria; Kazerani, Maryam; Nuber, Alexander; Ellwart, Joachim; Klein, Ludger; Busslinger, Meinrad; Schotta, Gunnar

    2016-05-15

    The H3K9me3-specific histone methyltransferase Setdb1 impacts on transcriptional regulation by repressing both developmental genes and retrotransposons. How impaired retrotransposon silencing may lead to developmental phenotypes is currently unclear. Here, we show that loss of Setdb1 in pro-B cells completely abrogates B cell development. In pro-B cells, Setdb1 is dispensable for silencing of lineage-inappropriate developmental genes. Instead, we detect strong derepression of endogenous murine leukemia virus (MLV) copies. This activation coincides with an unusual change in chromatin structure, with only partial loss of H3K9me3 and unchanged DNA methylation, but strongly increased H3K4me3. Production of MLV proteins leads to activation of the unfolded protein response pathway and apoptosis. Thus, our data demonstrate that B cell development depends on the proper repression of retrotransposon sequences through Setdb1. PMID:27013243

  6. Milk-derived tripeptides IPP (Ile-Pro-Pro and VPP (Val-Pro-Pro promote adipocyte differentiation and inhibit inflammation in 3T3-F442A cells.

    Directory of Open Access Journals (Sweden)

    Subhadeep Chakrabarti

    Full Text Available Milk derived tripeptides IPP (Ile-Pro-Pro and VPP (Val-Pro-Pro have shown promise as anti-hypertensive agents due to their inhibitory effects on angiotensin converting enzyme (ACE. Due to the key inter-related roles of hypertension, chronic inflammation and insulin resistance in the pathogenesis of metabolic syndrome, there is growing interest in investigating established anti-hypertensive agents for their effects on insulin sensitivity and inflammation. In this study, we examined the effects of IPP and VPP on 3T3-F442A murine pre-adipocytes, a widely used model for studying metabolic diseases. We found that both IPP and VPP induced beneficial adipogenic differentiation as manifested by intracellular lipid accumulation, upregulation of peroxisome proliferator-activated receptor gamma (PPARγ and secretion of the protective lipid hormone adiponectin by these cells. The observed effects were similar to those induced by insulin, suggesting potential benefits in the presence of insulin resistance. IPP and VPP also inhibited cytokine induced pro-inflammatory changes such as reduction in adipokine levels and activation of the nuclear factor kappa B (NF-κB pathway. Taken together, our findings suggest that IPP and VPP exert insulin-mimetic adipogenic effects and prevent inflammatory changes in adipocytes, which may offer protection against metabolic disease.

  7. File list: NoD.Bld.50.AllAg.Pro-B_cells [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  8. File list: NoD.Bld.10.AllAg.Pro-B_cells [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  9. Cis→Trans Isomerization of Pro(7) in Oxytocin Regulates Zn(2+) Binding.

    Science.gov (United States)

    Fuller, Daniel R; Glover, Matthew S; Pierson, Nicholas A; Kim, DoYong; Russell, David H; Clemmer, David E

    2016-08-01

    Ion mobility/mass spectrometry techniques are employed to investigate the binding of Zn(2+) to the nine-residue peptide hormone oxytocin (OT, Cys(1)-Tyr(2)-Ile(3)-Gln(4)-Asn(5)-Cys(6)-Pro(7)-Leu(8)-Gly(9)-NH2, having a disulfide bond between Cys(1) and Cys(6) residues). Zn(2+) binding to OT is known to increase the affinity of OT for its receptor [Pearlmutter, A. F., Soloff, M. S.: Characterization of the metal ion requirement for oxytocin-receptor interaction in rat mammary gland membranes. J. Biol. Chem. 254, 3899-3906 (1979)]. In the absence of Zn(2+), we find evidence for two primary OT conformations, which arise because the Cys(6)-Pro(7) peptide bond exists in both the trans- and cis-configurations. Upon addition of Zn(2+), we determine binding constants in water of KA = 1.43 ± 0.24 and 0.42 ± 0.12 μM(-1), for the trans- and cis-configured populations, respectively. The Zn(2+) bound form of OT, having a cross section of Ω = 235 Å(2), has Pro(7) in the trans-configuration, which agrees with a prior report [Wyttenbach, T., Liu, D., Bowers, M. T.: Interactions of the hormone oxytocin with divalent metal ions. J. Am. Chem. Soc. 130, 5993-6000 (2008)], in which it was proposed that Zn(2+) binds to the peptide ring and is further coordinated by interaction of the C-terminal, Pro(7)-Leu(8)-Gly(9)-NH2, tail. The present work shows that the cis-configuration of OT isomerizes to the trans-configuration upon binding Zn(2+). In this way, the proline residue regulates Zn(2+) binding to OT and, hence, is important in receptor binding. Graphical Abstract ᅟ. PMID:27154022

  10. Cis→Trans Isomerization of Pro7 in Oxytocin Regulates Zn2+ Binding

    Science.gov (United States)

    Fuller, Daniel R.; Glover, Matthew S.; Pierson, Nicholas A.; Kim, DoYong; Russell, David H.; Clemmer, David E.

    2016-08-01

    Ion mobility/mass spectrometry techniques are employed to investigate the binding of Zn2+ to the nine-residue peptide hormone oxytocin (OT, Cys1-Tyr2-Ile3-Gln4-Asn5-Cys6-Pro7-Leu8-Gly9-NH2, having a disulfide bond between Cys1 and Cys6 residues). Zn2+ binding to OT is known to increase the affinity of OT for its receptor [Pearlmutter, A. F., Soloff, M. S.: Characterization of the metal ion requirement for oxytocin-receptor interaction in rat mammary gland membranes. J. Biol. Chem. 254, 3899-3906 (1979)]. In the absence of Zn2+, we find evidence for two primary OT conformations, which arise because the Cys6-Pro7 peptide bond exists in both the trans- and cis-configurations. Upon addition of Zn2+, we determine binding constants in water of KA = 1.43 ± 0.24 and 0.42 ± 0.12 μM-1, for the trans- and cis-configured populations, respectively. The Zn2+ bound form of OT, having a cross section of Ω = 235 Å2, has Pro7 in the trans-configuration, which agrees with a prior report [Wyttenbach, T., Liu, D., Bowers, M. T.: Interactions of the hormone oxytocin with divalent metal ions. J. Am. Chem. Soc. 130, 5993-6000 (2008)], in which it was proposed that Zn2+ binds to the peptide ring and is further coordinated by interaction of the C-terminal, Pro7-Leu8-Gly9-NH2, tail. The present work shows that the cis-configuration of OT isomerizes to the trans-configuration upon binding Zn2+. In this way, the proline residue regulates Zn2+ binding to OT and, hence, is important in receptor binding.

  11. Cis→Trans Isomerization of Pro7 in Oxytocin Regulates Zn2+ Binding

    Science.gov (United States)

    Fuller, Daniel R.; Glover, Matthew S.; Pierson, Nicholas A.; Kim, DoYong; Russell, David H.; Clemmer, David E.

    2016-05-01

    Ion mobility/mass spectrometry techniques are employed to investigate the binding of Zn2+ to the nine-residue peptide hormone oxytocin (OT, Cys1-Tyr2-Ile3-Gln4-Asn5-Cys6-Pro7-Leu8-Gly9-NH2, having a disulfide bond between Cys1 and Cys6 residues). Zn2+ binding to OT is known to increase the affinity of OT for its receptor [Pearlmutter, A. F., Soloff, M. S.: Characterization of the metal ion requirement for oxytocin-receptor interaction in rat mammary gland membranes. J. Biol. Chem. 254, 3899-3906 (1979)]. In the absence of Zn2+, we find evidence for two primary OT conformations, which arise because the Cys6-Pro7 peptide bond exists in both the trans- and cis-configurations. Upon addition of Zn2+, we determine binding constants in water of KA = 1.43 ± 0.24 and 0.42 ± 0.12 μM-1, for the trans- and cis-configured populations, respectively. The Zn2+ bound form of OT, having a cross section of Ω = 235 Å2, has Pro7 in the trans-configuration, which agrees with a prior report [Wyttenbach, T., Liu, D., Bowers, M. T.: Interactions of the hormone oxytocin with divalent metal ions. J. Am. Chem. Soc. 130, 5993-6000 (2008)], in which it was proposed that Zn2+ binds to the peptide ring and is further coordinated by interaction of the C-terminal, Pro7-Leu8-Gly9-NH2, tail. The present work shows that the cis-configuration of OT isomerizes to the trans-configuration upon binding Zn2+. In this way, the proline residue regulates Zn2+ binding to OT and, hence, is important in receptor binding.

  12. γ-Synuclein confers both pro-invasive and doxorubicin-mediated pro-apoptotic properties to the colon adenocarcinoma LS 174T cell line.

    Science.gov (United States)

    Goh, Kai-Wey; Say, Yee-How

    2015-09-01

    γ-synuclein, a neuronal protein of the synuclein family, is involved in carcinogenesis. To investigate its role in colorectal cancer carcinogenesis, we overexpressed γ-synuclein in LS 174T colon adenocarcinoma cell line (termed LS 174T-γsyn). When compared with untransfected/mock transfectants, LS 174T-γsyn had higher mobility in scratch wound assay, tend to scatter more in cell-scattering assay, and had enhanced lamellipodia and filopodia formation in cell-spreading assay. Enhanced adhesion of LS 174T-γsyn to fibronectin and collagen and significantly higher proliferation rate showed that γ-synuclein was able to increase extracellular matrix interaction and promoted proliferation of LS 174T. Higher invasiveness of LS 174T-γsyn was evidenced by enhanced invasion to the bottom of the basement membrane in Boyden chamber assay. However, LS 174T-γsyn were significantly more vulnerable to doxorubicin, vincristine and hydrogen peroxide insults, via apoptotic cell death. LS 174T-γsyn also had reduced anchorage-independent growth as shown by reduced colony formation and reduced anoikis resistance. We found that overexpression of γ-synuclein confers both pro-invasive and doxorubicin-mediated pro-apoptotic properties to LS 174T, where the former was mediated through enhanced cyclic adenosine monophosphate response element binding protein (CREB) phosphorylation, while the latter involved hepatocyte growth factor (HGF) downregulation and subsequent downstream signalling pathways possibly involving extracellular signal-regulated kinases (ERK)1/2, p38α, c-Jun N-terminal kinase (JNK) pan and Signal Transducers and Activators of Transcription (STATs). This unexpected contrasting finding as compared to other similar studies on colon cancer cell lines might be correlated with the degree of tumour advancement from which the cell lines were derived from.

  13. A pro-inflammatory role of deubiquitinating enzyme cylindromatosis (CYLD) in vascular smooth muscle cells

    Energy Technology Data Exchange (ETDEWEB)

    Liu, Shuai [Shandong University Qilu Hospital Research Center for Cell Therapy, Key Laboratory of Cardiovascular Remodeling and Function Research, Qilu Hospital of Shandong University, Jinan 250012 (China); Department of Cell Biology and Anatomy, University of South Carolina School of Medicine, Columbia, SC 29208 (United States); Lv, Jiaju [Department of Urology, Shandong Provincial Hospital, Shandong University, Jinan 250021 (China); Han, Liping; Ichikawa, Tomonaga; Wang, Wenjuan; Li, Siying [Department of Cell Biology and Anatomy, University of South Carolina School of Medicine, Columbia, SC 29208 (United States); Wang, Xing Li [Shandong University Qilu Hospital Research Center for Cell Therapy, Key Laboratory of Cardiovascular Remodeling and Function Research, Qilu Hospital of Shandong University, Jinan 250012 (China); Tang, Dongqi, E-mail: tangdq@pathology.ufl.edu [Department of Pathology, Immunology, and Laboratory Medicine, University of Florida College of Medicine, Gainesville, FL 32610-0275 (United States); Cui, Taixing, E-mail: taixing.cui@uscmed.sc.edu [Department of Cell Biology and Anatomy, University of South Carolina School of Medicine, Columbia, SC 29208 (United States)

    2012-03-30

    Highlights: Black-Right-Pointing-Pointer Cyld deficiency suppresses pro-inflammatory phenotypic switch of VSMCs. Black-Right-Pointing-Pointer Cyld deficiency inhibits MAPK rather than NF-kB activity in inflamed VSMCs. Black-Right-Pointing-Pointer CYLD is up-regulated in the coronary artery with neointimal hyperplasia. -- Abstract: CYLD, a deubiquitinating enzyme (DUB), is a critical regulator of diverse cellular processes, ranging from proliferation and differentiation to inflammatory responses, via regulating multiple key signaling cascades such as nuclear factor kappa B (NF-{kappa}B) pathway. CYLD has been shown to inhibit vascular lesion formation presumably through suppressing NF-{kappa}B activity in vascular cells. However, herein we report a novel role of CYLD in mediating pro-inflammatory responses in vascular smooth muscle cells (VSMCs) via a mechanism independent of NF-{kappa}B activity. Adenoviral knockdown of Cyld inhibited basal and the tumor necrosis factor alpha (TNF{alpha})-induced mRNA expression of pro-inflammatory cytokines including monocyte chemotactic protein-1 (Mcp-1), intercellular adhesion molecule (Icam-1) and interleukin-6 (Il-6) in rat adult aortic SMCs (RASMCs). The CYLD deficiency led to increases in the basal NF-{kappa}B transcriptional activity in RASMCs; however, did not affect the TNF{alpha}-induced NF-{kappa}B activity. Intriguingly, the TNF{alpha}-induced I{kappa}B phosphorylation was enhanced in the CYLD deficient RASMCs. While knocking down of Cyld decreased slightly the basal expression levels of I{kappa}B{alpha} and I{kappa}B{beta} proteins, it did not alter the kinetics of TNF{alpha}-induced I{kappa}B protein degradation in RASMCs. These results indicate that CYLD suppresses the basal NF-{kappa}B activity and TNF{alpha}-induced I{kappa}B kinase activation without affecting TNF{alpha}-induced NF-{kappa}B activity in VSMCs. In addition, knocking down of Cyld suppressed TNF{alpha}-induced activation of mitogen activated protein

  14. MTOR regulates the pro-tumorigenic senescence-associated secretory phenotype by promoting IL1A translation

    Science.gov (United States)

    Laberge, Remi-Martin; Sun, Yu; Orjalo, Arturo V.; Patil, Christopher K.; Freund, Adam; Zhou, Lili; Curran, Samuel C.; Davalos, Albert R.; Wilson-Edell, Kathleen A.; Liu, Su; Limbad, Chandani; Demaria, Marco; Li, Patrick; Hubbard, Gene B.; Ikeno, Yuji; Javors, Martin; Desprez, Pierre-Yves; Benz, Christopher C.; Kapahi, Pankaj; Nelson, Peter S.; Campisi, Judith

    2015-01-01

    The TOR (target of rapamycin) kinase limits longevity by poorly understood mechanisms. Rapamycin suppresses the mammalian TORC1 complex, which regulates translation, and extends lifespan in diverse species, including mice. We show that rapamycin selectively blunts the pro-inflammatory phenotype of senescent cells. Cellular senescence suppresses cancer by preventing cell proliferation. However, as senescent cells accumulate with age, the senescence-associated secretory phenotype (SASP) can disrupt tissues and contribute to age-related pathologies, including cancer. MTOR inhibition suppressed the secretion of inflammatory cytokines by senescent cells. Rapamycin reduced IL6 and other cytokine mRNA levels, but selectively suppressed translation of the membrane-bound cytokine IL1A. Reduced IL1A diminished NF-κB transcriptional activity, which controls much of the SASP; exogenous IL1A restored IL6 secretion to rapamycin-treated cells. Importantly, rapamycin suppressed the ability of senescent fibroblasts to stimulate prostate tumour growth in mice. Thus, rapamycin might ameliorate age-related pathologies, including late-life cancer, by suppressing senescence-associated inflammation. PMID:26147250

  15. Regulation of beta cell replication

    DEFF Research Database (Denmark)

    Lee, Ying C; Nielsen, Jens Høiriis

    2008-01-01

    Beta cell mass, at any given time, is governed by cell differentiation, neogenesis, increased or decreased cell size (cell hypertrophy or atrophy), cell death (apoptosis), and beta cell proliferation. Nutrients, hormones and growth factors coupled with their signalling intermediates have been...... suggested to play a role in beta cell mass regulation. In addition, genetic mouse model studies have indicated that cyclins and cyclin-dependent kinases that determine cell cycle progression are involved in beta cell replication, and more recently, menin in association with cyclin-dependent kinase...... inhibitors has been demonstrated to be important in beta cell growth. In this review, we consider and highlight some aspects of cell cycle regulation in relation to beta cell replication. The role of cell cycle regulation in beta cell replication is mostly from studies in rodent models, but whether...

  16. Tissue kallikrein mediates pro-inflammatory pathways and activation of protease-activated receptor-4 in proximal tubular epithelial cells.

    Directory of Open Access Journals (Sweden)

    Wai Han Yiu

    Full Text Available Tissue kallikrein (KLK1 expression is up-regulated in human diabetic kidney tissue and induced by high glucose (HG in human proximal tubular epithelial cells (PTEC. Since the kallikrein-kinin system (KKS has been linked to cellular inflammatory process in many diseases, it is likely that KLK1 expression may mediate the inflammatory process during the development of diabetic nephropathy. In this study, we explored the role of KLK1 in tubular pro-inflammatory responses under the diabetic milieu. Recombinant KLK1 stimulated the production of inflammatory cytokines in PTEC via the activation of p42/44 and p38 MAPK signaling pathways. Molecular knockdown of endogenous KLK1 expression by siRNA transfection in PTEC attenuated advanced glycation end-products (AGE-induced IL-8 and ICAM-1 productions in vitro. Interestingly, exposure of PTEC to KLK1 induced the expression of protease-activated receptors (PARs. There was a 2.9-fold increase in PAR-4, 1.4-fold increase in PAR-1 and 1.2-fold increase in PAR-2 mRNA levels. Activation of PAR-4 by a selective agonist was found to elicit the pro-inflammatory and pro-fibrotic phenotypes in PTEC while blockade of the receptor by specific antagonist attenuated high glucose-induced IL-6, CCL-2, CTGF and collagen IV expression. Calcium mobilization by the PAR-4 agonist in PTEC was desensitized by pretreatment with KLK1. Consistent with these in vitro findings, there was a markedly up-regulation of tubular PAR-4 expression in human diabetic renal cortical tissues. Together, these results suggest that up-regulation of KLK1 in tubular epithelial cells may mediate pro-inflammatory pathway and PAR activation during diabetic nephropathy and provide a new therapeutic target for further investigation.

  17. Tissue kallikrein mediates pro-inflammatory pathways and activation of protease-activated receptor-4 in proximal tubular epithelial cells.

    Science.gov (United States)

    Yiu, Wai Han; Wong, Dickson W L; Chan, Loretta Y Y; Leung, Joseph C K; Chan, Kwok Wah; Lan, Hui Yao; Lai, Kar Neng; Tang, Sydney C W

    2014-01-01

    Tissue kallikrein (KLK1) expression is up-regulated in human diabetic kidney tissue and induced by high glucose (HG) in human proximal tubular epithelial cells (PTEC). Since the kallikrein-kinin system (KKS) has been linked to cellular inflammatory process in many diseases, it is likely that KLK1 expression may mediate the inflammatory process during the development of diabetic nephropathy. In this study, we explored the role of KLK1 in tubular pro-inflammatory responses under the diabetic milieu. Recombinant KLK1 stimulated the production of inflammatory cytokines in PTEC via the activation of p42/44 and p38 MAPK signaling pathways. Molecular knockdown of endogenous KLK1 expression by siRNA transfection in PTEC attenuated advanced glycation end-products (AGE)-induced IL-8 and ICAM-1 productions in vitro. Interestingly, exposure of PTEC to KLK1 induced the expression of protease-activated receptors (PARs). There was a 2.9-fold increase in PAR-4, 1.4-fold increase in PAR-1 and 1.2-fold increase in PAR-2 mRNA levels. Activation of PAR-4 by a selective agonist was found to elicit the pro-inflammatory and pro-fibrotic phenotypes in PTEC while blockade of the receptor by specific antagonist attenuated high glucose-induced IL-6, CCL-2, CTGF and collagen IV expression. Calcium mobilization by the PAR-4 agonist in PTEC was desensitized by pretreatment with KLK1. Consistent with these in vitro findings, there was a markedly up-regulation of tubular PAR-4 expression in human diabetic renal cortical tissues. Together, these results suggest that up-regulation of KLK1 in tubular epithelial cells may mediate pro-inflammatory pathway and PAR activation during diabetic nephropathy and provide a new therapeutic target for further investigation. PMID:24586431

  18. (Pro)insulin associates with Golgi membranes of pancreatic B cells.

    OpenAIRE

    Orci, L; Ravazzola, M; Perrelet, A

    1984-01-01

    The immunocytochemical demonstration of (pro)insulin on intracellular membrane compartments of the pancreatic B cell reveals that the immunolabeling detected by the protein A/gold method is associated, at the level of the Golgi apparatus, with the inner aspect of the cisternal membranes; on the secretory granules, by contrast, insulin immunoreactive sites predominate over the granule core, and very little immunoreactivity is associated with the granule membrane. The localization of (pro)insul...

  19. REDUCED TISSUE OSMOLARITY INCREASES TRPV4 EXPRESSION AND PRO-INFLAMMATORY CYTOKINES IN INTERVERTEBRAL DISC CELLS

    Science.gov (United States)

    Walter, B.A.; Purmessur, D; Moon, A.; Occhiogrosso, J.; Laudier, D.M.; Hecht, A.C.; Iatridis, J.C.

    2016-01-01

    The mechanical behaviour and cellular metabolism of intervertebral discs (IVDs) and articular cartilage are strongly influenced by their proteoglycan content and associated osmotic properties. This osmotic environment is a biophysical signal that changes with disease and may contribute to the elevated matrix breakdown and altered biologic response to loading observed in IVD degeneration and osteoarthritis. This study tested the hypothesis that changes in osmo-sensation by the transient receptor potential vallinoid-4 (TRPV4) ion channel occur with disease and contribute to the inflammatory environment found during degeneration. Immunohistochemistry on bovine IVDs from an inflammatory organ culture model were used to investigate if TRPV4 is expressed in the IVD and how expression changes with degeneration. Western blot, live-cell calcium imaging, and qRT-PCR were used to investigate whether osmolarity changes or tumour necrosis factor α (TNFα) regulate TRPV4 expression, and how altered TRPV4 expression influences calcium signalling and pro-inflammatory cytokine expression. TRPV4 expression correlated with TNFα expression, and was increased when cultured in reduced medium osmolarity and unaltered with TNFα-stimulation. Increased TRPV4 expression increased the calcium flux following TRPV4 activation and increased interleukin-1β (IL-1β) and IL-6 gene expression in IVD cells. TRPV4 expression was qualitatively elevated in regions of aggrecan depletion in degenerated human IVDs. Collectively, results suggest that reduced tissue osmolarity, likely following proteoglycan degradation, can increase TRPV4 signalling and enhance pro-inflammatory cytokine production, suggesting changes in TRPV4 mediated osmo-sensation may contribute to the progressive matrix breakdown in disease. PMID:27434269

  20. Fish and mammalian phagocytes differentially regulate pro-inflammatory and homeostatic responses in vivo.

    Directory of Open Access Journals (Sweden)

    Aja M Rieger

    Full Text Available Phagocytosis is a cellular mechanism that is important to the early induction of antimicrobial responses and the regulation of adaptive immunity. At an inflammatory site, phagocytes serve as central regulators for both pro-inflammatory and homeostatic anti-inflammatory processes. However, it remains unclear if this is a recent evolutionary development or whether the capacity to balance between these two seemingly contradictory processes is a feature already displayed in lower vertebrates. In this study, we used murine (C57BL/6 and teleost fish (C. auratus in vitro and in vivo models to assess the evolutionary conservation of this dichotomy at a site of inflammation. At the level of the macrophage, we found that teleost fish already displayed divergent pro-inflammatory and homeostatic responses following internalization of zymosan or apoptotic bodies, respectively, and that these were consistent with those of mice. However, fish and mice displayed significant differences in vivo with regards to the level of responsiveness to zymosan and apoptotic bodies, the identity of infiltrating leukocytes, their rate of infiltration, and the kinetics and strength of resulting antimicrobial responses. Unlike macrophages, significant differences were identified between teleost and murine neutrophilic responses. We report for the first time that activated murine, but not teleost neutrophils, possess the capacity to internalize apoptotic bodies. This internalization translates into reduction of neutrophil ROS production. This may play an important part in the recently identified anti-inflammatory activity that mammalian neutrophils display during the resolution phase of inflammation. Our observations are consistent with continued honing of inflammatory control mechanisms from fish to mammals, and provide added insights into the evolutionary path that has resulted in the integrated, multilayered responses that are characteristic of higher vertebrates.

  1. Local transplantation of osteogenic pre-differentiated autologous adipose-derived mesenchymal stem cells may accelerate non-union fracture healing with limited pro-metastatic potency.

    Science.gov (United States)

    Han, Duanyang; Han, Na; Zhang, Peixun; Jiang, Baoguo

    2015-01-01

    Fracture non-union is a serious complication in orthopedic clinical practice. Mesenchymal stem cells are believed to play a vital role in fracture healing process. Among various origins of mesenchymal stem cell, adipose derived stem cells hold great promise especially in clinical milieu. However, the wide spread application of mesenchymal stem cell based therapy is impeded by the pro-metastasis nature of the mesenchymal stem cell itself. Based on the findings from previous studies, we hypothesize that local transplanted osteogenic pre-differentiatiated adipose stem cell may promote the non-union fracture healing. Moreover, the pre-differnetiation stem cells by down-regulating the expression of CCL5 and CCL2. This novel osteogenic pre-differnetiation technique may help clinical orthopedists to resolve the refractory non-union cases and shed new light on other stem cell based therapies to counteract to avoid the pro-metastasis nature of the mesenchymal stem cells. PMID:25785146

  2. A latent pro-survival function for the mir-290-295 cluster in mouse embryonic stem cells.

    Directory of Open Access Journals (Sweden)

    Grace X Y Zheng

    2011-05-01

    Full Text Available MicroRNAs (miRNAs post-transcriptionally regulate the expression of thousands of distinct mRNAs. While some regulatory interactions help to maintain basal cellular functions, others are likely relevant in more specific settings, such as response to stress. Here we describe such a role for the mir-290-295 cluster, the dominant miRNA cluster in mouse embryonic stem cells (mESCs. Examination of a target list generated from bioinformatic prediction, as well as expression data following miRNA loss, revealed strong enrichment for apoptotic regulators, two of which we validated directly: Caspase 2, the most highly conserved mammalian caspase, and Ei24, a p53 transcriptional target. Consistent with these predictions, mESCs lacking miRNAs were more likely to initiate apoptosis following genotoxic exposure to gamma irradiation or doxorubicin. Knockdown of either candidate partially rescued this pro-apoptotic phenotype, as did transfection of members of the mir-290-295 cluster. These findings were recapitulated in a specific mir-290-295 deletion line, confirming that they reflect miRNA functions at physiological levels. In contrast to the basal regulatory roles previously identified, the pro-survival phenotype shown here may be most relevant to stressful gestations, where pro-oxidant metabolic states induce DNA damage. Similarly, this cluster may mediate chemotherapeutic resistance in a neoplastic context, making it a useful clinical target.

  3. Imbalances in Mobilization and Activation of Pro-Inflammatory and Vascular Reparative Bone Marrow-Derived Cells in Diabetic Retinopathy.

    Science.gov (United States)

    Chakravarthy, Harshini; Beli, Eleni; Navitskaya, Svetlana; O'Reilly, Sandra; Wang, Qi; Kady, Nermin; Huang, Chao; Grant, Maria B; Busik, Julia V

    2016-01-01

    Diabetic retinopathy is a sight-threatening complication of diabetes, affecting 65% of patients after 10 years of the disease. Diabetic metabolic insult leads to chronic low-grade inflammation, retinal endothelial cell loss and inadequate vascular repair. This is partly due to bone marrow (BM) pathology leading to increased activity of BM-derived pro-inflammatory monocytes and impaired function of BM-derived reparative circulating angiogenic cells (CACs). We propose that diabetes has a significant long-term effect on the nature and proportion of BM-derived cells that circulate in the blood, localize to the retina and home back to their BM niche. Using a streptozotocin mouse model of diabetic retinopathy with GFP BM-transplantation, we have demonstrated that BM-derived circulating pro-inflammatory monocytes are increased in diabetes while reparative CACs are trapped in the BM and spleen, with impaired release into circulation. Diabetes also alters activation of splenocytes and BM-derived dendritic cells in response to LPS stimulation. A majority of the BM-derived GFP cells that migrate to the retina express microglial markers, while others express endothelial, pericyte and Müller cell markers. Diabetes significantly increases infiltration of BM-derived microglia in an activated state, while reducing infiltration of BM-derived endothelial progenitor cells in the retina. Further, control CACs injected into the vitreous are very efficient at migrating back to their BM niche, whereas diabetic CACs have lost this ability, indicating that the in vivo homing efficiency of diabetic CACs is dramatically decreased. Moreover, diabetes causes a significant reduction in expression of specific integrins regulating CAC migration. Collectively, these findings indicate that BM pathology in diabetes could play a role in both increased pro-inflammatory state and inadequate vascular repair contributing to diabetic retinopathy.

  4. Imbalances in Mobilization and Activation of Pro-Inflammatory and Vascular Reparative Bone Marrow-Derived Cells in Diabetic Retinopathy.

    Directory of Open Access Journals (Sweden)

    Harshini Chakravarthy

    Full Text Available Diabetic retinopathy is a sight-threatening complication of diabetes, affecting 65% of patients after 10 years of the disease. Diabetic metabolic insult leads to chronic low-grade inflammation, retinal endothelial cell loss and inadequate vascular repair. This is partly due to bone marrow (BM pathology leading to increased activity of BM-derived pro-inflammatory monocytes and impaired function of BM-derived reparative circulating angiogenic cells (CACs. We propose that diabetes has a significant long-term effect on the nature and proportion of BM-derived cells that circulate in the blood, localize to the retina and home back to their BM niche. Using a streptozotocin mouse model of diabetic retinopathy with GFP BM-transplantation, we have demonstrated that BM-derived circulating pro-inflammatory monocytes are increased in diabetes while reparative CACs are trapped in the BM and spleen, with impaired release into circulation. Diabetes also alters activation of splenocytes and BM-derived dendritic cells in response to LPS stimulation. A majority of the BM-derived GFP cells that migrate to the retina express microglial markers, while others express endothelial, pericyte and Müller cell markers. Diabetes significantly increases infiltration of BM-derived microglia in an activated state, while reducing infiltration of BM-derived endothelial progenitor cells in the retina. Further, control CACs injected into the vitreous are very efficient at migrating back to their BM niche, whereas diabetic CACs have lost this ability, indicating that the in vivo homing efficiency of diabetic CACs is dramatically decreased. Moreover, diabetes causes a significant reduction in expression of specific integrins regulating CAC migration. Collectively, these findings indicate that BM pathology in diabetes could play a role in both increased pro-inflammatory state and inadequate vascular repair contributing to diabetic retinopathy.

  5. MTOR regulates the pro-tumorigenic senescence-associated secretory phenotype by promoting IL1A translation

    NARCIS (Netherlands)

    Laberge, Remi-Martin; Sun, Yu; Orjalo, Arturo V; Patil, Christopher K; Freund, Adam; Zhou, Lili; Curran, Samuel C; Davalos, Albert R; Wilson-Edell, Kathleen A; Liu, Su; Limbad, Chandani; Demaria, Marco; Li, Patrick; Hubbard, Gene B; Ikeno, Yuji; Javors, Martin; Desprez, Pierre-Yves; Benz, Christopher C; Kapahi, Pankaj; Nelson, Peter S; Campisi, Judith

    2015-01-01

    The TOR (target of rapamycin) kinase limits longevity by poorly understood mechanisms. Rapamycin suppresses the mammalian TORC1 complex, which regulates translation, and extends lifespan in diverse species, including mice. We show that rapamycin selectively blunts the pro-inflammatory phenotype of s

  6. Intrinsic, pro-apoptotic effects of IGFBP-3 on breast cancer cells are reversible: Involvement of PKA, Rho and ceramide.

    Directory of Open Access Journals (Sweden)

    Claire M Perks

    2011-05-01

    Full Text Available We established previously that IGFBP-3 could exert positive or negative effects on cell function depending upon the extracellular matrix composition and by interacting with integrin signalling. To elicit its pro-apoptotic effects IGFBP-3 bound to caveolin-1 and the beta 1 integrin receptor and increased their association culminating in MAPK activation. Disruption of these complexes or blocking the beta 1 integrin receptor reversed these intrinsic actions of IGFBP-3. In this study we have examined the signalling pathway between integrin receptor binding and MAPK activation that mediates the intrinsic, pro-apoptotic actions of IGFBP-3. We found on inhibiting protein kinase A(PKA, Rho associated kinase (ROCK and ceramide, the accentuating effects of IGFBP-3 on apoptotic triggers were reversed, such that IGFBP-3 then conferred cell survival. We established that IGFBP-3 activated Rho, the upstream regulator of ROCK and that beta1 integrin and PKA were upstream of Rho activation, whereas the involvement of ceramide was downstream. The beta 1 integrin, PKA, Rho and ceramide were all upstream of MAPK activation. These data highlight key components involved in the pro-apoptotic effects of IGFBP-3 and that inhibiting them leads to a reversal in the action of IGFBP-3.

  7. Expression of IP-10 Chemokine is Regulated by Pro-inflammatory Cytokines in Cultured Hepatocytes

    Directory of Open Access Journals (Sweden)

    Gholamhossein Hassanshahi

    2007-09-01

    Full Text Available Chemokines are classified in four distinct groups as CXC, CC, CX3C and C, depending on the presence or absence of a motif called ELR (Arg-Leu-Glu before the first cysteine residue in their structure. CXC chemokines are also subdivided into ELR+ and ELR-. Increasing evidence has indicated the existence of a chemokine network in the liver which is involved in both physiological responses and, under certain circumstances, pathological and repair processes following hepatic injury.  The CXC chemokines play a major role in both these processes, and much attention has been focused on their therapeutic applications to liver disease. The aim of this study was to examine the response of cultured hepatocytes to exogenous inflammatory cytokines (TNF-a and IFN-g regarding expression of IP-10 and growth regulatory oncogen (Gro chemokines. In this study we employed western and northern analysis to measure chemokines at the level of protein and mRNA by hepatocytes in response to pro-inflammatory cytokines. We found that, the pro-inflammatory cytokines, TNF-a and IFN-g, selectively stimulated expression of IP-10 but were without effect on Gro. This confirms a potential direct involvement of these cytokines in chemokine production by hepatocytes. Thus, IFN-g and TNF-a may play a role in hepatic injury and inflammation and produce some of their biological effects by localized induction of chemokines by hepatocytes. Given the similarity to an acute phase response, we were able to show that IFN-g and TNF-a mimicked the effects of cell isolation and culture on induction of IP-10 expression. Further, evidence for linkages between IFN-g and TNF-a and liver injuries is seen in hepatitis C and hepatitis B in which increased levels of TNF-a and its soluble receptor were reported. 

  8. p75 neurotrophin receptor and pro-BDNF promote cell survival and migration in clear cell renal cell carcinoma

    Science.gov (United States)

    Sánchez-Prieto, Ricardo; Saada, Sofiane; Naves, Thomas; Guillaudeau, Angélique; Perraud, Aurélie; Sindou, Philippe; Lacroix, Aurélie; Descazeaud, Aurélien; Lalloué, Fabrice; Jauberteau, Marie-Odile

    2016-01-01

    p75NTR, a member of TNF receptor family, is the low affinity receptor common to several mature neurotrophins and the high affinity receptor for pro-neurotrophins. Brain-Derived Neurotrophic Factor (BDNF), a member of neurotrophin family has been described to play an important role in development and progression of several cancers, through its binding to a high affinity tyrosine kinase receptor B (TrkB) and/or p75NTR. However, the functions of these two receptors in renal cell carcinoma (RCC) have never been investigated. An overexpression of p75NTR, pro-BDNF, and to a lesser extent for TrkB and sortilin, was detected by immunohistochemistry in a cohort of 83 clear cell RCC tumors. p75NTR, mainly expressed in tumor tissues, was significantly associated with higher Fuhrman grade in multivariate analysis. In two derived-RCC lines, 786-O and ACHN cells, we demonstrated that pro-BDNF induced cell survival and migration, through p75NTR as provided by p75NTR RNA silencing or blocking anti-p75NTR antibody. This mechanism is independent of TrkB activation as demonstrated by k252a, a tyrosine kinase inhibitor for Trk neurotrophin receptors. Taken together, these data highlight for the first time an important role for p75NTR in renal cancer and indicate a putative novel target therapy in RCC. PMID:27120782

  9. Retracted: Effects of pro-inflammatory cytokines on mineralization potential of rat dental pulp stem cells

    NARCIS (Netherlands)

    Yang, X.; Walboomers, X.F.; Bian, Z.; Jansen, J.A.; Fan, M.

    2011-01-01

    The following article from the Journal of Tissue Engineering and Regenerative Medicine, 'Effects of Pro-inflammatory Cytokines on Mineralization Potential of Rat Dental Pulp Stem Cells' by Yang X, Walboomers XF, Bian Z, Jansen JA, Fan M, published online on 11 July 2011 in Wiley Online Library (onli

  10. Identification of the sorting signal motif within pro-opiomelanocortin for the regulated secretory pathway

    DEFF Research Database (Denmark)

    Cool, D R; Fenger, M; Snell, C R;

    1995-01-01

    The NH2-terminal region of pro-opiomelanocortin (POMC) is highly conserved across species, having two disulfide bridges that cause the formation of an amphipathic hairpin loop structure between the 2nd and 3rd cysteine residues (Cys8 to Cys20). The role that the NH2-terminal region of pro-opiomel...

  11. VMP1 related autophagy and apoptosis in colorectal cancer cells: VMP1 regulates cell death

    Energy Technology Data Exchange (ETDEWEB)

    Qian, Qinyi [Department of Ultrasonograph, Changshu No. 2 People’s Hospital, Changshu (China); Zhou, Hao; Chen, Yan [Department of General Surgery, The First Affiliated Hospital of Soochow University, Suzhou (China); Shen, Chenglong [Department of General Surgery, Changshu No. 2 People’s Hospital, Changshu (China); He, Songbing; Zhao, Hua; Wang, Liang [Department of General Surgery, The First Affiliated Hospital of Soochow University, Suzhou (China); Wan, Daiwei, E-mail: 372710369@qq.com [Department of Hepatobiliary Surgery, The First Affiliated Hospital of Sun Yat-sen University, Guangzhou (China); Gu, Wen, E-mail: 505339704@qq.com [Department of General Surgery, The First Affiliated Hospital of Soochow University, Suzhou (China)

    2014-01-17

    Highlights: •This research confirmed VMP1 as a regulator of autophagy in colorectal cancer cell lines. •We proved the pro-survival role of VMP1-mediated autophagy in colorectal cancer cell lines. •We found the interaction between VMP1 and BECLIN1 also existing in colorectal cancer cell lines. -- Abstract: Vacuole membrane protein 1 (VMP1) is an autophagy-related protein and identified as a key regulator of autophagy in recent years. In pancreatic cell lines, VMP1-dependent autophagy has been linked to positive regulation of apoptosis. However, there are no published reports on the role of VMP1 in autophagy and apoptosis in colorectal cancers. Therefore, to address this gap of knowledge, we decided to interrogate regulation of autophagy and apoptosis by VMP1. We have studied the induction of autophagy by starvation and rapamycin treatment in colorectal cell lines using electron microscopy, immunofluorescence, and immunoblotting. We found that starvation-induced autophagy correlated with an increase in VMP1 expression, that VMP1 interacted with BECLIN1, and that siRNA mediated down-regulation of VMP1-reduced autophagy. Next, we examined the relationship between VMP1-dependent autophagy and apoptosis and found that VMP1 down-regulation sensitizes cells to apoptosis and that agents that induce apoptosis down-regulate VMP1. In conclusion, similar to its reported role in other cell types, VMP1 is an important regulator of autophagy in colorectal cell lines. However, in contrast to its role in pancreatic cell lines, in colorectal cancer cells, VMP1-dependent autophagy appears to be pro-survival rather than pro-cell death.

  12. Apigenin inhibits PMA-induced expression of pro-inflammatory cytokines and AP-1 factors in A549 cells.

    Science.gov (United States)

    Patil, Rajeshwari H; Babu, R L; Naveen Kumar, M; Kiran Kumar, K M; Hegde, Shubha M; Ramesh, Govindarajan T; Chidananda Sharma, S

    2015-05-01

    Acute and chronic alveolar or bronchial inflammation is thought to be central to the pathogenesis of many respiratory disorders. Cytokines and granulocyte macrophage colony-stimulating factors (GM-CSF) play an important role in chronic inflammation. Activator protein-1 (AP-1) the superfamily of transcription factors is involved in proliferation, differentiation, apoptosis, and transformation including inflammation. Understanding the function and regulation of proinflammatory factors involved in inflammation may provide the novel therapeutic strategies in the treatment of inflammatory diseases. Our aim of the present study is to investigate the pro-inflammatory cytokines and pattern of AP-1 factors expressed during activation of lung adenocarcinoma A549 cells by Phorbol-12-myristate-13-acetate (PMA) and to understand the anti-inflammatory effect of apigenin. A549 cells were treated with and without PMA or apigenin, and the cell viability was assessed by MTT assay. Expressions of inflammatory mediators and different AP-1 factors were analyzed by semi-quantitative RT-PCR. IL-6 protein secreted was analyzed by ELISA, and expressions of IL-1β, c-Jun, and c-Fos proteins were analyzed by Western blotting. Activation of A549 cells by PMA, induced the expression of pro-inflammatory cytokine (IL-1β, IL-2, IL-6, IL-8, and TNF-α) mRNAs and secretion of IL-6 and the expression of specific AP-1 factors (c-Jun, c-Fos, and Fra-1). Treatment of cells with apigenin, significantly inhibited PMA-stimulated mRNA expression of above pro-inflammatory cytokines, AP-1 factors, cyclooxygenase-2, and secretion of IL-6 protein. Results suggested that the AP-1 factors may be involved in inflammation and apigenin has anti-inflammatory effect, which may be useful for therapeutic management of lung inflammatory diseases. PMID:25666088

  13. Negative regulators of cell proliferation

    Science.gov (United States)

    Johnson, T. C.; Spooner, B. S. (Principal Investigator)

    1994-01-01

    Cell proliferation is governed by the influence of both mitogens and inhibitors. Although cell contact has long been thought to play a fundamental role in cell cycling regulation, and negative regulators have long been suspected to exist, their isolation and purification has been complicated by a variety of technical difficulties. Nevertheless, over recent years an ever-expanding list of putative negative regulators have emerged. In many cases, their biological inhibitory activities are consistent with density-dependent growth inhibition. Most likely their interactions with mitogenic agents, at an intracellular level, are responsible for either mitotic arrest or continued cell cycling. A review of naturally occurring cell growth inhibitors is presented with an emphasis on those factors shown to be residents of the cell surface membrane. Particular attention is focused on a cell surface sialoglycopeptide, isolated from intact bovine cerebral cortex cells, which has been shown to inhibit the proliferation of an unusually wide range of target cells. The glycopeptide arrest cells obtained from diverse species, both fibroblasts and epithelial cells, and a broad variety of transformed cells. Signal transduction events and a limited spectrum of cells that are refractory to the sialoglycopeptide have provided insight into the molecular events mediated by this cell surface inhibitor.

  14. Selenium supplementation in patients undergoing hematopoietic stem cell transplantation: effects on pro-inflammatory cytokines levels

    OpenAIRE

    Daeian, Nesa; Radfar, Mania; Jahangard-Rafsanjani, Zahra; Hadjibabaie, Molouk; Ghavamzadeh, Ardeshir

    2014-01-01

    Background Pro-inflammatory cytokines including tumor necrosis factor alpha (TNF-α), interleukin-1β (IL-1β) and interleukin-6 (IL-6) play an important role in the development of hematopoietic stem cell transplantation (HSCT) complications. We explored the effect of Selenium as an antioxidant and anti-inflammatory agent on pro-inflammatory cytokines levels in HSCT candidates. Findings Plasma concentrations of TNF-α, IL-1β and IL-6 were measured in 74 patients from a double-blind, randomized, p...

  15. Glutathione S-transferase pi modulates NF-κB activation and pro-inflammatory responses in lung epithelial cells

    Directory of Open Access Journals (Sweden)

    Jane T. Jones

    2016-08-01

    Full Text Available Nuclear Factor kappa B (NF-κB is a transcription factor family critical in the activation of pro- inflammatory responses. The NF-κB pathway is regulated by oxidant-induced post-translational modifications. Protein S-glutathionylation, or the conjugation of the antioxidant molecule, glutathione to reactive cysteines inhibits the activity of inhibitory kappa B kinase beta (IKKβ, among other NF-κB proteins. Glutathione S-transferase Pi (GSTP is an enzyme that has been shown to catalyze protein S-glutathionylation (PSSG under conditions of oxidative stress. The objective of the present study was to determine whether GSTP regulates NF-κB signaling, S-glutathionylation of IKK, and subsequent pro-inflammatory signaling. We demonstrated that, in unstimulated cells, GSTP associated with the inhibitor of NF-κB, IκBα. However, exposure to LPS resulted in a rapid loss of association between IκBα and GSTP, and instead led to a protracted association between IKKβ and GSTP. LPS exposure also led to increases in the S-glutathionylation of IKKβ. SiRNA-mediated knockdown of GSTP decreased IKKβ-SSG, and enhanced NF-κB nuclear translocation, transcriptional activity, and pro-inflammatory cytokine production in response to lipopolysaccharide (LPS. TLK117, an isotype-selective inhibitor of GSTP, also enhanced LPS-induced NF-κB transcriptional activity and pro-inflammatory cytokine production, suggesting that the catalytic activity of GSTP is important in repressing NF-κB activation. Expression of both wild-type and catalytically-inactive Y7F mutant GSTP significantly attenuated LPS- or IKKβ-induced production of GM-CSF. These studies indicate a complex role for GSTP in modulating NF-κB, which may involve S-glutathionylation of IKK proteins, and interaction with NF-κB family members. Our findings suggest that targeting GSTP is a potential avenue for regulating the activity of this prominent pro-inflammatory and immunomodulatory transcription factor.

  16. Basic fibroblast growth factor is pro-adipogenic in rat skeletal muscle progenitor clone, 2G11 cells.

    Science.gov (United States)

    Nakano, Shin-ichi; Nakamura, Katsuyuki; Teramoto, Naomi; Yamanouchi, Keitaro; Nishihara, Masugi

    2016-01-01

    Intramuscular adipose tissue (IMAT) formation is a hallmark of marbling in cattle. IMAT is considered to originate from skeletal muscle progenitor cells with adipogenic potential. However, the mechanism involved in IMAT formation from these progenitor cells in vivo remains unclear. In the present study, among the growth factors tested, which were known to be expressed in skeletal muscle, we found only basic fibroblast growth factor (bFGF) has a pro-adipogenic effect on skeletal muscle derived adipogenic progenitor clone, 2G11 cells. Pre-exposure of 2G11 cells to bFGF did not affect initial gene expressions of CCAAT/enhancer-binding protein (C/EBP)β and C/EBPδ, while resulting in an enhancement of subsequent expressions of C/EBPα and proliferator-activated receptor gamma (PPARγ) during adipogenesis, indicating that bFGF is acting on the transcriptional regulation of C/EBPα and PPARγ. In addition, the effect of bFGF is mediated via two types of FGF receptor (FGFR) isoforms: FGFR1 and FGFR2 IIIc, and both receptors are prerequisite for bFGF to express its pro-adipogenic effect. These results suggest that bFGF plays an important role as a key trigger of IMAT formation in vivo. PMID:26154243

  17. Cytotoxicity of carteolol to human corneal epithelial cells by inducing apoptosis via triggering the Bcl-2 family protein-mediated mitochondrial pro-apoptotic pathway.

    Science.gov (United States)

    Shan, Ming; Fan, Ting-Jun

    2016-09-01

    Carteolol is a frequently used nonselective β-adrenoceptor antagonist for glaucoma and ocular hypertension treatment, and its repeated/prolonged usage might be cytotoxic to the cornea, especially the outmost human corneal epithelium (HCEP). The aim of the present study was to characterize the cytotoxicity of carteolol to HCEP and its underlying cellular and molecular mechanisms using an in vitro model of HCEP cells. After HCEP cells were treated with carteolol at concentrations varying from 2% to 0.015625%, the cytotoxicity, apoptosis-inducing effect and pro-apoptotic pathway was investigated, respectively. Our results showed that carteolol at concentrations above 0.03125% induced time- and dose-dependent growth retardation, cytopathic morphological changes and viability decline of HCEP cells. Moreover, carteolol induced G1 phase arrest, plasma membrane permeability elevation, phosphatidylserine externalization, DNA fragmentation, and apoptotic body formation of HCEP cells. Furthermore, carteolol also induced activation of caspase-9 and -3, disruption of mitochondrial transmembrane potential, up-regulation the cytoplasmic amount of cytochrome c and apoptosis-inducing factor, and up-regulation of pro-apoptotic Bax and Bad, down-regulation of anti-apoptotic Bcl-2 and Bcl-xL. In conclusion, carteolol above 1/64 of its clinical therapeutic dosage has a time- and dose-dependent cytotoxicity to HCEP cells, which is achieved by inducing apoptosis via triggering Bcl-2 family protein-mediated mitochondrial pro-apoptotic pathway. PMID:27216471

  18. Synthesis and tritium labeling of the highly potent mast cell-degranulating substance P analog H-Arg-Pro-Lys-Pro-NH-C sub 12 H sub 25

    Energy Technology Data Exchange (ETDEWEB)

    Bienert, M.; Oehlke, J.; Niedrich, H. (Academy of Sciences of GDR, Berlin (Germany, F.R.). Inst. of Drug Research); Mittag, E. (Zentralinstitut fuer Kernforschung, Rossendorf (Germany, F.R.))

    1990-12-01

    Tritium labeling of the mast cell degranulating substance P analog H-Arg-Pro-Lys(3,4-{sup 3}H-Pro)-NH-C{sub 12}H{sub 25} by catalytic saturation of the dehydroproline (Dhp{sup 1}) double bond is described. Catalytic tritiation in water afforded the radioactive analog with a specific activity of 1.07 TBq/mmol. Tenfold enhancement of the catalyst-to-substrate ratio resulted in a reduced specific activity of 0.74 TBq/mmol. (author).

  19. p53 contributes to T cell homeostasis through the induction of pro-apoptotic SAP.

    Science.gov (United States)

    Madapura, Harsha S; Salamon, Daniel; Wiman, Klas G; Lain, Sonia; Klein, George; Klein, Eva; Nagy, Noémi

    2012-12-15

    Lack of functional SAP protein, due to gene deletion or mutation, is the cause of X-linked lymphoproliferative disease (XLP), characterized by functionally impaired T and NK cells and a high risk of lymphoma development. We have demonstrated earlier that SAP has a pro-apoptotic function in T and B cells. Deficiency of this function might contribute to the pathogenesis of XLP. We have also shown that SAP is a target of p53 in B cell lines. In the present study, we show that activated primary T cells express p53, which induces SAP expression. p53 is functional as a transcription factor in activated T cells and induces the expression of p21, PUMA and MDM2. PARP cleavage in the late phase of activation indicates that T cells expressing high levels of SAP undergo apoptosis. Modifying p53 levels using Nutlin-3, which specifically dissociates the MDM2-p53 interaction, was sufficient to upregulate SAP expression, indicating that SAP is a target of p53 in T cells. We also demonstrated p53's role as a transcription factor for SAP in activated T cells by ChIP assays. Our result suggests that p53 contributes to T cell homeostasis through the induction of the pro-apoptotic SAP. A high level of SAP is necessary for the activation-induced cell death that is pivotal in termination of the T cell response.

  20. Nivalenol induces oxidative stress and increases deoxynivalenol pro-oxidant effect in intestinal epithelial cells

    International Nuclear Information System (INIS)

    Mycotoxins are secondary fungal metabolites often found as contaminants in almost all agricultural commodities worldwide, and the consumption of food or feed contaminated by mycotoxins represents a major risk for human and animal health. Reactive oxygen species are normal products of cellular metabolism. However, disproportionate generation of reactive oxygen species poses a serious problem to bodily homeostasis and causes oxidative tissue damage. In this study we analyzed the effect of two trichothecenes mycotoxins: nivalenol and deoxynivalenol, alone and in combination, on oxidative stress in the non-tumorigenic intestinal epithelial cell line IEC-6. Our results indicate the pro-oxidant nivalenol effect in IEC-6, the stronger pro-oxidant effect of nivalenol when compared to deoxynivalenol and, interestingly, that nivalenol increases deoxynivalenol pro-oxidative effects. Mechanistic studies indicate that the observed effects were mediated by NADPH oxidase, calcium homeostasis alteration, NF-kB and Nrf2 pathways activation and by iNOS and nitrotyrosine formation. The toxicological interaction by nivalenol and deoxynivalenol reported in this study in IEC-6, points out the importance of the toxic effect of these mycotoxins, mostly in combination, further highlighting the risk assessment process of these toxins that are of growing concern. - Highlights: • Nivalenol induces oxidative stress in intestinal epithelial cells (IECs). • Nivalenol increases deoxynivalenol pro-oxidant effects in IECs. • Nivalenol and deoxynivalenol trigger antioxidant response IECs. • These results indicate the importance of mycotoxins co-contamination

  1. Nivalenol induces oxidative stress and increases deoxynivalenol pro-oxidant effect in intestinal epithelial cells

    Energy Technology Data Exchange (ETDEWEB)

    Del Regno, Marisanta; Adesso, Simona; Popolo, Ada [Department of Pharmacy, School of Pharmacy, University of Salerno, Via Giovanni Paolo II, 132–84084 Fisciano, Salerno (Italy); Quaroni, Andrea [Department of Biomedical Sciences, Cornell University, Veterinary Research Tower, Cornell University, Ithaca, NY 14853–6401 (United States); Autore, Giuseppina [Department of Pharmacy, School of Pharmacy, University of Salerno, Via Giovanni Paolo II, 132–84084 Fisciano, Salerno (Italy); Severino, Lorella [Department of Pathology and Animal Health, Division of Toxicology, School of Veterinary Medicine, University of Naples “Federico II”, Via Delpino 1, 80137 Naples (Italy); Marzocco, Stefania, E-mail: smarzocco@unisa.it [Department of Pharmacy, School of Pharmacy, University of Salerno, Via Giovanni Paolo II, 132–84084 Fisciano, Salerno (Italy)

    2015-06-01

    Mycotoxins are secondary fungal metabolites often found as contaminants in almost all agricultural commodities worldwide, and the consumption of food or feed contaminated by mycotoxins represents a major risk for human and animal health. Reactive oxygen species are normal products of cellular metabolism. However, disproportionate generation of reactive oxygen species poses a serious problem to bodily homeostasis and causes oxidative tissue damage. In this study we analyzed the effect of two trichothecenes mycotoxins: nivalenol and deoxynivalenol, alone and in combination, on oxidative stress in the non-tumorigenic intestinal epithelial cell line IEC-6. Our results indicate the pro-oxidant nivalenol effect in IEC-6, the stronger pro-oxidant effect of nivalenol when compared to deoxynivalenol and, interestingly, that nivalenol increases deoxynivalenol pro-oxidative effects. Mechanistic studies indicate that the observed effects were mediated by NADPH oxidase, calcium homeostasis alteration, NF-kB and Nrf2 pathways activation and by iNOS and nitrotyrosine formation. The toxicological interaction by nivalenol and deoxynivalenol reported in this study in IEC-6, points out the importance of the toxic effect of these mycotoxins, mostly in combination, further highlighting the risk assessment process of these toxins that are of growing concern. - Highlights: • Nivalenol induces oxidative stress in intestinal epithelial cells (IECs). • Nivalenol increases deoxynivalenol pro-oxidant effects in IECs. • Nivalenol and deoxynivalenol trigger antioxidant response IECs. • These results indicate the importance of mycotoxins co-contamination.

  2. Pro-apoptotic NOXA is implicated in atmospheric-pressure plasma-induced melanoma cell death

    Science.gov (United States)

    Ishaq, M.; Bazaka, K.; Ostrikov, K.

    2015-11-01

    Atmospheric-pressure plasma (APP) has been successfully used to treat several types of cancers in vivo and in vitro, with the effect being primarily attributed to the generation of reactive oxygen species (ROS). However, the mechanisms by which APP induces apoptosis in cancer cells require further elucidation. In this study, the effects of APP on the expression of 500 genes in melanoma Mel007 cancer cells were examined. Pro-apoptotic phorbol-12-myristate-13-acetate-induced protein (PMAIP1), also known as NOXA, was highly expressed as a result of APP treatment in a dose-dependent manner. Blocking of ROS using scavenger NAC or silencing of NOXA gene by RNA interference inhibited the APP-induced NOXA genes upregulation and impaired caspases 3/7 mediated apoptosis, confirming the important role plasma-generated ROS species and pro-apoptotic NOXA play in APP-induced cancer cell death.

  3. Antiproliferative and pro-apoptotic effects of Uncaria tomentosa in human medullary thyroid carcinoma cells.

    Science.gov (United States)

    Rinner, Beate; Li, Zeng Xia; Haas, Helga; Siegl, Veronika; Sturm, Sonja; Stuppner, Hermann; Pfragner, Roswitha

    2009-11-01

    Medullary thyroid carcinoma (MTC), a rare calcitonin-producing tumor, is derived from parafollicular C-cells of the thyroid and is characterized by constitutive Bcl-2 overexpression. The tumor is relatively insensitive to radiation therapy as well as conventional chemotherapy. To date, the only curative treatment is the early and complete surgical removal of all neoplastic tissue. In this study, the antiproliferative and pro-apoptotic effects of fractions obtained from Uncaria tomentosa (Willd.) DC, commonly known as uña de gato or cat's claw were investigated. Cell growth of MTC cells as well as enzymatic activity of mitochondrial dehydrogenase was markedly inhibited after treatment with different fractions of the plant. Furthermore, there was an increase in the expressions of caspase-3 and -7 and poly(ADP-ribose) polymerase (PARP) fraction, while bcl-2 overexpression remained constant. In particular, the alkaloids isopterpodine and pteropodine of U. tomentosa exhibited a significant pro-apoptotic effect on MTC cells, whereas the alkaloid-poor fraction inhibited cell proliferation but did not show any pro-apoptotic effects. These promising results indicate the growth-restraining and apoptotic potential of plant extracts against neuroendocrine tumors, which may add to existing therapies for cancer. PMID:20032400

  4. Antiproliferative and pro-apoptotic effects of Uncaria tomentosa in human medullary thyroid carcinoma cells.

    Science.gov (United States)

    Rinner, Beate; Li, Zeng Xia; Haas, Helga; Siegl, Veronika; Sturm, Sonja; Stuppner, Hermann; Pfragner, Roswitha

    2009-11-01

    Medullary thyroid carcinoma (MTC), a rare calcitonin-producing tumor, is derived from parafollicular C-cells of the thyroid and is characterized by constitutive Bcl-2 overexpression. The tumor is relatively insensitive to radiation therapy as well as conventional chemotherapy. To date, the only curative treatment is the early and complete surgical removal of all neoplastic tissue. In this study, the antiproliferative and pro-apoptotic effects of fractions obtained from Uncaria tomentosa (Willd.) DC, commonly known as uña de gato or cat's claw were investigated. Cell growth of MTC cells as well as enzymatic activity of mitochondrial dehydrogenase was markedly inhibited after treatment with different fractions of the plant. Furthermore, there was an increase in the expressions of caspase-3 and -7 and poly(ADP-ribose) polymerase (PARP) fraction, while bcl-2 overexpression remained constant. In particular, the alkaloids isopterpodine and pteropodine of U. tomentosa exhibited a significant pro-apoptotic effect on MTC cells, whereas the alkaloid-poor fraction inhibited cell proliferation but did not show any pro-apoptotic effects. These promising results indicate the growth-restraining and apoptotic potential of plant extracts against neuroendocrine tumors, which may add to existing therapies for cancer.

  5. Small molecule screening with laser cytometry can be used to identify pro-survival molecules in human embryonic stem cells.

    Directory of Open Access Journals (Sweden)

    Sean P Sherman

    Full Text Available Differentiated cells from human embryonic stem cells (hESCs provide an unlimited source of cells for use in regenerative medicine. The recent derivation of human induced pluripotent cells (hiPSCs provides a potential supply of pluripotent cells that avoid immune rejection and could provide patient-tailored therapy. In addition, the use of pluripotent cells for drug screening could enable routine toxicity testing and evaluation of underlying disease mechanisms. However, prior to establishment of patient specific cells for cell therapy it is important to understand the basic regulation of cell fate decisions in hESCs. One critical issue that hinders the use of these cells is the fact that hESCs survive poorly upon dissociation, which limits genetic manipulation because of poor cloning efficiency of individual hESCs, and hampers production of large-scale culture of hESCs. To address the problems associated with poor growth in culture and our lack of understanding of what regulates hESC signaling, we successfully developed a screening platform that allows for large scale screening for small molecules that regulate survival. In this work we developed the first large scale platform for hESC screening using laser scanning cytometry and were able to validate this platform by identifying the pro-survival molecule HA-1077. These small molecules provide targets for both improving our basic understanding of hESC survival as well as a tool to improve our ability to expand and genetically manipulate hESCs for use in regenerative applications.

  6. Entamoeba histolytica cysteine proteinase 5 binds integrin on colonic cells and stimulates NFkappaB-mediated pro-inflammatory responses.

    Science.gov (United States)

    Hou, Yongzhong; Mortimer, Leanne; Chadee, Kris

    2010-11-12

    Integrins are important mammalian receptors involved in normal cellular functions and the pathogenesis of inflammation and disease. Entamoeba histolytica is a protozoan parasite that colonizes the gut, and in 10% of infected individuals, causes amebic colitis and liver abscess resulting in 10(5) deaths/year. E. histolytica-induced host inflammatory responses are critical in the pathogenesis of the disease, yet the host and parasite factors involved in disease are poorly defined. Here we show that pro-mature cysteine proteinase 5 (PCP5), a major virulent factor that is abundantly secreted and/or present on the surface of ameba, binds via its RGD motif to α(V)β(3) integrin on Caco-2 colonic cells and stimulates NFκB-mediated pro-inflammatory responses. PCP5 RGD binding to α(V)β(3) integrin triggered integrin-linked kinase(ILK)-mediated phosphorylation of Akt-473 that bound and induced the ubiquitination of NF-κB essential modulator (NEMO). As NEMO is required for activation of the IKKα-IKKβ complex and NFκB signaling, these events markedly up-regulated pro-inflammatory mediator expressions in vitro in Caco-2 cells and in vivo in colonic loop studies in wild-type and Muc2(-/-) mice lacking an intact protective mucus barrier. These results have revealed that EhPCP5 RGD motif is a ligand for α(V)β(3) integrin-mediated adhesion on colonic cells and represents a novel mechanism that E. histolytica trophozoites use to trigger an inflammatory response in the pathogenesis of intestinal amebiasis.

  7. Pro-angiogenic Hematopoietic Progenitor Cells and Endothelial Colony Forming Cells in Pathological Angiogenesis of Bronchial and Pulmonary Circulation

    OpenAIRE

    Duong, Heng; Erzurum, Serpil; Asosingh, Kewal

    2011-01-01

    Dysregulation of angiogenesis is a common feature of many disease processes. Vascular remodeling is believed to depend on the participation of endothelial progenitor cells, but the identification of endothelial progenitors in postnatal neovascularization remains elusive. Current understanding posits a role for circulating pro-angiogenic hematopoietic cells, which interact with local endothelial cells to establish an environment that favors angiogenesis in physiologic and pathophysiologic resp...

  8. Expression of surface-associated 82kDa-proMMP-9 in primary acute leukemia blast cells inversely correlates with patients' risk.

    Science.gov (United States)

    Schmohl, Joerg; Santovito, Donato; Guenther, Thomas; Sutanto, Wishnu; Kroell, Tanja; Salih, Helmut; Pitsch, Thomas; Egea, Virginia; Weber, Christian; Schmetzer, Helga; Ries, Christian

    2016-05-01

    With its ability to degrade extracellular matrix proteins and activate growth factors and cytokines, matrix metalloproteinase (MMP)-9 is an important regulator of cell function. Previously, we reported that myeloid leukemic cells express a unique 82kDa-proMMP-9 variant on their cell surface that is not affected by its natural inhibitor. In this study, we generated monoclonal antibodies that specifically recognize 82kDa-proMMP-9. Flow cytometry analysis using these antibodies revealed significant surface expression of 82kDa-proMMP-9 in monocytes, but minimal amounts in T and B cells isolated from peripheral blood of nine healthy donors and 22 patients with acute myeloid leukemia (AML). In all AML patients, blasts expressed 82kDa-proMMP-9 at levels of 4%-46%, with significantly higher levels in patients with a better risk defined according to National Comprehensive Cancer Network (NCCN) guidelines (ρ = -0.748, p < 0.001) and favorable phenotype according to the French-American-British classification (p = 0.02) compared with patients with adverse prognoses. Receiver operating characteristic curve analysis confirmed the diagnostic accuracy of 82kDa-proMMP-9 measurement in AML blasts (area under the curve: 0.893 [0.739-1.000], p = 0.019). It led us to define a cutoff value of 11.5% for identifying patients with lower NCCN risk (p = 0.005) and with a tendency toward a higher probability of response to anthracycline-based therapy (p = 0.109) and increased event-free survival (p = 0.24). Thus, 82kDa-proMMP-9 expression on blasts may represent a novel independent marker of prognosis in patients with AML. PMID:26845021

  9. Processing of pro-CGRP in a rat medullary thyroid carcinoma cell line transfected with protease inhibitors

    DEFF Research Database (Denmark)

    Johansen, Teit Eliot; Schifter, S; Vogel, Charlotte Katrine;

    1991-01-01

    A rat medullary thyroid carcinoma cell line, CA77, was used to study the effect of a series of biosynthesized protease inhibitors on the proteolytic cleavage of the endogenously synthesized pro-CGRP. This cell line efficiently converted the pro-CGRP to mature CGRP as assessed by chromatography of...... could be measured in the CA77 cells transfected with plasmids encoding the three serine protease inhibitors. CA77 cells were also transfected with two constructs encoding chimeric proteins consisting of cystatin C and the precursor for neuropeptide Y. Release experiments using 8-bromo c......AMP as the secretagogue showed that the chimer was co-released with CGRP. However, no effect of this chimer upon pro-CGRP processing could be detected. It is concluded that the processing of pro-CGRP in the CA77 cell line was very efficient and that four different protease inhibitors and two cystatin C/NPY chimers...

  10. Early growth response gene-2 (Egr-2 regulates the development of B and T cells.

    Directory of Open Access Journals (Sweden)

    Suling Li

    Full Text Available BACKGROUND: Understanding of how transcription factors are involved in lymphocyte development still remains a challenge. It has been shown that Egr-2 deficiency results in impaired NKT cell development and defective positive selection of T cells. Here we investigated the development of T, B and NKT cells in Egr-2 transgenic mice and the roles in the regulation of distinct stages of B and T cell development. METHODS AND FINDINGS: The expression of Egr1, 2 and 3 were analysed at different stages of T and B cell development by RT-PCT and results showed that the expression was strictly regulated at different stages. Forced expression of Egr-2 in CD2(+ lymphocytes resulted in a severe reduction of CD4(+CD8(+ (DP cells in thymus and pro-B cells in bone marrow, which was associated with reduced expression of Notch1 in ISP thymocytes and Pax5 in pro-B cells, suggesting that retraction of Egr-2 at the ISP and pro-B cell stages is important for the activation of lineage differentiation programs. In contrast to reduction of DP and pro-B cells, Egr-2 enhanced the maturation of DP cells into single positive (SP T and NKT cells in thymus, and immature B cells into mature B cells in bone marrow. CONCLUSIONS: Our results demonstrate that Egr-2 expressed in restricted stages of lymphocyte development plays a dynamic, but similar role for the development of T, NKT and B cells.

  11. 5-HT2CRs expressed by pro-opiomelanocortin neurons regulate insulin sensitivity in liver

    Science.gov (United States)

    Mice lacking 5-HT 2C receptors displayed hepatic insulin resistance, a phenotype normalized by re-expression of 5-HT2CRs only in pro-opiomelanocortin (POMC) neurons. 5-HT2CR deficiency also abolished the anti-diabetic effects of meta-chlorophenylpiperazine (a 5-HT2CR agonist); these effects were re...

  12. Expression profiling of genes regulated by TGF-beta: Differential regulation in normal and tumour cells

    Directory of Open Access Journals (Sweden)

    Takahashi Takashi

    2007-04-01

    Full Text Available Abstract Background TGF-beta is one of the key cytokines implicated in various disease processes including cancer. TGF-beta inhibits growth and promotes apoptosis in normal epithelial cells and in contrast, acts as a pro-tumour cytokine by promoting tumour angiogenesis, immune-escape and metastasis. It is not clear if various actions of TGF-beta on normal and tumour cells are due to differential gene regulations. Hence we studied the regulation of gene expression by TGF-beta in normal and cancer cells. Results Using human 19 K cDNA microarrays, we show that 1757 genes are exclusively regulated by TGF-beta in A549 cells in contrast to 733 genes exclusively regulated in HPL1D cells. In addition, 267 genes are commonly regulated in both the cell-lines. Semi-quantitative and real-time qRT-PCR analysis of some genes agrees with the microarray data. In order to identify the signalling pathways that influence TGF-beta mediated gene regulation, we used specific inhibitors of p38 MAP kinase, ERK kinase, JNK kinase and integrin signalling pathways. The data suggest that regulation of majority of the selected genes is dependent on at least one of these pathways and this dependence is cell-type specific. Interestingly, an integrin pathway inhibitor, RGD peptide, significantly affected TGF-beta regulation of Thrombospondin 1 in A549 cells. Conclusion These data suggest major differences with respect to TGF-beta mediated gene regulation in normal and transformed cells and significant role of non-canonical TGF-beta pathways in the regulation of many genes by TGF-beta.

  13. Effect of ProNectin F derivatives on cell attachment and proliferation.

    OpenAIRE

    Somamoto, Satoshi; Tabata, Yasuhiko

    2013-01-01

    ProNectin F (PnF) was chemically modified by introducing some functional groups to prepare various derivatives of primary amino (PnF-N(1)), tertiary amino (PnF-N(3)), quaternary ammonium (PnF-N(4)), carboxyl (PnF-COOH) and sulfonyl groups (PnF-SO(3)H). When C3H10T1/2 cells were cultured on non-treated dishes coated with the derivatives, the number of mesenchymal cells attached to the culture dishes increased for the coating with PnF-COOH and PnF-SO(3)H, even at their low adsorption amount. Th...

  14. Methyl jasmonate induces apoptosis and pro-apoptotic autophagy via the ROS pathway in human non-small cell lung cancer.

    Science.gov (United States)

    Zhang, Mutian; Su, Ling; Xiao, Zhenna; Liu, Xianfang; Liu, Xiangguo

    2016-01-01

    Methyl jasmonate (MJ) is a botanical hormone that serves as a signal transduction intermediate and regulates cell death in stressed plants. MJ induces cell cycle arrest, apoptosis and non-apoptotic cell death selectively in cancer cells. However, the underlying mechanism of MJ-induced apoptosis remains unclear. In this study, we examined the molecular mechanism through which MJ induces apoptosis in human non-small cell lung cancer (NSCLC). We found that MJ triggered apoptosis via the DDIT3-TNFRSF10B-CASP axis. MJ treatment significantly decreased the expression of CFLAR (CASP8 and FADD-like apoptosis regulator, an inhibitor of CASP8) in NSCLC cells, and ectopic expression of CFLAR partly protected cells from MJ-induced apoptosis. MJ also induced pro-apoptotic autophagy in NSCLC cells. Importantly, inhibition of ROS suppressed both MJ-induced apoptosis and autophagy. Taken together, MJ induces apoptosis and pro-apoptotic autophagy in NSCLC cells through the ROS pathway. Thus, MJ and its derivative treatment may serve as a novel chemotherapeutic strategy for cancer therapy. PMID:27186395

  15. Phototherapy-treated apoptotic tumor cells induce pro-inflammatory cytokines production in macrophage

    Science.gov (United States)

    Lu, Cuixia; Wei, Yanchun; Xing, Da

    2014-09-01

    Our previous studies have demonstrated that as a mitochondria-targeting cancer phototherapy, high fluence low-power laser irradiation (HF-LPLI) induces mitochondrial superoxide anion burst, resulting in oxidative damage to tumor cells. In this study, we further explored the immunological effects of HF-LPLI-induced apoptotic tumor cells. When macrophages were co-incubated with apoptotic cells induced by HF-LPLI, we observed the increased levels of TNF-α secretion and NO production in macrophages. Further experiments showed that NF-κB was activated in macrophages after co-incubation with HF-LPLI-induced apoptotic cells, and inhibition of NF-κB activity by pyrrolidinedithiocarbamic acid (PDTC) reduced the elevated levels of TNF-α secretion and NO production. These data indicate that HF-LPLI-induced apoptotic tumor cells induce the secretion of pro-inflammatory cytokines in macrophages, which may be helpful for better understanding the biological effects of cancer phototherapy.

  16. Separation of plasmacytoid dendritic cells from B-cell-biased lymphoid progenitor (BLP and Pre-pro B cells using PDCA-1.

    Directory of Open Access Journals (Sweden)

    Kay L Medina

    Full Text Available B-cell-biased lymphoid progenitors (BLPs and Pre-pro B cells lie at a critical juncture between B cell specification and commitment. However, both of these populations are heterogenous, which hampers investigation into the molecular changes that occur as lymphoid progenitors commit to the B cell lineage. Here, we demonstrate that there are PDCA-1(+Siglec H(+ plasmacytoid dendritic cells (pDCs that co-purify with BLPs and Pre-pro B cells, which express little or no CD11c or Ly6C. Removal of PDCA-1(+ pDCs separates B cell progenitors that express high levels of a Rag1-GFP reporter from Rag1-GFP(low/neg pDCs within the BLP and Pre-pro B populations. Analysis of Flt3-ligand knockout and IL-7Rα knockout mice revealed that there is a block in B cell development at the all-lymphoid progenitor (ALP stage, as the majority of cells within the BLP or Pre-pro B gates were PDCA-1(+ pDCs. Thus, removal of PDCA-1(+ pDCs is critical for analysis of BLP and Pre-pro B cell populations. Analysis of B cell potential within the B220(+CD19(- fraction demonstrated that AA4.1(+Ly6D(+PDCA-1(- Pre-pro B cells gave rise to CD19(+ B cells at high frequency, while PDCA-1(+ pDCs in this fraction did not. Interestingly, the presence of PDCA-1(+ pDCs within CLPs may help to explain the conflicting results regarding the origin of these cells.

  17. Cell-free culture supernatant of Bifidobacterium breve CNCM I-4035 decreases pro-inflammatory cytokines in human dendritic cells challenged with Salmonella typhi through TLR activation.

    Directory of Open Access Journals (Sweden)

    Miriam Bermudez-Brito

    Full Text Available Dendritic cells (DCs constitute the first point of contact between gut commensals and our immune system. Despite growing evidence of the immunomodulatory effects of probiotics, the interactions between the cells of the intestinal immune system and bacteria remain largely unknown. Indeed,, the aim of this work was to determine whether the probiotic Bifidobacterium breve CNCM I-4035 and its cell-free culture supernatant (CFS have immunomodulatory effects in human intestinal-like dendritic cells (DCs and how they respond to the pathogenic bacterium Salmonella enterica serovar Typhi, and also to elucidate the molecular mechanisms involved in these interactions. Human DCs were directly challenged with B. breve/CFS, S. typhi or a combination of these stimuli for 4 h. The expression pattern of genes involved in Toll-like receptor (TLR signaling pathway and cytokine secretion was analyzed. CFS decreased pro-inflammatory cytokines and chemokines in human intestinal DCs challenged with S. typhi. In contrast, the B. breve CNCM I-4035 probiotic strain was a potent inducer of the pro-inflammatory cytokines and chemokines tested, i.e., TNF-α, IL-8 and RANTES, as well as anti-inflammatory cytokines including IL-10. CFS restored TGF-β levels in the presence of Salmonella. Live B.breve and its supernatant enhanced innate immune responses by the activation of TLR signaling pathway. These treatments upregulated TLR9 gene transcription. In addition, CFS was a more potent inducer of TLR9 expression than the probiotic bacteria in the presence of S. typhi. Expression levels of CASP8 and IRAK4 were also increased by CFS, and both treatments induced TOLLIP gene expression. Our results indicate that the probiotic strain B. breve CNCM I-4035 affects the intestinal immune response, whereas its supernatant exerts anti-inflammatory effects mediated by DCs. This supernatant may protect immune system from highly infectious agents such as Salmonella typhi and can down-regulate

  18. Specialized Pro-Resolving Mediators from Omega-3 Fatty Acids Improve Amyloid-β Phagocytosis and Regulate Inflammation in Patients with Minor Cognitive Impairment.

    Science.gov (United States)

    Fiala, Milan; Terrando, Niccolo; Dalli, Jesmond

    2015-01-01

    In this review we discuss the immunopathology of Alzheimer's disease (AD) and recent advances in the prevention of minor cognitive impairment (MCI) by nutritional supplementation with omega-3 fatty acids. Defective phagocytosis of amyloid-β (Aβ) and abnormal inflammatory activation of peripheral blood mononuclear cells (PBMCs) are the two key immune pathologies of MCI and AD patients. The phagocytosis of Aβ by PBMCs of MCI and AD patients is universally defective and the inflammatory gene transcription is heterogeneously deregulated in comparison to normal subjects. Recent studies have discovered a cornucopia of beneficial anti-inflammatory and pro-resolving effects of the specialized proresolving mediators (SPMs) resolvins, protectins, maresins, and their metabolic precursors. Resolvin D1 and other mediators switch macrophages from an inflammatory to a tissue protective/pro-resolving phenotype and increase phagocytosis of Aβ. In a recent study of AD and MCI patients, nutritional supplementation by omega-3 fatty acids individually increased resolvin D1, improved Aβ phagocytosis, and regulated inflammatory genes toward a physiological state, but only in MCI patients. Our studies are beginning to dissect positive factors (adherence to Mediterranean diet with omega-3 and exercise) and negative factors (high fat diet, infections, cancer, and surgeries) in each patient. The in vitro and in vivo effects of omega-3 fatty acids and SPMs suggest that defective phagocytosis and chronic inflammation are related to defective production and/or defective signaling by SPMs in immune cells.

  19. In vivo inhibition of c-MYC in myeloid cells impairs tumor-associated macrophage maturation and pro-tumoral activities.

    Directory of Open Access Journals (Sweden)

    Oscar M Pello

    Full Text Available Although tumor-associated macrophages (TAMs are involved in tumor growth and metastasis, the mechanisms controlling their pro-tumoral activities remain largely unknown. The transcription factor c-MYC has been recently shown to regulate in vitro human macrophage polarization and be expressed in macrophages infiltrating human tumors. In this study, we exploited the predominant expression of LysM in myeloid cells to generate c-Myc(fl/fl LysM(cre/+ mice, which lack c-Myc in macrophages, to investigate the role of macrophage c-MYC expression in cancer. Under steady-state conditions, immune system parameters in c-Myc(fl/fl LysM(cre/+ mice appeared normal, including the abundance of different subsets of bone marrow hematopoietic stem cells, precursors and circulating cells, macrophage density, and immune organ structure. In a model of melanoma, however, TAMs lacking c-Myc displayed a delay in maturation and showed an attenuation of pro-tumoral functions (e.g., reduced expression of VEGF, MMP9, and HIF1α that was associated with impaired tissue remodeling and angiogenesis and limited tumor growth in c-Myc(fl/fl LysM(cre/+ mice. Macrophage c-Myc deletion also diminished fibrosarcoma growth. These data identify c-Myc as a positive regulator of the pro-tumoral program of TAMs and suggest c-Myc inactivation as an attractive target for anti-cancer therapy.

  20. Phenotypic switch in blood: effects of pro-inflammatory cytokines on breast cancer cell aggregation and adhesion.

    Directory of Open Access Journals (Sweden)

    Yue Geng

    Full Text Available Hematogeneous metastasis can occur via a cascade of circulating tumor cell adhesion events to the endothelial lining of the vasculature, i.e. the metastatic cascade. Interestingly, the pro-inflammatory cytokines IL-6 and TNF-α, which play an important role in potentiating the inflammatory cascade, are significantly elevated in metastatic breast cancer (BCa patients. Despite their high metastatic potential, human breast carcinoma cells MDA-MB-231 lack interactions with E-selectin functionalized surfaces under physiological shear stresses. We hypothesized that human plasma, 3-D tumor spheroid culture, and cytokine-supplemented culture media could induce a phenotypic switch that allows BCa cells to interact with E-selectin coated surfaces under physiological flow. Flow cytometry, immunofluorescence imaging, and flow-based cell adhesion assay were utilized to investigate the phenotypic changes of MDA-MB-231 cells with various treatments. Our results indicate that plasma, IL-6, and TNF-α promote breast cancer cell growth as aggregates and induce adhesive recruitment of BCa cells on E-selectin coated surfaces under flow. 3-D tumor spheroid culture exhibits the most significant increases in the interactions between BCa and E-selectin coated surfaces by upregulating CD44V4 and sLe(x expression. Furthermore, we show that IL-6 and TNF-α concentrations in blood may regulate the recruitment of BCa cells to the inflamed endothelium. Finally, we propose a mechanism that could explain the invasiveness of 'triple-negative' breast cancer cell line MDA-MB-231 via a positive feedback loop of IL-6 secretion and maintenance. Taken together, our results suggest that therapeutic approaches targeting cytokine receptors and adhesion molecules on cancer cells may potentially reduce metastatic load and improve current cancer treatments.

  1. Cell swelling and volume regulation

    DEFF Research Database (Denmark)

    Hoffmann, Else Kay

    1992-01-01

    The extracellular space in the brain is typically 20% of the tissue volume and is reduced to at least half its size under conditions of neural insult. Whether there is a minimum size to the extracellular space was discussed. A general model for cell volume regulation was presented, followed...... by a discussion on how many of the generally involved mechanisms are identified in neural cells and (or) in astrocytes. There seems to be clear evidence suggesting that parallel K+ and Cl- channels mediate regulatory volume decrease in primary cultures of astrocytes, and a stretch-activated cation channel has...

  2. Retracted: Effects of pro-inflammatory cytokines on mineralization potential of rat dental pulp stem cells.

    Science.gov (United States)

    2011-10-01

    The following article from the Journal of Tissue Engineering and Regenerative Medicine, 'Effects of Pro-inflammatory Cytokines on Mineralization Potential of Rat Dental Pulp Stem Cells' by Yang X, Walboomers XF, Bian Z, Jansen JA, Fan M, published online on 11 July 2011 in Wiley Online Library (onlinelibrary.wiley.com), has been retracted by agreement between the authors, the journal Editor-in-Chief, and John Wiley & Sons, Ltd. The retraction has been agreed due to two authors (Walboomers XF, and Jansen JA) not having been involved in the research described, nor made aware of their names being listed on the manuscript, nor told of its submission to the journal.

  3. Thrombin cleavage of osteopontin disrupts a pro-chemotactic sequence for dendritic cells, which is compensated by the release of its pro-chemotactic C-terminal fragment.

    Science.gov (United States)

    Shao, Zhifei; Morser, John; Leung, Lawrence L K

    2014-09-26

    Thrombin cleavage alters the function of osteopontin (OPN) by exposing an integrin binding site and releasing a chemotactic C-terminal fragment. Here, we examined thrombin cleavage of OPN in the context of dendritic cell (DC) migration to define its functional domains. Full-length OPN (OPN-FL), thrombin-cleaved N-terminal fragment (OPN-R), thrombin- and carboxypeptidase B2-double-cleaved N-terminal fragment (OPN-L), and C-terminal fragment (OPN-CTF) did not have intrinsic chemotactic activity, but all potentiated CCL21-induced DC migration. OPN-FL possessed the highest potency, whereas OPNRAA-FL had substantially less activity, indicating the importance of RGD. We identified a conserved (168)RSKSKKFRR(176) sequence on OPN-FL that spans the thrombin cleavage site, and it demonstrated potent pro-chemotactic effects on CCL21-induced DC migration. OPN-FLR168A had reduced activity, and the double mutant OPNRAA-FLR168A had even lower activity, indicating that these functional domains accounted for most of the pro-chemotactic activity of OPN-FL. OPN-CTF also possessed substantial pro-chemotactic activity, which was fully expressed upon thrombin cleavage and its release from the intact protein, because OPN-CTF was substantially more active than OPNRAA-FLR168A containing the OPN-CTF sequence within the intact protein. OPN-R and OPN-L possessed similar potency, indicating that the newly exposed C-terminal SVVYGLR sequence in OPN-R was not involved in the pro-chemotactic effect. OPN-FL and OPN-CTF did not directly bind to the CD44 standard form or CD44v6. In conclusion, thrombin cleavage of OPN disrupts a pro-chemotactic sequence in intact OPN, and its loss of pro-chemotactic activity is compensated by the release of OPN-CTF, which assumes a new conformation and possesses substantial activity in enhancing chemokine-induced migration of DCs. PMID:25112870

  4. Downregulation of pro-inflammatory mediators by a water extract of Schisandra chinensis (Turcz.) Baill fruit in lipopolysaccharide-stimulated RAW 264.7 macrophage cells.

    Science.gov (United States)

    Dilshara, Matharage Gayani; Jayasooriya, Rajapaksha Gedara Prasad Tharanga; Kang, Chang-Hee; Lee, Seungheon; Park, Sang Rul; Jeong, Jin-Woo; Choi, Yung Hyun; Seo, Yong Taek; Jang, Young Pyo; Kim, Gi-Young

    2013-09-01

    Schisandra chinensis has a long-standing history of medicinal use as a tonic, a sedative, an anti-tussive, and an anti-aging drug. Nevertheless, the antagonistic effects of S. chinensis against lipopolysaccharide (LPS)-stimulated responses have not yet been studied. In this study, we investigated whether water extract of S. chinensis fruit (WESC) has the ability to attenuate the expression of pro-inflammatory mediators such as nitric oxide (NO), prostaglandin E2 (PGE2), and tumor necrosis factor-α (TNF-α) in LPS-stimulated RAW 264.7 macrophage cells. WESC inhibited the expression of LPS-induced pro-inflammatory mediators, namely, NO, PGE2, and TNF-α. Furthermore, gene expression of inducible NO synthase (iNOS), cyclooxygenase-2 (COX-2), and TNF-α was inhibited both at mRNA and protein synthesis levels, without any cytotoxic effect. Moreover, WESC significantly suppressed LPS-induced DNA-binding activity of NF-κB by inhibiting degradation of IκBα. It was found that pyrrolidine dithiocarbamate (PDTC), a specific NF-κB inhibitor, downregulates the expression of these pro-inflammatory genes to be closely regulated by NF-κB activity. Furthermore, we found that WESC retains dephosphorylation of Akt in response to LPS, and consequently suppressed the DNA-binding activity of NF-κB in RAW 264.7 macrophage cells. LY294002, a specific Akt inhibitor, attenuated LPS-induced pro-inflammatory gene expression via suppression of NF-κB activity. Taken together, our results indicate that WESC downregulates the expression of pro-inflammatory genes involved in the synthesis of NO, PGE2, and TNF-α in LPS-stimulated RAW 264.7 macrophage cells by suppressing Akt-dependent NF-κB activity.

  5. Effect of ethanol on pro-apoptotic mechanisms in polarized hepatic cells

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    Chronic ethanol consumption is associated with serious and potentially fatal alcohol-related liver injuries such as hepatomegaly, alcoholic hepatitis and cirrhosis. Moreover,it has been documented that the clinical progression of alcohol-induced liver damage may be associated with an increase in hepatocellular death that involves apoptotic mechanisms. Although much information has been learned about the clinical manifestations associated with alcohol-related diseases, the search continues for a better understanding of the molecular and/or cellular mechanisms by which ethanol exerts its deleterious effects such as the induction of pro-apoptotic mechanisms and related cell damaging events. As part of the effort to enhance our understanding of those particular cellular pathways and mechanisms associated with ethanol toxicity, researchers over the years have utilized a variety of model systems. Recently, work has come forth demonstrating the utility of a hybrid cell line (WIF-B) as a cell culture model system for the study of alcohol-associated alterations in hepatocellular mechanisms. Success with such emerging model systems could aid in the development of potential therapeutic treatments for the prevention of alcoholinduced apoptotic cell death that may ultimately serve as a significant target in delaying the onset and/or progression of clinical symptoms of alcohol-mediated liver disease. This review article summarizes the current understanding of ethanol-mediated modifications in cell survival and thus the promotion of pro-apoptotic events with emphasis on analyses made in various experimental model systems, particularly the more recently characterized WIF-B cell system.

  6. Haloperidol disrupts Akt signalling to reveal a phosphorylation-dependent regulation of pro-apoptotic Bcl-XS function.

    Science.gov (United States)

    Wei, Zelan; Qi, Ji; Dai, Yunxiu; Bowen, Wayne D; Mousseau, Darrell D

    2009-01-01

    The antipsychotic drug haloperidol is still used to treat psychosis and "agitation", often with devastating consequences, particularly in geriatric and pre-demented patients. Cytotoxicity induced by haloperidol has been associated with induction of Bcl-XS, a pro-apoptotic member of the Bcl-2 family, as well as with modulation of the Akt pro-survival pathway. Using preneuronal PC12 and primary neuronal cultures, we show that haloperidol inactivates Akt. This induces the dephosphorylation of serine residues in Bcl-XS and promotes its association with the mitochondrial voltage-dependent anion channel (VDAC), as well as with cytochrome c- and caspase-3-dependent events. These events are sensitive to expression of constitutively active Akt. Mutation of Serine106 (Ser106), which is flanked by a putative Akt motif, hinders the association of the Bcl-XS protein with Akt, but promotes its association with VDAC. The dephosphorylation mimic, Bcl-XS(Ser106Ala), induces caspase-dependent PC12 and neuronal cell apoptosis. In contrast, Bcl-XS(Ser106Ala) induces a significant loss of VDAC expression, and cytochrome c- and caspase-independent toxicity in the non-neuronal HEK293A cells. We link haloperidol and Akt to Bcl-XS-sensitive toxicity via cell line-dependent mitochondrial events centering on VDAC. This clearly mitigates the chronic use of haloperidol in neuropsychiatric populations, but supports its use as a potential acute therapeutic in cancer, where apoptosis is desirable.

  7. Regulation of T-helper-cell lineage development by osteopontin: the inside story

    OpenAIRE

    Cantor, Harvey; Shinohara, Mari L.

    2009-01-01

    Studies of osteopontin (OPN)-dependent regulation of immune responses have focused on the cytokine activities of the secreted form of this protein. Recent evidence has revealed that an intracellular form of OPN expressed by dendritic cells regulates the expression of pro-inflammatory cytokines and the differentiation of T helper (TH)-cell lineages. In this Opinion article, we discuss the properties of both OPN isoforms and their respective contributions to the immune response. We propose that...

  8. Apoptosis and pro-inflammatory cytokine response of mast cells induced by influenza A viruses.

    Directory of Open Access Journals (Sweden)

    Bo Liu

    Full Text Available The pathogenesis of the influenza A virus has been investigated heavily, and both the inflammatory response and apoptosis have been found to have a definitive role in this process. The results of studies performed by the present and other groups have indicated that mast cells may play a role in the severity of the disease. To further investigate cellular responses to influenza A virus infection, apoptosis and inflammatory response were studied in mouse mastocytoma cell line P815. This is the first study to demonstrate that H1N1 (A/WSN/33, H5N1 (A/Chicken/Henan/1/04, and H7N2 (A/Chicken/Hebei/2/02 influenza viruses can induce mast cell apoptosis. They were found to do this mainly through the mitochondria/cytochrome c-mediated intrinsic pathway, and the activation of caspase 8-mediated extrinsic pathway was here found to be weak. Two pro-apoptotic Bcl-2 homology domain 3 (BH3 -only molecules Bim and Puma appeared to be involved in the apoptotic pathways. When virus-induced apoptosis was inhibited in P815 cells using pan-caspase (Z-VAD-fmk and caspase-9 (Z-LEHD-fmk inhibitors, the replication of these three subtypes of viruses was suppressed and the secretions of pro-inflammatory cytokines and chemokines, including IL-6, IL-18, TNF-α, and MCP-1, decreased. The results of this study may further understanding of the role of mast cells in host defense and pathogenesis of influenza virus. They may also facilitate the development of novel therapeutic aids against influenza virus infection.

  9. Intrinsic pro-angiogenic status of cystic fibrosis airway epithelial cells

    International Nuclear Information System (INIS)

    Cystic fibrosis is a common genetic disorder characterized by a severe lung inflammation and fibrosis leading to the patient's death. Enhanced angiogenesis in cystic fibrosis (CF) tissue has been suggested, probably caused by the process of inflammation, as similarly described in asthma and chronic bronchitis. The present study demonstrates an intrinsic pro-angiogenic status of cystic fibrosis airway epithelial cells. Microarray experiments showed that CF airway epithelial cells expressed several angiogenic factors such as VEGF-A, VEGF-C, bFGF, and PLGF at higher levels than control cells. These data were confirmed by real-time quantitative PCR and, at the protein level, by ELISA. Conditioned media of these cystic fibrosis cells were able to induce proliferation, migration and sprouting of cultured primary endothelial cells. This report describes for the first time that cystic fibrosis epithelial cells have an intrinsic angiogenic activity. Since excess of angiogenesis is correlated with more severe pulmonary disease, our results could lead to the development of new therapeutic applications

  10. Ion Channels Involved in Cell Volume Regulation

    DEFF Research Database (Denmark)

    Hoffmann, Else Kay

    2011-01-01

    regulatory ion channels involved, and the mechanisms (cellular signalling pathways) that regulate these channels. Finally, I shall also briefly review current investigations in these two cell lines that focuses on how changes in cell volume can regulate cell functions such as cell migration, proliferation...

  11. Controlled Inhibition of the Mesenchymal Stromal Cell Pro-inflammatory Secretome via Microparticle Engineering

    Directory of Open Access Journals (Sweden)

    Sudhir H. Ranganath

    2016-06-01

    Full Text Available Mesenchymal stromal cells (MSCs are promising therapeutic candidates given their potent immunomodulatory and anti-inflammatory secretome. However, controlling the MSC secretome post-transplantation is considered a major challenge that hinders their clinical efficacy. To address this, we used a microparticle-based engineering approach to non-genetically modulate pro-inflammatory pathways in human MSCs (hMSCs under simulated inflammatory conditions. Here we show that microparticles loaded with TPCA-1, a small-molecule NF-κB inhibitor, when delivered to hMSCs can attenuate secretion of pro-inflammatory factors for at least 6 days in vitro. Conditioned medium (CM derived from TPCA-1-loaded hMSCs also showed reduced ability to attract human monocytes and prevented differentiation of human cardiac fibroblasts to myofibroblasts, compared with CM from untreated or TPCA-1-preconditioned hMSCs. Thus, we provide a broadly applicable bioengineering solution to facilitate intracellular sustained release of agents that modulate signaling. We propose that this approach could be harnessed to improve control over MSC secretome post-transplantation, especially to prevent adverse remodeling post-myocardial infarction.

  12. Regulation of YKL-40 expression by corticosteroids : effect on pro-inflammatory macrophages in vitro and its modulation in COPD in vivo

    NARCIS (Netherlands)

    Kunz, L. I. Z.; van't Wout, E. F. A.; van Schadewijk, A.; Postma, D. S.; Kerstjens, H. A. M.; Sterk, P. J.; Hiemstra, P. S.

    2015-01-01

    Background: Macrophages constitute a heterogeneous cell population with pro-(M Phi 1) and anti-inflammatory (M Phi 2) cells. The soluble chitinase-like-protein YKL-40 is expressed in macrophages and various other cell types, and has been linked to a variety of inflammatory diseases, including COPD.

  13. Val-boroPro accelerates T cell priming via modulation of dendritic cell trafficking resulting in complete regression of established murine tumors.

    Directory of Open Access Journals (Sweden)

    Meghaan P Walsh

    Full Text Available Although tumors naturally prime adaptive immune responses, tolerance may limit the capacity to control progression and can compromise effectiveness of immune-based therapies for cancer. Post-proline cleaving enzymes (PPCE modulate protein function through N-terminal dipeptide cleavage and inhibition of these enzymes has been shown to have anti-tumor activity. We investigated the mechanism by which Val-boroPro, a boronic dipeptide that inhibits post-proline cleaving enzymes, mediates tumor regression and tested whether this agent could serve as a novel immune adjuvant to dendritic cell vaccines in two different murine syngeneic murine tumors. In mice challenged with MB49, which expresses the HY antigen complex, T cell responses primed by the tumor with and without Val-boroPro were measured using interferon gamma ELISPOT. Antibody depletion and gene-deficient mice were used to establish the immune cell subsets required for tumor regression. We demonstrate that Val-boroPro mediates tumor eradication by accelerating the expansion of tumor-specific T cells. Interestingly, T cells primed by tumor during Val-boroPro treatment demonstrate increased capacity to reject tumors following adoptive transfer without further treatment of the recipient. Val-boroPro -mediated tumor regression requires dendritic cells and is associated with enhanced trafficking of dendritic cells to tumor draining lymph nodes. Finally, dendritic cell vaccination combined with Val-boroPro treatment results in complete regression of established tumors. Our findings demonstrate that Val-boroPro has antitumor activity and a novel mechanism of action that involves more robust DC trafficking with earlier priming of T cells. Finally, we show that Val-boroPro has potent adjuvant properties resulting in an effective therapeutic vaccine.

  14. Protective effects of matrine on experimental autoimmune encephalomyelitis via regulation of ProNGF and NGF signaling.

    Science.gov (United States)

    Zhu, Lin; Pan, Qing-xia; Zhang, Xiao-Jian; Xu, Yu-Ming; Chu, Yao-juan; Liu, Nan; Lv, Peng; Zhang, Guang-Xian; Kan, Quan-Cheng

    2016-04-01

    Inflammation, demyelination, oligodendrocyte (OLG) death, and axonal degeneration are primary characteristics of multiple sclerosis (MS) and its animal model, experimental autoimmune encephalomyelitis (EAE). OLGs generate myelin sheaths that surround axons, while damage to OLGs leads to demyelination and neurological functional deficit. Matrine (MAT), a quinolizidine alkaloid derived from the herb Radix Sophorae Flave, has been recently found to effectively ameliorate clinical signs in EAE. Its therapeutic mechanism has, however, not been completely elucidated. In the present study, we found that MAT retarded the disease process, attenuated the clinical severity of EAE rats, ameliorated inflammation and demyelination, and suppressed the apoptosis of OLGs in the central nervous system (CNS) of EAE rats. In addition, MAT markedly blocked increased expression of the proNGF-p75(NTR) death signaling complex, which is known to mediate OLG death in EAE animals. At the same time, MAT also prevented a decrease in the levels of NGF and its receptor TrkA, which together mediate the cell survival pathway and differentiation of OLGs. ProNGF, NGF, and the downstream effector proteins play an important role in the growth, differentiation, and apoptosis of OLGs as well as the reparative response to neuronal damage. These findings thus indicate that MAT improves clinical severity of EAE in part by reducing OLG apoptosis via restoring the ratios of proNGF:NGF and the respective receptors p75(NTR):TrkA in vivo. Taken together, these results suggest that MAT may be a promising agent for MS treatment based on its protective effect on OLGs.

  15. Thioredoxin ameliorates cutaneous inflammation by regulating the epithelial production and release of pro-Inflammatory cytokines

    Directory of Open Access Journals (Sweden)

    Hai eTian

    2013-09-01

    Full Text Available Human thioredoxin-1 (TRX is a 12-kDa protein with redox-active dithiol in the active site -Cys-Gly-Pro-Cys-. It has been demonstrated that systemic administration and transgenic overexpression of TRX ameliorate inflammation in various animal models, but its anti-inflammatory mechanism is not well characterized. We investigated the anti-inflammatory effects of topically applied recombinant human TRX (rhTRX in a murine irritant contact dermatitis (ICD induced by croton oil. Topically applied rhTRX was distributed only in the skin tissues under both non-inflammatory and inflammatory conditions, and significantly suppressed the inflammatory response by inhibiting the production of cytokines and chemokines, such as TNF-α, Il-1β, IL-6, CXCL-1, and MCP-1. In an in vitro study, rhTRX also significantly inhibited the formation of cytokines and chemokines produced by keratinocytes after exposure to croton oil and phorbol 12-myristate 13-acetate. These results indicate that TRX prevents skin inflammation via the inhibition of local formation of inflammatory cytokines and chemokines. As a promising new approach, local application of TRX may be useful for the treatment of various skin and mucosal inflammatory disorders.

  16. Do mechanical strain and TNF-α interact to amplify pro-inflammatory cytokine production in human annulus fibrosus cells?

    Science.gov (United States)

    Likhitpanichkul, Morakot; Torre, Olivia M; Gruen, Jadry; Walter, Benjamin A; Hecht, Andrew C; Iatridis, James C

    2016-05-01

    During intervertebral disc (IVD) injury and degeneration, annulus fibrosus (AF) cells experience large mechanical strains in a pro-inflammatory milieu. We hypothesized that TNF-α, an initiator of IVD inflammation, modifies AF cell mechanobiology via cytoskeletal changes, and interacts with mechanical strain to enhance pro-inflammatory cytokine production. Human AF cells (N=5, Thompson grades 2-4) were stretched uniaxially on collagen-I coated chambers to 0%, 5% (physiological) or 15% (pathologic) strains at 0.5Hz for 24h under hypoxic conditions with or without TNF-α (10ng/mL). AF cells were treated with anti-TNF-α and anti-IL-6. ELISA assessed IL-1β, IL-6, and IL-8 production and immunocytochemistry measured F-actin, vinculin and α-tubulin in AF cells. TNF-α significantly increased AF cell pro-inflammatory cytokine production compared to basal conditions (IL-1β:2.0±1.4-84.0±77.3, IL-6:10.6±9.9-280.9±214.1, IL-8:23.9±26.0-5125.1±4170.8pg/ml for basal and TNF-α treatment, respectively) as expected, but mechanical strain did not. Pathologic strain in combination with TNF-α increased IL-1β, and IL-8 but not IL-6 production of AF cells. TNF-α treatment altered F-actin and α-tubulin in AF cells, suggestive of altered cytoskeletal stiffness. Anti-TNF-α (infliximab) significantly inhibited pro-inflammatory cytokine production while anti-IL-6 (atlizumab) did not. In conclusion, TNF-α altered AF cell mechanobiology with cytoskeletal remodeling that potentially sensitized AF cells to mechanical strain and increased TNF-α-induced pro-inflammatory cytokine production. Results suggest an interaction between TNF-α and mechanical strain and future mechanistic studies are required to validate these observations.

  17. Decreased Regulatory T Cells in Vulnerable Atherosclerotic Lesions: Imbalance between Pro- and Anti-Inflammatory Cells in Atherosclerosis

    Directory of Open Access Journals (Sweden)

    Ilonka Rohm

    2015-01-01

    Full Text Available Atherosclerosis is a chronic inflammatory disease of the arterial wall in which presentation of autoantigens by dendritic cells (DCs leads to the activation of T cells. Anti-inflammatory cells like Tregs counterbalance inflammation in atherogenesis. In our study, human carotid plaque specimens were classified as stable (14 and unstable (15 according to established morphological criteria. Vessel specimens (n=12 without any signs of atherosclerosis were used as controls. Immunohistochemical staining was performed to detect different types of DCs (S100, fascin, CD83, CD209, CD304, and CD123, proinflammatory T cells (CD3, CD4, CD8, and CD161, and anti-inflammatory Tregs (FoxP3. The following results were observed: in unstable lesions, significantly higher numbers of proinflammatory cells like DCs, T helper cells, cytotoxic T cells, and natural killer cells were detected compared to stable plaques. Additionally, there was a significantly higher expression of HLA-DR and more T cell activation (CD25, CD69 in unstable lesions. On the contrary, unstable lesions contained significantly lower numbers of Tregs. Furthermore, a significant inverse correlation between myeloid DCs and Tregs was shown. These data suggest an increased inflammatory state in vulnerable plaques resulting from an imbalance of the frequency of local pro- and anti-inflammatory immune cells.

  18. Pro-apoptotic effect of Persea americana var. Hass (avocado) on Jurkat lymphoblastic leukemia cells.

    Science.gov (United States)

    Bonilla-Porras, Angelica R; Salazar-Ospina, Andrea; Jimenez-Del-Rio, Marlene; Pereañez-Jimenez, Andres; Velez-Pardo, Carlos

    2013-11-01

    Abstract Context: Therapy for leukemia has a limited efficacy. There is a need to search for alternative anti-leukemia therapies. Persea americana Mill var. Hass (Lauraceae) is a tropical fruit (avocado) that might be used against cancer. Objective: To investigate whether P. americana induces death in Jurkat lymphoblastic leukemia cells. Materials and methods: Four ethanol extracts (0.1, 0.5, 1, 2 and 5 mg/mL) from avocado fruit (endocarp, whole seed, seed and leaves) were analyzed against Jurkat cells. Hydrogen peroxide generation by oxidation of 2',7'-dichlorodihydrofluorescein diacetate to the fluorescent compound 2',7'-dichlorfluorescein assay, acridine orange/ethidium bromide staining, flow cytometry analysis of annexin-V/7-amino-actinomycin, mitochondrial membrane potential and immunocytochemistry detection of transcription factor p53, caspase-3 and apoptosis-inducing factor (AIF) were evaluated. Results: Endocarp, seed, whole seed, and leaf (0.1 mg/mL) extracts induced significant apoptosis in Jurkat cells (p americana extracts function as a pro-apoptotic compound. Leukemic cells are eliminated through an oxidative stress mechanism. This study contributes to the understanding of the molecular mechanism of the avocado and its therapeutic action on leukemia. PMID:24188375

  19. Serotonin 2c receptors in pro-opiomelanocortin neurons regulate energy and glucose homeostasis

    Science.gov (United States)

    Energy and glucose homeostasis are regulated by central serotonin 2C receptors. These receptors are attractive pharmacological targets for the treatment of obesity; however, the identity of the serotonin 2C receptor-expressing neurons that mediate the effects of serotonin and serotonin 2C receptor a...

  20. The pro-survival function of p53 in HeLa cells

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Jin Kyu; Kang, Mi Young; Jang, Eun Yeong; Kim, Jin Hong [Korea Atomic Energy Research Institute, Advanced Radiation Technology Institute, Jeongeup (Korea, Republic of)

    2014-11-15

    The rate of apoptosis and autophagy was variable with different p53 status after IR treatment of cells. The influence of p53 status on cell fate suggests a role of p53 in two fundamentally important cell biological pathways: autophagy and apoptosis. p53 coordinates cell cycle arrest and apoptosis to govern cell fate. This study was done to identify p53-mediated regulation of cell's fate. Autophagy induced by IR may prevent cells from undergoing apoptosis, implying an interlink modulation between autophagy and apoptosis. The rate of apoptosis and autophagy was determined with different p53 status after IR treatment of HeLa cells in this study. Our research on IR-induced cellular responses may provide new information about fate decision between the processes of apoptosis and autophagy.

  1. Purinergic Signaling as a Regulator of Th17 Cell Plasticity.

    Directory of Open Access Journals (Sweden)

    Dominique Fernández

    Full Text Available T helper type 17 (Th17 lymphocytes, characterized by the production of interleukin-17 and other pro-inflammatory cytokines, are present in intestinal lamina propria and have been described as important players driving intestinal inflammation. Recent evidence, supporting the notion of a functional and phenotypic instability of Th17 cells, has shown that Th17 differentiate into type 1 regulatory (Tr1 T cells during the resolution of intestinal inflammation. Moreover, it has been suggested that the expression of CD39 ectonucleotidase endows Th17 cells with immunosuppressive properties. However, the exact role of CD39 ectonucleotidase in Th17 cells has not been studied in the context of intestinal inflammation. Here we show that Th17 cells expressing CD39 ectonucleotidase can hydrolyze ATP and survive to ATP-induced cell death. Moreover, in vitro-generated Th17 cells expressing the CD39 ectonucleotidase produce IL-10 and are less pathogenic than CD39 negative Th17 cells in a model of experimental colitis in Rag-/- mice. Remarkably, we show that CD39 activity regulates the conversion of Th17 cells to IL-10-producing cells in vitro, which is abrogated in the presence of ATP and the CD39-specific inhibitor ARL67156. All these data suggest that CD39 expression by Th17 cells allows the depletion of ATP and is crucial for IL-10 production and survival during the resolution of intestinal inflammation.

  2. Purinergic Signaling as a Regulator of Th17 Cell Plasticity

    Science.gov (United States)

    Fernández, Dominique; Flores-Santibáñez, Felipe; Neira, Jocelyn; Osorio-Barrios, Francisco; Tejón, Gabriela; Nuñez, Sarah; Hidalgo, Yessia; Fuenzalida, Maria Jose; Meza, Daniel; Ureta, Gonzalo; Lladser, Alvaro; Pacheco, Rodrigo; Acuña-Castillo, Claudio; Guixé, Victoria; Quintana, Francisco J.; Bono, Maria Rosa; Rosemblatt, Mario; Sauma, Daniela

    2016-01-01

    T helper type 17 (Th17) lymphocytes, characterized by the production of interleukin-17 and other pro-inflammatory cytokines, are present in intestinal lamina propria and have been described as important players driving intestinal inflammation. Recent evidence, supporting the notion of a functional and phenotypic instability of Th17 cells, has shown that Th17 differentiate into type 1 regulatory (Tr1) T cells during the resolution of intestinal inflammation. Moreover, it has been suggested that the expression of CD39 ectonucleotidase endows Th17 cells with immunosuppressive properties. However, the exact role of CD39 ectonucleotidase in Th17 cells has not been studied in the context of intestinal inflammation. Here we show that Th17 cells expressing CD39 ectonucleotidase can hydrolyze ATP and survive to ATP-induced cell death. Moreover, in vitro-generated Th17 cells expressing the CD39 ectonucleotidase produce IL-10 and are less pathogenic than CD39 negative Th17 cells in a model of experimental colitis in Rag-/- mice. Remarkably, we show that CD39 activity regulates the conversion of Th17 cells to IL-10-producing cells in vitro, which is abrogated in the presence of ATP and the CD39-specific inhibitor ARL67156. All these data suggest that CD39 expression by Th17 cells allows the depletion of ATP and is crucial for IL-10 production and survival during the resolution of intestinal inflammation. PMID:27322617

  3. Bioprinting cell-laden matrigel for radioprotection study of liver by pro-drug conversion in a dual-tissue microfluidic chip

    International Nuclear Information System (INIS)

    The objective of this paper is to introduce a novel cell printing and microfluidic system to serve as a portable ground model for the study of drug conversion and radiation protection of living liver tissue analogs. The system is applied to study behavior in ground models of space stress, particularly radiation. A microfluidic environment is engineered by two cell types to prepare an improved higher fidelity in vitro micro-liver tissue analog. Cell-laden Matrigel printing and microfluidic chips were used to test radiation shielding to liver cells by the pro-drug amifostine. In this work, the sealed microfluidic chip regulates three variables of interest: radiation exposure, anti-radiation drug treatment and single- or dual-tissue culture environments. This application is intended to obtain a scientific understanding of the response of the multi-cellular biological system for long-term manned space exploration, disease models and biosensors.

  4. Bioprinting cell-laden matrigel for radioprotection study of liver by pro-drug conversion in a dual-tissue microfluidic chip

    Energy Technology Data Exchange (ETDEWEB)

    Snyder, J E; Hamid, Q; Wang, C; Chang, R; Sun, W [Department of Mechanical Engineering, Drexel University, Philadelphia, PA 19104 (United States); Emami, K; Wu, H, E-mail: sunwei@drexel.edu, E-mail: weisun@tsinghua.edu.cn [Radiation Biophysics Lab, NASA Johnson Space Center, Houston, TX 77586 (United States)

    2011-09-15

    The objective of this paper is to introduce a novel cell printing and microfluidic system to serve as a portable ground model for the study of drug conversion and radiation protection of living liver tissue analogs. The system is applied to study behavior in ground models of space stress, particularly radiation. A microfluidic environment is engineered by two cell types to prepare an improved higher fidelity in vitro micro-liver tissue analog. Cell-laden Matrigel printing and microfluidic chips were used to test radiation shielding to liver cells by the pro-drug amifostine. In this work, the sealed microfluidic chip regulates three variables of interest: radiation exposure, anti-radiation drug treatment and single- or dual-tissue culture environments. This application is intended to obtain a scientific understanding of the response of the multi-cellular biological system for long-term manned space exploration, disease models and biosensors.

  5. Receptor tyrosine kinase-like orphan receptor 1, a target of NKX2-1/TTF-1 lineage-survival oncogene, inhibits apoptosis signal-regulating kinase 1-mediated pro-apoptotic signaling in lung adenocarcinoma.

    Science.gov (United States)

    Ida, Lisa; Yamaguchi, Tomoya; Yanagisawa, Kiyoshi; Kajino, Taisuke; Shimada, Yukako; Suzuki, Motoshi; Takahashi, Takashi

    2016-02-01

    We previously identified receptor tyrosine kinase-like orphan receptor 1 (ROR1) as a transcriptional target of the NKX2-1/TTF-1 lineage-survival oncogene in lung adenocarcinoma. ROR1 consequently sustains a favorable balance between pro-survival phosphatidylinositol 3-kinase-protein kinase B and pro-apoptotic apoptosis signal-regulating kinase 1 (ASK1)-p38MAPK signaling. In contrast to recent advances in understanding how ROR1 sustains pro-survival signaling, the mechanism of ROR1 repression of pro-apoptotic signaling remains rather elusive. In the present study, we investigated the underlying mechanism of ROR1-mediated inhibition of the ASK1-p38MAPK signaling pathway. Growth inhibition mediated by siROR1 was partially but significantly alleviated by ASK1 co-knockdown in lung adenocarcinoma cell lines. Also, ASK1 phosphorylation at Thr845, which reflects its activated state, was clearly inhibited by ROR1 overexpression in both steady state and oxidative stress-elicited conditions in MSTO-211H cells. In addition, we found that ROR1 was physically associated with ASK1 at the C-terminal serine threonine-rich domain of ROR1. Furthermore, ROR1 kinase activity was shown to be required to repress the ASK1-p38 axis and oxidative stress-induced cell death. The present findings thus support our notion that ROR1 sustains lung adenocarcinoma survival, at least in part, through direct physical interaction with ASK1 and consequential repression of the pro-apoptotic ASK1-p38 axis in a ROR1 kinase activity-dependent manner. PMID:26661061

  6. Pro-oxidant effect of ALA is implicated in mitochondrial dysfunction of HepG2 cells.

    Science.gov (United States)

    Laafi, Jihane; Homedan, Chadi; Jacques, Caroline; Gueguen, Naig; Schmitt, Caroline; Puy, Hervé; Reynier, Pascal; Carmen Martinez, Maria; Malthièry, Yves

    2014-11-01

    Heme biosynthesis begins in the mitochondrion with the formation of delta-aminolevulinic acid (ALA). In acute intermittent porphyria, hereditary tyrosinemia type I and lead poisoning patients, ALA is accumulated in plasma and in organs, especially the liver. These diseases are also associated with neuromuscular dysfunction and increased incidence of hepatocellular carcinoma. Many studies suggest that this damage may originate from ALA-induced oxidative stress following its accumulation. Using the MnSOD as an oxidative stress marker, we showed here that ALA treatment of cultured cells induced ROS production, increasing with ALA concentration. The mitochondrial energetic function of ALA-treated HepG2 cells was further explored. Mitochondrial respiration and ATP content were reduced compared to control cells. For the 300 μM treatment, ALA induced a mitochondrial mass decrease and a mitochondrial network imbalance although neither necrosis nor apoptosis were observed. The up regulation of PGC-1, Tfam and ND5 genes was also found; these genes encode mitochondrial proteins involved in mitochondrial biogenesis activation and OXPHOS function. We propose that ALA may constitute an internal bioenergetic signal, which initiates a coordinated upregulation of respiratory genes, which ultimately drives mitochondrial metabolic adaptation within cells. The addition of an antioxidant, Manganese(III) tetrakis(1-methyl-4-pyridyl)porphyrin (MnTMPyP), resulted in improvement of maximal respiratory chain capacity with 300 μM ALA. Our results suggest that mitochondria, an ALA-production site, are more sensitive to pro-oxidant effect of ALA, and may be directly involved in pathophysiology of patients with inherited or acquired porphyria.

  7. A stable HeLa cell line that inducibly expresses poliovirus 2A(pro): effects on cellular and viral gene expression.

    Science.gov (United States)

    Barco, A; Feduchi, E; Carrasco, L

    2000-03-01

    A HeLa cell clone (2A7d) that inducibly expresses the gene for poliovirus protease 2A (2A(pro)) under the control of tetracycline has been obtained. Synthesis of 2A(pro) induces severe morphological changes in 2A7d cells. One day after tetracycline removal, cells round up and a few hours later die. Poliovirus 2A(pro) cleaves both forms of initiation factor eIF4G, causing extensive inhibition of capped-mRNA translation a few hours after protease induction. Methoxysuccinyl-Ala-Ala-Pro-Val-chloromethylketone, a selective inhibitor of 2A(pro), prevents both eIF4G cleavage and inhibition of translation but not cellular death. Expression of 2A(pro) still allows both the replication of poliovirus and the translation of mRNAs containing a picornavirus leader sequence, while vaccinia virus replication is drastically inhibited. Translation of transfected capped mRNA is blocked in 2A7d-On cells, while luciferase synthesis from a mRNA bearing a picornavirus internal ribosome entry site (IRES) sequence is enhanced by the presence of 2A(pro). Moreover, synthesis of 2A(pro) in 2A7d cells complements the translational defect of a poliovirus 2A(pro)-defective variant. These results show that poliovirus 2A(pro) expression mimics some phenotypical characteristics of poliovirus-infected cells, such as cell rounding, inhibition of protein synthesis and enhancement of IRES-driven translation. This cell line constitutes a useful tool to further analyze 2A(pro) functions, to complement poliovirus 2A(pro) mutants, and to test antiviral compounds.

  8. HBpF-proBDNF: A New Tool for the Analysis of Pro-Brain Derived Neurotrophic Factor Receptor Signaling and Cell Biology

    Science.gov (United States)

    Gaub, Perrine; de Léon, Andrès; Gibon, Julien; Soubannier, Vincent; Dorval, Geneviève; Séguéla, Philippe; Barker, Philip A.

    2016-01-01

    Neurotrophins activate intracellular signaling pathways necessary for neuronal survival, growth and apoptosis. The most abundant neurotrophin in the adult brain, brain-derived neurotrophic factor (BDNF), is first synthesized as a proBDNF precursor and recent studies have demonstrated that proBDNF can be secreted and that it functions as a ligand for a receptor complex containing p75NTR and sortilin. Activation of proBDNF receptors mediates growth cone collapse, reduces synaptic activity, and facilitates developmental apoptosis of motoneurons but the precise signaling cascades have been difficult to discern. To address this, we have engineered, expressed and purified HBpF-proBDNF, an expression construct containing a 6X-HIS tag, a biotin acceptor peptide (BAP) sequence, a PreScission™ Protease cleavage site and a FLAG-tag attached to the N-terminal part of murine proBDNF. Intact HBpF-proBDNF has activities indistinguishable from its wild-type counterpart and can be used to purify proBDNF signaling complexes or to monitor proBDNF endocytosis and retrograde transport. HBpF-proBDNF will be useful for characterizing proBDNF signaling complexes and for deciphering the role of proBDNF in neuronal development, synapse function and neurodegenerative disease. PMID:26950209

  9. HBpF-proBDNF: A New Tool for the Analysis of Pro-Brain Derived Neurotrophic Factor Receptor Signaling and Cell Biology.

    Science.gov (United States)

    Gaub, Perrine; de Léon, Andrès; Gibon, Julien; Soubannier, Vincent; Dorval, Geneviève; Séguéla, Philippe; Barker, Philip A

    2016-01-01

    Neurotrophins activate intracellular signaling pathways necessary for neuronal survival, growth and apoptosis. The most abundant neurotrophin in the adult brain, brain-derived neurotrophic factor (BDNF), is first synthesized as a proBDNF precursor and recent studies have demonstrated that proBDNF can be secreted and that it functions as a ligand for a receptor complex containing p75NTR and sortilin. Activation of proBDNF receptors mediates growth cone collapse, reduces synaptic activity, and facilitates developmental apoptosis of motoneurons but the precise signaling cascades have been difficult to discern. To address this, we have engineered, expressed and purified HBpF-proBDNF, an expression construct containing a 6X-HIS tag, a biotin acceptor peptide (BAP) sequence, a PreScission™ Protease cleavage site and a FLAG-tag attached to the N-terminal part of murine proBDNF. Intact HBpF-proBDNF has activities indistinguishable from its wild-type counterpart and can be used to purify proBDNF signaling complexes or to monitor proBDNF endocytosis and retrograde transport. HBpF-proBDNF will be useful for characterizing proBDNF signaling complexes and for deciphering the role of proBDNF in neuronal development, synapse function and neurodegenerative disease. PMID:26950209

  10. Cell cycle regulation in Trypanosoma brucei

    OpenAIRE

    Tansy C Hammarton

    2007-01-01

    Cell division is regulated by intricate and interconnected signal transduction pathways that precisely coordinate, in time and space, the complex series of events involved in replicating and segregating the component parts of the cell. In Trypanosoma brucei, considerable progress has been made over recent years in identifying molecular regulators of the cell cycle and elucidating their functions, although many regulators undoubtedly remain to be identified, and there is still a long way to go...

  11. Photoreceptor Differentiation following Transplantation of Allogeneic Retinal Progenitor Cells to the Dystrophic Rhodopsin Pro347Leu Transgenic Pig

    DEFF Research Database (Denmark)

    Klassen, H; Kiilgaard, Jens Folke; Warfvinge, K;

    2012-01-01

    . Methods. Retinal progenitor cells were derived from the neural retina of GFP-transgenic pigs and transplanted to the subretinal space of rhodopsin Pro347Leu-transgenic allorecipients, in the early stage of the degeneration and the absence of immune suppression. Results. Results confirm the survival of...

  12. Novel analogue of colchicine induces selective pro-death autophagy and necrosis in human cancer cells.

    Directory of Open Access Journals (Sweden)

    Kristen Larocque

    Full Text Available Colchicine, a natural product of Colchicum autumnae currently used for gout treatment, is a tubulin targeting compound which inhibits microtubule formation by targeting fast dividing cells. This tubulin-targeting property has lead researchers to investigate the potential of colchicine and analogs as possible cancer therapies. One major study conducted on an analogue of allocolchicine, ZD 6126, was halted in phase 2 clinical trials due to severe cardio-toxicity associated with treatment. This study involves the development and testing of novel allocolchicine analogues that hold non-toxic anti-cancer properties. Currently we have synthesized and evaluated the anti-cancer activities of two analogues; N-acetyl-O-methylcolchinol (NSC 51046 or NCME, which is structurally similar to ZD 6126, and (S-3,8,9,10-tetramethoxyallocolchicine (Green 1, which is a novel derivative of allocolchicine that is isomeric in the A ring. NSC 51046 was found to be non-selective as it induced apoptosis in both BxPC-3 and PANC-1 pancreatic cancer cells and in normal human fibroblasts. Interestingly, we found that Green 1 was able to modestly induce pro-death autophagy in these pancreatic cancer cells and E6-1 leukemia cells but not in normal human fibroblasts. Unlike colchicine and NSC 51046, Green 1 does not appear to affect tubulin polymerization indicating that it has a different molecular target. Green 1 also caused increased reactive oxygen species (ROS production in mitochondria isolated from pancreatic cancer cells. Furthermore, in vivo studies revealed that Green 1 was well tolerated in mice. Our findings suggest that a small change in the structure of colchicine has apparently changed the mechanism of action and lead to improved selectivity. This may lead to better selective treatments in cancer therapy.

  13. An activated set point of T-cell and monocyte inflammatory networks in recent-onset schizophrenia patients involves both pro- and anti-inflammatory forces

    NARCIS (Netherlands)

    Drexhage, Roosmarijn C.; Hoogenboezem, Thomas A.; Cohen, Dan; Versnel, Marjan A.; Nolen, Willem A.; van Beveren, Nico J. M.; Drexhage, Hemmo A.

    2011-01-01

    We recently described a pro-inflammatory gene expression signature in the monocytes of 60% of patients with recent-onset schizophrenia (SCZ). Here we investigated whether the T-cell system is also in a pro-inflammatory state. A detailed fluorescence-activated cell sorting (FACS) analysis, e. g. of C

  14. AKT activation drives the nuclear localization of CSE1L and a pro-oncogenic transcriptional activation in ovarian cancer cells

    International Nuclear Information System (INIS)

    The human homolog of the yeast cse1 gene (CSE1L) is over-expressed in ovarian cancer. CSE1L forms complex with Ran and importin-α and has roles in nucleocytoplasmic traffic and gene expression. CSE1L accumulated in the nucleus of ovarian cancer cell lines, while it was localized also in the cytoplasm of other cancer cell lines. Nuclear localization depended on AKT, which was constitutively active in ovarian cancer cells, as the CSE1L protein translocated to the cytoplasm when AKT was inactivated. Moreover, the expression of a constitutively active AKT forced the translocation of CSE1L from the cytoplasm to the nucleus in other cancer cells. Nuclear accrual of CSE1L was associated to the nuclear accumulation of the phosphorylated Ran Binding protein 3 (RanBP3), which depended on AKT as well. Also in samples of human ovarian cancer, AKT activation was associated to nuclear accumulation of CSE1L and phosphorylation of RanBP3. Expression profiling of ovarian cancer cells after CSE1L silencing showed that CSE1L was required for the expression of genes promoting invasion and metastasis. In agreement, CSE1L silencing impaired motility and invasiveness of ovarian cancer cells. Altogether these data show that in ovarian cancer cells activated AKT by affecting RanBP3 phosphorylation determines the nuclear accumulation of CSE1L and likely the nuclear concentration of transcription factors conveying pro-oncogenic signals. - highlights: • CSE1L is a key player in nucleocytoplasmic traffic by forming complex with Ran. • AKT phosphorylates RanBP3 that regulates the nucleocytoplasmic gradient of Ran. • The activated oncogenic AKT drives the nuclear accumulation of CSE1L. • CSE1L in the nucleus up-regulates genes conveying pro-oncogenic signals. • CSE1L might contribute to tumor progression driven by the activated oncogenic AKT

  15. AKT activation drives the nuclear localization of CSE1L and a pro-oncogenic transcriptional activation in ovarian cancer cells

    Energy Technology Data Exchange (ETDEWEB)

    Lorenzato, Annalisa; Biolatti, Marta [Department of Oncology, University of Torino School of Medicine, Torino (Italy); Institute for Cancer Research at Candiolo, Candiolo, Torino (Italy); Delogu, Giuseppe [Department of Biomedical Sciences-Histology, University of Sassari, Sassari (Italy); Capobianco, Giampiero [Department of Surgical, Microsurgical and Medical Sciences, University of Sassari, Sassari (Italy); Farace, Cristiano [Department of Biomedical Sciences-Histology, University of Sassari, Sassari (Italy); Dessole, Salvatore; Cossu, Antonio; Tanda, Francesco [Department of Surgical, Microsurgical and Medical Sciences, University of Sassari, Sassari (Italy); Madeddu, Roberto [Department of Biomedical Sciences-Histology, University of Sassari, Sassari (Italy); National Institute of Biostructures and Biosystems, Rome (Italy); Olivero, Martina [Department of Oncology, University of Torino School of Medicine, Torino (Italy); Institute for Cancer Research at Candiolo, Candiolo, Torino (Italy); Di Renzo, Maria Flavia, E-mail: mariaflavia.direnzo@unito.it [Department of Oncology, University of Torino School of Medicine, Torino (Italy); Institute for Cancer Research at Candiolo, Candiolo, Torino (Italy)

    2013-10-15

    The human homolog of the yeast cse1 gene (CSE1L) is over-expressed in ovarian cancer. CSE1L forms complex with Ran and importin-α and has roles in nucleocytoplasmic traffic and gene expression. CSE1L accumulated in the nucleus of ovarian cancer cell lines, while it was localized also in the cytoplasm of other cancer cell lines. Nuclear localization depended on AKT, which was constitutively active in ovarian cancer cells, as the CSE1L protein translocated to the cytoplasm when AKT was inactivated. Moreover, the expression of a constitutively active AKT forced the translocation of CSE1L from the cytoplasm to the nucleus in other cancer cells. Nuclear accrual of CSE1L was associated to the nuclear accumulation of the phosphorylated Ran Binding protein 3 (RanBP3), which depended on AKT as well. Also in samples of human ovarian cancer, AKT activation was associated to nuclear accumulation of CSE1L and phosphorylation of RanBP3. Expression profiling of ovarian cancer cells after CSE1L silencing showed that CSE1L was required for the expression of genes promoting invasion and metastasis. In agreement, CSE1L silencing impaired motility and invasiveness of ovarian cancer cells. Altogether these data show that in ovarian cancer cells activated AKT by affecting RanBP3 phosphorylation determines the nuclear accumulation of CSE1L and likely the nuclear concentration of transcription factors conveying pro-oncogenic signals. - highlights: • CSE1L is a key player in nucleocytoplasmic traffic by forming complex with Ran. • AKT phosphorylates RanBP3 that regulates the nucleocytoplasmic gradient of Ran. • The activated oncogenic AKT drives the nuclear accumulation of CSE1L. • CSE1L in the nucleus up-regulates genes conveying pro-oncogenic signals. • CSE1L might contribute to tumor progression driven by the activated oncogenic AKT.

  16. Regulation of programmed cell death by plasminogen activator inhibitor type 1 (PAI-1)

    DEFF Research Database (Denmark)

    Lademann, Ulrik Axel; Rømer, Maria Unni Koefoed

    2008-01-01

    PA) observed in tumours; however, several lines of evidence suggest that PAI-1 may contribute directly to the pathology of the disease. PAI-1 has been reported to have an effect on most of the basic cellular processes including cell adhesion, cell migration, cell invasion, and cell proliferation and increasing...... numbers of reports suggest that PAI-1 also can regulate programmed cell death (PCD) in cancer cells and normal cells. A number of reports suggest that PAI-1 can inhibit PCD through its pro-adhesive/anti-proteolytic property whereas other reports suggest that PAI-1 induces PCD through its anti......-adhesive property.Furthermore,it has been suggested that PAI-1 can either induce or inhibit PCD though activation of cell signalling pathways.This review will focus on the regulation of programmed cell death by PAI-1 in both normal cells and cancer cells....

  17. ProMMP-1 PRODUCTION BY CULTIVATED CELLS OF VASCULAR ENDОTHELIUM IN VITRO AND IN A HUMAN BODY

    Directory of Open Access Journals (Sweden)

    N. N. Scliankina

    2011-01-01

    Full Text Available Abstract. Matrix metalloproteinases (MMP are structurally related endopeptidase composed of active sites which include ions Zn2+ and Ca2+. Cultured cells of human blood vessels produce MMP-1, proteolytic effect is aimed at splitting the collagen I and III types, and subsequent vascular remodeling. MMP-1 is synthesized as an inactive zymogen proMMP-1. It was shown that interferon alpha, beta and gamma inhibited production by culture of HUVEC proenzyme MMP-1, which seems to characterize their anti-angiogenic action. The effect of immunomodulators is more difficult to explain: perhaps inhibiting effect of imunofan and, as well as activating effect of cycloferon due to their internal structural peculiarities. The action of interferon alpha, beta and gamma, used as HUVEC before infection with HSV-1, and after it led to decrease in production proMMP-1. Apparently, the antiangiogenic effect of IFN is saved in the case of infection of cultures of vascular endothelium with HSV-1. Scatter in the content of proMMP-1 in the serum of blood donors was 1.625–11.8 ng/ml and in patients with chronic microbial-viral infections was 1.22–21.16 ng/ml. Higher rates of proMMP-1 were in older patients group. To estimate the system of MMP in vitro, and in the body a comprehensive study must be conducted, including proMMP-1, the active form of proenzyme and specific inhibitor of MMP-1.

  18. TGFβ induces proHB-EGF shedding and EGFR transactivation through ADAM activation in gastric cancer cells

    International Nuclear Information System (INIS)

    Research highlights: → TGFβ induces EGFR transactivation through proHB-EGF shedding by activated ADAM members in gastric cancer cells. → TGFβ induces nuclear translocation of HB-EGF-CTF cleaved by ADAM members. → TGFβ enhances cell growth by EGFR transactivation and HB-EGF-CTF nuclear translocation and ADAM inhibitors block these effects. → Silencing of ADAM17 also blocks EGFR transactivation, HB-EGF-CTF nuclear translocation and cancer cell growth by TGFβ. → ADAM17 may play a crucial role in this TGFβ-HB-EGF signal transduction. -- Abstract: Background and aims: Transforming growth factor-beta (TGFβ) is known to potently inhibit cell growth. Loss of responsiveness to TGFβ inhibition on cell growth is a hallmark of many types of cancer, yet its mechanism is not fully understood. Membrane-anchored heparin-binding EGF-like growth factor (proHB-EGF) ectodomain is cleaved by a disintegrin and metalloproteinase (ADAM) members and is implicated in epidermal growth factor receptor (EGFR) transactivation. Recently, nuclear translocation of the C-terminal fragment (CTF) of pro-HB-EGF was found to induce cell growth. We investigated the association between TGFβ and HB-EGF signal transduction via ADAM activation. Materials and methods: The CCK-8 assay in two gastric cancer cell lines was used to determine the effect for cell growth by TGFβ. The effect of two ADAM inhibitors was also evaluated. Induction of EGFR phosphorylation by TGFβ was analyzed and the effect of the ADAM inhibitors was also examined. Nuclear translocation of HB-EGF-CTF by shedding through ADAM activated by TGFβ was also analyzed. EGFR transactivation, HB-EGF-CTF nuclear translocation, and cell growth were examined under the condition of ADAM17 knockdown. Result: TGFβ-induced EGFR phosphorylation of which ADAM inhibitors were able to inhibit. TGFβ induced shedding of proHB-EGF allowing HB-EGF-CTF to translocate to the nucleus. ADAM inhibitors blocked this nuclear translocation. TGF

  19. RAGE regulates immune cell infiltration and angiogenesis in choroidal neovascularization.

    Directory of Open Access Journals (Sweden)

    Mei Chen

    Full Text Available PURPOSE: RAGE regulates pro-inflammatory responses in diverse cells and tissues. This study has investigated if RAGE plays a role in immune cell mobilization and choroidal neovascular pathology that is associated with the neovascular form of age-related macular degeneration (nvAMD. METHODS: RAGE null (RAGE-/- mice and age-matched wild type (WT control mice underwent laser photocoagulation to generate choroidal neovascularization (CNV lesions which were then analyzed for morphology, S100B immunoreactivity and inflammatory cell infiltration. The chemotactic ability of bone marrow derived macrophages (BMDMs towards S100B was investigated. RESULTS: RAGE expression was significantly increased in the retina during CNV of WT mice (p<0.001. RAGE-/- mice exhibited significantly reduced CNV lesion size when compared to WT controls (p<0.05. S100B mRNA was upregulated in the lasered WT retina but not RAGE-/- retina and S100B immunoreactivity was present within CNV lesions although levels were less when RAGE-/- mice were compared to WT controls. Activated microglia in lesions were considerably less abundant in RAGE-/- mice when compared to WT counterparts (p<0.001. A dose dependent chemotactic migration was observed in BMDMs from WT mice (p<0.05-0.01 but this was not apparent in cells isolated from RAGE-/- mice. CONCLUSIONS: RAGE-S100B interactions appear to play an important role in CNV lesion formation by regulating pro-inflammatory and angiogenic responses. This study highlights the role of RAGE in inflammation-mediated outer retinal pathology.

  20. Mitochondria, calcium and pro-apoptotic proteins as mediators in cell death signaling

    Directory of Open Access Journals (Sweden)

    S.S. Smaili

    2003-02-01

    Full Text Available Cellular Ca2+ signals are crucial in the control of most physiological processes, cell injury and programmed cell death through the regulation of a number of Ca2+-dependent enzymes such as phospholipases, proteases, and nucleases. Mitochondria along with the endoplasmic reticulum play pivotal roles in regulating intracellular Ca2+ content. Mitochondria are endowed with multiple Ca2+ transport mechanisms by which they take up and release Ca2+ across their inner membrane. During cellular Ca2+ overload, mitochondria take up cytosolic Ca2+, which in turn induces opening of permeability transition pores and disrupts the mitochondrial membrane potential (Dym. The collapse of Dym along with the release of cytochrome c from mitochondria is followed by the activation of caspases, nuclear fragmentation and cell death. Members of the Bcl-2 family are a group of proteins that play important roles in apoptosis regulation. Members of this family appear to differentially regulate intracellular Ca2+ level. Translocation of Bax, an apoptotic signaling protein, from the cytosol to the mitochondrial membrane is another step in this apoptosis signaling pathway.

  1. Isoquercitrin suppresses the expression of histamine and pro-inflammatory cytokines by inhibiting the activation of MAP Kinases and NF-κB in human KU812 cells.

    Science.gov (United States)

    Li, Li; Zhang, Xiao-Hui; Liu, Guang-Rong; Liu, Chang; Dong, Yin-Mao

    2016-06-01

    Mast cells and basophils are multifunctional effector cells that contain abundant secretory granules in their cytoplasm. Both cell types are involved in a variety of inflammatory and immune events, producing an array of inflammatory mediators, such as cytokines. The aim of the study was to examine whether isoquercitrin modulates allergic and inflammatory reactions in the human basophilic KU812 cells and to elucidate its influence on the phosphorylation of mitogen-activated protein kinase (MAPK) and nuclear factor (NF)-κB activation. The KU812 cells were stimulated with phorbol-12-myristate 13-acetate plus the calcium ionophore A23187 (PMACI). The inhibitory effects of isoquercitrin on the productions of histamine and pro-inflammatory cytokines in the stimulated KU812 cells were measured using cytokine-specific enzyme-linked immunosorbent (ELISA) assays. Western blotting analysis was used to assess the effects of isoquercitrin on the MAPKs and NF-κB protein levels. Our results indicated that the isoquercitrin treatment of PMACI-stimulated KU812 cells significantly reduced the production of histamine and the pro-inflammatory cytokines, such as interleukin (IL)-6, IL-8, IL-1β, and tumor necrosis factor (TNF)-α. The treated cells exhibited decreased phosphorylation of extracellular signal-regulated kinase (ERK), revealing the role of ERK MAPK in isoquercitrin-mediated allergy inhibition. Furthermore, isoquercitrin suppressed the PMACI-mediated activation of NF-κB in the human basophil cells. In conclusion, the results from the present study provide insights into the potential therapeutic use of isoquercitrin for the treatment of inflammatory and allergic reactions. PMID:27473957

  2. Isoquercitrin suppresses the expression of histamine and pro-inflammatory cytokines by inhibiting the activation of MAP Kinases and NF-κB in human KU812 cells

    Institute of Scientific and Technical Information of China (English)

    LI Li; ZHANG Xiao-Hui; LIU Guang-Rong; LIU Chang; DONG Yin-Mao

    2016-01-01

    Mast cells and basophils are multifunctional effector cells that contain abundant secretory granules in their cytoplasm.Both cell types are involved in a variety of inflammatory and immune events,producing an array of inflammatory mediators,such as cytokines.The aim of the study was to examine whether isoquercitrin modulates allergic and inflammatory reactions in the human basophilic KU812 cells and to elucidate its influence on the phosphorylation of mitogen-activated protein kinase (MAPK) and nuclear factor (NF)-κB activation.The KU812 cells were stimulated with phorbol-12-myristate 13-acetate plus the calcium ionophore A23187 (PMACI).The inhibitory effects of isoquercitrin on the productions of histamine and pro-inflammatory cytokines in the stimulated KU812 cells were measured using cytokine-specific enzyme-linked immunosorbent (ELISA) assays.Western blotting analysis was used to assess the effects of isoquercitrin on the MAPKs and NF-κB protein levels.Our results indicated that the isoquercitrin treatment of PMACI-stimulated KU812 cells significantly reduced the production of histamine and the pro-inflammatory cytokines,such as interleukin (IL)-6,IL-8,IL-1β,and tumor necrosis factor (TNF)-α.The treated cells exhibited decreased phosphorylation of extracellular signal-regulated kinase (ERK),revealing the role of ERK MAPK in isoquercitrin-mediated allergy inhibition.Furthermore,isoquercitrin suppressed the PMACI-mediated activation of NF-κB in the human basophil cells.In conclusion,the results from the present study provide insights into the potential therapeutic use of isoquercitrin for the treatment of inflammatory and allergic reactions.

  3. Naegleria fowleri lysate induces strong cytopathic effects and pro-inflammatory cytokine release in rat microglial cells.

    Science.gov (United States)

    Lee, Yang-Jin; Park, Chang-Eun; Kim, Jong-Hyun; Sohn, Hae-Jin; Lee, Jinyoung; Jung, Suk-Yul; Shin, Ho-Joon

    2011-09-01

    Naegleria fowleri, a ubiquitous free-living ameba, causes fatal primary amebic meningoencephalitis in humans. N. fowleri trophozoites are known to induce cytopathic changes upon contact with microglial cells, including necrotic and apoptotic cell death and pro-inflammatory cytokine release. In this study, we treated rat microglial cells with amebic lysate to probe contact-independent mechanisms for cytotoxicity, determining through a combination of light microscopy and scanning and transmission electron microscopy whether N. fowleri lysate could effect on both necrosis and apoptosis on microglia in a time- as well as dose-dependent fashion. A (51)Cr release assay demonstrated pronounced lysate induction of cytotoxicity (71.5%) toward microglial cells by 24 hr after its addition to cultures. In an assay of pro-inflammatory cytokine release, microglial cells treated with N. fowleri lysate produced TNF-α, IL-6, and IL-1β, though generation of the former 2 cytokines was reduced with time, and that of the last increased throughout the experimental period. In summary, N. fowleri lysate exerted strong cytopathic effects on microglial cells, and elicited pro-inflammatory cytokine release as a primary immune response.

  4. Naegleria fowleri Lysate Induces Strong Cytopathic Effects and Pro-inflammatory Cytokine Release in Rat Microglial Cells

    Science.gov (United States)

    Lee, Yang-Jin; Park, Chang-Eun; Kim, Jong-Hyun; Sohn, Hae-Jin; Lee, Jinyoung; Jung, Suk-Yul

    2011-01-01

    Naegleria fowleri, a ubiquitous free-living ameba, causes fatal primary amebic meningoencephalitis in humans. N. fowleri trophozoites are known to induce cytopathic changes upon contact with microglial cells, including necrotic and apoptotic cell death and pro-inflammatory cytokine release. In this study, we treated rat microglial cells with amebic lysate to probe contact-independent mechanisms for cytotoxicity, determining through a combination of light microscopy and scanning and transmission electron microscopy whether N. fowleri lysate could effect on both necrosis and apoptosis on microglia in a time- as well as dose-dependent fashion. A 51Cr release assay demonstrated pronounced lysate induction of cytotoxicity (71.5%) toward microglial cells by 24 hr after its addition to cultures. In an assay of pro-inflammatory cytokine release, microglial cells treated with N. fowleri lysate produced TNF-α, IL-6, and IL-1β, though generation of the former 2 cytokines was reduced with time, and that of the last increased throughout the experimental period. In summary, N. fowleri lysate exerted strong cytopathic effects on microglial cells, and elicited pro-inflammatory cytokine release as a primary immune response. PMID:22072830

  5. Cbl negatively regulates JNK activation and cell death

    Institute of Scientific and Technical Information of China (English)

    Andrew A Sproul; Zhiheng Xu; Michael Wilhelm; Stephen Gire; Lloyd A Greene

    2009-01-01

    Here, we explore the role of Cbl proteins in regulation of neuronal apoptosis. In two paradigms of neuron apopto-sis--nerve growth factor (NGF) deprivation and DNA damage--cellular levels of c-Cbl and Cbl-b fell well before the onset of cell death. NGF deprivation also induced rapid loss of tyrosine phosphorylation (and most likely, activa-tion) of c-Cbl. Targeting e-Cbl and Cbl-b with siRNAs to mimic their loss/inactivation sensitized neuronal cells to death promoted by NGF deprivation or DNA damage. One potential mechanism by which Cbl proteins might affect neuronal death is by regulation of apoptotic c-Jun N-terminal kinase (JNK) signaling. We demonstrate that Cbl pro-teins interact with the JNK pathway components mixed lineage kinase (MLK) 3 and POSH and that knockdown of Cbl proteins is sufficient to increase JNK pathway activity. Furthermore, expression of c-Cbl blocks the ability of MLKs to signal to downstream components of the kinase cascade leading to JNK activation and protects neuronal cells from death induced by MLKs, but not from downstream JNK activators. On the basis of these findings, we propose that Cbls suppress cell death in healthy neurons at least in part by inhibiting the ability of MLKs to activate JNK signaling. Apoptotic stimuli lead to loss of Cbl protein/activity, thereby removing a critical brake on JNK acti-vation and on cell death.

  6. Insulin signaling regulates mitochondrial function in pancreatic beta-cells.

    Directory of Open Access Journals (Sweden)

    Siming Liu

    Full Text Available Insulin/IGF-I signaling regulates the metabolism of most mammalian tissues including pancreatic islets. To dissect the mechanisms linking insulin signaling with mitochondrial function, we first identified a mitochondria-tethering complex in beta-cells that included glucokinase (GK, and the pro-apoptotic protein, BAD(S. Mitochondria isolated from beta-cells derived from beta-cell specific insulin receptor knockout (betaIRKO mice exhibited reduced BAD(S, GK and protein kinase A in the complex, and attenuated function. Similar alterations were evident in islets from patients with type 2 diabetes. Decreased mitochondrial GK activity in betaIRKOs could be explained, in part, by reduced expression and altered phosphorylation of BAD(S. The elevated phosphorylation of p70S6K and JNK1 was likely due to compensatory increase in IGF-1 receptor expression. Re-expression of insulin receptors in betaIRKO cells partially restored the stoichiometry of the complex and mitochondrial function. These data indicate that insulin signaling regulates mitochondrial function and have implications for beta-cell dysfunction in type 2 diabetes.

  7. Materials as stem cell regulators

    Science.gov (United States)

    Murphy, William L.; McDevitt, Todd C.; Engler, Adam J.

    2014-06-01

    The stem cell/material interface is a complex, dynamic microenvironment in which the cell and the material cooperatively dictate one another's fate: the cell by remodelling its surroundings, and the material through its inherent properties (such as adhesivity, stiffness, nanostructure or degradability). Stem cells in contact with materials are able to sense their properties, integrate cues via signal propagation and ultimately translate parallel signalling information into cell fate decisions. However, discovering the mechanisms by which stem cells respond to inherent material characteristics is challenging because of the highly complex, multicomponent signalling milieu present in the stem cell environment. In this Review, we discuss recent evidence that shows that inherent material properties may be engineered to dictate stem cell fate decisions, and overview a subset of the operative signal transduction mechanisms that have begun to emerge. Further developments in stem cell engineering and mechanotransduction are poised to have substantial implications for stem cell biology and regenerative medicine.

  8. Use of SLAM and PVRL4 and identification of pro-HB-EGF as cell entry receptors for wild type phocine distemper virus.

    Directory of Open Access Journals (Sweden)

    Mary M Melia

    Full Text Available Signalling lymphocyte activation molecule (SLAM has been identified as an immune cell receptor for the morbilliviruses, measles (MV, canine distemper (CDV, rinderpest and peste des petits ruminants (PPRV viruses, while CD46 is a receptor for vaccine strains of MV. More recently poliovirus like receptor 4 (PVRL4, also known as nectin 4, has been identified as a receptor for MV, CDV and PPRV on the basolateral surface of polarised epithelial cells. PVRL4 is also up-regulated by MV in human brain endothelial cells. Utilisation of PVRL4 as a receptor by phocine distemper virus (PDV remains to be demonstrated as well as confirmation of use of SLAM. We have observed that unlike wild type (wt MV or wtCDV, wtPDV strains replicate in African green monkey kidney Vero cells without prior adaptation, suggesting the use of a further receptor. We therefore examined candidate molecules, glycosaminoglycans (GAG and the tetraspan proteins, integrin β and the membrane bound form of heparin binding epithelial growth factor (proHB-EGF,for receptor usage by wtPDV in Vero cells. We show that wtPDV replicates in Chinese hamster ovary (CHO cells expressing SLAM and PVRL4. Similar wtPDV titres are produced in Vero and VeroSLAM cells but more limited fusion occurs in the latter. Infection of Vero cells was not inhibited by anti-CD46 antibody. Removal/disruption of GAG decreased fusion but not the titre of virus. Treatment with anti-integrin β antibody increased rather than decreased infection of Vero cells by wtPDV. However, infection was inhibited by antibody to HB-EGF and the virus replicated in CHO-proHB-EGF cells, indicating use of this molecule as a receptor. Common use of SLAM and PVRL4 by morbilliviruses increases the possibility of cross-species infection. Lack of a requirement for wtPDV adaptation to Vero cells raises the possibility of usage of proHB-EGF as a receptor in vivo but requires further investigation.

  9. LGR5 regulates pro-survival MEK/ERK and proliferative Wnt/β-catenin signalling in neuroblastoma.

    Science.gov (United States)

    Vieira, Gabriella Cunha; Chockalingam, S; Melegh, Zsombor; Greenhough, Alexander; Malik, Sally; Szemes, Marianna; Park, Ji Hyun; Kaidi, Abderrahmane; Zhou, Li; Catchpoole, Daniel; Morgan, Rhys; Bates, David O; Gabb, Peter David; Malik, Karim

    2015-11-24

    LGR5 is a marker of normal and cancer stem cells in various tissues where it functions as a receptor for R-spondins and increases canonical Wnt signalling amplitude. Here we report that LGR5 is also highly expressed in a subset of high grade neuroblastomas. Neuroblastoma is a clinically heterogenous paediatric cancer comprising a high proportion of poor prognosis cases (~40%) which are frequently lethal. Unlike many cancers, Wnt pathway mutations are not apparent in neuroblastoma, although previous microarray analyses have implicated deregulated Wnt signalling in high-risk neuroblastoma. We demonstrate that LGR5 facilitates high Wnt signalling in neuroblastoma cell lines treated with Wnt3a and R-spondins, with SK-N-BE(2)-C, SK-N-NAS and SH-SY5Y cell-lines all displaying strong Wnt induction. These lines represent MYCN-amplified, NRAS and ALK mutant neuroblastoma subtypes respectively. Wnt3a/R-Spondin treatment also promoted nuclear translocation of β-catenin, increased proliferation and activation of Wnt target genes. Strikingly, short-interfering RNA mediated knockdown of LGR5 induces dramatic Wnt-independent apoptosis in all three cell-lines, accompanied by greatly diminished phosphorylation of mitogen/extracellular signal-regulated kinases (MEK1/2) and extracellular signal-regulated kinases (ERK1/2), and an increase of BimEL, an apoptosis facilitator downstream of ERK. Akt signalling is also decreased by a Rictor dependent, PDK1-independent mechanism. LGR5 expression is cell cycle regulated and LGR5 depletion triggers G1 cell-cycle arrest, increased p27 and decreased phosphorylated retinoblastoma protein. Our study therefore characterises new cancer-associated pathways regulated by LGR5, and suggest that targeting of LGR5 may be of therapeutic benefit for neuroblastomas with diverse etiologies, as well as other cancers expressing high LGR5.

  10. First Test Results of the bERLinPro 2-cell Booster Cavities

    Energy Technology Data Exchange (ETDEWEB)

    Burrill, Andrew [Helmholtz-Zentrum Berlin (HZB), (Germany); Anders, W. [Helmholtz-Zentrum Berlin (HZB), (Germany); Frahm, A. [Helmholtz-Zentrum Berlin (HZB), (Germany); Knobloch, Jens [Helmholtz-Zentrum Berlin (HZB), (Germany); Neumann, Axel [Helmholtz-Zentrum Berlin (HZB), (Germany); Ciovati, Gianluigi [Thomas Jefferson National Accelerator Facility, Newport News, VA (United States); Clemens, William [Thomas Jefferson National Accelerator Facility, Newport News, VA (United States); Kneisel, Peter [Thomas Jefferson National Accelerator Facility, Newport News, VA (United States); Turlington, Larry [Thomas Jefferson National Accelerator Facility, Newport News, VA (United States)

    2015-09-01

    The bERLinPro Energy Recovery Linac (ERL) is currently being built at Helmholtz-Zentrum Berlin in order to study the physics of operating a high-current, a 100 mA, 50 MeV ERL utilizing all SRF cavity technology. This machine will utilize three unique SRF cryomodules for the photoinjector, booster and linac cryomodules respectively. The focus of this paper will be on the cavities contained within the booster cryomodule. Here there will be three 2-cell SRF cavities, based on the original design by Cornell University, but optimized to meet the needs of the project. All of the cavity fabrication, processing and testing was carried out at Jefferson Laboratory, where 4 cavities were produced, and the 3 cavities with the best RF performance were fitted with helium vessels for installation in the cryomodule. This paper will report on the test results of the cavities as measured in the vertical testing dewar at JLab after fabrication and again after outfitting with the helium vessels.

  11. Computational model of vascular endothelial growth factor spatial distribution in muscle and pro-angiogenic cell therapy.

    Directory of Open Access Journals (Sweden)

    Feilim Mac Gabhann

    2006-09-01

    Full Text Available Members of the vascular endothelial growth factor (VEGF family of proteins are critical regulators of angiogenesis. VEGF concentration gradients are important for activation and chemotactic guidance of capillary sprouting, but measurement of these gradients in vivo is not currently possible. We have constructed a biophysically and molecularly detailed computational model to study microenvironmental transport of two isoforms of VEGF in rat extensor digitorum longus skeletal muscle under in vivo conditions. Using parameters based on experimental measurements, the model includes: VEGF secretion from muscle fibers; binding to the extracellular matrix; binding to and activation of endothelial cell surface VEGF receptors; and internalization. For 2-D cross sections of tissue, we analyzed predicted VEGF distributions, gradients, and receptor binding. Significant VEGF gradients (up to 12% change in VEGF concentration over 10 mum were predicted in resting skeletal muscle with uniform VEGF secretion, due to non-uniform capillary distribution. These relative VEGF gradients were not sensitive to extracellular matrix composition, or to the overall VEGF expression level, but were dependent on VEGF receptor density and affinity, and internalization rate parameters. VEGF upregulation in a subset of fibers increased VEGF gradients, simulating transplantation of pro-angiogenic myoblasts, a possible therapy for ischemic diseases. The number and relative position of overexpressing fibers determined the VEGF gradients and distribution of VEGF receptor activation. With total VEGF expression level in the tissue unchanged, concentrating overexpression into a small number of adjacent fibers can increase the number of capillaries activated. The VEGF concentration gradients predicted for resting muscle (average 3% VEGF/10 mum is sufficient for cellular sensing; the tip cell of a vessel sprout is approximately 50 mum long. The VEGF gradients also result in heterogeneity in

  12. Ionomycin-induced Ca2+ overload is not accompanied by mitochondrial membrane potential dissipation in murine pro-B cells

    OpenAIRE

    Gabriela Ildiko Zonda; Ancuta Goriuc; Marcel Costuleanu

    2010-01-01

    There are extremely few data concerning the involvement of Ca2+ fluxes in the apoptosis of the pro-B cell type Ba/F3. Thus, we aimed the characterization of ionomycin-induced effects on Ba/F3 cells in vitro. Our obtained data show that cytosolic Ca2+ increased in Ba/F3 cells by 1 μM ionomycin in the presence of 1 mM Ca2+ for 24 hours did not induced significant effects on the mitochondrial membrane potential as compared with control cells. The same effects were also associated by ...

  13. Pro-Apoptotic Activity of New Honokiol/Triphenylmethane Analogues in B-Cell Lymphoid Malignancies.

    Science.gov (United States)

    Mędra, Aleksandra; Witkowska, Magdalena; Majchrzak, Agata; Cebula-Obrzut, Barbara; Bonner, Michael Y; Robak, Tadeusz; Arbiser, Jack L; Smolewski, Piotr

    2016-01-01

    Honokiol and triphenylmethanes are small molecules with anti-tumor properties. Recently, we synthesized new honokiol analogues (HAs) that possess common features of both groups. We assessed the anti-tumor effectiveness of HAs in B-cell leukemia/lymphoma cells, namely in chronic lymphocytic leukemia (CLL) cells ex vivo and in pre-B-cell acute lymphoblastic leukemia (Nalm-6), Burkitt lymphoma (BL; Raji), diffuse large B-cell lymphoma (DLBCL; Toledo) and multiple myeloma (MM; RPMI 8226) cell lines. Four of these compounds appeared to be significantly active against the majority of cells examined, with no significant impact on healthy lymphocytes. These active HAs induced caspase-dependent apoptosis, causing significant deregulation of several apoptosis-regulating proteins. Overall, these compounds downregulated Bcl-2 and XIAP and upregulated Bax, Bak and survivin proteins. In conclusion, some of the HAs are potent tumor-selective inducers of apoptosis in ex vivo CLL and in BL, DLBCL and MM cells in vitro. Further preclinical studies of these agents are recommended. PMID:27483232

  14. Pro-nerve growth factor induces autocrine stimulation of breast cancer cell invasion through tropomyosin-related kinase A (TrkA) and sortilin protein.

    Science.gov (United States)

    Demont, Yohann; Corbet, Cyril; Page, Adeline; Ataman-Önal, Yasemin; Choquet-Kastylevsky, Genevieve; Fliniaux, Ingrid; Le Bourhis, Xuefen; Toillon, Robert-Alain; Bradshaw, Ralph A; Hondermarck, Hubert

    2012-01-13

    The precursor of nerve growth factor (proNGF) has been described as a biologically active polypeptide able to induce apoptosis in neuronal cells, via the neurotrophin receptor p75(NTR) and the sortilin receptor. Herein, it is shown that proNGF is produced and secreted by breast cancer cells, stimulating their invasion. Using Western blotting and mass spectrometry, proNGF was detected in a panel of breast cancer cells as well as in their conditioned media. Immunohistochemical analysis indicated an overproduction of proNGF in breast tumors, when compared with benign and normal breast biopsies, and a relationship to lymph node invasion in ductal carcinomas. Interestingly, siRNA against proNGF induced a decrease of breast cancer cell invasion that was restored by the addition of non-cleavable proNGF. The activation of TrkA, Akt, and Src, but not the MAP kinases, was observed. In addition, the proNGF invasive effect was inhibited by the Trk pharmacological inhibitor K252a, a kinase-dead TrkA, and siRNA against TrkA sortilin, neurotensin, whereas siRNA against p75(NTR) and the MAP kinase inhibitor PD98059 had no impact. These data reveal the existence of an autocrine loop stimulated by proNGF and mediated by TrkA and sortilin, with the activation of Akt and Src, for the stimulation of breast cancer cell invasion.

  15. Developing Anticancer Copper(II) Pro-drugs Based on the Nature of Cancer Cells and the Human Serum Albumin Carrier IIA Subdomain.

    Science.gov (United States)

    Gou, Yi; Qi, Jinxu; Ajayi, Joshua-Paul; Zhang, Yao; Zhou, Zuping; Wu, Xiaoyang; Yang, Feng; Liang, Hong

    2015-10-01

    To synergistically enhance the selectivity and efficiency of anticancer copper drugs, we proposed and built a model to develop anticancer copper pro-drugs based on the nature of human serum albumin (HSA) IIA subdomain and cancer cells. Three copper(II) compounds of a 2-hydroxy-1-naphthaldehyde benzoyl hydrazone Schiff-base ligand in the presence pyridine, imidazole, or indazole ligands were synthesized (C1-C3). The structures of three HSA complexes revealed that the Cu compounds bind to the hydrophobic cavity in the HSA IIA subdomain. Among them, the pyridine and imidazole ligands of C1 and C2 are replaced by Lys199, and His242 directly coordinates with Cu(II). The indazole and Br ligands of C3 are replaced by Lys199 and His242, respectively. Compared with the Cu(II) compounds alone, the HSA complexes enhance cytotoxicity in MCF-7 cells approximately 3-5-fold, but do not raise cytotoxicity levels in normal cells in vitro through selectively accumulating in cancer cells to some extent. We find that the HSA complex has a stronger capacity for cell cycle arrest in the G2/M phase of MCF-7 by targeting cyclin-dependent kinase 1 (CDK1) and down-regulating the expression of CDK1 and cyclin B1. Moreover, the HSA complex promotes MCF-7 cell apoptosis possibly through the intrinsic reactive oxygen species (ROS) mediated mitochondrial pathway, accompanied by the regulation of Bcl-2 family proteins.

  16. miR-211 is a pro-survival micro-RNA that regulates chop expression in a PERK-dependent manner

    Science.gov (United States)

    Chitnis, Nilesh; Pytel, Dariusz; Bobrovnikova-Marjon, Ekaterina; Pant, Dhruv; Zheng, Hui; Maas, Nancy; Frederick, Brian; Kushner, Jake A.; Chodosh, Lewis A.; Koumenis, Constantinos; Fuchs, Serge Y.; Diehl, J. Alan

    2012-01-01

    Micro-RNAs typically function at the level of post-transcriptional gene silencing within the cytoplasm; however increasing evidence suggests that they may also function in nuclear, Argonaut containing complexes, to directly repress target gene transcription. We have investigated the role of micro-RNAs in mediating endoplasmic reticulum (ER) stress responses. ER stress triggers the activation of three signaling molecules: Ire-1α/β, PERK and ATF6 whose function is to facilitate adaption to the ensuing stress. We demonstrate that PERK induces miR-211, which in turn attenuates stress-dependent expression of the pro-apoptotic transcription factor chop/gadd153. MiR-211 directly targets the proximal chop/gadd153 promoter where it increases histone methylation and represses chop expression. Maximal chop accumulation ultimately correlates with miR-211 down regulation. Our data suggests a model where PERK-dependent miR-211 induction prevents premature chop accumulation and thereby provides a window of opportunity for the cell to re-establish homeostasis prior to apoptotic commitment. PMID:23022383

  17. Regulation of the pro-inflammatory cytokine osteopontin by GIP in adipocytes – A role for the transcription factor NFAT and phosphodiesterase 3B

    International Nuclear Information System (INIS)

    Highlights: ► GIP stimulates lipogenesis and osteopontin expression in primary adipocytes. ► GIP-induced osteopontin expression is NFAT-dependent. ► Osteopontin expression is PDE3-dependent. ► Osteopontin expression is increased in PDE3B KO mice. -- Abstract: The incretin – glucose-dependent insulinotropic polypeptide (GIP) – and the pro-inflammatory cytokine osteopontin are known to have important roles in the regulation of adipose tissue functions. In this work we show that GIP stimulates lipogenesis and osteopontin expression in primary adipocytes. The GIP-induced increase in osteopontin expression was inhibited by the NFAT (the transcription factor nuclear factor of activated T-cells) inhibitor A-285222. Also, the NFAT kinase glycogen synthase kinase (GSK) 3 was upregulated by GIP. To test whether cAMP might be involved in GIP-mediated effects on osteopontin a number of strategies were used. Thus, the β3-adrenergic receptor agonist CL316,243 stimulated osteopontin expression, an effects which was mimicked by OPC3911, a specific inhibitor of phosphodiesterase 3. Furthermore, treatment of phosphodiesterase 3B knock-out mice with CL316,243 resulted in a dramatic upregulation of osteopontin in adipose tissue which was not the case in wild-type mice. In summary, we delineate mechanisms by which GIP stimulates osteopontin in adipocytes. Given the established link between osteopontin and insulin resistance, our data suggest that GIP by stimulating osteopontin expression, also could promote insulin resistance in adipocytes.

  18. Activated factor X signaling via protease-activated receptor 2 suppresses pro-inflammatory cytokine production from LPS-stimulated myeloid cells.

    LENUS (Irish Health Repository)

    Gleeson, Eimear M

    2013-07-19

    -associated protein-sensitive, protease-activated receptor 2-dependent regulator of myeloid cell pro-inflammatory cytokine production.

  19. Regulating cell differentiation at different layers

    Institute of Scientific and Technical Information of China (English)

    Jiarui Wu

    2011-01-01

    Cell differentiation is a basic behavior in the developmental process of multi-cellular organisms,through which various cell types are generated from one embryonic cell for further building different tissues and organs of animals or plants.It is estimated that there are more than two hundred cell types in a human body.To understand the molecular mechanisms of cell differentiation,researchers usually focus on a question how particular genes are selectively expressed during the differentiation process.However,more and more evidence indicates that the regulation of cell differentiation is far beyond simply controlling the expression of genetic program,which is supported by the collection of four research articles in this issue that the regulation of cell differentiation involves various factors at different layers,including epigenetics,metabolism and cell-cell interaction.

  20. Regulating the leukemia stem cell

    OpenAIRE

    Cleary, Michael L.

    2009-01-01

    Leukemia stem cells (LSCs) are responsible for sustaining and propagating malignant disease, and, as such, are promising targets for therapy. Studies of human LSCs have served an important role in defining the major tenets of the cancer stem cell model, which center on the frequencies of cancer stem cells, their potential hierarchical organization, and their degree of maturation. LSCs in acute myeloid leukemia (AML) have recently been studied using mouse syngeneic models of leukemia induced b...

  1. RETINOIDS REGULATE STEM CELL DIFFERENTIATION

    OpenAIRE

    Gudas, Lorraine J.; Wagner, John A.

    2011-01-01

    Retinoids are ubiquitous signaling molecules that influence nearly every cell type, exert profound effects on development, and complement cancer chemotherapeutic regimens. All-trans retinoic acid (RA) and other active retinoids are generated from vitamin A (retinol), but key aspects of the signaling pathways required to produce active retinoids remain unclear. Retinoids generated by one cell type can affect nearby cells, so retinoids also function in intercellular communication. RA induces di...

  2. Regulation of Power Conversion in Fuel Cells

    Institute of Scientific and Technical Information of China (English)

    SHEN Mu-zhong; ZHANG J.; K. Scott

    2004-01-01

    Here we report a regulation about power conversion in fuel cells. This regulation is expressed as that total power produced by fuel cells is always proportional to the square of the potential difference between the equilibrium potential and work potential. With this regulation we deduced fuel cell performance equation which can describe the potential vs. the current performance curves, namely, polarization curves of fuel cells with three power source parameters: equilibrium potential E0; internal resistance R; and power conversion coefficient K. The concept of the power conversion coefficient is a new criterion to evaluate and compare the characteristics and capacity of different fuel cells. The calculated values obtained with this equation agree with practical performance of different types of fuel cells.

  3. Protein Localization in Escherichia coli Cells: Comparison of the Cytoplasmic Membrane Proteins ProP, LacY, ProW, AqpZ, MscS, and MscL

    OpenAIRE

    Romantsov, Tatyana; Battle, Andrew R.; Hendel, Jenifer L.; Martinac, Boris; Wood, Janet M.

    2010-01-01

    Fluorescence microscopy has revealed that the phospholipid cardiolipin (CL) and FlAsH-labeled transporters ProP and LacY are concentrated at the poles of Escherichia coli cells. The proportion of CL among E. coli phospholipids can be varied in vivo as it is decreased by cls mutations and it increases with the osmolality of the growth medium. In this report we compare the localization of CL, ProP, and LacY with that of other cytoplasmic membrane proteins. The proportion of cells in which FlAsH...

  4. Antioxidants inhibit SAA formation and pro-inflammatory cytokine release in a human cell model of alkaptonuria

    OpenAIRE

    Spreafico, Adriano; Millucci, Lia; Ghezzi, Lorenzo; Geminiani, Michela; Braconi, Daniela; Amato, Loredana; Chellini, Federico; Frediani, Bruno; Moretti, Elena; Collodel, Giulia; Bernardini, Giulia; Santucci, Annalisa

    2013-01-01

    Objective. Alkaptonuria (AKU) is an ultra-rare autosomal recessive disease that currently lacks an appropriate therapy. Recently we provided experimental evidence that AKU is a secondary serum amyloid A (SAA)-based amyloidosis. The aim of the present work was to evaluate the use of antioxidants to inhibit SAA amyloid and pro-inflammatory cytokine release in AKU. Methods. We adopted a human chondrocytic cell AKU model to evaluate the anti-amyloid capacity of a set of antioxidants that had prev...

  5. Wedelolactone inhibits LPS-induced pro-inflammation via NF-kappaB Pathway in RAW 264.7 cells

    OpenAIRE

    Yuan, Fang; Chen, Jie; Sun, Ping-Ping; Guan, Su; Xu, Jing

    2013-01-01

    Background Wedelolactone (WEL), a major coumestan ingredient in Wedelia chinensis, has been used to treat septic shock, hepatitis and venom poisoning in traditional Chinese medicines. The objective of the study was to elucidate the anti-inflammatory effects and mechanism of WEL with a cellular model of lipopolysaccharide (LPS)-induced RAW 264.7 cells. Results To study the role of WEL in pro-inflammation, we measured key inflammation mediators and end products including nitric oxide (NO), pros...

  6. Intrinsic, pro-apoptotic effects of IGFBP-3 on breast cancer cells are reversible: Involvement of PKA, Rho and ceramide.

    OpenAIRE

    Perks, Claire M.; Carla eBurrows; Holly, Jeff M P

    2011-01-01

    We established previously that IGFBP-3 could exert positive or negative effects on cell function depending upon the extracellular matrix composition and by interacting with integrin signaling. To elicit its pro-apoptotic effects IGFBP-3 bound to caveolin-1 and the beta 1 integrin receptor and increased their association culminating in MAPK activation. Disruption of these complexes or blocking the beta 1 integrin receptor reversed these intrinsic actions of IGFBP-3. In this study we have exami...

  7. Co-Expression of Cancer Stem Cell Markers Corresponds to a Pro-Tumorigenic Expression Profile in Pancreatic Adenocarcinoma

    Science.gov (United States)

    Skoda, Jan; Hermanova, Marketa; Loja, Tomas; Nemec, Pavel; Neradil, Jakub; Karasek, Petr; Veselska, Renata

    2016-01-01

    Pancreatic ductal adenocarcinoma (PDAC) remains one of the most lethal malignancies. Its dismal prognosis is often attributed to the presence of cancer stem cells (CSCs) that have been identified in PDAC using various markers. However, the co-expression of all of these markers has not yet been evaluated. Furthermore, studies that compare the expression levels of CSC markers in PDAC tumor samples and in cell lines derived directly from those tumors are lacking. Here, we analyzed the expression of putative CSC markers—CD24, CD44, epithelial cell adhesion molecule (EpCAM), CD133, and nestin—by immunofluorescence, flow cytometry and quantitative PCR in 3 PDAC-derived cell lines and by immunohistochemistry in 3 corresponding tumor samples. We showed high expression of the examined CSC markers among all of the cell lines and tumor samples, with the exception of CD24 and CD44, which were enriched under in vitro conditions compared with tumor tissues. The proportions of cells positive for the remaining markers were comparable to those detected in the corresponding tumors. Co-expression analysis using flow cytometry revealed that CD24+/CD44+/EpCAM+/CD133+ cells represented a significant population of the cells (range, 43 to 72%) among the cell lines. The highest proportion of CD24+/CD44+/EpCAM+/CD133+ cells was detected in the cell line derived from the tumor of a patient with the shortest survival. Using gene expression profiling, we further identified the specific pro-tumorigenic expression profile of this cell line compared with the profiles of the other two cell lines. Together, CD24+/CD44+/EpCAM+/CD133+ cells are present in PDAC cell lines derived from primary tumors, and their increased proportion corresponds with a pro-tumorigenic gene expression profile. PMID:27414409

  8. Co-Expression of Cancer Stem Cell Markers Corresponds to a Pro-Tumorigenic Expression Profile in Pancreatic Adenocarcinoma.

    Science.gov (United States)

    Skoda, Jan; Hermanova, Marketa; Loja, Tomas; Nemec, Pavel; Neradil, Jakub; Karasek, Petr; Veselska, Renata

    2016-01-01

    Pancreatic ductal adenocarcinoma (PDAC) remains one of the most lethal malignancies. Its dismal prognosis is often attributed to the presence of cancer stem cells (CSCs) that have been identified in PDAC using various markers. However, the co-expression of all of these markers has not yet been evaluated. Furthermore, studies that compare the expression levels of CSC markers in PDAC tumor samples and in cell lines derived directly from those tumors are lacking. Here, we analyzed the expression of putative CSC markers-CD24, CD44, epithelial cell adhesion molecule (EpCAM), CD133, and nestin-by immunofluorescence, flow cytometry and quantitative PCR in 3 PDAC-derived cell lines and by immunohistochemistry in 3 corresponding tumor samples. We showed high expression of the examined CSC markers among all of the cell lines and tumor samples, with the exception of CD24 and CD44, which were enriched under in vitro conditions compared with tumor tissues. The proportions of cells positive for the remaining markers were comparable to those detected in the corresponding tumors. Co-expression analysis using flow cytometry revealed that CD24+/CD44+/EpCAM+/CD133+ cells represented a significant population of the cells (range, 43 to 72%) among the cell lines. The highest proportion of CD24+/CD44+/EpCAM+/CD133+ cells was detected in the cell line derived from the tumor of a patient with the shortest survival. Using gene expression profiling, we further identified the specific pro-tumorigenic expression profile of this cell line compared with the profiles of the other two cell lines. Together, CD24+/CD44+/EpCAM+/CD133+ cells are present in PDAC cell lines derived from primary tumors, and their increased proportion corresponds with a pro-tumorigenic gene expression profile. PMID:27414409

  9. Pro-apoptotic activity of α-bisabolol in preclinical models of primary human acute leukemia cells

    Directory of Open Access Journals (Sweden)

    Fato Romana

    2011-04-01

    Full Text Available Abstract Background We previously demonstrated that the plant-derived agent α-bisabolol enters cells via lipid rafts, binds to the pro-apoptotic Bcl-2 family protein BID, and may induce apoptosis. Here we studied the activity of α-bisabolol in acute leukemia cells. Methods We tested ex vivo blasts from 42 acute leukemias (14 Philadelphia-negative and 14 Philadelphia-positive B acute lymphoid leukemias, Ph-/Ph+B-ALL; 14 acute myeloid leukemias, AML for their sensitivity to α-bisabolol in 24-hour dose-response assays. Concentrations and time were chosen based on CD34+, CD33+my and normal peripheral blood cell sensitivity to increasing α-bisabolol concentrations for up to 120 hours. Results A clustering analysis of the sensitivity over 24 hours identified three clusters. Cluster 1 (14 ± 5 μM α-bisabolol IC50 included mainly Ph-B-ALL cells. AML cells were split into cluster 2 and 3 (45 ± 7 and 65 ± 5 μM IC50. Ph+B-ALL cells were scattered, but mainly grouped into cluster 2. All leukemias, including 3 imatinib-resistant cases, were eventually responsive, but a subset of B-ALL cells was fairly sensitive to low α-bisabolol concentrations. α-bisabolol acted as a pro-apoptotic agent via a direct damage to mitochondrial integrity, which was responsible for the decrease in NADH-supported state 3 respiration and the disruption of the mitochondrial membrane potential. Conclusion Our study provides the first evidence that α-bisabolol is a pro-apoptotic agent for primary human acute leukemia cells.

  10. Two cases of breast carcinoma with osteoclastic giant cells: Are the osteoclastic giant cells pro-tumoural differentiation of macrophages?

    Directory of Open Access Journals (Sweden)

    Shishido-Hara Yukiko

    2010-08-01

    Full Text Available Abstract Breast carcinoma with osteoclastic giant cells (OGCs is characterized by multinucleated OGCs, and usually displays inflammatory hypervascular stroma. OGCs may derive from tumor-associated macrophages, but their nature remains controversial. We report two cases, in which OGCs appear in common microenvironment despite different tumoural histology. A 44-year-old woman (Case 1 had OGCs accompanying invasive ductal carcinoma, and an 83-year-old woman (Case 2 with carcinosarcoma. Immunohistochemically, in both cases, tumoural and non-tumoural cells strongly expressed VEGF and MMP12, which promote macrophage migration and angiogenesis. The Chalkley count on CD-31-stained sections revealed elevated angiogenesis in both cases. The OGCs expressed bone-osteoclast markers (MMP9, TRAP, cathepsin K and a histiocyte marker (CD68, but not an MHC class II antigen, HLA-DR. The results indicate a pathogenesis: regardless of tumoural histology, OGCs derive from macrophages, likely in response to hypervascular microenvironments with secretion of common cytokines. The OGCs have acquired bone-osteoclast-like characteristics, but lost antigen presentation abilities as an anti-cancer defense. Appearance of OGCs may not be anti-tumoural immunological reactions, but rather pro-tumoural differentiation of macrophage responding to hypervascular microenvironments induced by breast cancer.

  11. Cotinine inhibits the pro-inflammatory response initiated by multiple cell surface Toll-like receptors in monocytic THP cells

    Directory of Open Access Journals (Sweden)

    Bagaitkar Juhi

    2012-11-01

    Full Text Available Abstract Background The primary, stable metabolite of nicotine [(S-3-(1-methyl-2-pyrrolidinyl pyridine] in humans is cotinine [(S-1-methyl-5-(3-pyridinyl-2-pyrrolidinone]. We have previously shown that cotinine exposure induces convergence and amplification of the GSK3β-dependent PI3 kinase and cholinergic anti-inflammatory systems. The consequence is reduced pro-inflammatory cytokine secretion by human monocytes responding to bacteria or LPS, a TLR4 agonist. Findings Here we show that cotinine-induced inflammatory suppression may not be restricted to individual Toll-like receptors (TLRs. Indeed, in monocytic cells, cotinine suppresses the cytokine production that is normally resultant upon agonist-specific engagement of all of the major surface exposed TLRs (TLR 2/1; 2/6; 4 and 5, although the degree of suppression varies by TLR. Conclusions These results provide further mechanistic insight into the increased susceptibility to multiple bacterial infections known to occur in smokers. They also establish THP-1 cells as a potentially suitable model with which to study the influence of tobacco components and metabolites on TLR-initiated inflammatory events.

  12. System-level modeling and simulation of the cell culture microfluidic biochip ProCell

    DEFF Research Database (Denmark)

    Minhass, Wajid Hassan; Pop, Paul; Madsen, Jan;

    2010-01-01

    Microfluidic biochips offer a promising alternative to a conventional biochemical laboratory. There are two technologies for the microfluidic biochips: droplet-based and flow-based. In this paper we are interested in flow-based microfluidic biochips, where the liquid flows continuously through pre...... as a 16 × 16 matrix). We use an inverted fluorescence microscope to observe the experiments in real-time, allowing kinetic data analysis. We are able to automatically adjust the current experimental setup thus allowing, for the first time, conditional experiments. We propose a biochip architecture model...... and a comprehensive fault model that captures permanent faults occurring during chip operation. Using the proposed modeling and simulation framework, we perform an architectural level evaluation of two cell culture chamber implementations. A qualitative success metric is also proposed to evaluate chip performance...

  13. Cell Size Regulation in Bacteria

    Science.gov (United States)

    Amir, Ariel

    2014-05-01

    Various bacteria such as the canonical gram negative Escherichia coli or the well-studied gram positive Bacillus subtilis divide symmetrically after they approximately double their volume. Their size at division is not constant, but is typically distributed over a narrow range. Here, we propose an analytically tractable model for cell size control, and calculate the cell size and interdivision time distributions, as well as the correlations between these variables. We suggest ways of extracting the model parameters from experimental data, and show that existing data for E. coli supports partial size control, and a particular explanation: a cell attempts to add a constant volume from the time of initiation of DNA replication to the next initiation event. This hypothesis accounts for the experimentally observed correlations between mother and daughter cells as well as the exponential dependence of size on growth rate.

  14. Biophysical regulation of stem cell differentiation.

    Science.gov (United States)

    Govey, Peter M; Loiselle, Alayna E; Donahue, Henry J

    2013-06-01

    Bone adaptation to its mechanical environment, from embryonic through adult life, is thought to be the product of increased osteoblastic differentiation from mesenchymal stem cells. In parallel with tissue-scale loading, these heterogeneous populations of multipotent stem cells are subject to a variety of biophysical cues within their native microenvironments. Bone marrow-derived mesenchymal stem cells-the most broadly studied source of osteoblastic progenitors-undergo osteoblastic differentiation in vitro in response to biophysical signals, including hydrostatic pressure, fluid flow and accompanying shear stress, substrate strain and stiffness, substrate topography, and electromagnetic fields. Furthermore, stem cells may be subject to indirect regulation by mechano-sensing osteocytes positioned to more readily detect these same loading-induced signals within the bone matrix. Such paracrine and juxtacrine regulation of differentiation by osteocytes occurs in vitro. Further studies are needed to confirm both direct and indirect mechanisms of biophysical regulation within the in vivo stem cell niche.

  15. Cell volume regulation: physiology and pathophysiology

    DEFF Research Database (Denmark)

    Lambert, I H; Hoffmann, E K; Pedersen, Stine Helene Falsig

    2008-01-01

    Cell volume perturbation initiates a wide array of intracellular signalling cascades, leading to protective and adaptive events and, in most cases, activation of volume-regulatory osmolyte transport, water loss, and hence restoration of cell volume and cellular function. Cell volume is challenged....../hypernatremia. On the other hand, it has recently become clear that an increase or reduction in cell volume can also serve as a specific signal in the regulation of physiological processes such as transepithelial transport, cell migration, proliferation and death. Although the mechanisms by which cell volume perturbations...

  16. Carboxiterminal pro-endothelin-1 as an endothelial cell biomarker in thrombotic thrombocytopenic purpura.

    Science.gov (United States)

    Mikes, Bálint; Sinkovits, György; Farkas, Péter; Csuka, Dorottya; Rázsó, Katalin; Réti, Marienn; Radványi, Gáspár; Demeter, Judit; Prohászka, Zoltán

    2016-05-01

    Thrombotic thrombocytopenic purpura (TTP) is characterised by the deficiency of the von Willebrand factor (VWF) cleaving protease (ADAMTS-13). Although several observations indicate an important role of endothelial activation in the pathogenesis of TTP, no reliable endothelial activation markers are available in the clinical management of TTP. Our aim was to investigate the presence of endothelial activation in TTP and to determine its connections with disease activity, therapy and complement activation. We enrolled 54 patients (median age 40.5; 44 females) and 57 healthy controls (median age 34; 30 females),VWF antigen, carboxiterminal-pro-endothelin-1 (CT-proET-1), complement Factor H and complement activation products (C3bBbP and SC5b-9) were measured. In both the acute and remission phase of TTP we found increased CT-proET-1 and VWF levels, while Factor H levels decreased compared with healthy controls. In remission, however, the elevated CT-proET-1 levels showed 22 % decrease when compared with the acute phase in paired samples (p=0.0031), whereas no changes for VWF and Factor H levels were observed. We also found positive correlations between CT-proET-1 levels and alternative pathway activation markers (C3bBbP; p=0.0360; r=0.4299). The data we present here demonstrate a role of endothelium activation in patients with acute TTP. The finding that CT-proET-1 levels decreased in remission compared with the acute phase further supports endothelial involvement. In addition, we show that endothelial activation also correlated with the activation of the alternative complement pathway. The data suggest that complement and endothelium activation jointly contribute to the development of TTP episodes in patients with predisposition to TTP. PMID:26763086

  17. Mast cells and Th17 cells contribute to the lymphoma-associated pro-inflammatory microenvironment of angioimmunoblastic T-cell lymphoma.

    Science.gov (United States)

    Tripodo, Claudio; Gri, Giorgia; Piccaluga, Pier Paolo; Frossi, Barbara; Guarnotta, Carla; Piconese, Silvia; Franco, Giovanni; Vetri, Valeria; Pucillo, Carlo Ennio; Florena, Ada Maria; Colombo, Mario Paolo; Pileri, Stefano Aldo

    2010-08-01

    Reports focusing on the immunological microenvironment of peripheral T-cell lymphomas (PTCL) are rare. Here we studied the reciprocal contribution of regulatory (Treg) and interleukin-17-producing (Th17) T-cells to the composition of the lymphoma-associated microenvironment of angioimmunoblastic T-cell lymphoma (AITL) and PTCL not otherwise specified on tissue microarrays from 30 PTCLs not otherwise specified and 37 AITLs. We found that Th17 but not Treg cells were differently represented in the two lymphomas and correlated with the amount of mast cells (MCs) and granulocytes, which preferentially occurred in the cellular milieu of AITL cases. We observed that MCs directly synthesized interleukin-6 and thus contribute to the establishment of a pro-inflammatory, Th17 permissive environment in AITL. We further hypothesized that the AITL clone itself could be responsible for the preferential accumulation of MCs at sites of infiltration through the synthesis of CXCL-13 and its interaction with the CXCR3 and CXCR5 receptors expressed on MCs. Consistent with this hypothesis, we observed MCs efficiently migrating in response to CXCL-13. On these bases, we conclude that MCs have a role in molding the immunological microenvironment of AITL toward the maintenance of pro-inflammatory conditions prone to Th17 generation and autoimmunity. PMID:20595635

  18. Diagnostic value of tumor marker pro-gastrin-releasing peptide in patients with small cell lung cancer: a systematic review

    Institute of Scientific and Technical Information of China (English)

    TANG Jian-hua; ZHANG Xiu-long; ZHANG Zhi-hua; WANG Rui; ZHANG He-ming; ZHANG Zhi-lin; WANG Jing-hui; REN Wei-dong

    2011-01-01

    Background Lung cancer is one of the most common malignancies in the world and one of the leading cancers that result in death. The aim of this study was to evaluate and compare the diagnostic value of the serum tumor marker pro-gastrin-releasing peptide 31-98 (ProGRP31-98) to pathological diagnosis as reference standard in patients with suspected small cell lung cancer (SCLC).Methods Literature searches covering 1978 through to 2009 were performed in Pubmed, OVID, MEDLINE, EMbase,Cancerlit, China National Knowledge Infrastructure (CNKI), and CBM using the key search words; 'small cell lung cancer','tumor marker', 'ProGRP31-98' and 'diagnostic tests', 'ELISA', 'EIA' and 'diagnostic accuracy'. Studies were collected and data analyzed to evaluate the diagnostic value of serum ProGRP31-98 levels for the diagnosis of SCLC compared with pathology. Eligibility criteria for inclusion in the analysis were based on criteria for diagnostic research published by the Cochrane Screening and Diagnostic Tests Methods Group (SDTMG). The characteristics of the included articles were appraised and the data were extracted from the original articles for further statistical analysis of study heterogeneity using Review Manager 4.2 software. Based on study heterogeneity analysis, a suitable 'effect' model was selected to calculate pooled sensitivity and specificity by meta-analysis. A Summary Receiver Operating Characteristic (SROC) curve and the area under the curve (AUC) were generated and sensitivity analysis conducted.Results A total of 22 articles were entered into this meta-review, including 11 English articles with a quality at level C. In total, the studies involved 6759 subjects, of which 1470 were diagnosed with SCLC by pathology, and 5289 subjects diagnosed with non-SCLC (NSCLC). The meta-analysis showed that heterogeneity among studies was high (P=0.00001,(I2)=86.8%). With ELISA, the pooled sensitivity was 0.72 (0.70 to 0.75 at 95% Cl) and the pooled specificity was 0.93 (0

  19. Recognition and Regulation of T Cells by NK Cells

    Science.gov (United States)

    Pallmer, Katharina; Oxenius, Annette

    2016-01-01

    Regulation of T cell responses by innate lymphoid cells (ILCs) is increasingly documented and studied. Direct or indirect crosstalk between ILCs and T cells early during and after T cell activation can affect their differentiation, polarization, and survival. Natural killer (NK) cells that belong to the ILC1 group were initially described for their function in recognizing and eliminating “altered self” and as source of early inflammatory cytokines, most notably type II interferon. Using signals conveyed by various germ-line encoded activating and inhibitory receptors, NK cells are geared to sense sudden cellular changes that can be caused by infection events, malignant transformation, or cellular stress responses. T cells, when activated by TCR engagement (signal 1), costimulation (signal 2), and cytokines (signal 3), commit to a number of cellular alterations, including entry into rapid cell cycling, metabolic changes, and acquisition of effector functions. These abrupt changes may alert NK cells, and T cells might thereby expose themselves as NK cell targets. Here, we review how activated T cells can be recognized and regulated by NK cells and what consequences such regulation bears for T cell immunity in the context of vaccination, infection, or autoimmunity. Conversely, we will discuss mechanisms by which activated T cells protect themselves against NK cell attack and outline the significance of this safeguard mechanism. PMID:27446081

  20. Regulation of cell proliferation by G proteins.

    Science.gov (United States)

    Dhanasekaran, N; Tsim, S T; Dermott, J M; Onesime, D

    1998-09-17

    G Proteins provide signal transduction mechanisms to seven transmembrane receptors. Recent studies have indicated that the alpha-subunits as well as the betagamma-subunits of these proteins regulate several critical signaling pathways involved in cell proliferation, differentiation and apoptosis. Of the 17 alpha-subunits that have been cloned, at least ten of them have been shown to couple mitogenic signaling in fibroblast cells. Activating mutations in G alpha(s), G alpha(i)2, and G alpha12 have been correlated with different types of tumors. In addition, the ability of the betagamma-subunits to activate mitogenic pathways in different cell-types has been defined. The present review briefly summarizes the diverse and novel signaling pathways regulated by the alpha- as well as the betagamma-subunits of G proteins in regulating cell proliferation. PMID:9779986

  1. Glial Cell Regulation of Rhythmic Behavior

    Science.gov (United States)

    Jackson, F. Rob; Ng, Fanny S.; Sengupta, Sukanya; You, Samantha; Huang, Yanmei

    2015-01-01

    Brain glial cells, in particular astrocytes and microglia, secrete signaling molecules that regulate glia–glia or glia–neuron communication and synaptic activity. While much is known about roles of glial cells in nervous system development, we are only beginning to understand the physiological functions of such cells in the adult brain. Studies in vertebrate and invertebrate models, in particular mice and Drosophila, have revealed roles of glia–neuron communication in the modulation of complex behavior. This chapter emphasizes recent evidence from studies of rodents and Drosophila that highlight the importance of glial cells and similarities or differences in the neural circuits regulating circadian rhythms and sleep in the two models. The chapter discusses cellular, molecular, and genetic approaches that have been useful in these models for understanding how glia–neuron communication contributes to the regulation of rhythmic behavior. PMID:25707272

  2. Dengue virus NS1 enhances viral replication and pro-inflammatory cytokine production in human dendritic cells.

    Science.gov (United States)

    Alayli, Farah; Scholle, Frank

    2016-09-01

    Dengue virus (DV) has become the most prevalent arthropod borne virus due to globalization and climate change. It targets dendritic cells during infection and leads to production of pro-inflammatory cytokines and chemokines. Several DV non-structural proteins (NS) modulate activation of human dendritic cells. We investigated the effect of DV NS1 on human monocyte-derived dendritic cells (mo-DCs) during dengue infection. NS1 is secreted into the serum of infected individuals where it interacts with various immune mediators and cell types. We purified secreted DV1 NS1 from supernatants of 293T cells that over-express the protein. Upon incubation with mo-DCs, we observed NS1 uptake and enhancement of early DV1 replication. As a consequence, mo-DCs that were pre-exposed to NS1 produced more pro-inflammatory cytokines in response to subsequent DV infection compared to DCs exposed to heat-inactivated NS1 (HNS1). Therefore the presence of exogenous NS1 is able to modulate dengue infection in mo-DCs. PMID:27348054

  3. Pro- and anti-tumour effects of B cells and antibodies in cancer: a comparison of clinical studies and preclinical models.

    Science.gov (United States)

    Guy, Thomas V; Terry, Alexandra M; Bolton, Holly A; Hancock, David G; Shklovskaya, Elena; Fazekas de St. Groth, Barbara

    2016-08-01

    The primary immune role of B cells is to produce antibodies, but they can also influence T cell function via antigen presentation and, in some contexts, immune regulation. Whether their roles in tumour immunity are similar to those in other chronic immune responses such as autoimmunity and chronic infection, where both pro- and anti-inflammatory roles have been described, remains controversial. Many studies have aimed to define the role of B cells in antitumor immune responses, but despite this considerable body of work, it is not yet possible to predict how they will affect immunity to any given tumour. In many human cancers, the presence of tumour-infiltrating B cells and tumour-reactive antibodies correlates with extended patient survival, and this clinical observation is supported by data from some animal models. On the other hand, T cell responses can be adversely affected by B cell production of immunoregulatory cytokines, a phenomenon that has been demonstrated in humans and in animal models. The isotype and concentration of tumour-reactive antibodies may also influence tumour progression. Recruitment of B cells into tumours may directly reflect the subtype and strength of the anti-tumour T cell response. As the response becomes chronic, B cells may attenuate T cell responses in an attempt to decrease host damage, similar to their described role in chronic infection and autoimmunity. Understanding how B cell responses in cancer are related to the effectiveness of the overall anti-tumour response is likely to aid in the development of new therapeutic interventions against cancer.

  4. c-Myc dependent expression of pro-apoptotic Bim renders HER2-overexpressing breast cancer cells dependent on anti-apoptotic Mcl-1

    Directory of Open Access Journals (Sweden)

    Jézéquel Pascal

    2011-09-01

    Full Text Available Abstract Background Anti-apoptotic signals induced downstream of HER2 are known to contribute to the resistance to current treatments of breast cancer cells that overexpress this member of the EGFR family. Whether or not some of these signals are also involved in tumor maintenance by counteracting constitutive death signals is much less understood. To address this, we investigated what role anti- and pro-apoptotic Bcl-2 family members, key regulators of cancer cell survival, might play in the viability of HER2 overexpressing breast cancer cells. Methods We used cell lines as an in vitro model of HER2-overexpressing cells in order to evaluate how anti-apoptotic Bcl-2, Bcl-xL and Mcl-1, and pro-apoptotic Puma and Bim impact on their survival, and to investigate how the constitutive expression of these proteins is regulated. Expression of the proteins of interest was confirmed using lysates from HER2-overexpressing tumors and through analysis of publicly available RNA expression data. Results We show that the depletion of Mcl-1 is sufficient to induce apoptosis in HER2-overexpressing breast cancer cells. This Mcl-1 dependence is due to Bim expression and it directly results from oncogenic signaling, as depletion of the oncoprotein c-Myc, which occupies regions of the Bim promoter as evaluated in ChIP assays, decreases Bim levels and mitigates Mcl-1 dependence. Consistently, a reduction of c-Myc expression by inhibition of mTORC1 activity abrogates occupancy of the Bim promoter by c-Myc, decreases Bim expression and promotes tolerance to Mcl-1 depletion. Western blot analysis confirms that naïve HER2-overexpressing tumors constitutively express detectable levels of Mcl-1 and Bim, while expression data hint on enrichment for Mcl-1 transcripts in these tumors. Conclusions This work establishes that, in HER2-overexpressing tumors, it is necessary, and maybe sufficient, to therapeutically impact on the Mcl-1/Bim balance for efficient induction of

  5. Regulation of the pro-inflammatory cytokine osteopontin by GIP in adipocytes - A role for the transcription factor NFAT and phosphodiesterase 3B

    Energy Technology Data Exchange (ETDEWEB)

    Omar, Bilal [Department of Experimental Medical Sciences, Diabetes, Metabolism and Endocrinology, Biomedical Center, Lund University, Lund (Sweden); Banke, Elin, E-mail: elin.banke@med.lu.se [Department of Experimental Medical Sciences, Diabetes, Metabolism and Endocrinology, Biomedical Center, Lund University, Lund (Sweden); Guirguis, Emilia [Cardiovascular Pulmonary Branch, NHLBI, NIH, Bethesda, MD (United States); Aakesson, Lina [Department of Clinical Sciences, Diabetes and Celiac Disease Unit, Clinical Research Centre, Lund University, Malmoe (Sweden); Manganiello, Vincent [Cardiovascular Pulmonary Branch, NHLBI, NIH, Bethesda, MD (United States); Lyssenko, Valeriya; Groop, Leif [Department of Clinical Sciences, Diabetes and Endocrinology, Clinical Research Centre, Lund University, Malmoe (Sweden); Gomez, Maria F. [Department of Clinical Sciences, Vascular ET Coupling, Clinical Research Centre, Lund University, Malmoe (Sweden); Degerman, Eva [Department of Experimental Medical Sciences, Diabetes, Metabolism and Endocrinology, Biomedical Center, Lund University, Lund (Sweden)

    2012-09-07

    Highlights: Black-Right-Pointing-Pointer GIP stimulates lipogenesis and osteopontin expression in primary adipocytes. Black-Right-Pointing-Pointer GIP-induced osteopontin expression is NFAT-dependent. Black-Right-Pointing-Pointer Osteopontin expression is PDE3-dependent. Black-Right-Pointing-Pointer Osteopontin expression is increased in PDE3B KO mice. -- Abstract: The incretin - glucose-dependent insulinotropic polypeptide (GIP) - and the pro-inflammatory cytokine osteopontin are known to have important roles in the regulation of adipose tissue functions. In this work we show that GIP stimulates lipogenesis and osteopontin expression in primary adipocytes. The GIP-induced increase in osteopontin expression was inhibited by the NFAT (the transcription factor nuclear factor of activated T-cells) inhibitor A-285222. Also, the NFAT kinase glycogen synthase kinase (GSK) 3 was upregulated by GIP. To test whether cAMP might be involved in GIP-mediated effects on osteopontin a number of strategies were used. Thus, the {beta}3-adrenergic receptor agonist CL316,243 stimulated osteopontin expression, an effects which was mimicked by OPC3911, a specific inhibitor of phosphodiesterase 3. Furthermore, treatment of phosphodiesterase 3B knock-out mice with CL316,243 resulted in a dramatic upregulation of osteopontin in adipose tissue which was not the case in wild-type mice. In summary, we delineate mechanisms by which GIP stimulates osteopontin in adipocytes. Given the established link between osteopontin and insulin resistance, our data suggest that GIP by stimulating osteopontin expression, also could promote insulin resistance in adipocytes.

  6. Angiotensin II facilitates fibrogenic effect of TGF-β1 through enhancing the down-regulation of BAMBI caused by LPS: a new pro-fibrotic mechanism of angiotensin II.

    Directory of Open Access Journals (Sweden)

    Yu-Sheng Li

    Full Text Available Angiotensin II has progressively been considered to play an important role in the development of liver fibrosis, although the mechanism isn't fully understood. The aim of this study was to investigate a possible pro-fibrotic mechanism, by which angiotensin II would enhance the pro-fibrotic effect of transforming growth factor beta 1 (TGF-β1 through up-regulation of toll-like receptor 4 (TLR4 and enhancing down-regulation of TGF-β1 inhibitory pseudo-receptor-BAMBI caused by LPS in hepatic stellate cells (HSCs. Firstly, the synergistic effects of angiotensin II, TGF-β1 and LPS on collagen 1α production were confirmed in vitro by ELISA, in which angiotensin II, LPS and TGF-β1 were treated sequentially, and in vivo by immunofluorescence, in the experiments single or multiple intra-peritoneally implanted osmotic mini-pumps administrating angiotensin II or LPS combined with intra-peritoneal injections of TGF-β1 were used. We also found that only LPS and TGF-β1 weren't enough to induce obvious fibrogenesis without angiotensin II. Secondly, to identify the reason of why angiotensin II is so important, the minute level of TLR4 in activated HSCs - T6 and primary quiescent HSCs of rat, up-regulation of TLR4 by angiotensin II and blockage by different angiotensin II receptor type 1 (AT1 blockers in HSCs were assayed by western blotting in vitro and immunofluorescence in vivo. Finally, BAMBI expression level, which is regulated by LPS-TLR4 pathway, was detected by qRT-PCR and results showed angiotensin II enhanced the down-regulation of BAMBI mRNA caused by LPS in vitro and in vivo, and TLR4 neutralization antibody blocked this interactive effect. These data demonstrated that angiotensin II enhances LPS-TLR4 pathway signaling and further down-regulates expression of BAMBI through up-regulation of TLR4, which results in facilitation of pro-fibrotic activity of TGF-β1. Angiotensin II, LPS and TGF-β1 act synergistically during hepatic fibrogenesis

  7. Advanced glycation end products promote differentiation of CD4(+) T helper cells toward pro-inflammatory response.

    Science.gov (United States)

    Han, Xiao-qun; Gong, Zuo-jiong; Xu, San-qing; Li, Xun; Wang, Li-kun; Wu, Shi-min; Wu, Jian-hong; Yang, Hua-fen

    2014-02-01

    This study investigated the effect of advanced glycation end products (AGEs) on differentiation of naïve CD4(+) T cells and the role of the receptor of AGEs (RAGE) and peroxisome proliferator-activated receptors (PPARs) activity in the process in order to gain insight into the mechanism of immunological disorders in diabetes. AGEs were prepared by the reaction of bovine serum albumin (BSA) with glucose. Human naïve CD4(+) T cells, enriched from blood of healthy adult volunteers with negative selection assay, were cultured in vitro and treated with various agents including AGEs, BSA, high glucose, PGJ2 and PD68235 for indicated time. In short hairpin (sh) RNA knock-down experiment, naïve CD4(+) T cells were transduced with media containing shRNA-lentivirus generated from lentiviral packaging cell line, Lent-X(TM) 293 T cells. Surface and intracellular cytokine stainings were used for examination of CD4(+) T cell phenotypes, and real-time PCR and Western blotting for detection of transcription factor mRNA and protein expression, respectively. The suppressive function of regulatory T (Treg) cells was determined by a [(3)H]-thymidine incorporation assay. The results showed that AGEs induced higher pro-inflammatory Th1/Th17 cells differentiated from naïve CD4(+) T cells than the controls, whereas did not affect anti-inflammatory Treg cells. However, AGEs eliminated suppressive function of Treg cells. In addition, AGEs increased RAGE mRNA expression in naïve CD4(+) T cells, and RAGE knock-down by shRNA eliminated the effect of AGEs on the differentiation of CD4(+) T cells and the reduction of suppressive function of Treg cells. Furthermore, AGEs inhibited the mRNA expression of PPARγ, not PPARα PPARγ agonist, PGJ2, inhibited the effect of AGEs on naïve CD4(+) T cell differentiation and reversed the AGE-reduced suppressive function of Treg cells; on the other hand, PPARγ antagonist, PD68235, attenuated the blocking effect of RAGE shRNA on the role of AGEs. It

  8. Tip cells: master regulators of tubulogenesis?

    Science.gov (United States)

    Weavers, Helen; Skaer, Helen

    2014-07-01

    The normal development of an organ depends on the coordinated regulation of multiple cell activities. Focusing on tubulogenesis, we review the role of specialised cells or groups of cells that are selected from within tissue primordia and differentiate at the outgrowing tips or leading edge of developing tubules. Tip or leading cells develop distinctive patterns of gene expression that enable them to act both as sensors and transmitters of intercellular signalling. This enables them to explore the environment, respond to both tissue intrinsic signals and extrinsic cues from surrounding tissues and to regulate the behaviour of their neighbours, including the setting of cell fate, patterning cell division, inducing polarity and promoting cell movement and cell rearrangements by neighbour exchange. Tip cells are also able to transmit mechanical tension to promote tissue remodelling and, by interacting with the extracellular matrix, they can dictate migratory pathways and organ shape. Where separate tubular structures fuse to form networks, as in the airways of insects or the vascular system of vertebrates, specialised fusion tip cells act to interconnect disparate elements of the developing network. Finally, we consider their importance in the maturation of mature physiological function and in the development of disease.

  9. Physiology of cell volume regulation in vertebrates

    DEFF Research Database (Denmark)

    Hoffmann, Else K; Lambert, Ian H; Pedersen, Stine F

    2009-01-01

    cases, activation of volume regulatory osmolyte transport. After acute swelling, cell volume is regulated by the process of regulatory volume decrease (RVD), which involves the activation of KCl cotransport and of channels mediating K(+), Cl(-), and taurine efflux. Conversely, after acute shrinkage...... and their regulation by, e.g., membrane deformation, ionic strength, Ca(2+), protein kinases and phosphatases, cytoskeletal elements, GTP binding proteins, lipid mediators, and reactive oxygen species, upon changes in cell volume. We also discuss the nature of the upstream elements in volume sensing in vertebrate...

  10. Cell shape regulation through mechanosensory feedback control.

    Science.gov (United States)

    Mohan, Krithika; Luo, Tianzhi; Robinson, Douglas N; Iglesias, Pablo A

    2015-08-01

    Cells undergo controlled changes in morphology in response to intracellular and extracellular signals. These changes require a means for sensing and interpreting the signalling cues, for generating the forces that act on the cell's physical material, and a control system to regulate this process. Experiments on Dictyostelium amoebae have shown that force-generating proteins can localize in response to external mechanical perturbations. This mechanosensing, and the ensuing mechanical feedback, plays an important role in minimizing the effect of mechanical disturbances in the course of changes in cell shape, especially during cell division, and likely in other contexts, such as during three-dimensional migration. Owing to the complexity of the feedback system, which couples mechanical and biochemical signals involved in shape regulation, theoretical approaches can guide further investigation by providing insights that are difficult to decipher experimentally. Here, we present a computational model that explains the different mechanosensory and mechanoresponsive behaviours observed in Dictyostelium cells. The model features a multiscale description of myosin II bipolar thick filament assembly that includes cooperative and force-dependent myosin-actin binding, and identifies the feedback mechanisms hidden in the observed mechanoresponsive behaviours of Dictyostelium cells during micropipette aspiration experiments. These feedbacks provide a mechanistic explanation of cellular retraction and hence cell shape regulation. PMID:26224568

  11. Mitochondrial Regulation of Cell Cycle and Proliferation

    OpenAIRE

    Antico Arciuch, Valeria Gabriela; Elguero, María Eugenia; Poderoso, Juan José; Carreras, María Cecilia

    2012-01-01

    Eukaryotic mitochondria resulted from symbiotic incorporation of α-proteobacteria into ancient archaea species. During evolution, mitochondria lost most of the prokaryotic bacterial genes and only conserved a small fraction including those encoding 13 proteins of the respiratory chain. In this process, many functions were transferred to the host cells, but mitochondria gained a central role in the regulation of cell proliferation and apoptosis, and in the modulation of metabolism; accordingly...

  12. Activation of pro-(matrix metalloproteinase-2) (pro-MMP-2) by thrombin is membrane-type-MMP-dependent in human umbilical vein endothelial cells and generates a distinct 63 kDa active species.

    Science.gov (United States)

    Lafleur, M A; Hollenberg, M D; Atkinson, S J; Knäuper, V; Murphy, G; Edwards, D R

    2001-01-01

    Thrombin, a critical enzyme in the coagulation cascade, has also been associated with angiogenesis and activation of the zymogen form of matrix metalloproteinase-2 (MMP-2 or gelatinase-A). We show that thrombin activated pro-MMP-2 in a dose- and time-dependent manner in cultured human umbilical-vein endothelial cells (HUVECs) to generate a catalytically active 63 kDa protein that accumulated as the predominant form in the conditioned medium. This 63 kDa thrombin-activated MMP-2 is distinct from the 62 kDa species found following concanavalin A or PMA stimulated pro-MMP-2 activation. Hirudin and leupeptin blocked thrombin-induced pro-MMP-2 activation, demonstrating that the proteolytic activity of thrombin is essential. However, activation was also dependent upon membrane-type-MMP (MT-MMP) action, since it was blocked by EDTA, o-phenanthroline, hydroxamate metalloproteinase inhibitors, tissue inhibitor of metalloproteinase-2 (TIMP-2) and TIMP-4, but not TIMP-1. Thrombin inefficiently cleaved recombinant 72 kDa pro-MMP-2, but efficiently cleaved the 64 kDa MT-MMP-processed intermediate form in the presence of cells. Thrombin also rapidly (within 1 h) increased cellular MT-MMP activity, and at longer time points (>6 h) it increased expression of MT1-MMP mRNA and protein. Thus signalling via proteinase-activated receptors (PARs) may play a role in thrombin-induced MMP-2 activation, though this does not appear to involve PAR1, PAR2, or PAR4 in HUVECs. These results indicate that in HUVECs the activation of pro-MMP-2 by thrombin involves increased MT-MMP activity and preferential cleavage of the MT-MMP-processed 64 kDa MMP-2 form in the presence of cells. The integration of these proteinase systems in the vascular endothelium may be important during thrombogenesis and tissue remodelling associated with neovascularization. PMID:11415441

  13. Selective accumulation of pro-inflammatory T cells in the intestine contributes to the resistance to autoimmune demyelinating disease.

    Directory of Open Access Journals (Sweden)

    Kerstin Berer

    Full Text Available Myelin-specific, pro-inflammatory TH17 cells are widely regarded as the drivers of experimental autoimmune encephalomyelitis (EAE, an animal model for Multiple sclerosis (MS. The factors, responsible for the generation and maintenance of TH17 cells as well as their participation in the pathogenic cascade leading to the demyelinating disease, have been studied extensively. However, how these harmful autoreactive cells are controlled in vivo remains unclear. By comparing TCR transgenic mice on a disease susceptible and a disease resistant genetic background, we show here that pathogenic TH17 cells are sequestered within the intestine of spontaneous EAE resistant B10.S mice. Disease resistant B10.S mice harbored higher frequencies of TH17 cells in the intestine compared to EAE susceptible SJL/J mice. Moreover, transferred TH17 cells selectively migrated to intestinal lymphoid organs of B10.S mice. The sequestration of TH17 cells in the gut was partially dependent on the gut homing receptor α4β7-mediated adhesion to the intestine. Administration of α4β7 blocking-antibodies increased the peripheral availability of TH17 cells, resulting in increased EAE severity after immunization in B10.S mice. Together, these results support the concept that the intestine is a check-point for controlling pathogenic, organ-specific T cells.

  14. Regulation of cell division in higher plants

    Energy Technology Data Exchange (ETDEWEB)

    Jacobs, T.W.

    1992-01-01

    Cell division is arguably the most fundamental of all developmental processes. In higher plants, mitotic activity is largely confined to foci of patterned cell divisions called meristems. From these perpetually embryonic tissues arise the plant's essential organs of light capture, support, protection and reproduction. Once an adequate understanding of plant cell mitotic regulation is attained, unprecedented opportunities will ensue for analyzing and genetically controlling diverse aspects of development, including plant architecture, leaf shape, plant height, and root depth. The mitotic cycle in a variety of model eukaryotic systems in under the control of a regulatory network of striking evolutionary conservation. Homologues of the yeast cdc2 gene, its catalytic product, p34, and the cyclin regulatory subunits of the MPF complex have emerged as ubiquitous mitotic regulators. We have cloned cdc2-like and cyclin genes from pea. As in other eukaryotic model systems, p34 of Pisum sativum is a subunit of a high molecular weight complex which binds the fission yeast p13 protein and displays histone H1 kinase activity in vitro. Our primary objective in this study is to gain baseline information about the regulation of this higher plant cell division control complex in non-dividing, differentiated cells as well as in synchronous and asynchronous mitotic cells. We are investigating cdc2 and cyclin expression at the levels of protein abundance, protein phosphorylation and quaternary associations.

  15. Epigenetic regulation of the mammalian cell.

    Directory of Open Access Journals (Sweden)

    Keith Baverstock

    Full Text Available BACKGROUND: Understanding how mammalian cells are regulated epigenetically to express phenotype is a priority. The cellular phenotypic transition, induced by ionising radiation, from a normal cell to the genomic instability phenotype, where the ability to replicate the genotype accurately is compromised, illustrates important features of epigenetic regulation. Based on this phenomenon and earlier work we propose a model to describe the mammalian cell as a self assembled open system operating in an environment that includes its genotype, neighbouring cells and beyond. Phenotype is represented by high dimensional attractors, evolutionarily conditioned for stability and robustness and contingent on rules of engagement between gene products encoded in the genetic network. METHODOLOGY/FINDINGS: We describe how this system functions and note the indeterminacy and fluidity of its internal workings which place it in the logical reasoning framework of predicative logic. We find that the hypothesis is supported by evidence from cell and molecular biology. CONCLUSIONS: Epigenetic regulation and memory are fundamentally physical, as opposed to chemical, processes and the transition to genomic instability is an important feature of mammalian cells with probable fundamental relevance to speciation and carcinogenesis. A source of evolutionarily selectable variation, in terms of the rules of engagement between gene products, is seen as more likely to have greater prominence than genetic variation in an evolutionary context. As this epigenetic variation is based on attractor states phenotypic changes are not gradual; a phenotypic transition can involve the changed contribution of several gene products in a single step.

  16. Corticosteroid-Induced MKP-1 Represses Pro-Inflammatory Cytokine Secretion by Enhancing Activity of Tristetraprolin (TTP) in ASM Cells.

    Science.gov (United States)

    Prabhala, Pavan; Bunge, Kristin; Ge, Qi; Ammit, Alaina J

    2016-10-01

    Exaggerated cytokine secretion drives pathogenesis of a number of chronic inflammatory diseases, including asthma. Anti-inflammatory pharmacotherapies, including corticosteroids, are front-line therapies and although they have proven clinical utility, the molecular mechanisms responsible for their actions are not fully understood. The corticosteroid-inducible gene, mitogen-activated protein kinase (MAPK) phosphatase 1 (MKP-1, DUSP1) has emerged as a key molecule responsible for the repressive effects of steroids. MKP-1 is known to deactivate p38 MAPK phosphorylation and can control the expression and activity of the mRNA destabilizing protein-tristetraprolin (TTP). But whether corticosteroid-induced MKP-1 acts via p38 MAPK-mediated modulation of TTP function in a pivotal airway cell type, airway smooth muscle (ASM), was unknown. While pretreatment of ASM cells with the corticosteroid dexamethasone (preventative protocol) is known to reduce ASM synthetic function in vitro, the impact of adding dexamethasone after stimulation (therapeutic protocol) had not been explored. Whether dexamethasone modulates TTP in a p38 MAPK-dependent manner in this cell type was also unknown. We address this herein and utilize an in vitro model of asthmatic inflammation where ASM cells were stimulated with the pro-asthmatic cytokine tumor necrosis factor (TNF) and the impact of adding dexamethasone 1 h after stimulation assessed. IL-6 mRNA expression and protein secretion was significantly repressed by dexamethasone acting in a temporally distinct manner to increase MKP-1, deactivate p38 MAPK, and modulate TTP phosphorylation status. In this way, dexamethasone-induced MKP-1 acts via p38 MAPK to switch on the mRNA destabilizing function of TTP to repress pro-inflammatory cytokine secretion from ASM cells. J. Cell. Physiol. 231: 2153-2158, 2016. © 2016 Wiley Periodicals, Inc. PMID:26825339

  17. Redox regulation in cancer stem cells

    Science.gov (United States)

    Reactive oxygen species (ROS) and ROS-dependent (redox regulation) signaling pathways and transcriptional activities are thought to be critical in stem cell self-renewal and differentiation during growth and organogenesis. Aberrant ROS burst and dysregulation of those ROS-dependent cellular processe...

  18. The evaluation of NT-proCNP, C-reactive protein and serum amyloid A protein concentration in patients with multiple myeloma undergoing stem cell transplantation.

    Science.gov (United States)

    Tomasiuk, Ryszard; Gawroński, Krzysztof; Rzepecki, Piotr; Rabijewski, Michał; Cacko, Marek

    2016-08-01

    The importance of proinflamatory cytokines and acute phase proteins in pathogenesis and progression of MM is well known. However, there are any studies evaluating the role of NT-proCN in management and treatment of MM. The aim of our study was to evaluate the concentration of NT-proCNP and acute phase proteins in patients with MM before and after stem cell transplantation. We involved 40 newly diagnosed MM patients in stage III according to the Durie-Salmon classification and treated with high dose of melphalan (200mg/m2) prior to ASCT. Concentration of NT-proCNP, hs-CRP and SAA were measured before conditioning treatment and every 4days until the 24th day after stem cell infusion. We observed low NT-proCNP levels before conditioning treatment (0.121±0.04pmol/l), the higher in day on ASCT (0.28±0.14pmol/l). Further we showed significant gradual increase concentration of NT-proCNP up to 12days after stem cells infusion (1.07±0.72pmol/l). The kinetics of hs-CRP and SAA levels were similar to NT-proCNP. We showed positive correlation between NT-proCNP levels and absolute neutrophil and platelets count in patients after ASCT. NT-proCNP can be useful parameter to assess effectiveness of treatment and monitoring of hematopoetic recovery time in patients with MM after stem cell transplantations. PMID:27322507

  19. Topological regulation of lipid balance in cells.

    Science.gov (United States)

    Drin, Guillaume

    2014-01-01

    Lipids are unevenly distributed within and between cell membranes, thus defining organelle identity. Such distribution relies on local metabolic branches and mechanisms that move lipids. These processes are regulated by feedback mechanisms that decipher topographical information in organelle membranes and then regulate lipid levels or flows. In the endoplasmic reticulum, the major lipid source, transcriptional regulators and enzymes sense changes in membrane features to modulate lipid production. At the Golgi apparatus, lipid-synthesizing, lipid-flippase, and lipid-transport proteins (LTPs) collaborate to control lipid balance and distribution within the membrane to guarantee remodeling processes crucial for vesicular trafficking. Open questions exist regarding LTPs, which are thought to be lipid sensors that regulate lipid synthesis or carriers that transfer lipids between organelles across long distances or in contact sites. A novel model is that LTPs, by exchanging two different lipids, exploit one lipid gradient between two distinct membranes to build a second lipid gradient. PMID:24606148

  20. Pro-apoptosis Effect of Survivin T34A Mutant on Cancer Cells in Vitro and Vivo

    Institute of Scientific and Technical Information of China (English)

    SUN Jing; MA Lan; WU Hao; LIU Lian-ke

    2014-01-01

    Objective:To explore the pro-apoptosis effect of Survivin T34A mutant on cancer cells in vitro and vivo. Methods: After highly-metastasized breast cancer cells (4T1 cells) were transfected with Survivin T34A plasmid and wild survivin plasmid, cell proliferation was analyzed using tetrazolium blue (MTT) assay and apoptosis was detected with lfow cytometry. In animal experiments, mice were vaccinated subcutaneously with 4T1 cells and treated with T34A plasmid and wild survivin plasmid. The tumor volume and weight, wet weight of the lung, number of pulmonary metastasis nodule were measured. H&E staining and TUNEL detection of tumor apoptosis were performed after mice were executed. Results: The cell survival rate was signiifcantly decreased (P<0.01) and apoptotic rate increased (P<0.01) after treatment with Survivin T34A plasmid in vitro. In vivo, 4 days after treatment, tumor volume was signiifcantly smaller, mean tumor weight and mean wet weight of the lung were obviously lighter, and pulmonary metastasis nodule was evidently fewer (P<0.05). The apoptotic cells and large areas of necrosis were observed, and apoptotic index was increased (P<0.05). Conclusion:Survivin T34A can induce the apoptosis of 4T1 cells with specificity and may become a new approach to breast cancer.

  1. Epigallocatechin-3-gallate (EGCG) as a pro-osteogenic agent to enhance osteogenic differentiation of mesenchymal stem cells from human bone marrow: an in vitro study.

    Science.gov (United States)

    Jin, Pan; Wu, Huayu; Xu, Guojie; Zheng, Li; Zhao, Jinmin

    2014-05-01

    The proliferation and osteogenic capacity of mesenchymal stem cells (MSCs) needs to be improved for their use in cell-based therapy for osteoporosis. (-)-Epigallocatechin-3-gallate (EGCG), one of the green tea catechins, has been widely investigated in studies of osteoblasts and osteoclasts. However, no consensus on its role as an osteogenic inducer has been reached, possibly because of the various types of cell lines examined and the range of concentrations of EGCG used. In this study, the osteogenic effects of EGCG are studied in primary human bone-marrow-derived MSCs (hBMSCs) by detecting cell proliferation, alkaline phosphatase (ALP) activity and the expression of relevant osteogenic markers. Our results show that EGCG has a strong stimulatory effect on hBMSCs developing towards the osteogenic lineage, especially at a concentration of 5 μM, as evidenced by an increased ALP activity, the up-regulated expression of osteogenic genes and the formation of bone-like nodules. Further exploration has indicated that EGCG directes osteogenic differentiation via the continuous up-regulation of Runx2. The underlying mechanism might involve EGCG affects on osteogenic differentiation through the modulation of bone morphogenetic protein-2 expression. EGCG has also been found to promote the proliferation of hBMSCs in a dose-dependent manner. This might be associated with its antioxidative effect leading to favorable amounts of reactive oxygen species in the cellular environment. Our study thus indicates that EGCG can be used as a pro-osteogenic agent for the stem-cell-based therapy of osteoporosis.

  2. Autophagic regulation of smooth muscle cell biology

    Science.gov (United States)

    Salabei, Joshua K.; Hill, Bradford G.

    2014-01-01

    Autophagy regulates the metabolism, survival, and function of numerous cell types, including those comprising the cardiovascular system. In the vasculature, changes in autophagy have been documented in atherosclerotic and restenotic lesions and in hypertensive vessels. The biology of vascular smooth muscle cells appears particularly sensitive to changes in the autophagic program. Recent evidence indicates that stimuli or stressors evoked during the course of vascular disease can regulate autophagic activity, resulting in modulation of VSMC phenotype and viability. In particular, certain growth factors and cytokines, oxygen tension, and pharmacological drugs have been shown to trigger autophagy in smooth muscle cells. Importantly, each of these stimuli has a redox component, typically associated with changes in the abundance of reactive oxygen, nitrogen, or lipid species. Collective findings support the hypothesis that autophagy plays a critical role in vascular remodeling by regulating smooth muscle cell phenotype transitions and by influencing the cellular response to stress. In this graphical review, we summarize current knowledge on the role of autophagy in the biology of the smooth muscle cell in (patho)physiology. PMID:25544597

  3. Autophagic regulation of smooth muscle cell biology

    Directory of Open Access Journals (Sweden)

    Joshua K. Salabei

    2015-04-01

    Full Text Available Autophagy regulates the metabolism, survival, and function of numerous cell types, including those comprising the cardiovascular system. In the vasculature, changes in autophagy have been documented in atherosclerotic and restenotic lesions and in hypertensive vessels. The biology of vascular smooth muscle cells appears particularly sensitive to changes in the autophagic program. Recent evidence indicates that stimuli or stressors evoked during the course of vascular disease can regulate autophagic activity, resulting in modulation of VSMC phenotype and viability. In particular, certain growth factors and cytokines, oxygen tension, and pharmacological drugs have been shown to trigger autophagy in smooth muscle cells. Importantly, each of these stimuli has a redox component, typically associated with changes in the abundance of reactive oxygen, nitrogen, or lipid species. Collective findings support the hypothesis that autophagy plays a critical role in vascular remodeling by regulating smooth muscle cell phenotype transitions and by influencing the cellular response to stress. In this graphical review, we summarize current knowledge on the role of autophagy in the biology of the smooth muscle cell in (pathophysiology.

  4. The regulation of apoptotic cell death

    Directory of Open Access Journals (Sweden)

    G.P. Amarante-Mendes

    1999-09-01

    Full Text Available Apoptosis is a fundamental biological phenomenon in which the death of a cell is genetically and biochemically regulated. Different molecules are involved in the regulation of the apoptotic process. Death receptors, coupled to distinct members of the caspases as well as other adapter molecules, are involved in the initiation of the stress signals (The Indictment. Members of the Bcl-2 family control at the mitochondrial level the decision between life and death (The Judgement. The effector caspases are responsible for all morphological and biochemical changes related to apoptosis including the "eat-me" signals perceived by phagocytes and neighboring cells (The Execution. Finally, apoptosis would have little biological significance without the recognition and removal of the dying cells (The Burial.

  5. The regulation of apoptotic cell death

    Directory of Open Access Journals (Sweden)

    Amarante-Mendes G.P.

    1999-01-01

    Full Text Available Apoptosis is a fundamental biological phenomenon in which the death of a cell is genetically and biochemically regulated. Different molecules are involved in the regulation of the apoptotic process. Death receptors, coupled to distinct members of the caspases as well as other adapter molecules, are involved in the initiation of the stress signals (The Indictment. Members of the Bcl-2 family control at the mitochondrial level the decision between life and death (The Judgement. The effector caspases are responsible for all morphological and biochemical changes related to apoptosis including the "eat-me" signals perceived by phagocytes and neighboring cells (The Execution. Finally, apoptosis would have little biological significance without the recognition and removal of the dying cells (The Burial.

  6. Mast cells as regulators of T cell responses

    Directory of Open Access Journals (Sweden)

    Silvia eBulfone-Paus

    2015-08-01

    Full Text Available Mast cells are recognized to participate in the regulation of innate and adaptive immune responses. Owing to their strategic location at the host-environment interface they control tissue homeostasis and are key cells for starting early host defence against intruders. Upon degranulation induced, e.g. by immunoglobulin E (IgE and allergen-mediated engagement of the high-affinity IgE receptor, complement or certain neuropeptide receptors, mast cells release a wide variety of preformed and newly synthesized products including proteases, lipid mediators, and many cytokines, chemokines, and growth factors. Interestingly, increasing evidence suggests a regulatory role for mast cells in inflammatory diseases via the regulation of T cell activities. Furthermore, rather than only serving as effector cells, mast cells are now recognized to induce T cell activation, recruitment, proliferation and cytokine secretion in an antigen-dependent manner and to impact on regulatory T cells. This review synthesizes recent developments in mast cell-T cell interactions, discusses their biological and clinical relevance, and explores recent controversies in this field of mast cell research.

  7. X-linked inhibitor of apoptosis regulates T cell effector function

    DEFF Research Database (Denmark)

    Zehntner, Simone P; Bourbonnière, Lyne; Moore, Craig S;

    2007-01-01

    To understand how the balance between pro- and anti-apoptotic signals influences effector function in the immune system, we studied the X-linked inhibitor of apoptosis (XIAP), an endogenous regulator of cellular apoptosis. Real-time PCR showed increased XIAP expression in blood of mice...... and oligodendrocytes were not affected; neither did apoptosis increase in liver, where XIAP knockdown also occurred. ASO-XIAP increased susceptibility of T cells to activation-induced apoptosis in vitro. Our results identify XIAP as a critical controller of apoptotic susceptibility of effector T cell function...

  8. GATA-3 promotes T-cell specification by repressing B-cell potential in pro-T cells in mice

    NARCIS (Netherlands)

    M.E. García (Marcos); R.G.J. Klein Wolterink (Roel); F. Lemâitre (Fabrice); C. Le Goff (Carine); M. Hasan (Milena); R.W. Hendriks (Rudi); A. Cumano (Ana); J.P. di Santo (James)

    2013-01-01

    textabstractTranscription factors orchestrate T-lineage differentiation in the thymus. One critical checkpoint involves Notch1 signaling that instructs T-cell commitment at the expense of the B-lineage program. While GATA-3 is required for T-cell specification, its mechanism of action is poorly unde

  9. Autophagy in immune cell regulation and dysregulation.

    Science.gov (United States)

    Chaturvedi, Akanksha; Pierce, Susan K

    2009-09-01

    Autophagy is an ancient pathway required for cell and tissue homeostasis and differentiation. Initially thought to be a process leading to cell death, autophagy is currently viewed as a beneficial catabolic process that promotes cell survival under starvation conditions by sequestering components of the cytoplasm, including misfolded proteins, protein aggregates, and damaged organelles, and targeting them for lysosome-mediated degradation. In this way, autophagy plays a role in maintaining a balance between degradation and recycling of cellular material. The importance of autophagy is underscored by the fact that malfunctioning of this pathway results in neurodegeneration, cancer, susceptibility to microbial infection, and premature aging. Autophagy occurs in almost all cell types, including immune cells. Recent advances in the field suggest that autophagy plays a central role in regulating the immune system at multiple levels. In this review, we focus on recent developments in the area of autophagy-mediated modulation of immune responses. PMID:19671376

  10. Co-stimulation-induced release of pro-inflammatory cytokine interleukin-8 by allergen-specific T cells.

    Science.gov (United States)

    Spinozzi, F; Agea, E; Piattoni, S; Bistoni, O; Grignani, F; Bertotto, A

    1996-07-01

    Chemokines, which include interleukin (IL)-8, are a family of pro-inflammatory molecules with potent chemoattractant activity on neutrophils, as well as other cell types. IL-8 can be recovered from many inflammatory sites. To test the hypothesis that Th2-type allergen-specific T cells, known to be the main cell type governing the allergic inflammation, are a source of IL-8 and to investigate whether IL-8 release is influenced by the nature of the in vitro mitogenic or co-mitogenic stimulation, cypress-specific T-cell clones (TCC) were generated from five allergic subjects during in vitro seasonal exposure to the allergen. Purified cypress extract was produced directly from freshly collected pollen and used for in vitro stimulation of PBMC bulk cultures. After 5 days priming and a further 7 day period of IL-2-driven cell expansion, monoclonal antibodies to CD3, CD2 and CD28 were adopted for in vitro restimulation of allergen-specific cell lines or, subsequently, secondary established TCC. The induction of apoptosis was detected by propidium iodide (PI) cytofluorimetric assay. Basal and co-stimulation-induced IL-8 production was measured by an ELISA method. Both cypress-specific T-cell lines and TCC secreted appreciable amounts of IL-8. By cross-linking T-cell lines or Th2 CD4+ TCC with CD3, CD2 or CD28 MoAbs, the authors observed a great stimulation-induced IL-8 secretion, preferentially after CD2 or combined CD2/CD28 stimulation. In addition, CD4+ clones released large amounts of IL-8 into culture supernatants after CD2 stimulation while undergoing programmed cell death (30-40% hypodiploid DNA profile of PI-stained cells). In contrast, CD3 crosslinking was unable to determine the release of IL-8 or the induction of apoptosis. Taken together, these results suggest that incomplete TcR engagement by allergen may lead to the secretion of pro-inflammatory cytokines with a contemporary induction of apoptosis in a significant number of target cells. This phenomenon may

  11. Self-Limited versus Delayed Resolution of Acute Inflammation: Temporal Regulation of Pro-Resolving Mediators and MicroRNA

    OpenAIRE

    Gabrielle Fredman; Yongsheng Li; Jesmond Dalli; Nan Chiang; Serhan, Charles N.

    2012-01-01

    Mechanisms underlying delays in resolution programs of inflammation are of interest for many diseases. Here, we addressed delayed resolution of inflammation and identified specific microRNA (miR)-metabolipidomic signatures. Delayed resolution initiated by high-dose challenges decreased miR-219-5p expression along with increased leukotriene B4 (5-fold) and decreased (~3-fold) specialized pro-resolving mediators, e.g. protectin D1. Resolvin (Rv)E1 and RvD1 (1 nM) reduced miR-219-5p in human mac...

  12. Nuclear myosin I regulates cell membrane tension

    Science.gov (United States)

    Venit, Tomáš; Kalendová, Alžběta; Petr, Martin; Dzijak, Rastislav; Pastorek, Lukáš; Rohožková, Jana; Malohlava, Jakub; Hozák, Pavel

    2016-01-01

    Plasma membrane tension is an important feature that determines the cell shape and influences processes such as cell motility, spreading, endocytosis and exocytosis. Unconventional class 1 myosins are potent regulators of plasma membrane tension because they physically link the plasma membrane with adjacent cytoskeleton. We identified nuclear myosin 1 (NM1) - a putative nuclear isoform of myosin 1c (Myo1c) - as a new player in the field. Although having specific nuclear functions, NM1 localizes predominantly to the plasma membrane. Deletion of NM1 causes more than a 50% increase in the elasticity of the plasma membrane around the actin cytoskeleton as measured by atomic force microscopy. This higher elasticity of NM1 knock-out cells leads to 25% higher resistance to short-term hypotonic environment and rapid cell swelling. In contrast, overexpression of NM1 in wild type cells leads to an additional 30% reduction of their survival. We have shown that NM1 has a direct functional role in the cytoplasm as a dynamic linker between the cell membrane and the underlying cytoskeleton, regulating the degree of effective plasma membrane tension. PMID:27480647

  13. Ulk4 Regulates Neural Stem Cell Pool.

    Science.gov (United States)

    Liu, Min; Guan, Zhenlong; Shen, Qin; Flinter, Frances; Domínguez, Laura; Ahn, Joo Wook; Collier, David A; O'Brien, Timothy; Shen, Sanbing

    2016-09-01

    The size of neural stem cell (NSC) pool at birth determines the starting point of adult neurogenesis. Aberrant neurogenesis is associated with major mental illness, in which ULK4 is proposed as a rare risk factor. Little is known about factors regulating the NSC pool, or function of the ULK4. Here, we showed that Ulk4(tm1a/tm1a) mice displayed a dramatically reduced NSC pool at birth. Ulk4 was expressed in a cell cycle-dependent manner and peaked in G2/M phases. Targeted disruption of the Ulk4 perturbed mid-neurogenesis and significantly reduced cerebral cortex in postnatal mice. Pathway analyses of dysregulated genes in Ulk4(tm1a/tm1a) mice revealed Ulk4 as a key regulator of cell cycle and NSC proliferation, partially through regulation of the Wnt signaling. In addition, we identified hemizygous deletion of ULK4 gene in 1.2/1,000 patients with pleiotropic symptoms including severe language delay and learning difficulties. ULK4, therefore, may significantly contribute to neurodevelopmental, neuropsychiatric, and neurodegenerative disorders. Stem Cells 2016;34:2318-2331.

  14. Anti Proliferative and Pro Apoptotic Effects of Flavonoid Quercetin Are Mediated by CB1 Receptor in Human Colon Cancer Cell Lines.

    Science.gov (United States)

    Refolo, Maria Grazia; D'Alessandro, Rosalba; Malerba, Natascia; Laezza, Chiara; Bifulco, Maurizio; Messa, Caterina; Caruso, Maria Gabriella; Notarnicola, Maria; Tutino, Valeria

    2015-12-01

    Quercetin, the major constituent of flavonoid and widely present in fruits and vegetables, is an attractive compound for cancer prevention due to its beneficial anti proliferative effects, showing a crucial role in the regulation of apoptosis and cell cycle signaling. In vitro studies have demonstrated that quercetin specifically influences colon cancer cell proliferation. Our experiments, using human colon adenocarcinoma cells, confirmed the anti proliferative effect of quercetin and gave intriguing new insight in to the knowledge of the mechanisms involved. We observed a significant increase in the expression of the endocannabinoids receptor (CB1-R) after quercetin treatment. CB1-R can be considered an estrogen responsive receptor and quercetin, having a structure similar to that of the estrogens, can interact with CB1-R leading to the regulation of cell growth. In order to clarify the contribution of the CB1-R to the quercetin action, we investigated some of the principal molecular pathways that are inhibited or activated by this natural compound. In particular we detected the inhibition of the major survival signals like the PI3K/Akt/mTOR and an induction of the pro apoptotic JNK/JUN pathways. Interestingly, the metabolism of β-catenin was modified by flavonoid both directly and through activated CB1-R. In all the experiments done, the quercetin action has proven to be reinforced by anandamide (Met-F-AEA), a CB1-R agonist, and partially counteracted by SR141716, a CB1-R antagonist. These findings open new perspectives for anticancer therapeutic strategies.

  15. Regulation of satellite cell function in sarcopenia

    Directory of Open Access Journals (Sweden)

    Stephen E Alway

    2014-09-01

    Full Text Available The mechanisms contributing to sarcopenia include reduced satellite cell (myogenic stem cell function that is impacted by the environment (niche of these cells. Satellite cell function is affected by oxidative stress, which is elevated in aged muscles, and this along with changes in largely unknown systemic factors, likely contribute to the manner in which satellite cells respond to stressors such as exercise, disuse or rehabilitation in sarcopenic muscles. Nutritional intervention provides one therapeutic strategy to improve the satellite cell niche and systemic factors, with the goal of improving satellite cell function in aging muscles. Although many elderly persons consume various nutraceuticals with the hope of improving health, most of these compounds have not been thoroughly tested, and the impacts that they might have on sarcopenia, and satellite cell function are not clear. This review discusses data pertaining to the satellite cell responses and function in aging skeletal muscle, and the impact that three compounds: resveratrol, green tea catechins and β-Hydroxy-β-methylbutyrate have on regulating satellite cell function and therefore contributing to reducing sarcopenia or improving muscle mass after disuse in aging. The data suggest that these nutraceutical compounds improve satellite cell function during rehabilitative loading in animal models of aging after disuse (i.e., muscle regeneration. While these compounds have not been rigorously tested in humans, the data from animal models of aging provide a strong basis for conducting additional focused work to determine if these or other nutraceuticals can offset the muscle losses, or improve regeneration in sarcopenic muscles of older humans via improving satellite cell function.

  16. Regulation of satellite cell function in sarcopenia.

    Science.gov (United States)

    Alway, Stephen E; Myers, Matthew J; Mohamed, Junaith S

    2014-01-01

    The mechanisms contributing to sarcopenia include reduced satellite cell (myogenic stem cell) function that is impacted by the environment (niche) of these cells. Satellite cell function is affected by oxidative stress, which is elevated in aged muscles, and this along with changes in largely unknown systemic factors, likely contribute to the manner in which satellite cells respond to stressors such as exercise, disuse, or rehabilitation in sarcopenic muscles. Nutritional intervention provides one therapeutic strategy to improve the satellite cell niche and systemic factors, with the goal of improving satellite cell function in aging muscles. Although many elderly persons consume various nutraceuticals with the hope of improving health, most of these compounds have not been thoroughly tested, and the impacts that they might have on sarcopenia and satellite cell function are not clear. This review discusses data pertaining to the satellite cell responses and function in aging skeletal muscle, and the impact that three compounds: resveratrol, green tea catechins, and β-Hydroxy-β-methylbutyrate have on regulating satellite cell function and therefore contributing to reducing sarcopenia or improving muscle mass after disuse in aging. The data suggest that these nutraceutical compounds improve satellite cell function during rehabilitative loading in animal models of aging after disuse (i.e., muscle regeneration). While these compounds have not been rigorously tested in humans, the data from animal models of aging provide a strong basis for conducting additional focused work to determine if these or other nutraceuticals can offset the muscle losses, or improve regeneration in sarcopenic muscles of older humans via improving satellite cell function. PMID:25295003

  17. Activation of pro-urokinase and plasminogen on human sarcoma cells

    DEFF Research Database (Denmark)

    Stephens, R W; Pöllänen, J; Tapiovaara, H;

    1989-01-01

    from the cells with tranexamic acid, an analogue of lysine. The bound plasmin was the result of plasminogen activation on the cell surface; plasmin activity was not taken up onto cells after deliberate addition of plasmin to the serum-containing medium. The cell surface plasmin formation was inhibited...

  18. Targeting cell cycle regulators in hematologic malignancies

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    Eiman eAleem

    2015-04-01

    Full Text Available Hematologic malignancies represent the fourth most frequently diagnosed cancer in economically developed countries. In hematologic malignancies normal hematopoiesis is interrupted by uncontrolled growth of a genetically altered stem or progenitor cell (HSPC that maintains its ability of self-renewal. Cyclin-dependent kinases (CDKs not only regulate the mammalian cell cycle, but also influence other vital cellular processes, such as stem cell renewal, differentiation, transcription, epigenetic regulation, apoptosis, and DNA repair. Chromosomal translocations, amplification, overexpression and altered CDK activities have been described in different types of human cancer, which have made them attractive targets for pharmacological inhibition. Mouse models deficient for one or more CDKs have significantly contributed to our current understanding of the physiological functions of CDKs, as well as their roles in human cancer. The present review focuses on selected cell cycle kinases with recent emerging key functions in hematopoiesis and in hematopoietic malignancies, such as CDK6 and its role in MLL-rearranged leukemia and acute lymphocytic leukemia, CDK1 and its regulator WEE-1 in acute myeloid leukemia, and cyclin C/CDK8/CDK19 complexes in T-cell acute lymphocytic leukemia. The knowledge gained from gene knockout experiments in mice of these kinases is also summarized. An overview of compounds targeting these kinases, which are currently in clinical development in various solid tumors and hematopoietic malignances, is presented. These include the CDK4/CDK6 inhibitors (palbociclib, LEE011, LY2835219, pan-CDK inhibitors that target CDK1 (dinaciclib, flavopiridol, AT7519, TG02, P276-00, terampeprocol and RGB 286638 as well as the WEE-1 kinase inhibitor, MK-1775. The advantage of combination therapy of cell cycle inhibitors with conventional chemotherapeutic agents used in the treatment of AML, such as cytarabine, is discussed.

  19. Flavonoids: from cell cycle regulation to biotechnology.

    Science.gov (United States)

    Woo, Ho-Hyung; Jeong, Byeong Ryong; Hawes, Martha C

    2005-03-01

    Flavonoids have been proposed to play diverse roles in plant growth and development, including defense, symbiosis, pollen development and male fertility, polar auxin transport, and protection against ultraviolet radiation. Recently, a new role in cell cycle regulation has emerged. Genetic alteration of glucuronide metabolism by altered expression of a Pisum sativum UDP-glucuronosyltransferase (PsUGT1) results in an altered cell cycle in pea, alfalfa, and Arabidopsis. In alfalfa, altered expression of PsUGT1 results in accumulation of a flavonoid-like compound that suppresses growth of cultured cells. The results are consistent with the hypothesis that PsUGT1 functions by controlling cellular levels of a factor controlling cell cycle (FCC). PMID:15834800

  20. Bar represses dPax2 and decapentaplegic to regulate cell fate and morphogenetic cell death in Drosophila eye.

    Directory of Open Access Journals (Sweden)

    Jongkyun Kang

    Full Text Available The coordinated regulation of cell fate and cell survival is crucial for normal pattern formation in developing organisms. In Drosophila compound eye development, crystalline arrays of hexagonal ommatidia are established by precise assembly of diverse cell types, including the photoreceptor cells, cone cells and interommatidial (IOM pigment cells. The molecular basis for controlling the number of cone and IOM pigment cells during ommatidial pattern formation is not well understood. Here we present evidence that BarH1 and BarH2 homeobox genes are essential for eye patterning by inhibiting excess cone cell differentiation and promoting programmed death of IOM cells. Specifically, we show that loss of Bar from the undifferentiated retinal precursor cells leads to ectopic expression of Prospero and dPax2, two transcription factors essential for cone cell specification, resulting in excess cone cell differentiation. We also show that loss of Bar causes ectopic expression of the TGFβ homolog Decapentaplegic (Dpp posterior to the morphogenetic furrow in the larval eye imaginal disc. The ectopic Dpp expression is not responsible for the formation of excess cone cells in Bar loss-of-function mutant eyes. Instead, it causes reduction in IOM cell death in the pupal stage by antagonizing the function of pro-apoptotic gene reaper. Taken together, this study suggests a novel regulatory mechanism in the control of developmental cell death in which the repression of Dpp by Bar in larval eye disc is essential for IOM cell death in pupal retina.

  1. Auxin regulation of cell polarity in plants.

    Science.gov (United States)

    Pan, Xue; Chen, Jisheng; Yang, Zhenbiao

    2015-12-01

    Auxin is well known to control pattern formation and directional growth at the organ/tissue levels via the nuclear TIR1/AFB receptor-mediated transcriptional responses. Recent studies have expanded the arena of auxin actions as a trigger or key regulator of cell polarization and morphogenesis. These actions require non-transcriptional responses such as changes in the cytoskeleton and vesicular trafficking, which are commonly regulated by ROP/Rac GTPase-dependent pathways. These findings beg for the question about the nature of auxin receptors that regulate these responses and renew the interest in ABP1 as a cell surface auxin receptor, including the work showing auxin-binding protein 1 (ABP1) interacts with the extracellular domain of the transmembrane kinase (TMK) receptor-like kinases in an auxin-dependent manner, as well as the debate on this auxin binding protein discovered about 40 years ago. This review highlights recent work on the non-transcriptional auxin signaling mechanisms underscoring cell polarity and shape formation in plants. PMID:26599954

  2. Homeobox A7 increases cell proliferation by up-regulation of epidermal growth factor receptor expression in human granulosa cells

    Directory of Open Access Journals (Sweden)

    Yanase Toshihiko

    2010-06-01

    Full Text Available Abstract Background Homeobox (HOX genes encode transcription factors, which regulate cell proliferation, differentiation, adhesion, and migration. The deregulation of HOX genes is frequently associated with human reproductive system disorders. However, knowledge regarding the role of HOX genes in human granulosa cells is limited. Methods To determine the role of HOXA7 in the regulation and associated mechanisms of cell proliferation in human granulosa cells, HOXA7 and epidermal growth factor receptor (EGFR expressions were examined in primary granulosa cells (hGCs, an immortalized human granulosa cell line, SVOG, and a granulosa tumor cell line, KGN, by real-time PCR and Western blotting. To manipulate the expression of HOXA7, the HOXA7 specific siRNA was used to knockdown HOXA7 in KGN. Conversely, HOXA7 was overexpressed in SVOG by transfection with the pcDNA3.1-HOAX7 vector. Cell proliferation was measured by the MTT assay. Results Our results show that HOXA7 and EGFR were overexpressed in KGN cells compared to hGCs and SVOG cells. Knockdown of HOXA7 in KGN cells significantly decreased cell proliferation and EGFR expression. Overexpression of HOXA7 in SVOG cells significantly promoted cell growth and EGFR expression. Moreover, the EGF-induced KGN proliferation was abrogated, and the activation of downstream signaling was diminished when HOXA7 was knocked down. Overexpression of HOXA7 in SVOG cells had an opposite effect. Conclusions Our present study reveals a novel mechanistic role for HOXA7 in modulating granulosa cell proliferation via the regulation of EGFR. This finding contributes to the knowledge of the pro-proliferation effect of HOXA7 in granulosa cell growth and differentiation.

  3. Elevated level of pro inflammatory cytokine and chemokine expression in chicken bone marrow and monocyte derived dendritic cells following LPS induced maturation.

    Science.gov (United States)

    Kalaiyarasu, Semmannan; Bhatia, Sandeep; Mishra, Niranjan; Sood, Richa; Kumar, Manoj; SenthilKumar, D; Bhat, Sushant; Dass Prakash, M

    2016-09-01

    The study was designed to characterize and compare chicken bone marrow and peripheral blood monocyte derived dendritic cells (chBM-DC and chMoDC) and to evaluate inflammatory cytokine and chemokine alterations in response upon LPS stimulation. Typical morphology was observed in DCs from 48h of culture using recombinant chicken GM-CSF and IL-4. Maturation of DCs with LPS (1μg/ml) showed significant up regulation of mRNA of surface markers (CD40, CD80, CD83, CD86, MHC-II and DC-LAMP (CD208)), pro-inflammatory cytokines (IL-1β, IL-6, TNF-α (LITAF)), iNOS, chemokine CXCli2 and TLRs4 and 15. Basal level of TLR1 mRNA expression was higher followed by TLR15 in both DCs irrespective of their origin. Expression of iNOS and CXCLi2 mRNA in mature DCs of both origins were higher than other surface molecules and cytokines studied. Hence, its level of expression can also be used as an additional maturation marker for LPS induced chicken dendritic cell maturation along with CD83 and CD40. LPS matured DCs of both origins upregulated IL-12 and IFN-γ. Based on CD40 and CD83 mRNA expression, it was observed that LPS induced the maturation in both DCs, but chMoDCs responded better in expression of surface markers and inflammatory mediator genes. PMID:27344111

  4. Wear particle-mediated expressions of pro-inflammatory cytokines,NF-κB and RANK were impacted by lanthanum chloride in RAW264.7 cells

    Institute of Scientific and Technical Information of China (English)

    DAI Min; JIANG Chuan; LIU Xiang; LI Zhe; CHENG Xigao; ZOU Yang; NIE Tao

    2013-01-01

    To explore the impact of different concentrations of lanthanum chloride (LaCl3) on critical components of wear particle-mediated signaling pathways in inflammation and osteoclastogenesis,RAW264.7 cells were naturally divided into eight groups and analyzed by CCK-8 assay,flow cytometry,ELISA,RT-PCR and western blot after treatments.The results showed that three concentrations of LaCl3 had no influence on viability of RAW264.7 cells and down-regulated receptor activator of nuclear factor κB (RANK) instead of macrophage colony-stimulating factor receptor (M-CSFR).Additionally,2.5 and 10 μmol/L LaCl3 could signifi-cantly inhibit gene and protein levels of pro-inflammatory cytokines (tumor necrosis factor-α and interleukin-lβ,i.e.,TNF-α and IL-1β) and NF-κB/p65,but 100 μrnol/L LaCl3 did not exert an obvious inflammation-inhibiting effect,and even induced inflammation.In conclusion,these findings demonstrated that LaC13 was able to suppress wear particle-induced inflammation and activation of NF-κB in a certain range of concentrations in vitro and mainly decrease the expression of RANK,but not M-CSFR,all of which were generally recognized to play a pivotal role in osteoclastogenesis.

  5. Pro-inflammatory profile of preeclamptic placental mesenchymal stromal cells: new insights into the etiopathogenesis of preeclampsia.

    Directory of Open Access Journals (Sweden)

    Alessandro Rolfo

    Full Text Available UNLABELLED: The objective of the present study was to evaluate whether placental mesenchymal stromal cells (PDMSCs derived from normal and preeclamptic (PE chorionic villous tissue presented differences in their cytokines expression profiles. Moreover, we investigated the effects of conditioned media from normal and PE-PDMSCs on the expression of pro-inflammatory Macrophage migration Inhibitory Factor (MIF, Vascular Endothelial Growth Factor (VEGF, soluble FMS-like tyrosine kinase-1 (sFlt-1 and free β-human Chorionic Gonadotropin (βhCG by normal term villous explants. This information will help to understand whether anomalies in PE-PDMSCs could cause or contribute to the anomalies typical of preeclampsia. METHODS: Chorionic villous PDMSCs were isolated from severe preeclamptic (n = 12 and physiological control term (n = 12 placentae. Control and PE-PDMSCs's cytokines expression profiles were determined by Cytokine Array. Control and PE-PDMSCs were plated for 72 h and conditioned media (CM was collected. Physiological villous explants (n = 48 were treated with control or PE-PDMSCs CM for 72 h and processed for mRNA and protein isolation. MIF, VEGF and sFlt-1 mRNA and protein expression were analyzed by Real Time PCR and Western Blot respectively. Free βhCG was assessed by immunofluorescent. RESULTS: Cytokine array showed increased release of pro-inflammatory cytokines by PE relative to control PDMSCs. Physiological explants treated with PE-PDMSCs CM showed significantly increased MIF and sFlt-1 expression relative to untreated and control PDMSCs CM explants. Interestingly, both control and PE-PDMSCs media induced VEGF mRNA increase while only normal PDMSCs media promoted VEGF protein accumulation. PE-PDMSCs CM explants released significantly increased amounts of free βhCG relative to normal PDMSCs CM ones. CONCLUSIONS: Herein, we reported elevated production of pro-inflammatory cytokines by PE-PDMSCs. Importantly, PE PDMSCs

  6. Redox Regulation in Cancer Stem Cells

    Directory of Open Access Journals (Sweden)

    Shijie Ding

    2015-01-01

    Full Text Available Reactive oxygen species (ROS and ROS-dependent (redox regulation signaling pathways and transcriptional activities are thought to be critical in stem cell self-renewal and differentiation during growth and organogenesis. Aberrant ROS burst and dysregulation of those ROS-dependent cellular processes are strongly associated with human diseases including many cancers. ROS levels are elevated in cancer cells partially due to their higher metabolism rate. In the past 15 years, the concept of cancer stem cells (CSCs has been gaining ground as the subpopulation of cancer cells with stem cell-like properties and characteristics have been identified in various cancers. CSCs possess low levels of ROS and are responsible for cancer recurrence after chemotherapy or radiotherapy. Unfortunately, how CSCs control ROS production and scavenging and how ROS-dependent signaling pathways contribute to CSCs function remain poorly understood. This review focuses on the role of redox balance, especially in ROS-dependent cellular processes in cancer stem cells (CSCs. We updated recent advances in our understanding of ROS generation and elimination in CSCs and their effects on CSC self-renewal and differentiation through modulating signaling pathways and transcriptional activities. The review concludes that targeting CSCs by manipulating ROS metabolism/dependent pathways may be an effective approach for improving cancer treatment.

  7. Pro-inflammatory action of MIF in acute myocardial infarction via activation of peripheral blood mononuclear cells.

    Directory of Open Access Journals (Sweden)

    David A White

    Full Text Available OBJECTIVES: Macrophage migration inhibitory factor (MIF, a pro-inflammatory cytokine, has been implicated in the pathogenesis of multiple inflammatory disorders. We determined changes in circulating MIF levels, explored the cellular source of MIF, and studied the role of MIF in mediating inflammatory responses following acute myocardial infarction (MI. METHODS AND RESULTS: We recruited 15 patients with MI, 10 patients with stable angina and 10 healthy volunteers and measured temporal changes of MIF in plasma. Expression of MIF, matrix metalloproteinase-9 (MMP-9 and interleukin-6 (IL-6 in cultured peripheral blood mononuclear cells (PBMCs and the media were measured by ELISA or real-time PCR. Compared to controls, plasma levels of MIF and IL-6 were significantly elevated at admission and 72 h post-MI. In contrast, expression of MIF, MMP-9 and IL-6 by PBMCs from MI patients was unchanged at admission, but significantly increased at 72 h. Addition of MIF activated cultured PBMCs by upregulating expression of inflammatory molecules and also synergistically enhanced stimulatory action of IL-1β which were inhibited by anti-MIF interventions. In a mouse MI model we observed similar changes in circulating MIF as seen in patients, with reciprocal significant increases in plasma MIF and reduction of MIF content in the infarct myocardium at 3 h after MI. MIF content in the infarct myocardium was restored at 72 h post-MI and was associated with robust macrophage infiltration. Further, anti-MIF intervention significantly reduced inflammatory cell infiltration and expression of monocyte chemoattractant protein-1 at 24 h and incidence of cardiac rupture in mice post-MI. CONCLUSION: MI leads to a rapid release of MIF from the myocardium into circulation. Subsequently MIF facilitates PBMC production of pro-inflammatory mediators and myocardial inflammatory infiltration. Attenuation of these events, and post-MI cardiac rupture, by anti-MIF interventions suggests

  8. Regulatory T cells with multiple suppressive and potentially pro-tumor activities accumulate in human colorectal cancer.

    Science.gov (United States)

    Timperi, Eleonora; Pacella, Ilenia; Schinzari, Valeria; Focaccetti, Chiara; Sacco, Luca; Farelli, Francesco; Caronna, Roberto; Del Bene, Gabriella; Longo, Flavia; Ciardi, Antonio; Morelli, Sergio; Vestri, Anna Rita; Chirletti, Piero; Barnaba, Vincenzo; Piconese, Silvia

    2016-07-01

    Tregs can contribute to tumor progression by suppressing antitumor immunity. Exceptionally, in human colorectal cancer (CRC), Tregs are thought to exert beneficial roles in controlling pro-tumor chronic inflammation. The goal of our study was to characterize CRC-infiltrating Tregs at multiple levels, by phenotypical, molecular and functional evaluation of Tregs from the tumor site, compared to non-tumoral mucosa and peripheral blood of CRC patients. The frequency of Tregs was higher in mucosa than in blood, and further significantly increased in tumor. Ex vivo, those Tregs suppressed the proliferation of tumor-infiltrating CD8(+) and CD4(+) T cells. A differential compartmentalization was detected between Helios(high) and Helios(low) Treg subsets (thymus-derived versus peripherally induced): while Helios(low) Tregs were enriched in both sites, only Helios(high) Tregs accumulated significantly and specifically in tumors, displayed a highly demethylated TSDR region and contained high proportions of cells expressing CD39 and OX40, markers of activation and suppression. Besides the suppression of T cells, Tregs may contribute to CRC progression also through releasing IL-17, or differentiating into Tfr cells that potentially antagonize a protective Tfh response, events that were both detected in tumor-associated Tregs. Overall, our data indicate that Treg accumulation may contribute through multiple mechanisms to CRC establishment and progression. PMID:27622025

  9. The presence, function and regulation of IL-17 and Th17 cells in periodontitis.

    Science.gov (United States)

    Cheng, Wan-Chien; Hughes, Francis J; Taams, Leonie S

    2014-06-01

    Periodontitis (PD) is a chronic inflammatory disease characterised by tissue inflammation and destruction of the associated alveolar bone. It is caused by the colonisation of the bacterial plaque biofilm and the resultant host immune responses in the surrounding periodontal tissues. The pro-inflammatory cytokine IL-17, and IL-17 producing CD4+ T cells (also called Th17 cells) have been shown to play an important role in many inflammatory diseases. There is increasing evidence of the presence of IL-17 and Th17 cells in human PD lesions and this may be associated with disease severity. Moreover, several animal studies indicate the potential role of IL-17 and Th17 cells in gingival inflammation and the resultant bone destruction in PD. Here we review recent findings regarding the presence, function and regulation of IL-17 and Th17 cells in PD, and we highlight potential areas of future research interest. PMID:24735470

  10. Effects ofPlasmodium falciparum-infected erythrocytes on matrix metalloproteinase-9 regulation in human microvascular endothelial cells

    Institute of Scientific and Technical Information of China (English)

    Sarah D Alessandro; Nicoletta Basilico; Mauro Prato

    2013-01-01

    Objective:To investigate the regulation of matrix metalloproteinases(MMPs) and tissue inhibitors of metalloproteinases(TIMPs) in human microvascular endothelium(HMEC-1) exposed to erythrocytes infected by different strains ofPlasmodium falciparum (P. falciparum).Methods:HMEC-1 cells were co-incubated for72 h with erythrocytes infected by late stage trophozoite of D10(chloroquine-sensitive) orW2(chloroquine-resistant)P. falciparum strains.Cell supernatants were then collected and the levels of pro- or active gelatinasesMMP-9 andMMP-2 were evaluated by gelatin zymography and densitometry.The release of pro-MMP-9,MMP-3,MMP-1 andTIMP-1 proteins was analyzed by western blotting and densitometry.Results:Infected erythrocytes inducedde novo proMMP-9 andMMP-9 release.Neither basal levels of proMMP-2 were altered, nor activeMMP-2 was found.MMP-3 andMMP-1 secretion was significantly enhanced, whereas basalTIMP-1 was unaffected.All effects were similar for both strains. Conclusions:P. falciparum parasites, either chloroquine-sensitive or -resistant, induce the release of activeMMP-9 protein from human microvascular endothelium, by impairing balances between proMMP-9 and its inhibitor, and by enhancing the levels of its activators.This work provides new evidence onMMP involvement in malaria, pointing atMMP-9 as a possible target in adjuvant therapy.

  11. EBV BART MicroRNAs Target Multiple Pro-apoptotic Cellular Genes to Promote Epithelial Cell Survival.

    Directory of Open Access Journals (Sweden)

    Dong Kang

    2015-06-01

    Full Text Available Epstein-Barr virus (EBV is a ubiquitous human γ-herpesvirus that can give rise to cancers of both B-cell and epithelial cell origin. In EBV-induced cancers of epithelial origin, including nasopharyngeal carcinomas (NPCs and gastric carcinomas, the latent EBV genome expresses very high levels of a cluster of 22 viral pre-miRNAs, called the miR-BARTs, and these have previously been shown to confer a degree of resistance to pro-apoptotic drugs. Here, we present an analysis of the ability of individual miR-BART pre-miRNAs to confer an anti-apoptotic phenotype and report that five of the 22 miR-BARTs demonstrate this ability. We next used photoactivatable ribonucleoside-enhanced crosslinking and immunoprecipitation (PAR-CLIP to globally identify the mRNA targets bound by these miR-BARTs in latently infected epithelial cells. This led to the identification of ten mRNAs encoding pro-apoptotic mRNA targets, all of which could be confirmed as valid targets for the five anti-apoptotic miR-BARTs by indicator assays and by demonstrating that ectopic expression of physiological levels of the relevant miR-BART in the epithelial cell line AGS resulted in a significant repression of the target mRNA as well as the encoded protein product. Using RNA interference, we further demonstrated that knockdown of at least seven of these cellular miR-BART target transcripts phenocopies the anti-apoptotic activity seen upon expression of the relevant EBV miR-BART miRNA. Together, these observations validate previously published reports arguing that the miR-BARTs can exert an anti-apoptotic effect in EBV-infected epithelial cells and provide a mechanistic explanation for this activity. Moreover, these results identify and validate a substantial number of novel mRNA targets for the anti-apoptotic miR-BARTs.

  12. Differential regulation of intracellular factors mediating cell cycle, DNA repair and inflammation following exposure to silver nanoparticles in human cells

    Directory of Open Access Journals (Sweden)

    AshaRani PV

    2012-02-01

    Full Text Available Abstract Background Investigating the cellular and molecular signatures in eukaryotic cells following exposure to nanoparticles will further our understanding on the mechanisms mediating nanoparticle induced effects. This study illustrates the molecular effects of silver nanoparticles (Ag-np in normal human lung cells, IMR-90 and human brain cancer cells, U251 with emphasis on gene expression, induction of inflammatory mediators and the interaction of Ag-np with cytosolic proteins. Results We report that silver nanoparticles are capable of adsorbing cytosolic proteins on their surface that may influence the function of intracellular factors. Gene and protein expression profiles of Ag-np exposed cells revealed up regulation of many DNA damage response genes such as Gadd 45 in both the cell types and ATR in cancer cells. Moreover, down regulation of genes necessary for cell cycle progression (cyclin B and cyclin E and DNA damage response/repair (XRCC1 and 3, FEN1, RAD51C, RPA1 was observed in both the cell lines. Double strand DNA damage was observed in a dose dependant manner as evidenced in γH2AX foci assay. There was a down regulation of p53 and PCNA in treated cells. Cancer cells in particular showed a concentration dependant increase in phosphorylated p53 accompanied by the cleavage of caspase 3 and PARP. Our results demonstrate the involvement of NFκB and MAP kinase pathway in response to Ag-np exposure. Up regulation of pro-inflammatory cytokines such as interleukins (IL-8, IL-6, macrophage colony stimulating factor, macrophage inflammatory protein in fibroblasts following Ag-np exposure were also observed. Conclusion In summary, Ag-np can modulate gene expression and protein functions in IMR-90 cells and U251 cells, leading to defective DNA repair, proliferation arrest and inflammatory response. The observed changes could also be due to its capability to adsorb cytosolic proteins on its surface.

  13. Polymyalgia rheumatica is characterized by pro-inflammatory, senescent CD8+ T cells

    NARCIS (Netherlands)

    Van Der Geest, K.; Abdulahad, W.; Huitema, M.; Kroesen, B.; Rutgers, A.; Brouwer, E.; Boots, A.

    2013-01-01

    Background Polymyalgia rheumatica (PMR) is a frequent, inflammatory rheumatic disease affecting elderly people. Previous studies suggest that T cell mediated immune responses contribute to PMR. However, little is known about CD4+ and CD8+ T cell subsets and their function in PMR. Furthermore, it rem

  14. RGDechi-hCit: αvβ3 selective pro-apoptotic peptide as potential carrier for drug delivery into melanoma metastatic cells.

    Directory of Open Access Journals (Sweden)

    Domenica Capasso

    Full Text Available αvβ3 integrin is an important tumor marker widely expressed on the surface of cancer cells. Recently, we reported some biological features of RGDechi-hCit, an αvβ3 selective peptide antagonist. In the present work, we mainly investigated the pro-apoptotic activity of the molecule and its ability to penetrate the membrane of WM266 cells, human malignant melanoma cells expressing high levels of αvβ3 integrin. For the first time we demonstrated the pro-apoptotic effect and the ability of RGDechi-hCit to enter into cell overexpressing αvβ3 integrin mainly by clathrin- and caveolin-mediated endocytosis. Furthermore, we deepened and confirmed the selectivity, anti-adhesion, and anti-proliferative features of the peptide. Altogether these experiments give insight into the biological behavior of RGDechi-hCit and have important implications for the employment of the peptide as a new selective carrier to deliver drugs into the cell and as a therapeutic and diagnostic tool for metastatic melanoma. Moreover, since the peptide shows a pro-apoptotic effect, a great perspective could be the development of a new class of selective systems containing RGDechi-hCit and pro-apoptotic molecules or other therapeutic agents to attain a synergic action.

  15. Stem cells can form gap junctions with cardiac myocytes and exert pro-arrhythmic effects

    Directory of Open Access Journals (Sweden)

    Nicoline Willemijn Smit

    2014-10-01

    Full Text Available Stem cell therapy has been suggested to be a promising option for regeneration of injured myocardium, for example following a myocardial infarction. For clinical use cell-based therapies have to be safe and applicable and are aimed to renovate the architecture of the heart. Yet for functional and coordinated activity synchronized with the host myocardium stem cells have to be capable of forming electrical connections with resident cardiomyocytes. In this paper we discuss whether stem cells are capable of establishing functional electrotonic connections with cardiomyocytes and whether these may generate a risk for arrhythmias. Application of stem cells in the clinical setting with outcomes concerning arrhythmogenic safety and future perspectives will also briefly be touched upon.

  16. B cell development in the bone marrow is regulated by homeostatic feedback exerted by mature B cells

    Directory of Open Access Journals (Sweden)

    Gitit eShahaf

    2016-03-01

    Full Text Available Cellular homeostasis in the B cell compartment is strictly imposed to balance cell production and cell loss. However, it is not clear whether B cell development in the bone marrow (BM is an autonomous process or subjected to regulation by the peripheral B cell compartment. To specifically address this question, we used mice transgenic for human CD20, where effective depletion of B lineage cells is obtained upon administration of mouse-anti-human CD20 antibodies, in the absence of any effect on other cell lineages and/or tissues. We followed the kinetics of B cell return to equilibrium by BrdU labeling and flow cytometry and analyzed the resulting data by mathematical modeling. Labeling was much faster in depleted mice. Compared to control mice, B cell-depleted mice exhibited a higher proliferation rate in the pro-/pre-B compartment, and higher cell death and lower differentiation in the immature B cell compartment. We validated the first result by analysis of the expression of Ki67, the nuclear protein expressed in proliferating cells, and the second using Annexin-V staining. Collectively, our results suggest that B lymphopoiesis is subjected to homeostatic feedback mechanisms imposed by mature B cells in the peripheral compartment.

  17. Inhibition of N-linked glycosylation impairs ALK phosphorylation and disrupts pro-survival signaling in neuroblastoma cell lines

    Directory of Open Access Journals (Sweden)

    Del Grosso Federica

    2011-12-01

    involved in cell death. Conclusions In this study we showed that inhibition of N-linked glycosylation affects ALK phosphorylation and disrupts downstream pro-survival signaling, indicating that inhibition of this post-translational modification may be a promising therapeutic approach. However, as tunicamycin is not a likely candidate for clinical use other approaches to alter N-linked glycosylation need to be explored. Future studies will assess whether the efficacy in inhibiting ALK activity might be enhanced by the combination of ALK specific small molecule and N-linked glycosylation inhibitors.

  18. Saturated free fatty acids and apoptosis in microvascular mesangial cells: palmitate activates pro-apoptotic signaling involving caspase 9 and mitochondrial release of endonuclease G

    Directory of Open Access Journals (Sweden)

    Simonson Michael S

    2005-01-01

    Full Text Available Abstract Background In type 2 diabetes, free fatty acids (FFA accumulate in microvascular cells, but the phenotypic consequences of FFA accumulation in the microvasculature are incompletely understood. Here we investigated whether saturated FFA induce apoptosis in human microvascular mesangial cells and analyzed the signaling pathways involved. Methods Saturated and unsaturated FFA-albumin complexes were added to cultured human mesangial cells, after which the number of apoptotic cells were quantified and the signal transduction pathways involved were delineated. Results The saturated FFA palmitate and stearate were apoptotic unlike equivalent concentrations of the unsaturated FFA oleate and linoleate. Palmitate-induced apoptosis was potentiated by etomoxir, an inhibitor of mitochondrial β-oxidation, but was prevented by an activator of AMP-kinase, which increases fatty acid β-oxidation. Palmitate stimulated an intrinsic pathway of pro-apoptotic signaling as evidenced by increased mitochondrial release of cytochrome-c and activation of caspase 9. A caspase 9-selective inhibitor blocked caspase 3 activation but incompletely blocked apoptosis in response to palmitate, suggesting an additional caspase 9-independent pathway. Palmitate stimulated mitochondrial release of endonuclease G by a caspase 9-independent mechanism, thereby implicating endonuclease G in caspase 9-indpendent regulation of apoptosis by saturated FFA. We also observed that the unsaturated FFA oleate and linoleate prevented palmitate-induced mitochondrial release of both cytochrome-c and endonuclease G, which resulted in complete protection from palmitate-induced apoptosis. Conclusions Taken together, these results demonstrate that palmitate stimulates apoptosis by evoking an intrinsic pathway of proapoptotic signaling and identify mitochondrial release of endonuclease G as a key step in proapoptotic signaling by saturated FFA and in the anti-apoptotic actions of unsaturated FFA.

  19. Human hepatoma cells exposed to estuarine sediment contaminant extracts permitted the differentiation between cytotoxic and pro-mutagenic fractions

    International Nuclear Information System (INIS)

    Complex toxicant mixtures present in estuarine sediments often render contaminant screening unfeasible and compromise determining causation. HepG2 cells were subjected to bioassays with sediment extracts obtained with a series of progressively polar solvents plus a crude extract. The sediments were collected from an impacted area of an estuary otherwise regarded as pristine, whose stressors result mostly from aquaculture effluents and hydrodynamic shifts that enhance particle deposition. Compared to a reference scenario, the most polar extracts yielded highest cytotoxicity while higher genotoxicity (including oxidative damage) was elicited by non-polar solvents. While the former caused effects similar to those expected from biocides, the latter triggered effects compatible with known pro-mutagens like PAHs, even though the overall levels of toxicants were considered of low risk. The results indicate that the approach may constitute an effective line-of-evidence to infer on the predominant set of hazardous contaminants present in complex environmental mixtures. -- Highlights: • Estuarine sediment contaminants were extracted with different organic solvents. • More polar solvents contained the most cytotoxic contaminant fraction. • Non-polar solvents extracted the main genotoxic component of the mixture. • DNA base oxidation was detected through FPG/Comet assay. • The contamination pattern could be inferred from cytoassays with HepG2 cells. -- Polar/non-polar sediment fractions elicited differential cytotoxic and genotoxic effects in human HepG2 cells

  20. 3'UTR Shortening Potentiates MicroRNA-Based Repression of Pro-differentiation Genes in Proliferating Human Cells.

    Directory of Open Access Journals (Sweden)

    Yonit Hoffman

    2016-02-01

    Full Text Available Most mammalian genes often feature alternative polyadenylation (APA sites and hence diverse 3'UTR lengths. Proliferating cells were reported to favor APA sites that result in shorter 3'UTRs. One consequence of such shortening is escape of mRNAs from targeting by microRNAs (miRNAs whose binding sites are eliminated. Such a mechanism might provide proliferation-related genes with an expression gain during normal or cancerous proliferation. Notably, miRNA sites tend to be more active when located near both ends of the 3'UTR compared to those located more centrally. Accordingly, miRNA sites located near the center of the full 3'UTR might become more active upon 3'UTR shortening. To address this conjecture we performed 3' sequencing to determine the 3' ends of all human UTRs in several cell lines. Remarkably, we found that conserved miRNA binding sites are preferentially enriched immediately upstream to APA sites, and this enrichment is more prominent in pro-differentiation/anti-proliferative genes. Binding sites of the miR17-92 cluster, upregulated in rapidly proliferating cells, are particularly enriched just upstream to APA sites, presumably conferring stronger inhibitory activity upon shortening. Thus 3'UTR shortening appears not only to enable escape from inhibition of growth promoting genes but also to potentiate repression of anti-proliferative genes.

  1. LNO3 AND L3 Are Associated With Antiproliferative And Pro-Apoptotic Action In Hepatoma Cells

    Directory of Open Access Journals (Sweden)

    Leonardo Campos Zanelatto

    2016-06-01

    Full Text Available Abstract The identification of antitumoral substances is the focus of intense biomedical research. Two structural analogues of thalidomide, LNO3 and L3, are two synthetic compounds that might possess such antitumor properties. We evaluated the toxicological effects of these substances, including cytotoxicity, genotoxicity and induction of apoptosis in HTC cells. Additionally, the production of free radicals (nitric oxide and superoxide was investigated, and the expression of caspases genes 3, 8, and 9 were determined by RT-qPCR. The compounds exhibited cytotoxic effects that resulted in inhibited cell proliferation. LNO3 showed to be more effective and toxic than L3 in all assays. LNO3 stimulated the release of NO and superoxide, which was accompanied by the formation of peroxynitrite. Apoptosis was induced in a dose-dependent manner by both compounds; however, the expression of caspases 3, 8 and 9 was unchanged. These results suggested that L3 and LNO3 possess antiproliferative and pro-apoptotic effects in HTC cells. Additionally, although they exhibited cytotoxicity, L3 and LNO3 might be useful coadjuvants in tumor treatment studies.

  2. Oxidative and pro-inflammatory impact of regular and denicotinized cigarettes on blood brain barrier endothelial cells: is smoking reduced or nicotine-free products really safe?

    Science.gov (United States)

    2014-01-01

    Background Both active and passive tobacco smoke (TS) potentially impair the vascular endothelial function in a causative and dose-dependent manner, largely related to the content of reactive oxygen species (ROS), nicotine, and pro-inflammatory activity. Together these factors can compromise the restrictive properties of the blood–brain barrier (BBB) and trigger the pathogenesis/progression of several neurological disorders including silent cerebral infarction, stroke, multiple sclerosis and Alzheimer’s disease. Based on these premises, we analyzed and assessed the toxic impact of smoke extract from a range of tobacco products (with varying levels of nicotine) on brain microvascular endothelial cell line (hCMEC/D3), a well characterized human BBB model. Results Initial profiling of TS showed a significant release of reactive oxygen (ROS) and reactive nitrogen species (RNS) in full flavor, nicotine-free (NF, “reduced-exposure” brand) and ultralow nicotine products. This release correlated with increased oxidative cell damage. In parallel, membrane expression of endothelial tight junction proteins ZO-1 and occludin were significantly down-regulated suggesting the impairment of barrier function. Expression of VE-cadherin and claudin-5 were also increased by the ultralow or nicotine free tobacco smoke extract. TS extract from these cigarettes also induced an inflammatory response in BBB ECs as demonstrated by increased IL-6 and MMP-2 levels and up-regulation of vascular adhesion molecules, such as VCAM-1 and PECAM-1. Conclusions In summary, our results indicate that NF and ultralow nicotine cigarettes are potentially more harmful to the BBB endothelium than regular tobacco products. In addition, this study demonstrates that the TS-induced toxicity at BBB ECs is strongly correlated to the TAR and NO levels in the cigarettes rather than the nicotine content. PMID:24755281

  3. Cell shape regulates global histone acetylation in human mammaryepithelial cells

    Energy Technology Data Exchange (ETDEWEB)

    Le Beyec, Johanne; Xu, Ren; Lee, Sun-Young; Nelson, Celeste M.; Rizki, Aylin; Alcaraz, Jordi; Bissell, Mina J.

    2007-02-28

    Extracellular matrix (ECM) regulates cell morphology and gene expression in vivo; these relationships are maintained in three-dimensional (3D) cultures of mammary epithelial cells. In the presence of laminin-rich ECM (lrECM), mammary epithelial cells round up and undergo global histone deacetylation, a process critical for their functional differentiation. However, it remains unclear whether lrECM-dependent cell rounding and global histone deacetylation are indeed part of a common physical-biochemical pathway. Using 3D cultures as well as nonadhesive and micropatterned substrata, here we showed that the cell 'rounding' caused by lrECM was sufficient to induce deacetylation of histones H3 and H4 in the absence of biochemical cues. Microarray and confocal analysis demonstrated that this deacetylation in 3D culture is associated with a global increase in chromatin condensation and a reduction in gene expression. Whereas cells cultured on plastic substrata formed prominent stress fibers, cells grown in 3D lrECM or on micropatterns lacked these structures. Disruption of the actin cytoskeleton with cytochalasin D phenocopied the lrECM-induced cell rounding and histone deacetylation. These results reveal a novel link between ECM-controlled cell shape and chromatin structure, and suggest that this link is mediated by changes in the actin cytoskeleton.

  4. Mechanical regulation of mesenchymal stem cell differentiation.

    Science.gov (United States)

    Steward, Andrew J; Kelly, Daniel J

    2015-12-01

    Biophysical cues play a key role in directing the lineage commitment of mesenchymal stem cells or multipotent stromal cells (MSCs), but the mechanotransductive mechanisms at play are still not fully understood. This review article first describes the roles of both substrate mechanics (e.g. stiffness and topography) and extrinsic mechanical cues (e.g. fluid flow, compression, hydrostatic pressure, tension) on the differentiation of MSCs. A specific focus is placed on the role of such factors in regulating the osteogenic, chondrogenic, myogenic and adipogenic differentiation of MSCs. Next, the article focuses on the cellular components, specifically integrins, ion channels, focal adhesions and the cytoskeleton, hypothesized to be involved in MSC mechanotransduction. This review aims to illustrate the strides that have been made in elucidating how MSCs sense and respond to their mechanical environment, and also to identify areas where further research is needed.

  5. Exercise regulates breast cancer cell viability

    DEFF Research Database (Denmark)

    Dethlefsen, Christine; Lillelund, Christian; Midtgaard, Julie;

    2016-01-01

    Purpose: Exercise decreases breast cancer risk and disease recurrence, but the underlying mechanisms are unknown. Training adaptations in systemic factors have been suggested as mediating causes. We aimed to examine if systemic adaptations to training over time, or acute exercise responses......, in breast cancer survivors could regulate breast cancer cell viability in vitro. Methods: Blood samples were collected from breast cancer survivors, partaking in either a 6-month training intervention or across a 2 h acute exercise session. Changes in training parameters and systemic factors were evaluated...... and pre/post exercise-conditioned sera from both studies were used to stimulate breast cancer cell lines (MCF-7, MDA-MB-231) in vitro. Results: Six months of training increased VO2peak (16.4 %, p

  6. Pro-apoptotic effect of cecropin AD on nasopharyngeal carcinoma cells

    Institute of Scientific and Technical Information of China (English)

    XIAO Ye-chen; HUANG Ya-dong; XU Pei-lin; ZHOU Zhen-qing; LI Xiao-kun

    2006-01-01

    @@ Nasopharyngeal carcinoma (NPC) is one of the highly prevalent malignancies in Southeast Asia.1 Chemotherapy and radiotherapy are commonly administrated to treat nasopharyngeal carcinoma, but these therapies can not prevent the recurrence and metastasis of tumor cells.

  7. Hedgehog pathway regulators influence cervical cancer cell proliferation, survival and migration

    Energy Technology Data Exchange (ETDEWEB)

    Samarzija, Ivana [Ecole Polytechnique Federale Lausanne (EPFL), Department of Life Sciences, Swiss Institute for Experimental Cancer Research (ISREC), 1015 Lausanne (Switzerland); Beard, Peter, E-mail: peter.beard@epfl.ch [Ecole Polytechnique Federale Lausanne (EPFL), Department of Life Sciences, Swiss Institute for Experimental Cancer Research (ISREC), 1015 Lausanne (Switzerland)

    2012-08-17

    Highlights: Black-Right-Pointing-Pointer Unknown cellular mutations complement papillomavirus-induced carcinogenesis. Black-Right-Pointing-Pointer Hedgehog pathway components are expressed by cervical cancer cells. Black-Right-Pointing-Pointer Hedgehog pathway activators and inhibitors regulate cervical cancer cell biology. Black-Right-Pointing-Pointer Cell immortalization by papillomavirus and activation of Hedgehog are independent. -- Abstract: Human papillomavirus (HPV) infection is considered to be a primary hit that causes cervical cancer. However, infection with this agent, although needed, is not sufficient for a cancer to develop. Additional cellular changes are required to complement the action of HPV, but the precise nature of these changes is not clear. Here, we studied the function of the Hedgehog (Hh) signaling pathway in cervical cancer. The Hh pathway can have a role in a number of cancers, including those of liver, lung and digestive tract. We found that components of the Hh pathway are expressed in several cervical cancer cell lines, indicating that there could exists an autocrine Hh signaling loop in these cells. Inhibition of Hh signaling reduces proliferation and survival of the cervical cancer cells and induces their apoptosis as seen by the up-regulation of the pro-apoptotic protein cleaved caspase 3. Our results indicate that Hh signaling is not induced directly by HPV-encoded proteins but rather that Hh-activating mutations are selected in cells initially immortalized by HPV. Sonic Hedgehog (Shh) ligand induces proliferation and promotes migration of the cervical cancer cells studied. Together, these results indicate pro-survival and protective roles of an activated Hh signaling pathway in cervical cancer-derived cells, and suggest that inhibition of this pathway may be a therapeutic option in fighting cervical cancer.

  8. The Pro-Fibrotic Factor IGFBP-5 Induces Lung Fibroblast and Mononuclear Cell Migration

    OpenAIRE

    Yasuoka, Hidekata; Yamaguchi, Yukie; Feghali-Bostwick, Carol A.

    2009-01-01

    We have previously shown that insulin-like growth factor–binding protein-5 (IGFBP-5) is overexpressed in fibrotic lung tissues and that it induces production of extracellular matrix components such as collagen and fibronectin both in vitro and in vivo. We recently observed mononuclear cell infiltration in lung tissues of mice expressing IGFBP-5. We therefore examined the role of IGFBP-5 on the migration of immune cells. Migration assays demonstrated that IGFBP-5 induced migration of periphera...

  9. GATA2 regulates dendritic cell differentiation.

    Science.gov (United States)

    Onodera, Koichi; Fujiwara, Tohru; Onishi, Yasushi; Itoh-Nakadai, Ari; Okitsu, Yoko; Fukuhara, Noriko; Ishizawa, Kenichi; Shimizu, Ritsuko; Yamamoto, Masayuki; Harigae, Hideo

    2016-07-28

    Dendritic cells (DCs) are critical immune response regulators; however, the mechanism of DC differentiation is not fully understood. Heterozygous germ line GATA2 mutations induce GATA2-deficiency syndrome, characterized by monocytopenia, a predisposition to myelodysplasia/acute myeloid leukemia, and a profoundly reduced DC population, which is associated with increased susceptibility to viral infections, impaired phagocytosis, and decreased cytokine production. To define the role of GATA2 in DC differentiation and function, we studied Gata2 conditional knockout and haploinsufficient mice. Gata2 conditional deficiency significantly reduced the DC count, whereas Gata2 haploinsufficiency did not affect this population. GATA2 was required for the in vitro generation of DCs from Lin(-)Sca-1(+)Kit(+) cells, common myeloid-restricted progenitors, and common dendritic cell precursors, but not common lymphoid-restricted progenitors or granulocyte-macrophage progenitors, suggesting that GATA2 functions in the myeloid pathway of DC differentiation. Moreover, expression profiling demonstrated reduced expression of myeloid-related genes, including mafb, and increased expression of T-lymphocyte-related genes, including Gata3 and Tcf7, in Gata2-deficient DC progenitors. In addition, GATA2 was found to bind an enhancer element 190-kb downstream region of Gata3, and a reporter assay exhibited significantly reduced luciferase activity after adding this enhancer region to the Gata3 promoter, which was recovered by GATA sequence deletion within Gata3 +190. These results suggest that GATA2 plays an important role in cell-fate specification toward the myeloid vs T-lymphocyte lineage by regulating lineage-specific transcription factors in DC progenitors, thereby contributing to DC differentiation. PMID:27259979

  10. Effects of p53 overexpression on neoplastic cell pro-liferation and apoptosis in thymic carcinoma

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    To investigate p53 overexpression and its correlation with neoplastic cell proliferation and apoptosis in 20 thymic carcinomas. Methods: 20 surgical samples of thymic carcinoma were collected randomly during the past 15 years in the Guangzhou area. Immunohistochemical staining was performed using LSAB method with anti-p53 monoclonal antibody (DO-7) and proliferating cell nuclear antigen (clone PC 10) as primary antibodies. The p53 index was indicated by the number of p53 positive cells among 100 carcinoma cells. More than 25 percentage of p53 positive cells found in tissue sections was recognized as p53 overexpression. Carcinoma cell proliferation activity was assayed by PCNA index (PI), and apoptosis degree was evaluated by TUNEL (TdT-mediated dUTP-X nick end labeling) index (TI) using Boehringer Mannheim In Situ Death Detection Kit. Results: P53 positive cells could be found in vast majority of thymic carcinomas (19/20) and the overexpression rate reached 35% (7/20). The median PI (40%) of 7 cases with p53 overexpression was higher than that (31%) of 13 cases without p53 overexpression, but there was no statistical significance that existed between these two data (P>0.05). The median TI (0.5/HPF) of 7 p53 overexpression cases was much lower than that (4.5/HPF) of 13 non-overexpression cases, and there was a significant difference statistically (P<0.05). Conclusion: p53 expression was a frequent finding in thymic carcinoma cells, and the p53 overexpression which might represent p53 inactivation or gene mutation was often involved in thymic carcino-genesis. The median PCNA index of p53 overexpression group was higher than that of non-overexpression group though there existed no statistical difference. This indicates that the inhibiting function of p53 on cell proliferation seemed lost in p53 overexpressed thymic carcinomas. It is worthy to be specially mentioned that the inducing function of p53 on cell apoptosis was markedly lost in p53 overexpressed thymic

  11. Situational aldehyde dehydrogenase expression by regulatory T cells may explain the contextual duality of cyclophosphamide as both a pro-inflammatory and tolerogenic agent

    OpenAIRE

    Kanakry, Christopher G.; Ganguly, Sudipto; Luznik, Leo

    2015-01-01

    In two recent publications, we demonstrated that after allogeneic stimulation, regulatory T cells (Tregs) increase expression of aldehyde dehydrogenase (ALDH), the major in vivo mechanism of cyclophosphamide detoxification, thereby becoming cyclophosphamide resistant. Differential ALDH expression may explain why cyclophosphamide has pro- and anti-inflammatory effects that are temporally and contextually dependent.

  12. Epigenetic regulation of hematopoietic stem cell aging

    International Nuclear Information System (INIS)

    Aging is invariably associated with alterations of the hematopoietic stem cell (HSC) compartment, including loss of functional capacity, altered clonal composition, and changes in lineage contribution. Although accumulation of DNA damage occurs during HSC aging, it is unlikely such consistent aging phenotypes could be solely attributed to changes in DNA integrity. Another mechanism by which heritable traits could contribute to the changes in the functional potential of aged HSCs is through alterations in the epigenetic landscape of adult stem cells. Indeed, recent studies on hematopoietic stem cells have suggested that altered epigenetic profiles are associated with HSC aging and play a key role in modulating the functional potential of HSCs at different stages during ontogeny. Even small changes of the epigenetic landscape can lead to robustly altered expression patterns, either directly by loss of regulatory control or through indirect, additive effects, ultimately leading to transcriptional changes of the stem cells. Potential drivers of such changes in the epigenetic landscape of aged HSCs include proliferative history, DNA damage, and deregulation of key epigenetic enzymes and complexes. This review will focus largely on the two most characterized epigenetic marks – DNA methylation and histone modifications – but will also discuss the potential role of non-coding RNAs in regulating HSC function during aging

  13. Prostaglandin E2 regulates hematopoietic stem cell

    International Nuclear Information System (INIS)

    Prostaglandin E2 (PGE2) is a bioactive lipid molecule produced by cyclooxygenase (COX), which plays an important role on hematopoiesis. While it can block differentiation of myeloid progenitors but enhance proliferation of erythroid progenitors. Recent research found that PGE2 have the effects on hematopoietic stem cell (HSC) function and these effects were independent from effects on progenitor cells. Exposure of HSC cells to PGE2 in vitro can increase homing efficiency of HSC to the murine bone marrow compartment and decrease HSC apoptosis, meanwhile increase long-term stem cell engraftment. In-vivo treatment with PGE2 expands short-term HSC and engraftment in murine bone marrow but not long-term HSC.In addition, PGE2 increases HSC survival after radiation injury and enhance hematopoietic recovery, resulting maintains hematopoietic homeostasis. PGE2 regulates HSC homeostasis by reactive oxygen species and Wnt pathway. Clinical beneficial of 16, 16-dimethyl-prostaglandin E2 treatment to enhance engraftment of umbilical cord blood suggest important improvements to therapeutic strategies. (authors)

  14. Molecular regulation of pancreatic stellate cell function

    Directory of Open Access Journals (Sweden)

    Jaster Robert

    2004-10-01

    Full Text Available Abstract Until now, no specific therapies are available to inhibit pancreatic fibrosis, a constant pathological feature of chronic pancreatitis and pancreatic cancer. One major reason is the incomplete knowledge of the molecular principles underlying fibrogenesis in the pancreas. In the past few years, evidence has been accumulated that activated pancreatic stellate cells (PSCs are the predominant source of extracellular matrix (ECM proteins in the diseased organ. PSCs are vitamin A-storing, fibroblast-like cells with close morphological and biochemical similarities to hepatic stellate cells (also known as Ito-cells. In response to profibrogenic mediators such as various cytokines, PSCs undergo an activation process that involves proliferation, exhibition of a myofibroblastic phenotype and enhanced production of ECM proteins. The intracellular mediators of activation signals, and their antagonists, are only partially known so far. Recent data suggest an important role of enzymes of the mitogen-activated protein kinase family in PSC activation. On the other hand, ligands of the nuclear receptor PPARγ (peroxisome proliferator-activated receptor γ stimulate maintenance of a quiescent PSC phenotype. In the future, targeting regulators of the PSC activation process might become a promising approach for the treatment of pancreatic fibrosis.

  15. Epigenetic regulation of hematopoietic stem cell aging

    Energy Technology Data Exchange (ETDEWEB)

    Beerman, Isabel, E-mail: isabel.beerman@childrens.harvard.edu [Department of Stem Cell and Regenerative Biology, Harvard University, Cambridge, MA 02138 (United States); Department of Pediatrics, Harvard Medical School, Boston, MA 02115 (United States); Program in Cellular and Molecular Medicine, Division of Hematology/Oncology, Boston Children' s Hospital, MA 02116 (United States); Rossi, Derrick J. [Department of Stem Cell and Regenerative Biology, Harvard University, Cambridge, MA 02138 (United States); Department of Pediatrics, Harvard Medical School, Boston, MA 02115 (United States); Program in Cellular and Molecular Medicine, Division of Hematology/Oncology, Boston Children' s Hospital, MA 02116 (United States)

    2014-12-10

    Aging is invariably associated with alterations of the hematopoietic stem cell (HSC) compartment, including loss of functional capacity, altered clonal composition, and changes in lineage contribution. Although accumulation of DNA damage occurs during HSC aging, it is unlikely such consistent aging phenotypes could be solely attributed to changes in DNA integrity. Another mechanism by which heritable traits could contribute to the changes in the functional potential of aged HSCs is through alterations in the epigenetic landscape of adult stem cells. Indeed, recent studies on hematopoietic stem cells have suggested that altered epigenetic profiles are associated with HSC aging and play a key role in modulating the functional potential of HSCs at different stages during ontogeny. Even small changes of the epigenetic landscape can lead to robustly altered expression patterns, either directly by loss of regulatory control or through indirect, additive effects, ultimately leading to transcriptional changes of the stem cells. Potential drivers of such changes in the epigenetic landscape of aged HSCs include proliferative history, DNA damage, and deregulation of key epigenetic enzymes and complexes. This review will focus largely on the two most characterized epigenetic marks – DNA methylation and histone modifications – but will also discuss the potential role of non-coding RNAs in regulating HSC function during aging.

  16. Methamphetamine decreases CD4 T cell frequency and alters pro-inflammatory cytokine production in a model of drug abuse.

    Science.gov (United States)

    Mata, Mariana M; Napier, T Celeste; Graves, Steven M; Mahmood, Fareeha; Raeisi, Shohreh; Baum, Linda L

    2015-04-01

    The reason co-morbid methamphetamine use and HIV infection lead to more rapid progression to AIDS is unclear. We used a model of methamphetamine self-administration to measure the effect of methamphetamine on the systemic immune system to better understand the co-morbidity of methamphetamine and HIV. Catheters were implanted into the jugular veins of male, Sprague Dawley rats so they could self-administer methamphetamine (n=18) or be given saline (control; n=16) for 14 days. One day after the last operant session, blood and spleens were collected. We measured serum levels of pro-inflammatory cytokines, intracellular IFN-γ and TNF-α, and frequencies of CD4(+), CD8(+), CD200(+) and CD11b/c(+) lymphocytes in the spleen. Rats that self-administered methamphetamine had a lower frequency of CD4(+) T cells, but more of these cells produced IFN-γ. Methamphetamine did not alter the frequency of TNF-α-producing CD4(+) T cells. Methamphetamine using rats had a higher frequency of CD8(+) T cells, but fewer of them produced TNF-α. CD11b/c and CD200 expression were unchanged. Serum cytokine levels of IFN-γ, TNF-α and IL-6 in methamphetamine rats were unchanged. Methamphetamine lifetime dose inversely correlated with serum TNF-α levels. Our data suggest that methamphetamine abuse may exacerbate HIV disease progression by activating CD4 T cells, making them more susceptible to HIV infection, and contributing to their premature demise. Methamphetamine may also increase susceptibility to HIV infection, explaining why men who have sex with men (MSM) and frequently use methamphetamine are at the highest risk of HIV infection.

  17. HLA-B27-Homodimer-Specific Antibody Modulates the Expansion of Pro-Inflammatory T-Cells in HLA-B27 Transgenic Rats.

    Directory of Open Access Journals (Sweden)

    Osiris Marroquin Belaunzaran

    Full Text Available HLA-B27 is a common genetic risk factor for the development of Spondyloarthritides (SpA. HLA-B27 can misfold to form cell-surface heavy chain homodimers (B272 and induce pro-inflammatory responses that may lead to SpA pathogenesis. The presence of B272 can be detected on leukocytes of HLA-B27+ Ankylosing spondylitis (AS patients and HLA-B27 transgenic rats. We characterized a novel B272-specific monoclonal antibody to study its therapeutic use in HLA-B27 associated disorders.The monoclonal HD5 antibody was selected from a phage library to target cell-surface B272 homodimers and characterized for affinity, specificity and ligand binding. The immune modulating effect of HD5 was tested in HLA-B27 transgenic rats. Onset and progression of disease profiles were monitored during therapy. Cell-surface B272 and expansion of pro-inflammatory cells from blood, spleen and draining lymph nodes were assessed by flow cytometry.HD5 bound B272 with high specificity and affinity (Kd = 0.32 nM. HD5 blocked cell-surface interaction of B272 with immune regulatory receptors KIR3DL2, LILRB2 and Pirb. In addition, HD5 modulated the production of TNF from CD4+ T-cells by limiting B272 interactions in vitro. In an HLA-B27 transgenic rat model repetitive dosing of HD5 reduced the expansion of pro-inflammatory CD4+ T-cells, and decreased the levels of soluble TNF and number of cell-surface B272 molecules.HD5 predominantly inhibits early TNF production and expansion of pro-inflammatory CD4+ T-cells in HLA-B27 transgenic rats. Monoclonal antibodies targeting cell-surface B272 propose a new concept for the modulation of inflammatory responses in HLA-B27 related disorders.

  18. N-acetylcysteine increases the frequency of bone marrow pro-B/pre-B cells, but does not reverse cigarette smoking-induced loss of this subset.

    Directory of Open Access Journals (Sweden)

    Victoria L Palmer

    Full Text Available BACKGROUND: We previously showed that mice exposed to cigarette smoke for three weeks exhibit loss of bone marrow B cells at the Pro-B-to-pre-B cell transition, but the reason for this is unclear. The antioxidant N-acetylcysteine (NAC, a glutathione precursor, has been used as a chemopreventive agent to reduce adverse effects of cigarette smoke exposure on lung function. Here we determined whether smoke exposure impairs B cell development by inducing cell cycle arrest or apoptosis, and whether NAC treatment prevents smoking-induced loss of developing B cells. METHODOLOGY/PRINCIPAL FINDINGS: Groups of normal mice were either exposed to filtered room air or cigarette smoke with or without concomitant NAC treatment for 5 days/week for three weeks. Bone marrow B cell developmental subsets were enumerated, and sorted pro-B (B220(+CD43(+ and pre-B (B220(+CD43(- cell fractions were analyzed for cell cycle status and the percentage of apoptotic cells. We find that, compared to sham controls, smoke-exposed mice have ∼60% fewer pro-B/pre-B cells, regardless of NAC treatment. Interestingly, NAC-treated mice show a 21-38% increase in total bone marrow cellularity and lymphocyte frequency and about a 2-fold increase in the pro-B/pre-B cell subset, compared to sham-treated controls. No significant smoking- or NAC-dependent differences were detected in frequency of apoptotic cells or the percentage cells in the G1, S, or G2 phases of the cycle. CONCLUSIONS/SIGNIFICANCE: The failure of NAC treatment to prevent smoking-induced loss of bone marrow pre-B cells suggests that oxidative stress is not directly responsible for this loss. The unexpected expansion of the pro-B/pre-B cell subset in response to NAC treatment suggests oxidative stress normally contributes to cell loss at this developmental stage, and also reveals a potential side effect of therapeutic administration of NAC to prevent smoking-induced loss of lung function.

  19. p70S6 kinase signals cell survival as well as growth, inactivating the pro-apoptotic molecule BAD

    DEFF Research Database (Denmark)

    Harada, H; Andersen, Jens S.; Mann, M;

    2001-01-01

    Cytokines often deliver simultaneous, yet distinct, cell growth and cell survival signals. The 70-kDa ribosomal protein S6 kinase (p70S6K) is known to regulate cell growth by inducing protein synthesis components. We purified membrane-based p70S6K as a kinase responsible for site......-specific phosphorylation of BAD, which inactivates this proapoptotic molecule. Rapamycin inhibited mitochondrial-based p70S6K, which prevented phosphorylation of Ser-136 on BAD and blocked cell survival induced by insulin-like growth factor 1 (IGF-1). Moreover, IGF-1-induced phosphorylation of BAD Ser-136 was abolished in...... p70S6K-deficient cells. Thus, p70S6K is itself a dual pathway kinase, signaling cell survival as well as growth through differential substrates which include mitochondrial BAD and the ribosomal subunit S6, respectively....

  20. SIRT1 regulates MAPK pathways in vitiligo skin: insight into the molecular pathways of cell survival.

    Science.gov (United States)

    Becatti, Matteo; Fiorillo, Claudia; Barygina, Victoria; Cecchi, Cristina; Lotti, Torello; Prignano, Francesca; Silvestro, Agrippino; Nassi, Paolo; Taddei, Niccolò

    2014-03-01

    Vitiligo is an acquired and progressive hypomelanotic disease that manifests as circumscribed depigmented patches on the skin. The aetiology of vitiligo remains unclear, but recent experimental data underline the interactions between melanocytes and other typical skin cells, particularly keratinocytes. Our previous results indicate that keratinocytes from perilesional skin show the features of damaged cells. Sirtuins (silent mating type information regulation 2 homolog) 1, well-known modulators of lifespan in many species, have a role in gene repression, metabolic control, apoptosis and cell survival, DNA repair, development, inflammation, neuroprotection and healthy ageing. In the literature there is no evidence for SIRT1 signalling in vitiligo and its possible involvement in disease progression. Here, biopsies were taken from the perilesional skin of 16 patients suffering from non-segmental vitiligo and SIRT1 signalling was investigated in these cells. For the first time, a new SIRT1/Akt, also known as Protein Kinase B (PKB)/mitogen-activated protein kinase (MAPK) signalling has been revealed in vitiligo. SIRT1 regulates MAPK pathway via Akt-apoptosis signal-regulating kinase-1 and down-regulates pro-apoptotic molecules, leading to decreased oxidative stress and apoptotic cell death in perilesional vitiligo keratinocytes. We therefore propose SIRT1 activation as a novel way of protecting perilesional vitiligo keratinocytes from damage.

  1. A TLR4/MD2 fusion protein inhibits LPS-induced pro-inflammatory signaling in hepatic stellate cells

    International Nuclear Information System (INIS)

    Activated hepatic stellate cells (HSCs) play a key role in hepatic fibrogenesis. In injured liver they are the main extracellular matrix protein producing cell type and further perpetuate hepatic injury by secretion of pro-inflammatory mediators. Since LPS-mediated signaling through toll-like receptor 4 (TLR4) has been identified as key fibrogenic signal in HSCs we aimed to test TLR4 as potential target of therapy via ligand-binding soluble receptors. Incubation of human HSCs with a fusion protein between the extracellular domain of TLR4 and MD2 which binds LPS inhibited LPS-induced NFκB and JNK activation. TLR4/MD2 abolished LPS-induced secretion of IL-6, IL-8, MCP1, and RANTES in HSCs. In addition, TLR4/MD2 fused to human IgG-Fc neutralized LPS activity. Since TLR4 mutant mice are resistant to liver fibrosis, the TLR4/MD2 soluble receptor might represent a new therapeutic molecule for liver fibrogenesis in vivo

  2. Deep Proteomics of Breast Cancer Cells Reveals that Metformin Rewires Signaling Networks Away from a Pro-growth State.

    Science.gov (United States)

    Sacco, Francesca; Silvestri, Alessandra; Posca, Daniela; Pirrò, Stefano; Gherardini, Pier Federico; Castagnoli, Luisa; Mann, Matthias; Cesareni, Gianni

    2016-03-23

    Metformin is the most frequently prescribed drug for type 2 diabetes. In addition to its hypoglycemic effects, metformin also lowers cancer incidence. This anti-cancer activity is incompletely understood. Here, we profiled the metformin-dependent changes in the proteome and phosphoproteome of breast cancer cells using high-resolution mass spectrometry. In total, we quantified changes of 7,875 proteins and 15,813 phosphosites after metformin changes. To interpret these datasets, we developed a generally applicable strategy that overlays metformin-dependent changes in the proteome and phosphoproteome onto a literature-derived network. This approach suggested that metformin treatment makes cancer cells more sensitive to apoptotic stimuli and less sensitive to pro-growth stimuli. These hypotheses were tested in vivo; as a proof-of-principle, we demonstrated that metformin inhibits the p70S6K-rpS6 axis in a PP2A-phosphatase dependent manner. In conclusion, analysis of deep proteomics reveals both detailed and global mechanisms that contribute to the anti-cancer activity of metformin. PMID:27135362

  3. Endothelial progenitor cells give rise to pro-angiogenic smooth muscle-like progeny

    NARCIS (Netherlands)

    Moonen, Jan-Renier A. J.; Krenning, Guido; Brinker, Marja G. L.; Koerts, Jasper A.; van Luyn, Marja J. A.; Harmsen, Martin C.

    2010-01-01

    Reciprocal plasticity exists between endothelial and mesenchymal lineages. For instance, mature endothelial cells adopt a smooth muscle-like phenotype through transforming growth factor beta-1 (TGF beta 1)-driven endothelial-to-mesenchymal transdifferentiation (EndMT). Peripheral blood contains circ

  4. PAMPs and DAMPs stimulate the expression of pro-inflammatory cytokines in vitro in a fibroblast cell-line from rainbow trout (Oncorhynchus mykiss)

    DEFF Research Database (Denmark)

    Ingerslev, Hans-Christian; Ossum, C.G.; Nielsen, Michael Engelbrecht

    activates downstream signalling pathways, which subsequently leads to expression of pro-inflammatory cytokines and chemokines. DAMPs released from necrotic cells may also bind to and activate similar downstream signalling events. In telosts was found that mechanical damage of the muscle tissue using sterile...... needles induced a very rapid expression of the pro-inflammatory cytokines IL-1β, IL-8 and IL-10 as measured by real-time PCR. The results imply that cells located in the muscular tissue in addition to recruited cells are involved in the observed increased cytokine / chemokine expression. It is believed....... The present study reinforce the idea that fibroblasts are able to react to PAMPs and DAMPs and that non-immune cell-types play an important role in the inflammatory reaction per se. From an evolutionary perspective the facilitation of an inflammatory response through recognition of PAMPs and DAMPs by non...

  5. Pro Hadoop

    CERN Document Server

    Venner, J

    2009-01-01

    You've heard the hype about Hadoop: it runs petabyte--scale data mining tasks insanely fast, it runs gigantic tasks on clouds for absurdly cheap, it's been heavily committed to by tech giants like IBM, Yahoo!, and the Apache Project, and it's completely open source (thus free). But what exactly is it, and more importantly, how do you even get a Hadoop cluster up and running? From Apress, the name you've come to trust for hands--on technical knowledge, Pro Hadoop brings you up to speed on Hadoop. You learn the ins and outs of MapReduce; how to structure a cluster, design, and implement the Hado

  6. Inflammatory responses in epithelia: endotoxin-induced IL-6 secretion and iNOS/NO production are differentially regulated in mouse mammary epithelial cells

    OpenAIRE

    2010-01-01

    Background IL-6 is a pro-inflammatory cytokine that signals via binding to a soluble or membrane bound receptor, while nitric oxide (NO), an oxidative stress molecule, diffuses through the cell membrane without a receptor. Both mediators signal through different mechanisms, yet they are dependent on NFκB. We proposed that both mediators are co-induced and co-regulated in inflamed mammary epithelial cells. Methods SCp2 mammary epithelial cells were treated with bacterial endotoxin (ET) for dif...

  7. Molecular regulation of plant cell wall extensibility

    Science.gov (United States)

    Cosgrove, D. J.

    1998-01-01

    Gravity responses in plants often involve spatial and temporal changes in cell growth, which is regulated primarily by controlling the ability of the cell wall to extend. The wall is thought to be a cellulose-hemicellulose network embedded in a hydrated matrix of complex polysaccharides and a small amount of structural protein. The wall extends by a form of polymer creep, which is mediated by expansins, a novel group of wall-loosening proteins. Expansins were discovered during a molecular dissection of the "acid growth" behavior of cell walls. Expansin alters the rheology of plant walls in profound ways, yet its molecular mechanism of action is still uncertain. It lacks detectable hydrolytic activity against the major components of the wall, but it is able to disrupt noncovalent adhesion between wall polysaccharides. The discovery of a second family of expansins (beta-expansins) sheds light on the biological role of a major group of pollen allergens and implies that expansins have evolved for diverse developmental functions. Finally, the contribution of other processes to wall extensibility is briefly summarized.

  8. Mixed Pro- and Anti-Oxidative Effects of Pomegranate Polyphenols in Cultured Cells

    OpenAIRE

    Francesca Danesi; Kroon, Paul A.; Shikha Saha; Dario de Biase; Luigi Filippo D'Antuono; Alessandra Bordoni

    2014-01-01

    In recent years, the number of scientific papers concerning pomegranate (Punica granatum L.) and its health properties has increased greatly, and there is great potential for the use of bioactive-rich pomegranate extracts as ingredients in functional foods and nutraceuticals. To translate this potential into effective strategies it is essential to further elucidate the mechanisms of the reported bioactivity. In this study HepG2 cells were supplemented with a pomegranate fruit extract or with...

  9. Pro-apoptotic effect of Persea americana var. Hass (avocado) on Jurkat lymphoblastic leukemia cells.

    Science.gov (United States)

    Bonilla-Porras, Angelica R; Salazar-Ospina, Andrea; Jimenez-Del-Rio, Marlene; Pereañez-Jimenez, Andres; Velez-Pardo, Carlos

    2013-11-01

    Abstract Context: Therapy for leukemia has a limited efficacy. There is a need to search for alternative anti-leukemia therapies. Persea americana Mill var. Hass (Lauraceae) is a tropical fruit (avocado) that might be used against cancer. Objective: To investigate whether P. americana induces death in Jurkat lymphoblastic leukemia cells. Materials and methods: Four ethanol extracts (0.1, 0.5, 1, 2 and 5 mg/mL) from avocado fruit (endocarp, whole seed, seed and leaves) were analyzed against Jurkat cells. Hydrogen peroxide generation by oxidation of 2',7'-dichlorodihydrofluorescein diacetate to the fluorescent compound 2',7'-dichlorfluorescein assay, acridine orange/ethidium bromide staining, flow cytometry analysis of annexin-V/7-amino-actinomycin, mitochondrial membrane potential and immunocytochemistry detection of transcription factor p53, caspase-3 and apoptosis-inducing factor (AIF) were evaluated. Results: Endocarp, seed, whole seed, and leaf (0.1 mg/mL) extracts induced significant apoptosis in Jurkat cells (p avocado and its therapeutic action on leukemia.

  10. Mixed Pro- and Anti-Oxidative Effects of Pomegranate Polyphenols in Cultured Cells

    Directory of Open Access Journals (Sweden)

    Francesca Danesi

    2014-10-01

    Full Text Available In recent years, the number of scientific papers concerning pomegranate (Punica granatum L. and its health properties has increased greatly, and there is great potential for the use of bioactive-rich pomegranate extracts as ingredients in functional foods and nutraceuticals. To translate this potential into effective strategies it is essential to further elucidate the mechanisms of the reported bioactivity. In this study HepG2 cells were supplemented with a pomegranate fruit extract or with the corresponding amount of pure punicalagin, and then subjected to an exogenous oxidative stress. Overall, upon the oxidative stress the gene expression and activity of the main antioxidant enzymes appeared reduced in supplemented cells, which were more prone to the detrimental effects than unsupplemented ones. No differences were detected between cells supplemented with the pomegranate juice or the pure punicalagin. Although further studies are needed due to the gaps existing between in vitro and in vivo studies, our results suggest caution in the administration of high concentrations of nutraceutical molecules, particularly when they are administered in concentrated form.

  11. The amelioration of phagocytic ability in microglial cells by curcumin through the inhibition of EMF-induced pro-inflammatory responses

    OpenAIRE

    He, Gen-Lin; Liu, Yong; Min LI; Chen, Chun-Hai; Gao, Peng; Yu, Zheng-Ping; Yang, Xue-Sen

    2014-01-01

    Background Insufficient clearance by microglial cells, prevalent in several neurological conditions and diseases, is intricately intertwined with MFG-E8 expression and inflammatory responses. Electromagnetic field (EMF) exposure can elicit the pro-inflammatory activation and may also trigger an alteration of the clearance function in microglial cells. Curcumin has important roles in the anti-inflammatory and phagocytic process. Here, we evaluated the ability of curcumin to ameliorate the phag...

  12. Melatonin Regulates Angiogenic Factors under Hypoxia in Breast Cancer Cell Lines.

    Science.gov (United States)

    Jardim-Perassi, Bruna Victorasso; Lourenço, Mateus Repolês; Doho, Gabriel Mandarini; Grígolo, Ingrid Helen; Gelaleti, Gabriela Bottaro; Ferreira, Lívia Carvalho; Borin, Thaiz Ferraz; Moschetta, Marina Gobbe; Pires de Campos Zuccari, Debora Aparecida

    2016-01-01

    Angiogenesis is the process of new blood vessel formation, regulated by a number of pro- and antiangiogenic factors and usually begins in response to hypoxia. Exogenous administration of melatonin has shown numerous anti-tumor effects and appears to inhibit tumor angiogenesis. However, many factors involved in the anti-angiogenic effect of melatonin are still under investigation. Here, we evaluate the effects of melatonin on cell viability and expression of angiogenic factors in MCF-7 and MDA-MB-231 breast cancer cells under hypoxic conditions. Cell viability was investigated by MTT and gene and protein expression of the hypoxia-inducible factor-1α (HIF-1α) and vascular endothelial growth factor (VEGF-A) were verified by qPCR and immunocytochemistry after melatonin treatment (1 mM) under hypoxic conditions. Additionally, a protein array with 20 different cytokines/factors was performed on tumor cell lysates. The results showed that 1 mM of melatonin reduced the viability of MCF-7 and MDA-MB-231 cells (p melatonin treatment during hypoxia reduced VEGF-C, VEGFR receptors (VEGFR2 and VEGFR3), matrix metalloproteinase 9 (MMP9) and Angiogenin in MCF-7 cells. In MDA-MB-231 cells, a significant decrease was observed in VEGFR2, epidermal growth factor receptor (EGFR) and Angiogenin (p melatonin acts in the regulation of angiogenic factors in breast tumor cells and suggests an anti-angiogenic activity, particularly under hypoxic conditions. PMID:25963143

  13. Sphingosine kinase 1 serves as a pro-viral factor by regulating viral RNA synthesis and nuclear export of viral ribonucleoprotein complex upon influenza virus infection.

    Directory of Open Access Journals (Sweden)

    Young-Jin Seo

    Full Text Available Influenza continues to pose a threat to humans by causing significant morbidity and mortality. Thus, it is imperative to investigate mechanisms by which influenza virus manipulates the function of host factors and cellular signal pathways. In this study, we demonstrate that influenza virus increases the expression and activation of sphingosine kinase (SK 1, which in turn regulates diverse cellular signaling pathways. Inhibition of SK suppressed virus-induced NF-κB activation and markedly reduced the synthesis of viral RNAs and proteins. Further, SK blockade interfered with activation of Ran-binding protein 3 (RanBP3, a cofactor of chromosome region maintenance 1 (CRM1, to inhibit CRM1-mediated nuclear export of the influenza viral ribonucleoprotein complex. In support of this observation, SK inhibition altered the phosphorylation of ERK, p90RSK, and AKT, which is the upstream signal of RanBP3/CRM1 activation. Collectively, these results indicate that SK is a key pro-viral factor regulating multiple cellular signal pathways triggered by influenza virus infection.

  14. Eurycomanone induce apoptosis in HepG2 cells via up-regulation of p53

    Directory of Open Access Journals (Sweden)

    Zakaria Yusmazura

    2009-06-01

    Full Text Available Abstract Background Eurycomanone is a cytotoxic compound found in Eurycoma longifolia Jack. Previous studies had noted the cytotoxic effect against various cancer cell lines. The aim of this study is to investigate the cytotoxicity against human hepato carcinoma cell in vitro and the mode of action. The cytotoxicity of eurycomanone was evaluated using MTT assay and the mode of cell death was detected by Hoechst 33258 nuclear staining and flow cytometry with Annexin-V/propidium iodide double staining. The protein expression Bax, Bcl-2, p53 and cytochrome C were studied by flow cytometry using a spesific antibody conjugated fluorescent dye to confirm the up-regulation of p53 and Bax in cancer cells. Results The findings suggested that eurycomanone was cytotoxic on cancerous liver cell, HepG2 and less toxic on normal cells Chang's liver and WLR-68. Furthermore, various methods proved that apoptosis was the mode of death in eurycomanone-treated HepG2 cells. The characteristics of apoptosis including chromatin condensation, DNA fragmentation and apoptotic bodies were found following eurycomanone treatment. This study also found that apoptotic process triggered by eurycomanone involved the up-regulation of p53 tumor suppressor protein. The up-regulation of p53 was followed by the increasing of pro-apoptotic Bax and decreasing of anti-apoptotic Bcl-2. The increased of cytochrome C levels in cytosol also results in induction of apoptosis. Conclusion The data suggest that eurycomanone was cytotoxic on HepG2 cells by inducing apoptosis through the up-regulation of p53 and Bax, and down-regulation of Bcl-2.

  15. Tumour-Derived Interleukin-1 Beta Induces Pro-inflammatory Response in Human Mesenchymal Stem Cells

    DEFF Research Database (Denmark)

    Alajez, Nehad M; Al-toub, Mashael; Almusa, Abdulaziz;

    ’ secreted factors as represented by a panel of human cancer cell lines (breast (MCF7 and MDA-MB-231); prostate (PC-3); lung (NCI-H522); colon (HT-29) and head & neck (FaDu)) on the biological characteristics of MSCs. Background Over the past several years, significant amount of research has emerged......98059 were used to inhibit TGFb, FAK, and MAPKK pathways, respectively. IL1b was measured using ELISA. Observations MSCs exposed to secreted factors present in conditioned media (CM) from FaDu, MDA-MB-231, PC-3, and NCI-H522, but not from MCF7 and HT-29, developed an elongated, spindle-shaped morphology...

  16. Secreted phospholipase A2-IIA-induced a phenotype of activated microglia in BV-2 cells requires epidermal growth factor receptor transactivation and proHB-EGF shedding

    Directory of Open Access Journals (Sweden)

    Martín Rubén

    2012-07-01

    Full Text Available Abstract Background Activation of microglia, the primary component of the innate immune response in the brain, is a hallmark of neuroinflammation in neurodegenerative disorders, including Alzheimer’s disease (AD and other pathological conditions such as stroke or CNS infection. In response to a variety of insults, microglial cells produce high levels of inflammatory cytokines that are often involved in neuronal injury, and play an important role in the recognition, engulfment, and clearance of apoptotic cells and/or invading microbes. Secreted phospholipase A2-IIA (sPLA2-IIA, an enzyme that interacts with cells involved in the systemic immune/inflammatory response, has been found up-regulated in the cerebrospinal fluid and brain of AD patients. However, despite several approaches, its functions in mediating CNS inflammation remain unknown. In the present study, the role of sPLA2-IIA was examined by investigating its direct effects on microglial cells. Methods Primary and immortalized microglial cells were stimulated by sPLA2-IIA in order to characterize the cytokine-like actions of the phospholipase. The hallmarks of activated microglia analyzed include: mitogenic response, phagocytic capabilities and induction of inflammatory mediators. In addition, we studied several of the potential molecular mechanisms involved in those events. Results The direct exposure of microglial cells to sPLA2-IIA stimulated, in a time- and dose-dependent manner, their phagocytic and proliferative capabilities. sPLA2-IIA also triggered the synthesis of the inflammatory proteins COX-2 and TNFα. In addition, EGFR phosphorylation and shedding of the membrane-anchored heparin-binding EGF-like growth factor (pro-HB-EGF ectodomain, as well as a rapid activation/phosphorylation of the classical survival proteins ERK, P70S6K and rS6 were induced upon sPLA2-IIA treatment. We further demonstrated that the presence of an EGFR inhibitor (AG1478, a matrix metalloproteinase

  17. Regulation of Arabidopsis Early Anther Development by Putative Cell-Cell Signaling Molecules and Transcriptional Regulators

    Institute of Scientific and Technical Information of China (English)

    Yu-Jin Sun; Carey LH Hord; Chang-Bin Chen; Hong Ma

    2007-01-01

    Anther development in flowering plants involves the formation of several cell types, including the tapetal and pollen mother cells. The use of genetic and molecular tools has led to the identification and characterization of genes that are critical for normal cell division and differentiation in Arabidopsis early anther development. We review here several recent studies on these genes, including the demonstration that the putative receptor protein kinases BAM1 and BAM2 together play essential roles in the control of early cell division and differentiation. In addition, we discuss the hypothesis that BAM1/2 may form a positive-negative feedback regulatory loop with a previously identified key regulator, SPOROCYTELESS (also called NOZZLE),to control the balance between sporogenous and somatic cell types in the anther. Furthermore, we summarize the isolation and functional analysis of the DYSFUNCTIONAL TAPETUM1 (DYT1) gene in promoting proper tapetal cell differentiation. Our finding that DYT1 encodes a putative transcription factor of the bHLH family, as well as relevant expression analyses, strongly supports a model that DYT1 serves as a critical link between upstream factors and downstream target genes that are critical for normal tapetum development and function. These studies, together with other recently published works, indicate that cell-cell communication and transcriptional control are key processes essential for cell fate specification in anther development.

  18. Registered report: Melanoma exosomes educate bone marrow progenitor cells toward a pro-metastatic phenotype through MET.

    Science.gov (United States)

    Lesnik, Jake; Antes, Travis; Kim, Jeewon; Griner, Erin; Pedro, Luisa

    2016-01-29

    The Reproducibility Project: Cancer Biology seeks to address growing concerns about reproducibility in scientific research by conducting replications of selected experiments from a number of high-profile papers in the field of cancer biology. The papers, which were published between 2010 and 2012, were selected on the basis of citations and Altmetric scores (Errington et al., 2014). This Registered Report describes the proposed replication plan of key experiments from "Melanoma exosomes educate bone marrow progenitor cells toward a pro-metastatic phenotype through MET" by Peinado and colleagues, published in Nature Medicine in 2012 (Peinado et al., 2012). The key experiments being replicated are from Figures 4E, as well as Supplementary Figures 1C and 5A. In these experiments, Peinado and colleagues show tumor exosomes enhance metastasis to bones and lungs, which is diminished by reducing Met expression in exosomes (Peinado et al., 2012). The Reproducibility Project: Cancer Biology is a collaboration between the Center for Open Science and Science Exchange and the results of the replications will be published in eLife.

  19. Activation of Pro-uPA is Critical for Initial Escape from the Primary Tumor and Hematogenous Dissemination of Human Carcinoma Cells

    DEFF Research Database (Denmark)

    Bekes, Erin; Deryugina, Elena; Kuprianova, Tatyana;

    2011-01-01

    dissemination may be enhanced by cleavage of stromal fibronectin by uPA-generated plasmin. Together, our findings point to inhibition of pro-uPA activation at the apex of the uPA/plasmin cascade as a therapy-valid approach to control onset of tumor escape and ensuing metastatic spread......Urokinase-type plasminogen activator (uPA) and plasmin have long been implicated in cancer progression. However, the precise contributions of the uPA/plasmin system to specific steps involved in cancer cell dissemination have not been fully established. Herein, we have employed a highly...... disseminating variant of the human PC-3 prostate carcinoma cell line, PC-hi/diss, as a prototype of aggressive carcinomas to investigate the mechanisms whereby pro-uPA activation and uPA-generated plasmin functionally contribute to specific stages of metastasis. The PC-hi/diss cells secrete and activate...

  20. Common stemness regulators of embryonic and cancer stem cells

    OpenAIRE

    Hadjimichael, Christiana; Chanoumidou, Konstantina; Papadopoulou, Natalia; Arampatzi, Panagiota; Papamatheakis, Joseph; Kretsovali, Androniki

    2015-01-01

    Pluripotency of embryonic stem cells (ESCs) and induced pluripotent stem cells is regulated by a well characterized gene transcription circuitry. The circuitry is assembled by ESC specific transcription factors, signal transducing molecules and epigenetic regulators. Growing understanding of stem-like cells, albeit of more complex phenotypes, present in tumors (cancer stem cells), provides a common conceptual and research framework for basic and applied stem cell biology. In this review, we h...

  1. Regulation of IL-2 gene expression by Siva and FOXP3 in human T cells

    Directory of Open Access Journals (Sweden)

    Hench Virginia K

    2011-09-01

    Full Text Available Abstract Background Severe autoinflammatory diseases are associated with mutations in the Foxp3 locus in both mice and humans. Foxp3 is required for the development, function, and maintenance of regulatory T cells (Tregs, a subset of CD4 cells that suppress T cell activation and inflammatory processes. Siva is a pro-apoptotic gene that is expressed across a range of tissues, including CD4 T cells. Siva interacts with three tumor necrosis factor receptor (TNFR family members that are constitutively expressed on Treg cells: CD27, GITR, and OX40. Results Here we report a biophysical interaction between FOXP3 and Siva. We mapped the interaction domains to Siva's C-terminus and to a central region of FOXP3. We showed that Siva repressed IL-2 induction by suppressing IL-2 promoter activity during T cell activation. Siva-1's repressive effect on IL-2 gene expression appears to be mediated by inhibition of NFkappaB, whereas FOXP3 repressed both NFkappaB and NFAT activity. Conclusions In summary, our data suggest that both FOXP3 and Siva function as negative regulators of IL-2 gene expression in Treg cells, via suppression of NFAT by FOXP3 and of NFkappaB by both FOXP3 and Siva. Our work contributes evidence for Siva's role as a T cell signalling mediator in addition to its known pro-apoptotic function. Though further investigations are needed, evidence for the biophysical interaction between FOXP3 and Siva invites the possibility that Siva may be important for proper Treg cell function.

  2. Pro-inflammatory interleukin-18 increases Alzheimer’s disease-associated amyloid-β production in human neuron-like cells

    Directory of Open Access Journals (Sweden)

    Sutinen Elina M

    2012-08-01

    Full Text Available Abstract Background Alzheimer’s disease (AD involves increased accumulation of amyloid-β (Aβ plaques and neurofibrillary tangles as well as neuronal loss in various regions of the neocortex. Neuroinflammation is also present, but its role in AD is not fully understood. We previously showed increased levels of pro-inflammatory cytokine interleukin-18 (IL-18 in different regions of AD brains, where it co-localized with Aβ-plaques, as well as the ability of IL-18 to increase expression of glycogen synthase kinase-3β (GSK-3β and cyclin dependent kinase 5, involved in hyperphosphorylation of tau-protein. Elevated IL-18 has been detected in several risk conditions for AD, including obesity, type-II diabetes, and cardiovascular diseases as well as in stress. Methods We differentiated SH-SY5Y neuroblastoma cells as neuron-like and exposed them to IL-18 for various times. We examined the protein levels of amyloid-β precursor protein (APP and its processing products, its cleaving enzymes, involved in amyloidogenic processing of APP, and markers of apoptosis. Results IL-18 increased protein levels of the β-site APP-cleaving enzyme BACE-1, the N-terminal fragment of presenilin-1 and slightly presenilin enhancer 2, both of which are members of the γ-secretase complex, as well as Fe65, which is a binding protein of the C-terminus of APP and one regulator for GSK-3β. IL-18 also increased APP expression and phosphorylation, which preceded increased BACE-1 levels. Further, IL-18 altered APP processing, increasing Aβ40 production in particular, which was inhibited by IL-18 binding protein. Increased levels of soluble APPβ were detected in culture medium after the IL-18 exposure. IL-18 also increased anti-apoptotic bcl-xL levels, which likely counteracted the minor increase of the pro-apoptotic caspase-3. Lactate dehydrogenase activity in culture medium was unaffected. Conclusions The IL-18 induction of BACE-1, APP processing, and Aβ is likely to be

  3. Exogenous intervention modulates expression of radiosensitive proteins predominantly by regulating cell death pathway: study in mouse spleen

    International Nuclear Information System (INIS)

    Ionizing radiation (IR) affects cellular macromolecule inclusive of proteins by regulating transcription, translation and signal transduction pathways. A combination of active principles, isolated from high altitude plant Podophyllum hexandrum, extensively studied in our group for its radioprotective efficacy in various in-vitro and in-vivo model system, has shown its high radioprotective potential. To study the modulatory effect of P. hexandrum on radiation mediated cell death pathway in mice spleen by measuring status of various apoptotic proteins in different experimental groups. Strain 'A' female mice, divided into four groups: control, drug only, radiation only (9 Gy) and drug+radiation, were sacrificed at 6,12, and 24 h post experimentation. Spleen were excised from the animal and processed for western blotting and flow cytometric based expression of various pro and anti-apoptotic proteins (bak, bax, caspase, p53, puma bcl-2 and bcl-xl). Drug is a combination of three active principles, isolated from the dried rhizome of P. hexandrum. These three components coded as A (Lignan), B (Glucoside) and C (Rutin), were mixed in 3:1:1 ratio and freshly dissolved in the DMSO at the time of experimentation. In IR group we observed time dependent up-regulation of various pro-apoptotic proteins (bak, bax, p53, puma, caspases) in contrast to untreated controls. Time dependent studies indicated expression of these proteins were maximum at 24 h in IR group. In G-003M treated and irradiated mice, pro-apoptotic proteins were found significantly down-regulated when compared to corresponding radiation treated group. In the same group, we observed that the protein responsible for the cytoprotection and antioxidation (Nrf-2, Bcl-2,) got up regulated in comparison to radiation alone group and untreated control. Present study clearly demonstrates the ability of G-003M to attenuate radiation induced cell death in mice spleen by altering the levels of pro-apoptotic and anti

  4. Searching in mother nature for anti-cancer activity: anti-proliferative and pro-apoptotic effect elicited by green barley on leukemia/lymphoma cells.

    Directory of Open Access Journals (Sweden)

    Elisa Robles-Escajeda

    Full Text Available Green barley extract (GB was investigated for possible anti-cancer activity by examining its anti-proliferative and pro-apoptotic properties on human leukemia/lymphoma cell lines. Our results indicate that GB exhibits selective anti-proliferative activity on a panel of leukemia/lymphoma cells in comparison to non-cancerous cells. Specifically, GB disrupted the cell-cycle progression within BJAB cells, as manifested by G2/M phase arrest and DNA fragmentation, and induced apoptosis, as evidenced by phosphatidylserine (PS translocation to the outer cytoplasmic membrane in two B-lineage leukemia/lymphoma cell lines. The pro-apoptotic effect of GB was found to be independent of mitochondrial depolarization, thus implicating extrinsic cell death pathways to exert its cytotoxicity. Indeed, GB elicited an increase of TNF-α production, caspase-8 and caspase-3 activation, and PARP-1 cleavage within pre-B acute lymphoblastic leukemia Nalm-6 cells. Moreover, caspase-8 and caspase-3 activation and PARP-1 cleavage were strongly inhibited/blocked by the addition of the specific caspase inhibitors Z-VAD-FMK and Ac-DEVD-CHO. Furthermore, intracellular signaling analyses determined that GB treatment enhanced constitutive activation of Lck and Src tyrosine kinases in Nalm-6 cells. Taken together, these findings indicate that GB induced preferential anti-proliferative and pro-apoptotic signals within B-lineage leukemia/lymphoma cells, as determined by the following biochemical hallmarks of apoptosis: PS externalization, enhanced release of TNF-α, caspase-8 and caspase-3 activation, PARP-1 cleavage and DNA fragmentation Our observations reveal that GB has potential as an anti-leukemia/lymphoma agent alone or in combination with standard cancer therapies and thus warrants further evaluation in vivo to support these findings.

  5. Bim and Mcl-1 exert key roles in regulating JAK2V617F cell survival

    International Nuclear Information System (INIS)

    The JAK2V617F mutation plays a major role in the pathogenesis of myeloproliferative neoplasms and is found in the vast majority of patients suffering from polycythemia vera and in roughly every second patient suffering from essential thrombocythemia or from primary myelofibrosis. The V617F mutation is thought to provide hematopoietic stem cells and myeloid progenitors with a survival and proliferation advantage. It has previously been shown that activated JAK2 promotes cell survival by upregulating the anti-apoptotic STAT5 target gene Bcl-xL. In this study, we have investigated the role of additional apoptotic players, the pro-apoptotic protein Bim as well as the anti-apoptotic protein Mcl-1. Pharmacological inhibition of JAK2/STAT5 signaling in JAK2V617F mutant SET-2 and MB-02 cells was used to study effects on signaling, cell proliferation and apoptosis by Western blot analysis, WST-1 proliferation assays and flow cytometry. Cells were transfected with siRNA oligos to deplete candidate pro- and anti-apoptotic proteins. Co-immunoprecipitation assays were performed to assess the impact of JAK2 inhibition on complexes of pro- and anti-apoptotic proteins. Treatment of JAK2V617F mutant cell lines with a JAK2 inhibitor was found to trigger Bim activation. Furthermore, Bim depletion by RNAi suppressed JAK2 inhibitor-induced cell death. Bim activation following JAK2 inhibition led to enhanced sequestration of Mcl-1, besides Bcl-xL. Importantly, Mcl-1 depletion by RNAi was sufficient to compromise JAK2V617F mutant cell viability and sensitized the cells to JAK2 inhibition. We conclude that Bim and Mcl-1 have key opposing roles in regulating JAK2V617F cell survival and propose that inactivation of aberrant JAK2 signaling leads to changes in Bim complexes that trigger cell death. Thus, further preclinical evaluation of combinations of JAK2 inhibitors with Bcl-2 family antagonists that also tackle Mcl-1, besides Bcl-xL, is warranted to assess the therapeutic potential for

  6. Bim and Mcl-1 exert key roles in regulating JAK2V617F cell survival

    Directory of Open Access Journals (Sweden)

    Guthy Daniel A

    2011-01-01

    Full Text Available Abstract Background The JAK2V617F mutation plays a major role in the pathogenesis of myeloproliferative neoplasms and is found in the vast majority of patients suffering from polycythemia vera and in roughly every second patient suffering from essential thrombocythemia or from primary myelofibrosis. The V617F mutation is thought to provide hematopoietic stem cells and myeloid progenitors with a survival and proliferation advantage. It has previously been shown that activated JAK2 promotes cell survival by upregulating the anti-apoptotic STAT5 target gene Bcl-xL. In this study, we have investigated the role of additional apoptotic players, the pro-apoptotic protein Bim as well as the anti-apoptotic protein Mcl-1. Methods Pharmacological inhibition of JAK2/STAT5 signaling in JAK2V617F mutant SET-2 and MB-02 cells was used to study effects on signaling, cell proliferation and apoptosis by Western blot analysis, WST-1 proliferation assays and flow cytometry. Cells were transfected with siRNA oligos to deplete candidate pro- and anti-apoptotic proteins. Co-immunoprecipitation assays were performed to assess the impact of JAK2 inhibition on complexes of pro- and anti-apoptotic proteins. Results Treatment of JAK2V617F mutant cell lines with a JAK2 inhibitor was found to trigger Bim activation. Furthermore, Bim depletion by RNAi suppressed JAK2 inhibitor-induced cell death. Bim activation following JAK2 inhibition led to enhanced sequestration of Mcl-1, besides Bcl-xL. Importantly, Mcl-1 depletion by RNAi was sufficient to compromise JAK2V617F mutant cell viability and sensitized the cells to JAK2 inhibition. Conclusions We conclude that Bim and Mcl-1 have key opposing roles in regulating JAK2V617F cell survival and propose that inactivation of aberrant JAK2 signaling leads to changes in Bim complexes that trigger cell death. Thus, further preclinical evaluation of combinations of JAK2 inhibitors with Bcl-2 family antagonists that also tackle Mcl-1

  7. Cytotoxic and Pro-Apoptotic Effects of Cassane Diterpenoids from the Seeds of Caesalpinia sappan in Cancer Cells.

    Science.gov (United States)

    Bao, Han; Zhang, Le-Le; Liu, Qian-Yu; Feng, Lu; Ye, Yang; Lu, Jin-Jian; Lin, Li-Gen

    2016-01-01

    The chemical study on the seeds of Caesalpinia sappan led to the isolation of five new cassane diterpenoids, phanginins R‒T (1-3) and caesalsappanins M and N (4 and 5), together with seven known compounds 6-12. Their structures were elucidated on the basis of NMR and HRESIMS analyses. The absolute configurations of compounds 1 and 4 were determined by the corresponding CD spectra. All the isolated compounds were tested for their cytotoxicity against ovarian cancer A2780 and HEY, gastric cancer AGS, and non-small cell lung cancer A549 cells. Compound 1 displayed significant toxicity against the four cell lines with the IC50 values of 9.9 ± 1.6 µM, 12.2 ± 6.5 µM, 5.3 ± 1.9 µM, and 12.3 ± 3.1 µM, respectively. Compound 1 induced G1 phase cell cycle arrest in A2780 cells. Furthermore, compound 1 dose-dependently induced A2780 cells apoptosis as evidenced by Hoechst 33342 staining, Annexin V positive cells, the up-regulated cleaved-PARP and the enhanced Bax/Bcl-2 ratio. What's more, compound 1 also promoted the expression of the tumor suppressor p53 protein. These findings indicate that cassane diterpenoids might have potential as anti-cancer agents, and further in vivo animal studies and structural modification investigation are needed. PMID:27322234

  8. Cytotoxic and Pro-Apoptotic Effects of Cassane Diterpenoids from the Seeds of Caesalpinia sappan in Cancer Cells

    Directory of Open Access Journals (Sweden)

    Han Bao

    2016-06-01

    Full Text Available The chemical study on the seeds of Caesalpinia sappan led to the isolation of five new cassane diterpenoids, phanginins R‒T (1–3 and caesalsappanins M and N (4 and 5, together with seven known compounds 6–12. Their structures were elucidated on the basis of NMR and HRESIMS analyses. The absolute configurations of compounds 1 and 4 were determined by the corresponding CD spectra. All the isolated compounds were tested for their cytotoxicity against ovarian cancer A2780 and HEY, gastric cancer AGS, and non-small cell lung cancer A549 cells. Compound 1 displayed significant toxicity against the four cell lines with the IC50 values of 9.9 ± 1.6 µM, 12.2 ± 6.5 µM, 5.3 ± 1.9 µM, and 12.3 ± 3.1 µM, respectively. Compound 1 induced G1 phase cell cycle arrest in A2780 cells. Furthermore, compound 1 dose-dependently induced A2780 cells apoptosis as evidenced by Hoechst 33342 staining, Annexin V positive cells, the up-regulated cleaved-PARP and the enhanced Bax/Bcl-2 ratio. What’s more, compound 1 also promoted the expression of the tumor suppressor p53 protein. These findings indicate that cassane diterpenoids might have potential as anti-cancer agents, and further in vivo animal studies and structural modification investigation are needed.

  9. Nitric oxide modulates hypoxic pulmonary smooth muscle cell proliferation and apoptosis by regulating carbon monoxide pathway

    Institute of Scientific and Technical Information of China (English)

    Yan-fei WANG; Hong TIAN; Chao-shu TANG; Hong-fang JIN; Jun-bao DU

    2007-01-01

    Aim: To explore the role of carbon monoxide (CO) in the regulation of hypoxic pulmonary artery smooth muscle cell (PASMC) proliferation and apoptosis by nitric oxide (NO). Methods: PASMC of Wistar rats was cultured in vitro in the presence of a NO donor, sodium nitroprusside, or an inhibitor of heme oxygenase (HO), zinc protoporphyrin-IX, or under both normoxic and hypoxic conditions.Nitrite and carboxyhemoglobin in PASMC medium were detected with spectrophotometry. The proliferating and apoptotic percentage of PASMC was measured by flow cytometry. The expression of HO-1 mRNA in PASMC was analyzed by fluorescent real-time quantitative PCR, and the proliferating cell nuclear antigen and caspase-3 were examined by immunocytochemical analysis. Results: The results showed that hypoxia suppressed NO generation from PASMC, which promoted hypoxic PASMC proliferation and induced apoptosis. Meanwhile, hy-poxia induced HO-1 expression in PASMC and promoted CO production from PASMC, which inhibited PASMC proliferation and regulated PASMC apoptosis. NO upregulated the expression of HO-1 mRNA in hypoxic PASMC; NO also inhib-ited proliferation and promoted apoptosis of hypoxic PASMC, possibly by regu-lating the production of CO. Conclusion: The results indicated that CO could inhibit proliferation and regulate apoptosis of PASMC, and NO inhibited prolifera-tion and promoted apoptosis of hypoxic PASMC, possibly by regulating the pro-duction of CO.

  10. Nitric oxide synthase 2 is involved in the pro-tumorigenic potential of γδ17 T cells in melanoma.

    Science.gov (United States)

    Douguet, Laetitia; Bod, Lloyd; Lengagne, Renée; Labarthe, Laura; Kato, Masashi; Avril, Marie-Françoise; Prévost-Blondel, Armelle

    2016-08-01

    γδ T lymphocytes may exert either protective or tumor-promoting functions in cancer, mostly based on their polarization toward interferon (IFN)-γ or interleukin (IL)-17 productions, respectively. Here, we demonstrate that γδ T cells accelerate the spontaneous metastatic melanoma development in a model of transgenic mice for the human RET oncogene (Ret mice). We identify unanticipated roles of inducible nitric oxide synthase (NOS2) in favoring the recruitment of pro-tumor γδ T cells within the primary tumor. γδ T cells isolated from Ret mice deficient for NOS2 produced more IFNγ and less IL-17 than their counterparts from Ret mice. By supporting IL-17 production by γδ T cells, NOS2 leads to the recruitment of polymorphonuclear myeloid-derived suppressor cells (PMN-MDSCs) and metastasis formation. NOS2 also reduces the cytotoxicity of γδ T cells toward melanoma cells. Finally, we detected NOS2 expressing γδ T cells in the primary tumor and tumor-draining lymph nodes in Ret mice, but also in human melanoma. Overall our results support that this NOS2 autocrine expression is responsible for the polarization of γδ T cells toward a pro-tumor profile.

  11. Matrix-degrading and pro-inflammatory changes in human vascular endothelial cells exposed to cigarette smoke condensate.

    Science.gov (United States)

    Nordskog, Brian K; Blixt, Allison D; Morgan, Walter T; Fields, Wanda R; Hellmann, Gary M

    2003-01-01

    Cigarette smoking has been associated with an increase in the severity and prevalence of atherosclerosis in the abdominal aorta. To begin our investigation of this finding, we used an integrated approach combining gene expression profiling, protein analysis, cytokine measurements, and cytotoxicity determinations to examine molecular responses of cultured human aortic and coronary endothelial cells exposed to cigarette smoke condensate (CSC) and nicotine. Exposure of endothelial cells to CSC (30 and 60 microg/mL TPM) for 24 h resulted in minimal cytotoxicity, and the upregulation of genes involved in matrix degradation (MMP-1, MMP-8, and MMP-9), xenobiotic metabolism (HO-1 and CYP1A2), and downregulation of genes involved in cell cycle regulation (including TOP2A, CCNB1, CCNA, CDKN3). Exposure of cells to a high physiological concentration of nicotine resulted in few differentially expressed genes. Immunoblot analysis of proteins selected from genes shown to be differentially regulated by microarray analysis revealed similar responses. Finally, a number of inflammatory cytokines measured in culture media were elevated in response to CSC. Together, these results describe a complex proinflammatory response, possibly mediating the recruitment of leukocytes through cytokine signaling. Additionally, fibrous cap destabilization may be facilitated by matrix metalloproteinase upregulation. PMID:14501029

  12. Matrix-degrading and pro-inflammatory changes in human vascular endothelial cells exposed to cigarette smoke condensate.

    Science.gov (United States)

    Nordskog, Brian K; Blixt, Allison D; Morgan, Walter T; Fields, Wanda R; Hellmann, Gary M

    2003-01-01

    Cigarette smoking has been associated with an increase in the severity and prevalence of atherosclerosis in the abdominal aorta. To begin our investigation of this finding, we used an integrated approach combining gene expression profiling, protein analysis, cytokine measurements, and cytotoxicity determinations to examine molecular responses of cultured human aortic and coronary endothelial cells exposed to cigarette smoke condensate (CSC) and nicotine. Exposure of endothelial cells to CSC (30 and 60 microg/mL TPM) for 24 h resulted in minimal cytotoxicity, and the upregulation of genes involved in matrix degradation (MMP-1, MMP-8, and MMP-9), xenobiotic metabolism (HO-1 and CYP1A2), and downregulation of genes involved in cell cycle regulation (including TOP2A, CCNB1, CCNA, CDKN3). Exposure of cells to a high physiological concentration of nicotine resulted in few differentially expressed genes. Immunoblot analysis of proteins selected from genes shown to be differentially regulated by microarray analysis revealed similar responses. Finally, a number of inflammatory cytokines measured in culture media were elevated in response to CSC. Together, these results describe a complex proinflammatory response, possibly mediating the recruitment of leukocytes through cytokine signaling. Additionally, fibrous cap destabilization may be facilitated by matrix metalloproteinase upregulation.

  13. Regulation of lovastatin on a key inflammation-related microRNA in myocardial cells

    Institute of Scientific and Technical Information of China (English)

    Guo Weizao; Liu Huichen; Li Lin; Yang Man; Du Aihua

    2014-01-01

    Background Advances in the understanding of cardiovascular pathogenesis have highlighted that inflammation plays a central role in athemsclemtic coronary heart disease.Therefore,exploring pharmacologically based anti-inflammatory treatments to be used in cardiovascular therapeutics is worthwhile to promote the discovery of novel ways of treating cardiovascular disorders.Methods The myocardial cell line H9c2(2-1) was exposed to lipopolysacchadde (LPS) in culture and resulted in a cellular pro-inflammation status,miR-21 microRNA levels were detected using quantitative real-time polymerase chain reaction (Q-RT-PCR).The influence of Iovastatin on miR-21 under normal and pro-inflammatory conditions was tested after being added to the cell culture mixture for 24 hours.Conditional gene function of two predicted cardiovascular system relevant downstream targets of miR-21,protein phosphatase 1 regulatory subunit 3A (PPP1R3A) and signal transducer and activator of transcription 3 (STAT3),were analyzed with immunoblotting.Results Forty-eight hours of LPS treatment significantly increased the miR-21 to 170.71%±34.32% of control levels (P=0.002).Co-treatment with Iovastatin for 24 hours before harvesting attenuated the up-regulation of miR-21 (P=0.013).Twenty-four hours of Iovastatin exposure up-regulated PPP1R3A to 143.85%±21.89% of control levels in cardiomyocytes (P=0.023).Lovastatin up-regulated the phosphorylation level of STAT3 compared to the background LPS pretreatment (P=0.0077),this effect was significantly (P=0.018) blunted when miR-21 was functionally inhibited.Conclusions miR-21 plays a major role in the regulation of the cellular anti-inflammation effects of Iovastatin.

  14. Cellular Pressure and Volume Regulation and Implications for Cell Mechanics

    OpenAIRE

    Jiang, Hongyuan; Sun, Sean X.

    2013-01-01

    In eukaryotic cells, small changes in cell volume can serve as important signals for cell proliferation, death, and migration. Volume and shape regulation also directly impacts the mechanics of cells and tissues. Here, we develop a mathematical model of cellular volume and pressure regulation, incorporating essential elements such as water permeation, mechanosensitive channels, active ion pumps, and active stresses in the cortex. The model can fully explain recent experimental data, and it pr...

  15. LGR4 and LGR5 Regulate Hair Cell Differentiation in the Sensory Epithelium of the Developing Mouse Cochlea

    Science.gov (United States)

    Żak, Magdalena; van Oort, Thijs; Hendriksen, Ferry G.; Garcia, Marie-Isabelle; Vassart, Gilbert; Grolman, Wilko

    2016-01-01

    In the developing cochlea, Wnt/β-catenin signaling positively regulates the proliferation of precursors and promotes the formation of hair cells by up-regulating Atoh1 expression. Not much, however, is known about the regulation of Wnt/β-catenin activity in the cochlea. In multiple tissues, the activity of Wnt/β-catenin signaling is modulated by an interaction between LGR receptors and their ligands from the R-spondin family. The deficiency in Lgr4 and Lgr5 genes leads to developmental malformations and lethality. Using the Lgr5 knock-in mouse line we show that loss of LGR5 function increases Wnt/β-catenin activity in the embryonic cochlea, resulting in a mild overproduction of inner and outer hair cells (OHC). Supernumerary hair cells are likely formed due to an up-regulation of the “pro-hair cell” transcription factors Atoh1, Nhlh1, and Pou4f3. Using a hypomorphic Lgr4 mouse model we showed a mild overproduction of OHCs in the heterozygous and homozygous Lgr4 mice. The loss of LGR4 function prolonged the proliferation in the mid-basal turn of E13 cochleae, causing an increase in the number of SOX2-positive precursor cells within the pro-sensory domain. The premature differentiation of hair cells progressed in a medial to lateral gradient in Lgr4 deficient embryos. No significant up-regulation of Atoh1 was observed following Lgr4 deletion. Altogether, our findings suggest that LGR4 and LGR5 play an important role in the regulation of hair cell differentiation in the embryonic cochlea.

  16. PU.1-Bim axis is involved in Trichostatin A-induced apoptosis in murine pro-B lymphoma FL5.12 cells.

    Science.gov (United States)

    Li, Chao; Tao, Yufen; Li, Chao; Liu, Bo; Liu, Jiansheng; Wang, Guanlin; Liu, Hongqi

    2016-09-01

    Trichostatin A (TSA) is a well-known histone deacetylases (HDACs) inhibitor that has been reported to show potent anti-tumor capabilities in some types of cancer cell lines. However, detailed mechanism of TSA action on lymphoma remains to be described. In the present study, anti-proliferative effects of TSA were investigated using a murine pro-B lymphoma cell line FL5.12. MTT assay revealed that TSA potently inhibited the proliferation of FL5.12 cells in a time- and dose-dependent manner. Bright-field microscopy of FL5.12 cells showed apoptotic morphology at 24 h after TSA treatment. Consistently, TSA treatment led to DNA fragmentation and increased the protein levels of cleaved caspase 3 and PARP as revealed by western blot analysis. To explore the underlying mechanism of TSA-induced apoptosis of FL5.12 cells, we further analyzed the hematopoietic transcription factor Purine Rich Box-1 (PU.1) by western blot analysis. TSA treatment resulted in the inhibition of PU.1 in FL5.12 cells. In contrast, apoptotic protein Bim was induced by TSA, which was inversely correlated with the survival of FL5.12 cells. These results suggest the possible mechanism of TSA-induced apoptosis in murine pro-B lymphoma FL5.12 cells via the PU.1-Bim axis. PMID:27451443

  17. Cyanide-induced Death of Dopaminergic Cells is Mediated by Uncoupling Protein-2 Up-regulation and Reduced Bcl-2 Expression

    OpenAIRE

    Zhang, X.; Li, L.; Zhang, L.; Borowitz, J.L.; Isom, G.E.

    2009-01-01

    Cyanide is a potent inhibitor of mitochondrial oxidative metabolism and produces mitochondria-mediated death of dopaminergic neurons and sublethal intoxications are associated with a Parkinson-like syndrome. Cyanide toxicity is enhanced when mitochondrial uncoupling is stimulated following up-regulation of uncoupling protein-2 (UCP-2). In this study, the role of a pro-survival protein, Bcl-2, in cyanide-mediated cell death was determined in a rat dopaminergic immortalized mesencephalic cell l...

  18. Regulation of Water in Plant Cells

    Science.gov (United States)

    Kowles, Richard V.

    2010-01-01

    Cell water relationships are important topics to be included in cell biology courses. Differences exist in the control of water relationships in plant cells relative to control in animal cells. One important reason for these differences is that turgor pressure is a consideration in plant cells. Diffusion and osmosis are the underlying factors…

  19. Glycogen synthase kinase 3 regulates expression of nuclear factor-erythroid-2 related transcription factor-1 (Nrf1) and inhibits pro-survival function of Nrf1

    International Nuclear Information System (INIS)

    Nuclear factor E2-related factor-1 (Nrf1) is a basic leucine zipper transcription factor that is known to regulate antioxidant and cytoprotective gene expression. It was recently shown that Nrf1 is regulated by SCF–Fbw7 ubiquitin ligase. However our knowledge of upstream signals that targets Nrf1 for degradation by the UPS is not known. We report here that Nrf1 expression is negatively regulated by glycogen synthase kinase 3 (GSK3) in Fbw7-dependent manner. We show that GSK3 interacts with Nrf1 and phosphorylates the Cdc4 phosphodegron domain (CPD) in Nrf1. Mutation of serine residue in the CPD of Nrf1 to alanine (S350A), blocks Nrf1 from phosphorylation by GSK3, and stabilizes Nrf1. Knockdown of Nrf1 and expression of a constitutively active form of GSK3 results in increased apoptosis in neuronal cells in response to ER stress, while expression of the GSK3 phosphorylation resistant S350A–Nrf1 attenuates apoptotic cell death. Together these data suggest that GSK3 regulates Nrf1 expression and cell survival function in response to stress activation. Highlights: • The effect of GSK3 on Nrf1 expression was examined. • GSK3 destabilizes Nrf1 protein via Fbw7 ubiquitin ligase. • GSK3 binds and phosphorylates Nrf1. • Protection from stress-induced apoptosis by Nrf1 is inhibited by GSK3

  20. PKCη confers protection against apoptosis by inhibiting the pro-apoptotic JNK activity in MCF-7 cells

    International Nuclear Information System (INIS)

    Apoptosis is frequently regulated by different protein kinases including protein kinase C family enzymes. Both inhibitory and stimulatory effects were demonstrated for several of the different PKC isoforms. Here we show that the novel PKC isoform, PKCη, confers protection against apoptosis induced by the DNA damaging agents, UVC irradiation and the anti-cancer drug - Camptothecin, of the breast epithelial adenocarcinoma MCF-7 cells. The induced expression of PKCη in MCF-7 cells, under the control of the tetracycline-responsive promoter, resulted in increased cell survival and inhibition of cleavage of the apoptotic marker PARP-1. Activation of caspase-7 and 9 and the release of cytochrome c were also inhibited by the inducible expression of PKCη. Furthermore, JNK activity, required for apoptosis in MCF-7, as indicated by the inhibition of both caspase-7 cleavage and cytochrome c release from the mitochondria in the presence of the JNK inhibitor SP600125, was also suppressed by PKCη expression. Hence, in contrast to most PKC isoforms enhancing JNK activation, our studies show that PKCη is an anti-apoptotic protein, acting as a negative regulator of JNK activity. Thus, PKCη could represent a target for intervention aimed to reduce resistance to anti-cancer treatments.

  1. Role of autophagy in the regulation of epithelial cell junctions.

    Science.gov (United States)

    Nighot, Prashant; Ma, Thomas

    2016-01-01

    Autophagy is a cell survival mechanism by which bulk cytoplasmic material, including soluble macromolecules and organelles, is targeted for lysosomal degradation. The role of autophagy in diverse cellular processes such as metabolic stress, neurodegeneration, cancer, aging, immunity, and inflammatory diseases is being increasingly recognized. Epithelial cell junctions play an integral role in the cell homeostasis via physical binding, regulating paracellular pathways, integrating extracellular cues into intracellular signaling, and cell-cell communication. Recent data indicates that cell junction composition is very dynamic. The junctional protein complexes are actively regulated in response to various intra- and extra-cellular clues by intracellular trafficking and degradation pathways. This review discusses the recent and emerging information on how autophagy regulates various epithelial cell junctions. The knowledge of autophagy regulation of epithelial junctions will provide further rationale for targeting autophagy in a wide variety of human disease conditions. PMID:27583189

  2. Gangliosides in cell recognition and membrane protein regulation

    OpenAIRE

    Lopez, Pablo H. H.; Schnaar, Ronald L.

    2009-01-01

    Gangliosides, sialic acid-bearing glycosphingolipids, are expressed on all vertebrate cells, and are the major glycans on nerve cells. They are anchored to the plasma membrane through their ceramide lipids with their varied glycans extending into the extracellular space. Through sugar-specific interactions with glycan binding proteins on apposing cells, gangliosides function as receptors in cell-cell recognition, regulating natural killer cell cytotoxicity via Siglec-7 binding, myelin-axon in...

  3. Tetracycline regulator expression alters the transcriptional program of mammalian cells

    OpenAIRE

    Hackl, Hubert; Rommer, Anna; Konrad, Torsten A; Nassimbeni, Christine; Wieser, Rotraud

    2010-01-01

    Tetracycline regulated ectopic gene expression is a widely used tool to study gene function. However, the tetracycline regulator (tetR) itself has been reported to cause certain phenotypic changes in mammalian cells. We, therefore, asked whether human myeloid U937 cells expressing the tetR in an autoregulated manner would exhibit alterations in gene expression upon removal of tetracycline.

  4. The mRNA expression of pro- and anti-inflammatory cytokines in T regulatory cells in children with type 1 diabetes.

    Directory of Open Access Journals (Sweden)

    Maria Górska

    2010-06-01

    Full Text Available Type 1 diabetes mellitus (T1DM is caused by the autoimmune-mediated destruction of insulin-producing beta cells in the pancreas. T regulatory cells (Tregs represent an active mechanism of suppressing autoreactive T cells that escape central tolerance. The aim of our study was to test the hypothesis that T regulatory cells express pro- and anti-inflammatory cytokines, elements of cytotoxicity and OX40/4-1BB molecules. The examined group consisted of 50 children with T1DM. Fifty two healthy individuals (control group were enrolled into the study. A flow cytometric analysis of T-cell subpopulations was performed using the following markers: anti-CD3, anti-CD4, anti-CD25, anti-CD127, anti-CD134 and anti-CD137. Concurrently with the flow cytometric assessment of Tregs we separated CD4+CD25+CD127dim/- cells for further mRNA analysis. mRNA levels for transcription factor FoxP3, pro- and anti-inflammatory cytokines (interferon gamma, interleukin-2, interleukin-4, interleukin-10, transforming growth factor beta1 and tumor necrosis factor alpha, activatory molecules (OX40, 4-1BB and elements of cytotoxicity (granzyme B, perforin 1 were determined by real-time PCR technique. We found no alterations in the frequency of CD4+CD25highCD127low cells between diabetic and control children. Treg cells expressed mRNA for pro- and anti-inflammatory cytokines. Lower OX40 and higher 4-1BB mRNA but not protein levels in Treg cells in diabetic patients compared to the healthy children were noted. Our observations confirm the presence of mRNA for pro- and anti-inflammatory cytokines in CD4+CD25+CD127dim/- cells in the peripheral blood of children with T1DM. Further studies with the goal of developing new strategies to potentiate Treg function in autoimmune diseases are warranted.

  5. The Effect of Pro-Neurogenic Gene Expression on Adult Subventricular Zone Precursor Cell Recruitment and Fate Determination After Excitotoxic Brain Injury

    Science.gov (United States)

    Jones, Kathryn S; Connor, Bronwen J

    2016-01-01

    Despite the presence of on-going neurogenesis in the adult mammalian brain, neurons are generally not replaced after injury. Using a rodent model of excitotoxic cell loss and retroviral (RV) lineage tracing, we previously demonstrated transient recruitment of precursor cells from the subventricular zone (SVZ) into the lesioned striatum. In the current study we determined that these cells included migratory neuroblasts and oligodendrocyte precursor cells (OPC), with the predominant response from glial cells. We attempted to override this glial response by ectopic expression of the pro-neurogenic genes Pax6 or Dlx2 in the adult rat SVZ following quinolinic acid lesioning. RV-Dlx2 over-expression stimulated repair at a previously non-neurogenic time point by enhancing neuroblast recruitment and the percentage of cells that retained a neuronal fate within the lesioned area, compared to RV-GFP controls. RV-Pax6 expression was unsuccessful at inhibiting glial fate and intriguingly, increased OPC cell numbers with no change in neuronal recruitment. These findings suggest that gene choice is important when attempting to augment endogenous repair as the lesioned environment can overcome pro-neurogenic gene expression. Dlx2 over-expression however was able to partially overcome an anti-neuronal environment and therefore is a promising candidate for further study of striatal regeneration.

  6. Coordinated regulation of myeloid cells by tumours.

    Science.gov (United States)

    Gabrilovich, Dmitry I; Ostrand-Rosenberg, Suzanne; Bronte, Vincenzo

    2012-03-22

    Myeloid cells are the most abundant nucleated haematopoietic cells in the human body and are a collection of distinct cell populations with many diverse functions. The three groups of terminally differentiated myeloid cells - macrophages, dendritic cells and granulocytes - are essential for the normal function of both the innate and adaptive immune systems. Mounting evidence indicates that the tumour microenvironment alters myeloid cells and can convert them into potent immunosuppressive cells. Here, we consider myeloid cells as an intricately connected, complex, single system and we focus on how tumours manipulate the myeloid system to evade the host immune response.

  7. Pro farmer, pro industry land acquisition act

    OpenAIRE

    Varma, Vijaya Krushna Varma

    2015-01-01

    Varma suggests a new land acquisition act which is pro farmer, pro industry and pro development, and which leads to faster economic growth. My suggestions for new land acquisition act consist of 6 segments 1. Land acquisition 2. Categorisation of Land allotment 3. Time table for completion of projects 4. Compensation 5. Rehabilitation 6. Land management

  8. The Role of Nrf2 and Cytoprotection in Regulating Chemotherapy Resistance of Human Leukemia Cells

    Energy Technology Data Exchange (ETDEWEB)

    Rushworth, Stuart A., E-mail: s.rushworth@uea.ac.uk; MacEwan, David J. [School of Pharmacy, University of East Anglia, Norwich NR4 7TJ (United Kingdom)

    2011-03-29

    The Nrf2 anti-oxidant response element (ARE) pathway plays an important role in regulating cellular anti-oxidants. Under normal cellular conditions Nrf2 can be described as an anti-tumor molecule due to its induction of cytoprotective genes which protect cells from electrophile and oxidative damage. However in cancerous cells, Nrf2 takes on a pro-tumoral identity as the same cytoprotective genes can enhance resistance of those cancer cells to chemotherapeutic drugs. Such Nrf2-regulated cytoprotective genes include heme oxygenase-1 (HO-1), which has been shown to protect human leukemia cells from apoptotic signals. Moreover, a relationship between Nrf2 and the nuclear factor-κB (NF-κB) signaling pathway has been recently identified, and is now recognized as an important cross-talk mechanism by which Nrf2 can overcome apoptosis and provide cells with reduced sensitivity towards chemotherapeutic agents. In recent years a number of important research papers have highlighted the role of Nrf2 in providing protection against both current and new chemotherapeutic drugs in blood cancer. This review will provide a synopsis of these research papers with an aim to carefully consider if targeting Nrf2 in combination with current or new chemotherapeutics is a viable strategy in the more effective treatment of blood cancers.

  9. Regulation of Exacerbated Immune Responses in Human Peripheral Blood Cells by Hydrolysed Egg White Proteins.

    Science.gov (United States)

    Lozano-Ojalvo, Daniel; Molina, Elena; López-Fandiño, Rosina

    2016-01-01

    The anti-allergic potential of egg white protein hydrolysates (from ovalbumin, lysozyme and ovomucoid) was evaluated as their ability to hinder cytokine and IgE production by Th2-skewed human peripheral blood mononuclear cells (PBMCs), as well as the release of pro-inflammatory factors and generation of reactive oxygen species from Th1-stimulated peripheral blood leukocytes (PBLs). The binding to IgE of egg allergic patients was determined and the peptides present in the hydrolysates were identified. The hydrolysates with alcalase down-regulated the production of Th2-biased cytokines and the secretion of IgE to the culture media of Th2-skewed PBMCs, and they significantly neutralized oxidative stress in PBLs. The hydrolysates of ovalbumin and ovomucoid with pepsin helped to re-establish the Th1/Th2 balance in Th2-biased PBMCs, while they also inhibited the release of pro-inflammatory mediators and reduced oxidative stress in PBLs treated with inflammatory stimuli. The hydrolysates with alcalase, in addition to equilibrating Th2 differentiation, exhibited a low IgE-binding. Therefore, they would elicit mild allergic reactions while retaining T cell-stimulating abilities, which might correlate with an anti-allergic benefit. PMID:27007699

  10. Regulation of Exacerbated Immune Responses in Human Peripheral Blood Cells by Hydrolysed Egg White Proteins.

    Directory of Open Access Journals (Sweden)

    Daniel Lozano-Ojalvo

    Full Text Available The anti-allergic potential of egg white protein hydrolysates (from ovalbumin, lysozyme and ovomucoid was evaluated as their ability to hinder cytokine and IgE production by Th2-skewed human peripheral blood mononuclear cells (PBMCs, as well as the release of pro-inflammatory factors and generation of reactive oxygen species from Th1-stimulated peripheral blood leukocytes (PBLs. The binding to IgE of egg allergic patients was determined and the peptides present in the hydrolysates were identified. The hydrolysates with alcalase down-regulated the production of Th2-biased cytokines and the secretion of IgE to the culture media of Th2-skewed PBMCs, and they significantly neutralized oxidative stress in PBLs. The hydrolysates of ovalbumin and ovomucoid with pepsin helped to re-establish the Th1/Th2 balance in Th2-biased PBMCs, while they also inhibited the release of pro-inflammatory mediators and reduced oxidative stress in PBLs treated with inflammatory stimuli. The hydrolysates with alcalase, in addition to equilibrating Th2 differentiation, exhibited a low IgE-binding. Therefore, they would elicit mild allergic reactions while retaining T cell-stimulating abilities, which might correlate with an anti-allergic benefit.

  11. Genetic regulation of programmed cell death in Drosophila

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    Programmed cell death plays an important role in maintaining homeostasis during animal development, and has been conserved in animals as different as nematodes and humans. Recent studies of Drosophila have provided valuable information toward our understanding of genetic regulation of death. Different signals trigger the novel death regulators rpr, hid, and grim, that utilize the evolutionarily conserved iap and ark genes to modulate caspase function. Subsequent removal of dying cells also appears to be accomplished by conserved mechanisms. The similarity between Drosophila and human in cell death signaling pathways illustrate the promise of fruit flies as a model system to elucidate the mechanisms underlying regulation of programmed cell death.

  12. Conserved Pro-Glu (PE) and Pro-Pro-Glu (PPE) Protein Domains Target LipY Lipases of Pathogenic Mycobacteria to the Cell Surface via the ESX-5 Pathway*

    Science.gov (United States)

    Daleke, Maria H.; Cascioferro, Alessandro; de Punder, Karin; Ummels, Roy; Abdallah, Abdallah M.; van der Wel, Nicole; Peters, Peter J.; Luirink, Joen; Manganelli, Riccardo; Bitter, Wilbert

    2011-01-01

    The type VII secretion system ESX-5 is a major pathway for export of PE and PPE proteins in pathogenic mycobacteria. These mycobacteria-specific protein families are characterized by conserved N-terminal domains of 100 and 180 amino acids, which contain the proline-glutamic acid (PE) and proline-proline-glutamic acid (PPE) motifs after which they are named. Here we investigated secretion of the triacylglycerol lipase LipY, which in fast-growing mycobacteria contains a signal sequence, but in slow-growing species appears to have replaced the signal peptide with a PE or PPE domain. Selected LipY homologues were expressed in wild-type Mycobacterium marinum and its corresponding ESX-5 mutant, and localization of the proteins was investigated by immunoblotting and electron microscopy. Our study shows that Mycobacterium tuberculosis PE-LipY (LipYtub) and M. marinum PPE-LipY (LipYmar) are both secreted to the bacterial surface in an ESX-5-dependent fashion. After transport, the PE/PPE domains are removed by proteolytic cleavage. In contrast, Mycobacterium gilvum LipY, which has a signal sequence, is not transported to the cell surface. Furthermore, we show that LipYtub and LipYmar require their respective PE and PPE domains for ESX-5-dependent secretion. The role of the PE domain in ESX-5 secretion was confirmed in a whole cell lipase assay, in which wild-type bacteria expressing full-length LipYtub, but not LipYtub lacking its PE domain, were shown to hydrolyze extracellular lipids. In conclusion, both PE and PPE domains contain a signal required for secretion of LipY by the ESX-5 system, and these domains are proteolytically removed upon translocation. PMID:21471225

  13. CNS expression of B7-H1 regulates pro-inflammatory cytokine production and alters severity of Theiler's virus-induced demyelinating disease.

    Directory of Open Access Journals (Sweden)

    D'Anne S Duncan

    Full Text Available The CNS is a unique organ due to its limited capacity for immune surveillance. As macrophages of the CNS, microglia represent a population originally known for the ability to assist neuronal stability, are now appreciated for their role in initiating and regulating immune responses in the brain. Theiler's murine encephalomyelitis virus (TMEV-induced demyelinating disease is a mouse model of multiple sclerosis (MS. In response to TMEV infection in vitro, microglia produce high levels of inflammatory cytokines and chemokines, and are efficient antigen-presenting cells (APCs for activating CD4(+ T cells. However, the regulatory function of microglia and other CNS-infiltrating APCs in response to TMEV in vivo remains unclear. Here we demonstrate that microglia increase expression of proliferating cell nuclear antigen (PCNA, and phenotypically express high levels of major histocompatibility complex (MHC-Class I and II in response to acute infection with TMEV in SJL/J mice. Microglia increase expression of the inhibitory co-stimulatory molecule, B7-H1 as early as day 5 post-infection, while CNS-infiltrating CD11b(+CD11c(-CD45(HIGH monocytes/macrophages and CD11b(+CD11c(+CD45(HIGH dendritic cells upregulate expression of B7-H1 by day 3 post-infection. Utilizing a neutralizing antibody, we demonstrate that B7-H1 negatively regulates TMEV-specific ex vivo production of interferon (IFN-γ, interleukin (IL-17, IL-10, and IL-2 from CD4(+ and CD8(+ T cells. In vivo blockade of B7-H1 in SJL/J mice significantly exacerbates clinical disease symptoms during the chronic autoimmune stage of TMEV-IDD, but only has minimal effects on viral clearance. Collectively, these results suggest that CNS expression of B7-H1 regulates activation of TMEV-specific T cells, which affects protection against TMEV-IDD.

  14. Pro-inflammatory wnt5a and anti-inflammatory sFRP5 are differentially regulated by nutritional factors in obese human subjects.

    Directory of Open Access Journals (Sweden)

    Dominik M Schulte

    Full Text Available BACKGROUND: Obesity is associated with macrophage infiltration of adipose tissue. These inflammatory cells affect adipocytes not only by classical cytokines but also by the secreted glycopeptide wnt5a. Healthy adipocytes are able to release the wnt5a inhibitor sFRP5. This protective effect, however, was found to be diminished in obesity. The aim of the present study was to examine (1 whether obese human subjects exhibit increased serum concentrations of wnt5a and (2 whether wnt5a and/or sFRP5 serum concentrations in obese subjects can be influenced by caloric restriction. METHODOLOGY: 23 obese human subjects (BMI 44.1 ± 1.1 kg/m(2 and 12 age- and sex-matched lean controls (BMI 22.3 ± 0.4 kg/m(2 were included in the study. Obese subjects were treated with a very low-calorie diet (approximately 800 kcal/d for 12 weeks. Body composition was assessed by impedance analysis, insulin sensitivity was estimated by HOMA-IR and the leptin-to-adiponectin ratio and wnt5a and sFRP5 serum concentrations were measured by ELISA. sFRP5 expression in human adipose tissue biopsies was further determined on protein level by immunohistology. PRINCIPAL FINDINGS: Pro-inflammatory wnt5a was not measurable in any serum sample of lean control subjects. In patients with obesity, however, wnt5a became significantly detectable consistent with low grade inflammation in such subjects. Caloric restriction resulted in a weight loss from 131.9 ± 4.0 to 112.3 ± 3.2 kg in the obese patients group. This was accompanied by a significant decrease of HOMA-IR and leptin-to-adiponectin ratio, indicating improved insulin sensitivity. Interestingly, these metabolic improvements were associated with a significant increase in serum concentrations of the anti-inflammatory factor and wnt5a-inhibitor sFRP5. CONCLUSIONS/SIGNIFICANCE: Obesity is associated with elevated serum levels of pro-inflammatory wnt5a in humans. Furthermore, caloric restriction beneficially affects serum concentrations

  15. Microphthalmia transcription factor regulates pancreatic β-cell function.

    Science.gov (United States)

    Mazur, Magdalena A; Winkler, Marcus; Ganic, Elvira; Colberg, Jesper K; Johansson, Jenny K; Bennet, Hedvig; Fex, Malin; Nuber, Ulrike A; Artner, Isabella

    2013-08-01

    Precise regulation of β-cell function is crucial for maintaining blood glucose homeostasis. Pax6 is an essential regulator of β-cell-specific factors like insulin and Glut2. Studies in the developing eye suggest that Pax6 interacts with Mitf to regulate pigment cell differentiation. Here, we show that Mitf, like Pax6, is expressed in all pancreatic endocrine cells during mouse postnatal development and in the adult islet. A Mitf loss-of-function mutation results in improved glucose tolerance and enhanced insulin secretion but no increase in β-cell mass in adult mice. Mutant β-cells secrete more insulin in response to glucose than wild-type cells, suggesting that Mitf is involved in regulating β-cell function. In fact, the transcription of genes critical for maintaining glucose homeostasis (insulin and Glut2) and β-cell formation and function (Pax4 and Pax6) is significantly upregulated in Mitf mutant islets. The increased Pax6 expression may cause the improved β-cell function observed in Mitf mutant animals, as it activates insulin and Glut2 transcription. Chromatin immunoprecipitation analysis shows that Mitf binds to Pax4 and Pax6 regulatory regions, suggesting that Mitf represses their transcription in wild-type β-cells. We demonstrate that Mitf directly regulates Pax6 transcription and controls β-cell function. PMID:23610061

  16. THAP5 is a DNA-binding transcriptional repressor that is regulated in melanoma cells during DNA damage-induced cell death

    International Nuclear Information System (INIS)

    Research highlights: → THAP5 is a DNA-binding protein and a transcriptional repressor. → THAP5 is induced in melanoma cells upon exposure to UV or treatment with cisplatin. → THAP5 induction correlates with the degree of apoptosis in melanoma cell population. → THAP5 is a pro-apoptotic protein involved in melanoma cell death. -- Abstract: THAP5 was originally isolated as a specific interactor and substrate of the mitochondrial pro-apoptotic Omi/HtrA2 protease. It is a human zinc finger protein characterized by a restricted pattern of expression and the lack of orthologs in mouse and rat. The biological function of THAP5 is unknown but our previous studies suggest it could regulate G2/M transition in kidney cells and could be involved in human cardiomyocyte cell death associated with coronary artery disease (CAD). In this report, we expanded our studies on the properties and function of THAP5 in human melanoma cells. THAP5 was expressed in primary human melanocytes as well as in all melanoma cell lines that were tested. THAP5 protein level was significantly induced by UV irradiation or cisplatin treatment, conditions known to cause DNA damage. The induction of THAP5 correlated with a significant increase in apoptotic cell death. In addition, we show that THAP5 is a nuclear protein that could recognize and bind a specific DNA motif. THAP5 could also repress the transcription of a reporter gene in a heterologous system. Our work suggests that THAP5 is a DNA-binding protein and a transcriptional repressor. Furthermore, THAP5 has a pro-apoptotic function and it was induced in melanoma cells under conditions that promoted cell death.

  17. THAP5 is a DNA-binding transcriptional repressor that is regulated in melanoma cells during DNA damage-induced cell death

    Energy Technology Data Exchange (ETDEWEB)

    Balakrishnan, Meenakshi P.; Cilenti, Lucia; Ambivero, Camilla [Biomolecular Science Center, Burnett School of Biomedical Sciences, College of Medicine, University of Central Florida, Orlando, FL (United States); Goto, Yamafumi [Department of Dermatology, Shinshu University School of Medicine, Matsumoto (Japan); Takata, Minoru [Department of Dermatology, Okayama University Graduate School of Medical Dentistry and Pharmaceutical Sciences, Okayama (Japan); Turkson, James; Li, Xiaoman Shawn [Biomolecular Science Center, Burnett School of Biomedical Sciences, College of Medicine, University of Central Florida, Orlando, FL (United States); Zervos, Antonis S., E-mail: azervos@mail.ucf.edu [Biomolecular Science Center, Burnett School of Biomedical Sciences, College of Medicine, University of Central Florida, Orlando, FL (United States)

    2011-01-07

    Research highlights: {yields} THAP5 is a DNA-binding protein and a transcriptional repressor. {yields} THAP5 is induced in melanoma cells upon exposure to UV or treatment with cisplatin. {yields} THAP5 induction correlates with the degree of apoptosis in melanoma cell population. {yields} THAP5 is a pro-apoptotic protein involved in melanoma cell death. -- Abstract: THAP5 was originally isolated as a specific interactor and substrate of the mitochondrial pro-apoptotic Omi/HtrA2 protease. It is a human zinc finger protein characterized by a restricted pattern of expression and the lack of orthologs in mouse and rat. The biological function of THAP5 is unknown but our previous studies suggest it could regulate G2/M transition in kidney cells and could be involved in human cardiomyocyte cell death associated with coronary artery disease (CAD). In this report, we expanded our studies on the properties and function of THAP5 in human melanoma cells. THAP5 was expressed in primary human melanocytes as well as in all melanoma cell lines that were tested. THAP5 protein level was significantly induced by UV irradiation or cisplatin treatment, conditions known to cause DNA damage. The induction of THAP5 correlated with a significant increase in apoptotic cell death. In addition, we show that THAP5 is a nuclear protein that could recognize and bind a specific DNA motif. THAP5 could also repress the transcription of a reporter gene in a heterologous system. Our work suggests that THAP5 is a DNA-binding protein and a transcriptional repressor. Furthermore, THAP5 has a pro-apoptotic function and it was induced in melanoma cells under conditions that promoted cell death.

  18. MITF-independent pro-survival role of BRG1-containing SWI/SNF complex in melanoma cells.

    Directory of Open Access Journals (Sweden)

    Lubica Ondrušová

    Full Text Available Metastasized malignant melanoma has a poor prognosis because of its intrinsic resistance to chemotherapy and radiotherapy. The central role in the melanoma transcriptional network has the transcription factor MITF (microphthalmia-associated transcription factor. It has been shown recently that the expression of MITF and some of its target genes require the SWI/SNF chromatin remodeling complex. Here we demonstrate that survival of melanoma cells requires functional SWI/SNF complex not only by supporting expression of MITF and its targets and but also by activating expression of prosurvival proteins not directly regulated by MITF. Microarray analysis revealed that besides the MITF-driven genes, expression of proteins like osteopontin, IGF1, TGFß2 and survivin, the factors known to be generally associated with progression of tumors and the antiapoptotic properties, were reduced in acute BRG1-depleted 501mel cells. Western blots and RT-PCR confirmed the microarray findings. These proteins have been verified to be expressed independently of MITF, because MITF depletion did not impair their expression. Because these genes are not regulated by MITF, the data suggests that loss of BRG1-based SWI/SNF complexes negatively affects survival pathways beyond the MITF cascade. Immunohistochemistry showed high expression of both BRM and BRG1 in primary melanomas. Exogenous CDK2, osteopontin, or IGF1 each alone partly relieved the block of proliferation imposed by BRG1 depletion, implicating that more factors, besides the MITF target genes, are involved in melanoma cell survival. Together these results demonstrate an essential role of SWI/SNF for the expression of MITF-dependent and MITF-independent prosurvival factors in melanoma cells and suggest that SWI/SNF may be a potential and effective target in melanoma therapy.

  19. NFBD1/MDC1 participates in the regulation of G2/M transition in mammalian cells

    Energy Technology Data Exchange (ETDEWEB)

    Bu, Youquan [Division of Biochemistry and Anti-tumor Research, Chiba Cancer Center Research Institute, Chiba 260-8717 (Japan); Department of Biochemistry and Molecular Biology, Chongqing Medical University, Chongqing 400016 (China); Suenaga, Yusuke [Division of Biochemistry and Innovative Cancer Therapeutics, Chiba Cancer Center Research Institute, Chiba 260-8717 (Japan); Okoshi, Rintaro; Sang, Meixiang; Kubo, Natsumi [Division of Biochemistry and Anti-tumor Research, Chiba Cancer Center Research Institute, Chiba 260-8717 (Japan); Song, Fangzhou [Department of Biochemistry and Molecular Biology, Chongqing Medical University, Chongqing 400016 (China); Nakagawara, Akira [Division of Biochemistry and Innovative Cancer Therapeutics, Chiba Cancer Center Research Institute, Chiba 260-8717 (Japan); Ozaki, Toshinori, E-mail: tozaki@chiba-cc.jp [Division of Biochemistry and Anti-tumor Research, Chiba Cancer Center Research Institute, Chiba 260-8717 (Japan)

    2010-06-25

    NFBD1/MDC1 is a large nuclear protein involved in the early cellular response to DNA damage. Upon DNA damage, NFBD1 has an ability to facilitate the efficient DNA repair. In the present study, we have found that, in addition to DNA damage response, NFBD1 plays a critical role in the regulation of G2/M transition. Expression study using synchronized HeLa cells demonstrated that, like the mitotic kinase Plk1, NFBD1 expression level is maximal in G2/M-phase of the cell cycle. siRNA-mediated knockdown of NFBD1 resulted in G2/M arrest as well as simultaneous apoptosis in association with a significant increase in the amounts of {gamma}H2AX and pro-apoptotic p73. Since a remarkable down-regulation of mitotic phospho-histone H3 was detectable in NFBD1-knocked down cells, it is likely that knocking down of NFBD1 inhibits G2/M transition. Taken together, our present findings suggest that NFBD1 has a pivotal role in the regulation of proper mitotic entry.

  20. Regulation of satellite cell function in sarcopenia

    OpenAIRE

    Alway, Stephen E.; Myers, Matthew J.; Mohamed, Junaith S.

    2014-01-01

    The mechanisms contributing to sarcopenia include reduced satellite cell (myogenic stem cell) function that is impacted by the environment (niche) of these cells. Satellite cell function is affected by oxidative stress, which is elevated in aged muscles, and this along with changes in largely unknown systemic factors, likely contribute to the manner in which satellite cells respond to stressors such as exercise, disuse or rehabilitation in sarcopenic muscles. Nutritional intervention provides...

  1. Regulation of Satellite Cell Function in Sarcopenia

    OpenAIRE

    Alway, Stephen E.; Myers, Matthew J.; Mohamed, Junaith S.

    2014-01-01

    The mechanisms contributing to sarcopenia include reduced satellite cell (myogenic stem cell) function that is impacted by the environment (niche) of these cells. Satellite cell function is affected by oxidative stress, which is elevated in aged muscles, and this along with changes in largely unknown systemic factors, likely contribute to the manner in which satellite cells respond to stressors such as exercise, disuse, or rehabilitation in sarcopenic muscles. Nutritional intervention provide...

  2. Cellular pressure and volume regulation and implications for cell mechanics.

    Science.gov (United States)

    Jiang, Hongyuan; Sun, Sean X

    2013-08-01

    In eukaryotic cells, small changes in cell volume can serve as important signals for cell proliferation, death, and migration. Volume and shape regulation also directly impacts the mechanics of cells and tissues. Here, we develop a mathematical model of cellular volume and pressure regulation, incorporating essential elements such as water permeation, mechanosensitive channels, active ion pumps, and active stresses in the cortex. The model can fully explain recent experimental data, and it predicts cellular volume and pressure for several models of cell cortical mechanics. Moreover, we show that when cells are subjected to an externally applied load, such as in an atomic force microscopy indentation experiment, active regulation of volume and pressure leads to a complex cellular response. Instead of the passive mechanics of the cortex, the observed cell stiffness depends on several factors working together. This provides a mathematical explanation of rate-dependent response of cells under force. PMID:23931309

  3. Deletion of caspase-8 in mouse myeloid cells blocks microglia pro-inflammatory activation and confers protection in MPTP neurodegeneration model.

    Science.gov (United States)

    Kavanagh, Edel; Burguillos, Miguel Angel; Carrillo-Jimenez, Alejandro; Oliva-Martin, María José; Santiago, Martiniano; Rodhe, Johanna; Joseph, Bertrand; Venero, Jose Luis

    2015-09-01

    Increasing evidence involves sustained pro-inflammatory microglia activation in the pathogenesis of different neurodegenerative diseases, particularly Parkinson's disease (PD). We recently uncovered a completely novel and unexpected role for caspase-8 and its downstream substrates caspase-3/7 in the control of microglia activation and associated neurotoxicity to dopaminergic cells. To demonstrate the genetic evidence, mice bearing a floxed allele ofCASP8 were crossed onto a transgenic line expressing Cre under the control of Lysozyme 2 gene. Analysis of caspase-8 gene deletion in brain microglia demonstrated a high efficiency in activated but not in resident microglia. Mice were challenged with lipopolysaccharide, a potent inducer of microglia activation, or with MPTP, which promotes specific dopaminergic cell damage and consequent reactive microgliosis. In neither of these models, CASP8 deletion appeared to affect the overall number of microglia expressing the pan specific microglia marker, Iba1. In contrast, CD16/CD32 expression, a microglial pro-inflammatory marker, was found to be negatively affected upon CASP8 deletion. Expression of additional proinflammatory markers were also found to be reduced in response to lipopolysaccharide. Of importance, reduced pro-inflammatory microglia activation was accompanied by a significant protection of the nigro-striatal dopaminergic system in the MPTP mouse model of PD.

  4. Glycyrrhizin exerts antioxidative effects in H5N1 influenza A virus-infected cells and inhibits virus replication and pro-inflammatory gene expression.

    Directory of Open Access Journals (Sweden)

    Martin Michaelis

    Full Text Available Glycyrrhizin is known to exert antiviral and anti-inflammatory effects. Here, the effects of an approved parenteral glycyrrhizin preparation (Stronger Neo-Minophafen C were investigated on highly pathogenic influenza A H5N1 virus replication, H5N1-induced apoptosis, and H5N1-induced pro-inflammatory responses in lung epithelial (A549 cells. Therapeutic glycyrrhizin concentrations substantially inhibited H5N1-induced expression of the pro-inflammatory molecules CXCL10, interleukin 6, CCL2, and CCL5 (effective glycyrrhizin concentrations 25 to 50 µg/ml but interfered with H5N1 replication and H5N1-induced apoptosis to a lesser extent (effective glycyrrhizin concentrations 100 µg/ml or higher. Glycyrrhizin also diminished monocyte migration towards supernatants of H5N1-infected A549 cells. The mechanism by which glycyrrhizin interferes with H5N1 replication and H5N1-induced pro-inflammatory gene expression includes inhibition of H5N1-induced formation of reactive oxygen species and (in turn reduced activation of NFκB, JNK, and p38, redox-sensitive signalling events known to be relevant for influenza A virus replication. Therefore, glycyrrhizin may complement the arsenal of potential drugs for the treatment of H5N1 disease.

  5. The Essential Role of Phosphoinositide 3-Kinases (PI3Ks) in Regulating Pro-Inflammatory Responses and the Progression of Cancer

    Institute of Scientific and Technical Information of China (English)

    Keqiang Chen; Pablo Iribarren; Wanghua Gong; Ji-Ming Wang

    2005-01-01

    Phosphoinositide 3-Kinases (PI3Ks) are proteins coupled to a variety of cell surface receptors and play a key role in signal transduction cascade regulating fundamental cellular functions such as transcription, proliferation, and survival. PI3Ks also are important in disease processes such as inflammation and cancer. The aim of this review is to outline current understandings of the PI3K family, mechanism of their activation, their role in inflammatory responses and the development of malignant tumors.

  6. Complement regulator CD46 temporally regulates cytokine production by conventional and unconventional T cells

    OpenAIRE

    Kemper, Claudia; Cardone, John; Le Friec, Gaelle; Vantourout, Pierre; Roberts, Andrew; Fuchs, Anja; Jackson, Ian; Suddason, Tesha; Lord, Graham; Atkinson, John Petterson; Cope, Andrew; Hayday, Adrian C.

    2010-01-01

    Abstract This study reveals a novel form of immunoregulation: engagement on CD4+ T cells of the complement regulator CD46 promotes TH1 effector potential, but as interleukin-2 (IL-2) accumulates, "switches" cells toward a regulatory phenotype, attenuating IL-2 production via the transcriptional regulator ICER/CREM, and upregulating IL-10 following interaction of the CD46-tail with SPAKinase. Activated CD4+ T cells produce CD46 ligands, and blocking CD46 inhibits IL-10 production. F...

  7. Efficient expression of recombinant human pro-urokinase in CHO cells and its purification%重组人尿激酶原在CHO细胞中的高效表达及其纯化

    Institute of Scientific and Technical Information of China (English)

    李世崇; 叶玲玲; 刘红; 张正光; 高丽华; 胡显文; 陈昭烈

    2012-01-01

    The efficient expression vector for recombinant human pro-urokinase (rhPro-UK) was constructed with the combination of chicken β-globin MAR sequences, human elongation factor alphal ( hEF-1α) housekeeping gene regulatory sequences, and RNA stability and the output sequence. The rhPro-UK expression level reached 1299 IU/cells6/d in CHO ( Chinese hamster ovary) cell lines. The rhPro-UK containing supernatant purified with cation exchange chromatography, hydrophobic interaction chromatog-raphy, gel exclusion chromatography process resulted in rhPro-UK purity over 95% with recovery rate of 60% -70%.%用鸡β- globin的MAR序列和人看家基因延伸因子1α(hEF-1α)的调控序列以及旱獭RNA稳定与输出序列,构建了重组人尿激酶原(recombinant human pro-urokinase,rhPro - UK)的高效表达载体,在CHO细胞中获得了rhPro - UK的高效稳定表达,rhPro - UK的表达水平达到1299 IU(以百万细胞1d的表达量计).采用阳离子交换层析、疏水层析和凝胶排阻层析的三步工艺纯化表达rhPro - UK的CHO细胞培养上清液,rhPro - UK的纯度达到98%、回收率为60% ~70%.

  8. Regulation of Stem Cell Differentiation by Histone Methyltransferases and Demethylases

    DEFF Research Database (Denmark)

    Pasini, D; Bracken, A P; Agger, K;

    2008-01-01

    The generation of different cell types from stem cells containing identical genetic information and their organization into tissues and organs during development is a highly complex process that requires defined transcriptional programs. Maintenance of such programs is epigenetically regulated...... and the factors involved in these processes are often essential for development. The activities required for cell-fate decisions are frequently deregulated in human tumors, and the elucidation of the molecular mechanisms that regulate these processes is therefore important for understanding both developmental...

  9. Variability of doublecortin-associated dendrite maturation in adult hippocampal neurogenesis is independent of the regulation of precursor cell proliferation

    Directory of Open Access Journals (Sweden)

    Jessberger Sebastian

    2006-11-01

    Full Text Available Abstract Background In the course of adult hippocampal neurogenesis most regulation takes place during the phase of doublecortin (DCX expression, either as pro-proliferative effect on precursor cells or as survival-promoting effect on postmitotic cells. We here obtained quantitative data about the proliferative population and the dynamics of postmitotic dendrite development during the period of DCX expression. The question was, whether any indication could be obtained that the initiation of dendrite development is timely bound to the exit from the cell cycle. Alternatively, the temporal course of morphological maturation might be subject to additional regulatory events. Results We found that (1 20% of the DCX population were precursor cells in cell cycle, whereas more than 70% were postmitotic, (2 the time span until newborn cells had reached the most mature stage associated with DCX expression varied between 3 days and several weeks, (3 positive or negative regulation of precursor cell proliferation did not alter the pattern and dynamics of dendrite development. Dendrite maturation was largely independent of close contacts to astrocytes. Conclusion These data imply that dendrite maturation of immature neurons is initiated at varying times after cell cycle exit, is variable in duration, and is controlled independently of the regulation of precursor cell proliferation. We conclude that in addition to the major regulatory events in cell proliferation and selective survival, additional micro-regulatory events influence the course of adult hippocampal neurogenesis.

  10. EPAS1/HIF-2 alpha-mediated downregulation of tissue factor pathway inhibitor leads to a pro-thrombotic potential in endothelial cells.

    Science.gov (United States)

    Stavik, Benedicte; Espada, Sandra; Cui, Xue Yan; Iversen, Nina; Holm, Sverre; Mowinkel, Marie-Christine; Halvorsen, Bente; Skretting, Grethe; Sandset, Per Morten

    2016-04-01

    Neovascularization and hemorrhaging are evident in advanced atherosclerotic plaques due to hypoxic conditions, and mediate the accumulation of metabolic substrates, inflammatory cells, lipids, and other blood born factors inside the plaque. Tissue factor (TF) pathway inhibitor (TFPI) is mainly expressed by endothelial cells and is the endogenous inhibitor of the coagulation activator TF, which together with the downstream product thrombin can drive plaque progression and atherogenesis. We aimed to investigate the effect of hypoxic conditions on endothelial cell expression and activity of TFPI and TF that are important in coagulation initiation. Hypoxia was induced in primary human umbilical vein endothelial cells using chemicals or 1% oxygen tension, and mRNA and protein expressions were measured using qRT-PCR, ELISA, and Western blot analysis. Microscopy of fluorescence-labeled cells was used to visualize cell-associated TFPI. Cell-surface factor Xa (FXa) activity was measured using a two-stage chromogenic substrate method. We found that hypoxia reduced the TFPI mRNA and protein levels and increased the TF mRNA expression in a dose-dependent manner. The effect on TFPI was apparent on all the protein pools of TFPI, i.e., secreted TFPI, cell-surface associated TFPI, and intracellular TFPI, and seemed to be dependent upon hypoxia inducible factor-2α (HIF-2α). An increase in FXa activity was also observed on the endothelial cell surface, reflecting an increase in pro-thrombotic potential of the cells. Our findings indicate that hypoxic conditions may enhance the pro-coagulant activity of endothelial cells, which may promote atherogenesis in addition to clinical events and thus the severity of atherosclerotic disorders. PMID:26826018

  11. Exon-skipping strategy by ratio modulation between cytoprotective versus pro-apoptotic clusterin forms increased sensitivity of LNCaP to cell death.

    Directory of Open Access Journals (Sweden)

    Abdellatif Essabbani

    Full Text Available BACKGROUND: In prostate cancer the secreted form of clusterin (sCLU has been described as an anti-apoptotic protein whose expression is increased after therapeutic intervention, whereas, the nuclear protein form nCLU was reported to have pro-apoptotic properties. METHODOLOGY: In order to provide new therapeutic approaches targeting CLU, we developed a strategy based on exon skipping by using a lentiviral construct to preferentially induce the nuclear spliced form of the protein. The molecular construct was transduced in LNCaP cells for testing the modulation of sensitivity of the transduced cells to pro-apoptotic stress. RESULTS AND CONCLUSIONS: We showed an increase of nCLU/sCLU expression ratio in the prostate cancer cell line "LNCaP" after lentiviral vector-U7 nCLU transduction. Moreover, we showed a significant inhibition of cell proliferation in nCLU-U7 LNCaP cells after treatment with cisplatin and after exposure to ionizing radiation compared to control cells. Finally, we showed that nCLU-U7 LNCaP cells exposure to UV-C significantly reduced an increase of cell death compared to control. Finally, we showed that modulating nCLU expression had profound impact on Ku70/Bax interaction as well as Rad17 expression which could be a key mechanism in sensitizing cells to cell death. In conclusion, this is the first report showing that increasing of nCLU/sCLU expression ratio by using an "on demand alternative splicing" strategy successfully increased sensitivity to radiotherapy and chemotherapy of prostate cancer cells.

  12. Creatine kinase in cell cycle regulation and cancer.

    Science.gov (United States)

    Yan, Yong-Bin

    2016-08-01

    The phosphocreatine-creatine kinase (CK) shuttle system is increasingly recognized as a fundamental mechanism for ATP homeostasis in both excitable and non-excitable cells. Many intracellular processes are ATP dependent. Cell division is a process requiring a rapid rate of energy turnover. Cell cycle regulation is also a key point to understanding the mechanisms underlying cancer progression. It has been known for about 40 years that aberrant CK levels are associated with various cancers and for over 30 years that CK is involved in mitosis regulation. However, the underlying molecular mechanisms have not been investigated sufficiently until recently. By maintaining ATP at sites of high-energy demand, CK can regulate cell cycle progression by affecting the intracellular energy status as well as by influencing signaling pathways that are essential to activate cell division and cytoskeleton reorganization. Aberrant CK levels may impair cell viability under normal or stressed conditions and induce cell death. The involvement of CK in cell cycle regulation and cellular energy metabolism makes it a potential diagnostic biomarker and therapeutic target in cancer. To understand the multiple physiological/pathological functions of CK, it is necessary to identify CK-binding partners and regulators including proteins, non-coding RNAs and participating endogenous small molecular weight chemical compounds. This review will focus on molecular mechanisms of CK in cell cycle regulation and cancer progression. It will also discuss the implications of recent mechanistic studies, the emerging problems and future challenges of the multifunctional enzyme CK. PMID:27020776

  13. Oncogenic KRAS Regulates Tumor Cell Signaling via Stromal Reciprocation.

    Science.gov (United States)

    Tape, Christopher J; Ling, Stephanie; Dimitriadi, Maria; McMahon, Kelly M; Worboys, Jonathan D; Leong, Hui Sun; Norrie, Ida C; Miller, Crispin J; Poulogiannis, George; Lauffenburger, Douglas A; Jørgensen, Claus

    2016-05-01

    Oncogenic mutations regulate signaling within both tumor cells and adjacent stromal cells. Here, we show that oncogenic KRAS (KRAS(G12D)) also regulates tumor cell signaling via stromal cells. By combining cell-specific proteome labeling with multivariate phosphoproteomics, we analyzed heterocellular KRAS(G12D) signaling in pancreatic ductal adenocarcinoma (PDA) cells. Tumor cell KRAS(G12D) engages heterotypic fibroblasts, which subsequently instigate reciprocal signaling in the tumor cells. Reciprocal signaling employs additional kinases and doubles the number of regulated signaling nodes from cell-autonomous KRAS(G12D). Consequently, reciprocal KRAS(G12D) produces a tumor cell phosphoproteome and total proteome that is distinct from cell-autonomous KRAS(G12D) alone. Reciprocal signaling regulates tumor cell proliferation and apoptosis and increases mitochondrial capacity via an IGF1R/AXL-AKT axis. These results demonstrate that oncogene signaling should be viewed as a heterocellular process and that our existing cell-autonomous perspective underrepresents the extent of oncogene signaling in cancer. VIDEO ABSTRACT. PMID:27087446

  14. Decidual-secreted factors alter invasive trophoblast membrane and secreted proteins implying a role for decidual cell regulation of placentation.

    Science.gov (United States)

    Menkhorst, Ellen Melaleuca; Lane, Natalie; Winship, Amy Louise; Li, Priscilla; Yap, Joanne; Meehan, Katie; Rainczuk, Adam; Stephens, Andrew; Dimitriadis, Evdokia

    2012-01-01

    Inadequate or inappropriate implantation and placentation during the establishment of human pregnancy is thought to lead to first trimester miscarriage, placental insufficiency and other obstetric complications. To create the placental blood supply, specialized cells, the 'extravillous trophoblast' (EVT) invade through the differentiated uterine endometrium (the decidua) to engraft and remodel uterine spiral arteries. We hypothesized that decidual factors would regulate EVT function by altering the production of EVT membrane and secreted factors. We used a proteomics approach to identify EVT membrane and secreted proteins regulated by decidual cell factors. Human endometrial stromal cells were decidualized in vitro by treatment with estradiol (10(-8) M), medroxyprogesterone acetate (10(-7) M) and cAMP (0.5 mM) for 14 days. Conditioned media (CM) was collected on day 2 (non-decidualized CM) and 14 (decidualized CM) of treatment. Isolated primary EVT cultured on Matrigel™ were treated with media control, non-decidualized or decidualized CM for 16 h. EVT CM was fractionated for proteins HPLC-MS/MS. 43 proteins produced by EVT were identified; 14 not previously known to be expressed in the placenta and 12 which had previously been associated with diseases of pregnancy including preeclampsia. Profilin 1, lysosome associated membrane glycoprotein 1 (LAMP1), dipeptidyl peptidase 1 (DPP1/cathepsin C) and annexin A2 expression by interstitial EVT in vivo was validated by immunhistochemistry. Decidual CM regulation in vitro was validated by western blotting: decidualized CM upregulated profilin 1 in EVT CM and non-decidualized CM upregulated annexin A2 in EVT CM and pro-DPP1 in EVT cell lysate. Here, non-decidualized factors induced protease expression by EVT suggesting that non-decidualized factors may induce a pro-inflammatory cascade. Preeclampsia is a pro-inflammatory condition. Overall, we have demonstrated the potential of a proteomics approach to identify novel

  15. Identification of cisplatin-regulated metabolic pathways in pluripotent stem cells.

    Directory of Open Access Journals (Sweden)

    Louise von Stechow

    Full Text Available The chemotherapeutic compound, cisplatin causes various kinds of DNA lesions but also triggers other pertubations, such as ER and oxidative stress. We and others have shown that treatment of pluripotent stem cells with cisplatin causes a plethora of transcriptional and post-translational alterations that, to a major extent, point to DNA damage response (DDR signaling. The orchestrated DDR signaling network is important to arrest the cell cycle and repair the lesions or, in case of damage beyond repair, eliminate affected cells. Failure to properly balance the various aspects of the DDR in stem cells contributes to ageing and cancer. Here, we performed metabolic profiling by mass spectrometry of embryonic stem (ES cells treated for different time periods with cisplatin. We then integrated metabolomics with transcriptomics analyses and connected cisplatin-regulated metabolites with regulated metabolic enzymes to identify enriched metabolic pathways. These included nucleotide metabolism, urea cycle and arginine and proline metabolism. Silencing of identified proline metabolic and catabolic enzymes indicated that altered proline metabolism serves as an adaptive, rather than a toxic response. A group of enriched metabolic pathways clustered around the metabolite S-adenosylmethionine, which is a hub for methylation and transsulfuration reactions and polyamine metabolism. Enzymes and metabolites with pro- or anti-oxidant functions were also enriched but enhanced levels of reactive oxygen species were not measured in cisplatin-treated ES cells. Lastly, a number of the differentially regulated metabolic enzymes were identified as target genes of the transcription factor p53, pointing to p53-mediated alterations in metabolism in response to genotoxic stress. Altogether, our findings reveal interconnecting metabolic pathways that are responsive to cisplatin and may serve as signaling modules in the DDR in pluripotent stem cells.

  16. Regulation of Natural Killer Cell Function by STAT3

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    Nicholas eCacalano

    2016-04-01

    Full Text Available Natural killer (NK cells, key members of a distinct hempatopoietic lineage, innate lymphoid cells (ILCs, are critical effectors that mediate cytotoxicity toward tumor and virally-infected cells but also regulate inflammation, antigen presentation and the adaptive immune response. It has been shown that NK cells can regulate the development and activation of many other components of the immune response such as dendritic cells, which in turn, modulate the function of NK cells in multiple synergistic feed back loops driven by cell-cell contact and the secretion of cytokines and chemokines that control effector function and migration of cells to sites of immune activation. The Signal Transducer and Activator of Transcription (STAT-3 is involved in driving almost all of the pathways that control NK cytolytic activity as well as the reciprocal regulatory interactions between NK cells and other components of the immune system. In the context of tumor immunology, NK cells are a first line of defense that eliminates pre-cancerous and transformed cells early in the process of carcinogenesis, through a mechanism of immune surveillance. Even after tumors become established, NK cells are critical components of anti-cancer immunity: dysfunctional NK cells are often found in the peripheral blood of cancer patients and the lack of NK cells in the tumor microenvironment often correlates with poor prognosis. The pathways and soluble factors activated in tumor-associated NK cells, cancer cells, and regulatory myeloid cells which determine the outcome of cancer immunity are all critically regulated by STAT3. Using the tumor microenvironment as a paradigm, we present here an overview of the research that has revealed fundamental mechanisms through which STAT3 regulates all aspects of natural killer cell biology, including NK development, activation, target cell killing, and fine tuning of the innate and adaptive immune responses.

  17. Regulation of cell death in cancer - possible implications for immunotherapy

    OpenAIRE

    Simone eFulda

    2013-01-01

    Since most anticancer therapies including immunotherapy trigger programmed cell death in cancer cells, defective cell death programs can lead to treatment resistance and tumor immune escape. Therefore, evasion of programmed cell death may provide one possible explanation as to why cancer immunotherapy has so far only shown modest clinical benefits for children with cancer. A better understanding of the molecular mechanisms that regulate sensitivity and resistance to programmed cell death is e...

  18. The low density lipoprotein receptor-related protein/alpha2-macroglobulin receptor regulates cell surface plasminogen activator activity on human trophoblast cells.

    Science.gov (United States)

    Zhang, J C; Sakthivel, R; Kniss, D; Graham, C H; Strickland, D K; McCrae, K R

    1998-11-27

    The low density lipoprotein receptor-related protein/alpha2-macroglobulin receptor (LRP/alpha2MR) mediates the internalization of numerous ligands, including prourokinase (pro-UK) and complexes between two-chain urokinase (tc-u-PA) and plasminogen activator inhibitor type-1 (PAI-1). It has been suggested that through its ability to internalize these ligands, LRP/alpha2MR may regulate the expression of plasminogen activator activity on cell surfaces; this hypothesis, however, has not been experimentally confirmed. To address this issue, we assessed the ability of LRP/alpha2MR to regulate plasminogen activator activity on human trophoblast cells, which express both LRP/alpha2MR and the urokinase receptor (uPAR). Trophoblasts internalized and degraded exogenous 125I-pro-UK (primarily following its conversion to tc-u-PA and incorporation into tc-u-PA.PAI complexes) in an LRP/alpha2MR-dependent manner, which was inhibited by the LRP/alpha2MR receptor-associated protein. Receptor-associated protein also caused a approximately 50% reduction in cell surface plasminogen activator activity and delayed the regeneration of unoccupied uPAR by cells on which uPAR were initially saturated with pro-UK. Identical effects were caused by anti-LRP/alpha2MR antibodies. These results demonstrate that LRP/alpha2MR promotes the expression of cell surface plasminogen activator activity on trophoblasts by facilitating the clearance of tc-u-PA.PAI complexes and regeneration of unoccupied cell surface uPAR. PMID:9822706

  19. The mechanism of pleural inflammation by long carbon nanotubes: interaction of long fibres with macrophages stimulates them to amplify pro-inflammatory responses in mesothelial cells

    Directory of Open Access Journals (Sweden)

    Murphy Fiona A

    2012-04-01

    Full Text Available Abstract Carbon nanotubes (CNT are high aspect ratio nanoparticles with diameters in the nanometre range but lengths extending up to hundreds of microns. The structural similarities between CNT and asbestos have raised concern that they may pose a similar inhalation hazard. Recently CNT have been shown to elicit a length-dependent, asbestos-like inflammatory response in the pleural cavity of mice, where long fibres caused inflammation but short fibres did not. However the cellular mechanisms governing this response have yet to be elucidated. This study examined the in vitro effects of a range of CNT for their ability to stimulate the release of the acute phase cytokines; IL-1β, TNFα, IL-6 and the chemokine, IL-8 from both Met5a mesothelial cells and THP-1 macrophages. Results showed that direct exposure to CNT resulted in significant cytokine release from the macrophages but not mesothelial cells. This pro-inflammatory response was length dependent but modest and was shown to be a result of frustrated phagocytosis. Furthermore the indirect actions of the CNT were examined by treating the mesothelial cells with conditioned media from CNT-treated macrophages. This resulted in a dramatic amplification of the cytokine release from the mesothelial cells, a response which could be attenuated by inhibition of phagocytosis during the initial macrophage CNT treatments. We therefore hypothesise that long fibres elicit an inflammatory response in the pleural cavity via frustrated phagocytosis in pleural macrophages. The activated macrophages then stimulate an amplified pro-inflammatory cytokine response from the adjacent pleural mesothelial cells. This mechanism for producing a pro-inflammatory environment in the pleural space exposed to long CNT has implications for the general understanding of fibre-related pleural disease and design of safe nanofibres.

  20. Stage-specific expression of TNFα regulates bad/bid-mediated apoptosis and RIP1/ROS-mediated secondary necrosis in Birnavirus-infected fish cells.

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    Wei-Lun Wang

    Full Text Available Infectious pancreatic necrosis virus (IPNV can induce Bad-mediated apoptosis followed by secondary necrosis in fish cells, but it is not known how these two types of cell death are regulated by IPNV. We found that IPNV infection can regulate Bad/Bid-mediated apoptotic and Rip1/ROS-mediated necrotic death pathways via the up-regulation of TNFα in zebrafish ZF4 cells. Using a DNA microarray and quantitative RT-PCR analyses, two major subsets of differentially expressed genes were characterized, including the innate immune response gene TNFα and the pro-apoptotic genes Bad and Bid. In the early replication stage (0-6 h post-infection, or p.i., we observed that the pro-inflammatory cytokine TNFα underwent a rapid six-fold induction. Then, during the early-middle replication stages (6-12 h p.i., TNFα level was eight-fold induction and the pro-apoptotic Bcl-2 family members Bad and Bid were up-regulated. Furthermore, specific inhibitors of TNFα expression (AG-126 or TNFα-specific siRNA were used to block apoptotic and necrotic death signaling during the early or early-middle stages of IPNV infection. Inhibition of TNFα expression dramatically reduced the Bad/Bid-mediated apoptotic and Rip1/ROS-mediated necrotic cell death pathways and rescued host cell viability. Moreover, we used Rip1-specific inhibitors (Nec-1 and Rip1-specific siRNA to block Rip1 expression. The Rip1/ROS-mediated secondary necrotic pathway appeared to be reduced in IPNV-infected fish cells during the middle-late stage of infection (12-18 h p.i.. Taken together, our results indicate that IPNV triggers two death pathways via up-stream induction of the pro-inflammatory cytokine TNFα, and these results may provide new insights into the pathogenesis of RNA viruses.

  1. Telomere regulation in pluripotent stem cells

    OpenAIRE

    Huang, Yan; Liang, Puping; Liu, Dan; Huang, Junjiu; Songyang, Zhou

    2014-01-01

    Pluripotent stem cells (PSCs) have the potential to produce any types of cells from all three basic germ layers and the capacity to self-renew and proliferate indefinitely in vitro. The two main types of PSCs, embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), share common features such as colony morphology, high expression of Oct4 and Nanog, and strong alkaline phosphatase activity. In recent years, increasing evidences suggest that telomere length represents another imp...

  2. Common stemness regulators of embryonic and cancer stem cells

    Institute of Scientific and Technical Information of China (English)

    Christiana; Hadjimichael; Konstantina; Chanoumidou; Natalia; Papadopoulou; Panagiota; Arampatzi; Joseph; Papamatheakis; Androniki; Kretsovali

    2015-01-01

    Pluripotency of embryonic stem cells(ESCs) and induced pluripotent stem cells is regulated by a well characterized gene transcription circuitry. The circuitry is assembled by ESC specific transcription factors, signal trans-ducing molecules and epigenetic regulators. Growing understanding of stem-like cells, albeit of more complex phenotypes, present in tumors(cancer stem cells), provides a common conceptual and research frame-work for basic and applied stem cell biology. In this review, we highlight current results on biomarkers, gene signatures, signaling pathways and epigenetic regulators that are common in embryonic and cancer stem cells. We discuss their role in determining the cell phenotype and finally, their potential use to design next generation biological and pharmaceutical approaches for regenerative medicine and cancer therapies.

  3. Laminin regulates PDGFRβ(+) cell stemness and muscle development.

    Science.gov (United States)

    Yao, Yao; Norris, Erin H; E Mason, Christopher; Strickland, Sidney

    2016-01-01

    Muscle-resident PDGFRβ(+) cells, which include pericytes and PW1(+) interstitial cells (PICs), play a dual role in muscular dystrophy. They can either undergo myogenesis to promote muscle regeneration or differentiate into adipocytes and other cells to compromise regeneration. How the differentiation and fate determination of PDGFRβ(+) cells are regulated, however, remains unclear. Here, by utilizing a conditional knockout mouse line, we report that PDGFRβ(+) cell-derived laminin inhibits their proliferation and adipogenesis, but is indispensable for their myogenesis. In addition, we show that laminin alone is able to partially reverse the muscle dystrophic phenotype in these mice at the molecular, structural and functional levels. Further RNAseq analysis reveals that laminin regulates PDGFRβ(+) cell differentiation/fate determination via gpihbp1. These data support a critical role of laminin in the regulation of PDGFRβ(+) cell stemness, identify an innovative target for future drug development and may provide an effective treatment for muscular dystrophy. PMID:27138650

  4. The small GTPase RhoB regulates TNFα signaling in endothelial cells.

    Directory of Open Access Journals (Sweden)

    Jeffrey Kroon

    Full Text Available The inflammatory response of endothelial cells triggered by cytokines such as TNFα and IL1β plays a pivotal role in innate immunity. Upon pro-inflammatory cytokine stimulation, endothelial cells produce chemokines and cytokines that attract and activate leukocytes, and express high levels of leukocyte adhesion molecules. This process is mediated by intracellular signaling cascades triggered by activation of e.g. the TNFα receptor (TNFR that lead to the activation of the NFκB transcription factor and of MAP kinases, which in turn activate inflammatory gene transcription. We found that the small GTPase RhoB was strongly and rapidly upregulated in primary human endothelial cells by TNFα, IL1β and LPS. We subsequently investigated the role of RhoB in the regulation of TNFR signaling in endothelial cells by silencing RhoB expression with siRNA. We provide evidence that the TNFα-induced activation of p38 MAP kinase is strongly dependent on RhoB, but not on RhoA, while JNK activation is regulated by both RhoB and RhoA. Consistent with the important role of p38 MAP kinase in inflammation, we demonstrate that loss of RhoB impairs TNFα-induced ICAM-1 expression and reduces cell production of IL6 and IL8. In addition, we show that RhoB silencing alters the intracellular traffic of TNFα after endocytosis. Since RhoB is a known regulator of the intracellular traffic of membrane receptors, our data suggest that RhoB controls TNFα signaling through the regulation of the TNFR traffic.

  5. Activation of AKT negatively regulates the pro-apoptotic function of death-associated protein kinase 3 (DAPK3) in prostate cancer.

    Science.gov (United States)

    Das, Trinath P; Suman, Suman; Papu John, A M Sashi; Pal, Deeksha; Edwards, Angelena; Alatassi, Houda; Ankem, Murali K; Damodaran, Chendil

    2016-07-28

    The activation of AKT governs many signaling pathways and promotes cell growth and inhibits apoptosis in human malignancies including prostate cancer (CaP). Here, we investigated the molecular association between AKT activation and the function of death-associated protein kinase 3 (DAPK3) in CaP. An inverse correlation of pAKT and DAPK3 expression was seen in a panel of CaP cell lines. Inhibition of AKT by wortmannin/LY294002 or overexpression of DAPK3 reverts the proliferative function of AKT in CaP cells. On the other hand, ectopic expression of AKT inhibited DAPK3 function and induced proliferation of CaP cells. In addition, AKT over-expressed tumors exhibit aggressive growth when compared to control vector in xenograft models. The immunohistochemistry results revealed a down-regulation of DAPK3 expression in AKT over-expressed tumors as compared to control tumors. Finally, we examined the expression pattern of AKT and DAPK3 in human CaP specimens - the expected gradual increase and nuclear localization of pAKT was seen in higher Gleason score samples versus benign hyperplasia (BPH). On the contrary, reduced expression of DAPK3 was seen in higher Gleason stages versus BPH. This suggests that inhibition of DAPK3 may be a contributing factor to the carcinogenesis of the prostate. Understanding the mechanism by which AKT negatively regulates DAPK3 function may suggest whether DAPK3 can be a therapeutic target for CaP. PMID:27126362

  6. Autoimmunity: regulatory B cells--IL-35 and IL-21 regulate the regulators.

    Science.gov (United States)

    Tedder, Thomas F; Leonard, Warren J

    2014-08-01

    IL-21 regulates the activity and number of IL-10-producing regulatory B cells (B10 cells) that modulate immune responses and limit diverse autoimmune diseases. A new study demonstrates that IL-35 has a similar function. Identifying regulatory circuits that control B10-cell function in vivo might open the door to future treatments for autoimmune diseases.

  7. Nanotechnology in the regulation of stem cell behavior

    Science.gov (United States)

    Wu, King-Chuen; Tseng, Ching-Li; Wu, Chi-Chang; Kao, Feng-Chen; Tu, Yuan-Kun; So, Edmund C.; Wang, Yang-Kao

    2013-10-01

    Stem cells are known for their potential to repair damaged tissues. The adhesion, growth and differentiation of stem cells are likely controlled by the surrounding microenvironment which contains both chemical and physical cues. Physical cues in the microenvironment, for example, nanotopography, were shown to play important roles in stem cell fate decisions. Thus, controlling stem cell behavior by nanoscale topography has become an important issue in stem cell biology. Nanotechnology has emerged as a new exciting field and research from this field has greatly advanced. Nanotechnology allows the manipulation of sophisticated surfaces/scaffolds which can mimic the cellular environment for regulating cellular behaviors. Thus, we summarize recent studies on nanotechnology with applications to stem cell biology, including the regulation of stem cell adhesion, growth, differentiation, tracking and imaging. Understanding the interactions of nanomaterials with stem cells may provide the knowledge to apply to cell-scaffold combinations in tissue engineering and regenerative medicine.

  8. Cell adhesion in regulation of asymmetric stem cell division

    OpenAIRE

    Yamashita, Yukiko M

    2010-01-01

    Adult stem cells inevitably communicate with their cellular neighbors within the tissues they sustain. Indeed, such communication, particularly with components of the stem cell niche, is essential for many aspects of stem cell behavior, including the maintenance of stem cell identity and asymmetric cell division. Cell adhesion mediates this communication by placing stem cells in close proximity to the signaling source and by providing a polarity cue that orients stem cells. Here, I review the...

  9. Regulated expression of erythropoietin by two human hepatoma cell lines

    Energy Technology Data Exchange (ETDEWEB)

    Goldberg, M.A.; Glass, G.A.; Cunningham, J.M.; Bunn, H.F.

    1987-11-01

    The development of a cell culture system that produces erythropoietin (Epo) in a regulated manner has been the focus of much effort. The authors have screened multiple renal and hepatic cell lines for either constitutive or regulated expression of Epo. Only the human hepatoma cell lines, Hep3B and HepG2, made significant amounts of Epo as measured both by radioimmunoassay and in vitro bioassay (as much as 330 milliunits per 10/sup 6/ cells in 24 hr). The constitutive production of Epo increased dramatically as a function of cell density in both cell lines. At cell densities < 3.3 x 10/sup 5/ cells per cm/sup 2/, there was little constitutive release of Epo in the medium. With Hep3B cells grown at low cell densities, a mean 18-fold increase in Epo expression was seen in response to hypoxia and a 6-fold increase was observed in response to incubation in medium containing 50 ..mu..M cobalt(II) chloride. At similar low cell densities, Epo production in HepG2 cells could be enhanced an average of about 3-fold by stimulation with either hypoxia or cobalt(II) chloride. Upon such stimulation, both cell lines demonstrated markedly elevated levels of Epo mRNA. Hence, both Hep3B and HepG2 cell lines provide an excellent in vitro system in which to study the physiological regulation of Epo expression.

  10. IL-1beta-induced pro-apoptotic signalling is facilitated by NCAM/FGF receptor signalling and inhibited by the C3d ligand in the INS-1E rat beta cell line

    DEFF Research Database (Denmark)

    Petersen, L G; Størling, J; Heding, P;

    2006-01-01

    AIMS/HYPOTHESIS: IL-1beta released from immune cells induces beta cell pro-apoptotic signalling via mitogen-activated protein kinases (MAPKs) and nuclear factor-kappaB (NF-kappaB). In neurons, the neural cell adhesion molecule (NCAM) signals to several elements involved in IL-1beta-induced pro......-apoptotic signalling in beta cells. Pancreatic beta cells express NCAM, but its biological effects in these cells are unclear. The aim of this study was to investigate whether there is cross-talk between NCAM signalling and cytokine-induced pro-apoptotic signalling. MATERIALS AND METHODS: Western blotting was used...... to investigate levels of NCAM and inducible nitric oxide synthase, phosphorylation of Src and MAPKs, and cleavage of caspase-3. MAPK activity was investigated with an in vitro kinase assay. Apoptosis was detected by cleaved caspase-3 and a Cell Death Detection ELISA(plus) assay. NCAM-induced fibroblast growth...

  11. Differential effect of immune cells on non-pathogenic Gram-negative bacteria-induced nuclear factor-kappaB activation and pro-inflammatory gene expression in intestinal epithelial cells

    DEFF Research Database (Denmark)

    Haller, D.; Holt, L.; Parlesak, Alexandr;

    2004-01-01

    We have previously shown that non-pathogenic Gram negative bacteria induce RelA phosphorylation, nuclear factor (NF)-kappaB transcriptional activity and pro-inflammatory gene expression in intestinal epithelial cells (IEC) in vivo and in vitro. In this study, we investigated the molecular mechani...

  12. Miro1 regulates intercellular mitochondrial transport & enhances mesenchymal stem cell rescue efficacy.

    Science.gov (United States)

    Ahmad, Tanveer; Mukherjee, Shravani; Pattnaik, Bijay; Kumar, Manish; Singh, Suchita; Kumar, Manish; Rehman, Rakhshinda; Tiwari, Brijendra K; Jha, Kumar A; Barhanpurkar, Amruta P; Wani, Mohan R; Roy, Soumya S; Mabalirajan, Ulaganathan; Ghosh, Balaram; Agrawal, Anurag

    2014-05-01

    There is emerging evidence that stem cells can rejuvenate damaged cells by mitochondrial transfer. Earlier studies show that epithelial mitochondrial dysfunction is critical in asthma pathogenesis. Here we show for the first time that Miro1, a mitochondrial Rho-GTPase, regulates intercellular mitochondrial movement from mesenchymal stem cells (MSC) to epithelial cells (EC). We demonstrate that overexpression of Miro1 in MSC (MSCmiro(Hi)) leads to enhanced mitochondrial transfer and rescue of epithelial injury, while Miro1 knockdown (MSCmiro(Lo)) leads to loss of efficacy. Treatment with MSCmiro(Hi) was associated with greater therapeutic efficacy, when compared to control MSC, in mouse models of rotenone (Rot) induced airway injury and allergic airway inflammation (AAI). Notably, airway hyperresponsiveness and remodeling were reversed by MSCmiro(Hi) in three separate allergen-induced asthma models. In a human in vitro system, MSCmiro(Hi) reversed mitochondrial dysfunction in bronchial epithelial cells treated with pro-inflammatory supernatant of IL-13-induced macrophages. Anti-inflammatory MSC products like NO, TGF-β, IL-10 and PGE2, were unchanged by Miro1 overexpression, excluding non-specific paracrine effects. In summary, Miro1 overexpression leads to increased stem cell repair. PMID:24431222

  13. THAP5 is a DNA binding transcriptional repressor that is regulated in melanoma cells during DNA damage-induced cell death

    Science.gov (United States)

    Balakrishnan, Meenakshi P.; Cilenti, Lucia; Ambivero, Camilla; Goto, Yamafumi; Takata, Minoru; Turkson, James; Li, Xiaoman Shawn; Zervos, Antonis S.

    2011-01-01

    THAP5 was originally isolated as a specific interactor and substrate of the mitochondrial pro-apoptotic Omi/HtrA2 protease. It is a human zinc finger protein characterized by a restricted pattern of expression and the lack of orthologs in mouse and rat. The biological function of THAP5 is unknown but our previous studies suggest it could regulate G2/M transition in kidney cells and could be involved in human cardiomyocyte cell death associated with coronary artery disease (CAD). In this report, we expanded our studies on the properties and function of THAP5 in human melanoma cells. THAP5 was expressed in primary human melanocytes as well as in all melanoma cell lines that were tested. THAP5 protein level was significantly induced by UV irradiation or cisplatin treatment, conditions known to cause DNA damage. The induction of THAP5 correlated with a significant increase in apoptotic cell death. In addition, we show that THAP5 is a nuclear protein that could recognize and bind a specific DNA motif. THAP5 could also repress the transcription of a reporter gene in a heterologous system. Our work suggests that THAP5 is a DNA binding protein and a transcriptional repressor. Furthermore, THAP5 has a pro-apoptotic function and it was induced in melanoma cells under conditions that promoted cell death. PMID:21110952

  14. American Ginseng Stimulates Insulin Production and Prevents Apoptosis through Regulation of Uncoupling Protein-2 in Cultured β Cells

    Directory of Open Access Journals (Sweden)

    John Zeqi Luo

    2006-01-01

    Full Text Available American ginseng root displays the ability to achieve glucose homeostasis both experimentally and clinically but the unknown mechanism used by ginseng to achieve its therapeutic effects on diabetes limits its application. Disruption in the insulin secretion of pancreatic β cells is considered the major cause of diabetes. A mitochondrial protein, uncoupling protein-2 (UCP-2 has been found to play a critical role in insulin synthesis and β cell survival. Our preliminary studies found that the extracts of American ginseng inhibit UCP-2 expression which may contribute to the ability of ginseng protecting β cell death and improving insulin synthesis. Therefore, we hypothesized that ginseng extracts suppress UCP-2 in the mitochondria of pancreatic β cells, promoting insulin synthesis and anti-apoptosis (a programmed cell-death mechanism. To test the hypothesis, the serum-deprived quiescent β cells were cultured with or without interleukin-1β (IL-1β, (200 pg ml−1, a cytokine to induce β cell apoptosis and water extracts of American ginseng (25 μg per 5 μl administered to wells of 0.5 ml culture for 24 h. We evaluated effects of ginseng on UCP-2 expression, insulin production, anti-/pro-apoptotic factors Bcl-2/caspase-9 expression and cellular ATP levels. We found that ginseng suppresses UCP-2, down-regulates caspase-9 while increasing ATP and insulin production/secretion and up-regulates Bcl-2, reducing apoptosis. These findings suggest that stimulation of insulin production and prevention of β cell loss by American ginseng extracts can occur via the inhibition of mitochondrial UCP-2, resulting in increase in the ATP level and the anti-apoptotic factor Bcl-2, while down-regulation of pro-apoptotic factor caspase-9 occurs, lowering the occurrence of apoptosis, which support the hypothesis.

  15. Epigenetic Regulation of Adaptive NK Cell Diversification.

    Science.gov (United States)

    Tesi, Bianca; Schlums, Heinrich; Cichocki, Frank; Bryceson, Yenan T

    2016-07-01

    Natural killer (NK) cells were previously considered to represent short-lived, innate lymphocytes. However, mouse models have revealed expansion and persistence of differentiated NK cell subsets in response to cytomegalovirus (CMV) infection, paralleling antigen-specific T cell differentiation. Congruently, analyses of humans have uncovered CMV-associated NK cell subsets characterized by epigenetic diversification processes that lead to altered target cell specificities and functional capacities. Here, focusing on responses to viruses, we review similarities and differences between mouse and human adaptive NK cells, identifying molecular analogies that may be key to transcriptional reprogramming and functional alterations. We discuss possible molecular mechanisms underlying epigenetic diversification and hypothesize that processes driving epigenetic diversification may represent a more widespread mechanism for fine-tuning and optimization of cellular immunity.

  16. Regulation of Germinal Center Reactions by B and T Cells

    Directory of Open Access Journals (Sweden)

    Yeonseok Chung

    2013-10-01

    Full Text Available Break of B cell tolerance to self-antigens results in the development of autoantibodies and, thus, leads to autoimmunity. How B cell tolerance is maintained during active germinal center (GC reactions is yet to be fully understood. Recent advances revealed several subsets of T cells and B cells that can positively or negatively regulate GC B cell responses in vivo. IL-21-producing CXCR5+ CD4+ T cells comprise a distinct lineage of helper T cells—termed follicular helper T cells (TFH—that can provide help for the development of GC reactions where somatic hypermutation and affinity maturation take place. Although the function of TFH cells is beneficial in generating high affinity antibodies against infectious agents, aberrant activation of TFH cell or B cell to self-antigens results in autoimmunity. At least three subsets of immune cells have been proposed as regulatory cells that can limit such antibody-mediated autoimmunity, including follicular regulatory T cells (TFR, Qa-1 restricted CD8+ regulatory T cells (CD8+TREG, and regulatory B cells (BREG. In this review, we will discuss our current understanding of GC B cell regulation with specific emphasis on the newly identified immune cell subsets involved in this process.

  17. Anti-Inflammatory Effect of Apigenin on LPS-Induced Pro-Inflammatory Mediators and AP-1 Factors in Human Lung Epithelial Cells.

    Science.gov (United States)

    Patil, Rajeshwari H; Babu, R L; Naveen Kumar, M; Kiran Kumar, K M; Hegde, Shubha M; Nagesh, Rashmi; Ramesh, Govindarajan T; Sharma, S Chidananda

    2016-02-01

    Apigenin is one of the plant flavonoids present in fruits and vegetables, acting as an important nutraceutical component. It is recognized as a potential antioxidant, antimicrobial, and anti-inflammatory molecule. In the present study, the mechanism of anti-inflammatory action of apigenin on lipopolysaccharide (LPS)-induced pro-inflammatory cytokines and activator protein-1 (AP-1) factors in human lung A549 cells was investigated. The anti-inflammatory activity of apigenin on LPS-induced inflammation was determined by analyzing the expression of pro-inflammatory cytokines, nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), and different AP-1 factors. Apigenin significantly inhibited the LPS-induced expression of iNOS, COX-2, expression of pro-inflammatory cytokines (IL-1β, IL-2, IL-6, IL-8, and TNF-α), and AP-1 proteins (c-Jun, c-Fos, and JunB) including nitric oxide production. Study confirms the anti-inflammatory effect of apigenin by inhibiting the expression of inflammatory mediators and AP-1 factors involved in the inflammation and its importance in the treatment of lung inflammatory diseases. PMID:26276128

  18. Exosomes from HIV-1-infected Cells Stimulate Production of Pro-inflammatory Cytokines through Trans-activating Response (TAR) RNA.

    Science.gov (United States)

    Sampey, Gavin C; Saifuddin, Mohammed; Schwab, Angela; Barclay, Robert; Punya, Shreya; Chung, Myung-Chul; Hakami, Ramin M; Zadeh, Mohammad Asad; Lepene, Benjamin; Klase, Zachary A; El-Hage, Nazira; Young, Mary; Iordanskiy, Sergey; Kashanchi, Fatah

    2016-01-15

    HIV-1 infection results in a chronic illness because long-term highly active antiretroviral therapy can lower viral titers to an undetectable level. However, discontinuation of therapy rapidly increases virus burden. Moreover, patients under highly active antiretroviral therapy frequently develop various metabolic disorders, neurocognitive abnormalities, and cardiovascular diseases. We have previously shown that exosomes containing trans-activating response (TAR) element RNA enhance susceptibility of undifferentiated naive cells to HIV-1 infection. This study indicates that exosomes from HIV-1-infected primary cells are highly abundant with TAR RNA as detected by RT-real time PCR. Interestingly, up to a million copies of TAR RNA/μl were also detected in the serum from HIV-1-infected humanized mice suggesting that TAR RNA may be stable in vivo. Incubation of exosomes from HIV-1-infected cells with primary macrophages resulted in a dramatic increase of proinflammatory cytokines, IL-6 and TNF-β, indicating that exosomes containing TAR RNA could play a direct role in control of cytokine gene expression. The intact TAR molecule was able to bind to PKR and TLR3 effectively, whereas the 5' and 3' stems (TAR microRNAs) bound best to TLR7 and -8 and none to PKR. Binding of TAR to PKR did not result in its phosphorylation, and therefore, TAR may be a dominant negative decoy molecule in cells. The TLR binding through either TAR RNA or TAR microRNA potentially can activate the NF-κB pathway and regulate cytokine expression. Collectively, these results imply that exosomes containing TAR RNA could directly affect the proinflammatory cytokine gene expression and may explain a possible mechanism of inflammation observed in HIV-1-infected patients under cART.

  19. YAP regulates S-phase entry in endothelial cells.

    Directory of Open Access Journals (Sweden)

    Zhewei Shen

    Full Text Available The Hippo pathway regulates cell proliferation and apoptosis through the Yes-associated protein (YAP transcriptional activator. YAP has a well-described role in promoting cell proliferation and survival, but the precise mechanisms and transcriptional targets that underlie these properties are still unclear and likely context-dependent. We found, using siRNA-mediated knockdown, that YAP is required for proliferation in endothelial cells but not HeLa cells. Specifically, YAP is required for S-phase entry and its absence causes cells to accumulate in G1. Microarray analysis suggests that YAP mediates this effect by regulating the transcription of genes involved in the assembly and/or firing of replication origins and homologous recombination of DNA. These findings thus provide insight into the molecular mechanisms by which YAP regulates cell cycle progression.

  20. E2F-1 induces melanoma cell apoptosis via PUMA up-regulation and Bax translocation

    International Nuclear Information System (INIS)

    PUMA is a pro-apoptotic Bcl-2 family member that has been shown to be involved in apoptosis in many cell types. We sought to ascertain whether induction of PUMA plays a crucial role in E2F-1-induced apoptosis in melanoma cells. PUMA gene and protein expression levels were detected by real-time PCR and Western blot in SK-MEL-2 and HCT116 cell lines after Ad-E2F-1 infection. Activation of the PUMA promoter by E2F-1 overexpression was detected by dual luciferase reporter assay. E2F-1-induced Bax translocation was shown by immunocytochemistry. The induction of caspase-9 activity was measured by caspase-9 colorimetric assay kit. Up-regulation of the PUMA gene and protein by E2F-1 overexpression was detected by real-time PCR and Western blot analysis in the SK-MEL-2 melanoma cell line. In support of this finding, we found six putative E2F-1 binding sites within the PUMA promoter. Subsequent dual luciferase reporter assay showed that E2F-1 expression could increase the PUMA gene promoter activity 9.3 fold in SK-MEL-2 cells. The role of PUMA in E2F-1-induced apoptosis was further investigated in a PUMA knockout cell line. Cell viability assay showed that the HCT116 PUMA-/- cell line was more resistant to Ad-E2F-1-mediated cell death than the HCT116 PUMA+/+ cell line. Moreover, a 2.2-fold induction of the PUMA promoter was also noted in the HCT116 PUMA+/+ colon cancer cell line after Ad-E2F-1 infection. Overexpression of a truncated E2F-1 protein that lacks the transactivation domain failed to up-regulate PUMA promoter, suggesting that PUMA may be a transcriptional target of E2F-1. E2F-1-induced cancer cell apoptosis was accompanied by Bax translocation from the cytosol to mitochondria and the induction of caspase-9 activity, suggesting that E2F-1-induced apoptosis is mediated by PUMA through the cytochrome C/Apaf-1-dependent pathway. Our studies strongly demonstrated that E2F-1 induces melanoma cell apoptosis via PUMA up-regulation and Bax translocation. The signaling

  1. Role of ethanol in the regulation of hepatic stellate cell function

    Institute of Scientific and Technical Information of China (English)

    Jian-Hua Wang; Robert G Batey; Jacob George

    2006-01-01

    Evidence has accumulated to suggest an important role of ethanol and/or its metabolites in the pathogenesis of alcohol-related liver disease. In this review, the fibrogenic effects of ethanol and its metabolites on hepatic stellate cells (HSCs) are discussed. In brief,ethanol interferes with retinoid metabolism and its signaling, induces the release of fibrogenic cytokines such as transforming growth factor β-1 (TGFβ-1) from HSCs, up-regulates the gene expression of collagen I and enhances type Ⅰ collagen protein production by HSCs.Ethanol further perpetuates an activated HSC phenotype through extracellular matrix remodeling. The underlying pathophysiologic mechanisms by which ethanol exerts these pro-fibrogenic effects on HSCs are reviewed.

  2. Nanotechnology in the regulation of stem cell behavior

    International Nuclear Information System (INIS)

    Stem cells are known for their potential to repair damaged tissues. The adhesion, growth and differentiation of stem cells are likely controlled by the surrounding microenvironment which contains both chemical and physical cues. Physical cues in the microenvironment, for example, nanotopography, were shown to play important roles in stem cell fate decisions. Thus, controlling stem cell behavior by nanoscale topography has become an important issue in stem cell biology. Nanotechnology has emerged as a new exciting field and research from this field has greatly advanced. Nanotechnology allows the manipulation of sophisticated surfaces/scaffolds which can mimic the cellular environment for regulating cellular behaviors. Thus, we summarize recent studies on nanotechnology with applications to stem cell biology, including the regulation of stem cell adhesion, growth, differentiation, tracking and imaging. Understanding the interactions of nanomaterials with stem cells may provide the knowledge to apply to cell–scaffold combinations in tissue engineering and regenerative medicine. (review)

  3. Nanotechnology in the regulation of stem cell behavior

    Directory of Open Access Journals (Sweden)

    King-Chuen Wu, Ching-Li Tseng, Chi-Chang Wu, Feng-Chen Kao, Yuan-Kun Tu, Edmund C So and Yang-Kao Wang

    2013-01-01

    Full Text Available Stem cells are known for their potential to repair damaged tissues. The adhesion, growth and differentiation of stem cells are likely controlled by the surrounding microenvironment which contains both chemical and physical cues. Physical cues in the microenvironment, for example, nanotopography, were shown to play important roles in stem cell fate decisions. Thus, controlling stem cell behavior by nanoscale topography has become an important issue in stem cell biology. Nanotechnology has emerged as a new exciting field and research from this field has greatly advanced. Nanotechnology allows the manipulation of sophisticated surfaces/scaffolds which can mimic the cellular environment for regulating cellular behaviors. Thus, we summarize recent studies on nanotechnology with applications to stem cell biology, including the regulation of stem cell adhesion, growth, differentiation, tracking and imaging. Understanding the interactions of nanomaterials with stem cells may provide the knowledge to apply to cell–scaffold combinations in tissue engineering and regenerative medicine.

  4. Growth regulation of cultured human nevus cells.

    Science.gov (United States)

    Mancianti, M L; Györfi, T; Shih, I M; Valyi-Nagy, I; Levengood, G; Menssen, H D; Halpern, A C; Elder, D E; Herlyn, M

    1993-03-01

    Cells isolated from congenital melanocytic nevi and cultured in vitro have growth characteristics that resemble their premalignant stage in situ. A serum-free, chemically defined medium has been developed that allows continuous growth of established nevus cultures for up to several months. Like primary melanoma cells, nevus cells in high-calcium-containing W489 medium require insulin for growth. In contrast to melanoma cells, nevus cells in serum-free medium require the presence of alpha-melanocyte-stimulating hormone, which enhanced intracellular levels of cyclic adenosine monophosphate. In contrast to the requirements of normal human melanocytes from newborn foreskin, congenital nevus cells grow with less dependency on basic fibroblast growth factor (bFGF). Nevus cultures contain bFGF-like activity, and they express bFGF mRNA. Nevic cells of compound nevi also express bFGF mRNA in situ but only in the junctional areas. These results indicate that bFGF plays an important growth regulatory role for nevus cells in vitro and in vivo. PMID:8440904

  5. Mevalonate cascade regulation of airway mesenchymal cell autophagy and apoptosis: a dual role for p53.

    Directory of Open Access Journals (Sweden)

    Saeid Ghavami

    Full Text Available Statins inhibit the proximal steps of cholesterol biosynthesis, and are linked to health benefits in various conditions, including cancer and lung disease. We have previously investigated apoptotic pathways triggered by statins in airway mesenchymal cells, and identified reduced prenylation of small GTPases as a primary effector mechanism leading to p53-mediated cell death. Here, we extend our studies of statin-induced cell death by assessing endpoints of both apoptosis and autophagy, and investigating their interplay and coincident regulation. Using primary cultured human airway smooth muscle (HASM and human airway fibroblasts (HAF, autophagy, and autophagosome formation and flux were assessed by transmission electron microscopy, cytochemistry (lysosome number and co-localization with LC3 and immunoblotting (LC3 lipidation and Atg12-5 complex formation. Chemical inhibition of autophagy increased simvastatin-induced caspase activation and cell death. Similarly, Atg5 silencing with shRNA, thus preventing Atg5-12 complex formation, increased pro-apoptotic effects of simvastatin. Simvastatin concomitantly increased p53-dependent expression of p53 up-regulated modulator of apoptosis (PUMA, NOXA, and damage-regulated autophagy modulator (DRAM. Notably both mevalonate cascade inhibition-induced autophagy and apoptosis were p53 dependent: simvastatin increased nuclear p53 accumulation, and both cyclic pifithrin-α and p53 shRNAi partially inhibited NOXA, PUMA expression and caspase-3/7 cleavage (apoptosis and DRAM expression, Atg5-12 complex formation, LC3 lipidation, and autophagosome formation (autophagy. Furthermore, the autophagy response is induced rapidly, significantly delaying apoptosis, suggesting the existence of a temporally coordinated p53 regulation network. These findings are relevant for the development of statin-based therapeutic approaches in obstructive airway disease.

  6. Akt regulates drug-induced cell death through Bcl-w downregulation.

    Directory of Open Access Journals (Sweden)

    Michela Garofalo

    Full Text Available Akt is a serine threonine kinase with a major role in transducing survival signals and regulating proteins involved in apoptosis. To find new interactors of Akt involved in cell survival, we performed a two-hybrid screening in yeast using human full-length Akt c-DNA as bait and a murine c-DNA library as prey. Among the 80 clones obtained, two were identified as Bcl-w. Bcl-w is a member of the Bcl-2 family that is essential for the regulation of cellular survival, and that is up-regulated in different human tumors, such as gastric and colorectal carcinomas. Direct interaction of Bcl-w with Akt was confirmed by immunoprecipitation assays. Subsequently, we addressed the function of this interaction: by interfering with the activity or amount of Akt, we have demonstrated that Akt modulates the amount of Bcl-w protein. We have found that inhibition of Akt activity may promote apoptosis through the downregulation of Bcl-w protein and the consequential reduction in interaction of Bcl-w with pro-apoptotic members of the Bcl-2 family. Our data provide evidence that Bcl-w is a new member of the Akt pathway and that Akt may induce anti-apoptotic signals at least in part through the regulation of the amount and activity of Bcl-w.

  7. Histone Demethylase LSD1 Regulates Neural Stem Cell Proliferation▿

    OpenAIRE

    Sun, Guoqiang; Alzayady, Kamil; Stewart, Richard; Ye, Peng; Yang, Su; Li, Wendong; Shi, Yanhong

    2010-01-01

    Lysine-specific demethylase 1 (LSD1) functions as a transcriptional coregulator by modulating histone methylation. Its role in neural stem cells has not been studied. We show here for the first time that LSD1 serves as a key regulator of neural stem cell proliferation. Inhibition of LSD1 activity or knockdown of LSD1 expression led to dramatically reduced neural stem cell proliferation. LSD1 is recruited by nuclear receptor TLX, an essential neural stem cell regulator, to the promoters of TLX...

  8. TNFalpha and IL-8 regulate the expression and function of CD44 variant proteins in human colon carcinoma cells.

    Science.gov (United States)

    Barshishat, Michal; Ariel, Amiram; Cahalon, Liora; Chowers, Yehuda; Lider, Ofer; Schwartz, Betty

    2002-01-01

    The mechanisms underlying the inflammatory and metastatic processes share a number of similar pathways, such as those involving adhesion, migration and extravasation. In this article, the effects of pro-inflammatory cytokines on metastatic-related activities of colon cancer cells were tested. The expression and biological activity of the proteoglycan CD44 in low (LS174T) and high metastatic (HM7) cell lines following exposure to TNFalpha and IL-8 were assessed. Treated cells expressed more CD44 splice variants (CD44v), while CD44 standard protein (CD44s) expression remained unchanged. Treatment with TNFalpha induced IL-8 secretion and IL-8 gene transcription in a time-dependent manner. Both cytokines enhanced the ability of the cells to adhere to the CD44-specific ligand hyaluronic acid, an effect that was specifically blocked by an anti-IL-8 antibody. These results suggest that the effect of TNFalpha on IL-8 is responsible for the regulation of the expression of CD44 isoforms. Additional experiments showed that neither of the cytokines tested regulate the expression of CD44 gene regulation via activation of a well-characterized specific 22-bp epidermal growth factor regulatory element present in the CD44 promoter sequence, suggesting that this is not the mechanism of activation. We conclude that immuno-modulatory mediators can modify the expression of cell-to-cell or cell-to-matrix adhesion proteins, implicated in the determination of phenotypes associated with aggressiveness and metastasis of colon cancer cells. PMID:12090473

  9. Cell fate regulation in early mammalian development

    Science.gov (United States)

    Oron, Efrat; Ivanova, Natalia

    2012-08-01

    Preimplantation development in mammals encompasses a period from fertilization to implantation and results in formation of a blastocyst composed of three distinct cell lineages: epiblast, trophectoderm and primitive endoderm. The epiblast gives rise to the organism, while the trophectoderm and the primitive endoderm contribute to extraembryonic tissues that support embryo development after implantation. In many vertebrates, such as frog or fish, maternally supplied lineage determinants are partitioned within the egg. Cell cleavage that follows fertilization results in polarization of these factors between the individual blastomeres, which become restricted in their developmental fate. In contrast, the mouse oocyte and zygote lack clear polarity and, until the eight-cell stage, individual blastomeres retain the potential to form all lineages. How are cell lineages specified in the absence of a maternally supplied blueprint? This is a fundamental question in the field of developmental biology. The answer to this question lies in understanding the cell-cell interactions and gene networks involved in embryonic development prior to implantation and using this knowledge to create testable models of the developmental processes that govern cell fates. We provide an overview of classic and contemporary models of early lineage development in the mouse and discuss the emerging body of work that highlights similarities and differences between blastocyst development in the mouse and other mammalian species.

  10. Inhibitory effect of carnosine on interleukin-8 production in intestinal epithelial cells through translational regulation.

    Science.gov (United States)

    Son, Dong Ok; Satsu, Hideo; Kiso, Yoshinobu; Totsuka, Mamoru; Shimizu, Makoto

    2008-05-01

    The enhanced intestinal production of pro-inflammatory cytokines leads to inflammation and carcinogenesis, and therefore its down-regulation by nutrients could represent a promising therapeutic approach. We found for the first time that the secretion of interleukin-8 (IL-8) in intestinal epithelial cells stimulated by hydrogen peroxide or TNF-alpha was suppressed in the presence of carnosine (beta-Ala-His), a dietary dipeptide. Interestingly, carnosine had no influence on the stimulus-induced IL-8 mRNA expression, although the intracellular production and secretion of IL-8 were significantly inhibited by carnosine. The inhibitory effect of carnosine on the IL-8 secretion differed from that of other histidine-containing dipeptides like Gly-His, Ala-His, and anserine (beta-Ala-1-methyl-His), which inhibited both the hydrogen peroxide-induced secretion and mRNA expression of IL-8. These observations indicate that carnosine inhibited IL-8 secretion along a unique pathway, in which IL-8 production was suppressed at a post-transcriptional level, for instance, translation. The hypothesis that carnosine inhibited the translation of IL-8 mRNA is supported by the finding that the phosphorylation of eIF4E, an initiation factor, in stimulated Caco-2 cells was inhibited by carnosine. These results suggest that carnosine is a novel type of anti-inflammatory agent that down-regulates the inflammatory response in intestinal epithelial cells by a unique mechanism. PMID:18397832

  11. ARP2, a novel pro-apoptotic protein expressed in epithelial prostate cancer LNCaP cells and epithelial ovary CHO transformed cells.

    Directory of Open Access Journals (Sweden)

    Jaime Mas-Oliva

    Full Text Available Neoplastic epithelial cells generate the most aggressive types of cancers such as those located in the lung, breast, colon, prostate and ovary. During advanced stages of prostate cancer, epithelial cells are associated to the appearance of androgen-independent tumors, an apoptotic-resistant phenotype that ultimately overgrows and promotes metastatic events. We have previously identified and electrophysiologically characterized a novel Ca(2+-permeable channel activated during apoptosis in the androgen-independent prostate epithelial cancer cell line, LNCaP. In addition, we reported for the first time the cloning and characterization of this channel-like molecule named apoptosis regulated protein 2 (ARP2 associated to a lethal influx of Ca(2+ in Xenopus oocytes. In the present study, LNCaP cells and Chinese hamster ovary cells (CHO cell line transfected with arp2-cDNA are induced to undergo apoptosis showing an important impact on cell viability and activation of caspases 3 and 7 when compared to serum deprived grown cells and ionomycin treated cells. The subcellular localization of ARP2 in CHO cells undergoing apoptosis was studied using confocal microscopy. While apoptosis progresses, ARP2 initially localized in the peri-nuclear region of cells migrates with time towards the plasma membrane region. Based on the present results and those of our previous studies, the fact that ARP2 constitutes a novel cation channel is supported. Therefore, ARP2 becomes a valuable target to modulate the influx and concentration of calcium in the cytoplasm of epithelial cancer cells showing an apoptotic-resistant phenotype during the onset of an apoptotic event.

  12. Differential effect of immune cells on non-pathogenic Gram-negative bacteria-induced nuclear factor-kappaB activation and pro-inflammatory gene expression in intestinal epithelial cells

    DEFF Research Database (Denmark)

    Haller, D.; Holt, L.; Parlesak, Alexandr;

    2004-01-01

    We have previously shown that non-pathogenic Gram negative bacteria induce RelA phosphorylation, nuclear factor (NF)-kappaB transcriptional activity and pro-inflammatory gene expression in intestinal epithelial cells (IEC) in vivo and in vitro. In this study, we investigated the molecular mechanism...... of immune-epithelial cell cross-talk on Gram-negative enteric bacteria-induced NF-kappaB signalling and pro-inflammatory gene expression in IEC using HT-29/MTX as well as CaCO-2 transwell cultures Interestingly, while differentiated HT-29/MTX cells are unresponsive to non-pathogenic Gram negative bacterial...... in the presence of PBMC. Interestingly, B. vulgatus- and E. coli-derived lipopolysaccharide-induced similar IL-8 mRNA expression in epithelial cells after basolateral stimulation of HT-29/PBMC cocultures. Although luminal enteric bacteria have adjuvant and antigenic properties in chronic intestinal inflammation...

  13. Antiproliferative and pro-apoptotic effects of quercetin on human pancreatic carcinoma cell lines EPP85-181P and EPP85-181RDB.

    Directory of Open Access Journals (Sweden)

    Malgorzata Dumanska

    2010-08-01

    Full Text Available Polyphenols are present in several edible plants and for many years induce high interest mainly due to their antioxidative and anti-inflammatory influence. At present, numerous studies are conducted on antineoplastic effects of the compounds. One of most effective biopolyphenols involves the flavonol quercetin. Our studies aimed at evaluation of antiproliferative and pro-apoptotic effects of quercetin alone and in combinations with daunorubicin on cells of human pancreatic carcinoma lines. The experiments were conducted on two cell lines, sensitive to daunorubicin EPP85-181P line, and its resistant variant EPP85-181RDB. Effect of studied substances on cell proliferation was detected using sulphorhodamine B (SRB protein staining method. Apoptotic damage was estimated using comet and TUNEL techniques. Our data demonstrated that quercetin exerted cytotoxic action on cells of the both neoplastic cell lines in concentration-dependent manner. In the case of EPP85-181RDB cell line, quercetin seemed to sensitize resistant cells to daunorubicin. In parallel, the effect of both substances on the sensitive cell line was synergistic. Results of the studies confirmed that quercetin may probably break resistance of neoplastic cells to chemotherapy. On the other side, studied flavonol augmented action of cytostatic drug in case of sensitive tumour cells what suggest, that it might allow to decrease dosage of cytostatic drugs and reduce negative side effects of the treatment.

  14. Induction of RET dependent and independent pro-inflammatory programs in human peripheral blood mononuclear cells from Hirschsprung patients.

    Directory of Open Access Journals (Sweden)

    Marta Rusmini

    Full Text Available Hirschsprung disease (HSCR is a rare congenital anomaly characterized by the absence of enteric ganglia in the distal intestinal tract. While classified as a multigenic disorder, the altered function of the RET tyrosine kinase receptor is responsible for the majority of the pathogenesis of HSCR. Recent evidence demonstrate a strong association between RET and the homeostasis of immune system. Here, we utilize a unique cohort of fifty HSCR patients to fully characterize the expression of RET receptor on both innate (monocytes and Natural Killer lymphocytes and adaptive (B and T lymphocytes human peripheral blood mononuclear cells (PBMCs and to explore the role of RET signaling in the immune system. We show that the increased expression of RET receptor on immune cell subsets from HSCR individuals correlates with the presence of loss-of-function RET mutations. Moreover, we demonstrate that the engagement of RET on PBMCs induces the modulation of several inflammatory genes. In particular, RET stimulation with glial-cell line derived neurotrophic factor family (GDNF and glycosyl-phosphatidylinositol membrane anchored co-receptor α1 (GFRα1 trigger the up-modulation of genes encoding either for chemokines (CCL20, CCL2, CCL3, CCL4, CCL7, CXCL1 and cytokines (IL-1β, IL-6 and IL-8 and the down-regulation of chemokine/cytokine receptors (CCR2 and IL8-Rα. Although at different levels, the modulation of these "RET-dependent genes" occurs in both healthy donors and HSCR patients. We also describe another set of genes that, independently from RET stimulation, are differently regulated in healthy donors versus HSCR patients. Among these "RET-independent genes", there are CSF-1R, IL1-R1, IL1-R2 and TGFβ-1, whose levels of transcripts were lower in HSCR patients compared to healthy donors, thus suggesting aberrancies of inflammatory responses at mucosal level. Overall our results demonstrate that immune system actively participates in the physiopathology of

  15. HLA-B27-homodimer-specific antibody modulates the expansion of pro-inflammatory T-cells in HLA-B27 transgenic rats

    OpenAIRE

    Osiris Marroquin Belaunzaran; Sascha Kleber; Stefan Schauer; Martin Hausmann; Flora Nicholls; Maries Van den Broek; Sravan Payeli; Adrian Ciurea; Simon Milling; Frank Stenner; Jackie Shaw; Simon Kollnberger; Paul Bowness; Ulf Petrausch; Christoph Renner

    2015-01-01

    Objectives: HLA-B27 is a common genetic risk factor for the development of Spondyloarthritides (SpA). HLA-B27 can misfold to form cell-surface heavy chain homodimers (B272) and induce pro-inflammatory responses that may lead to SpA pathogenesis. The presence of B272 can be detected on leukocytes of HLA-B27+ Ankylosing spondylitis (AS) patients and HLA-B27 transgenic rats. We characterized a novel B272–specific monoclonal antibody to study its therapeutic use in HLA-B27 associated disorder...

  16. Melatonin-mediated Bim up-regulation and cyclooxygenase-2 (COX-2) down-regulation enhances tunicamycin-induced apoptosis in MDA-MB-231 cells.

    Science.gov (United States)

    Woo, Seon Min; Min, Kyoung-jin; Kwon, Taeg Kyu

    2015-04-01

    Melatonin is involved in many physiological functions, and it has differential effects on apoptosis in normal and cancer cells. However, the mechanism of its antitumor roles is not well understood. In this study, we show that melatonin enhances tunicamycin-induced apoptosis in human breast carcinoma MDA-MB-231 cells. Melatonin up-regulates pro-apoptotic protein Bim expression at the transcriptional levels in the presence of tunicamycin. Melatonin inhibits tunicamycin-induced COX-2 expression in MDA-MB-231 cells. Furthermore, inhibition of COX-2 activity using the COX-2 inhibitor, NS398, increases tunicamycin-induced apoptosis. Interestingly, these effects were not associated with melatonin receptor signal pathways. Pertussis toxin (a general Gi protein inhibitor) or luzindole (a nonspecific melatonin receptor antagonist) did not reverse the effect of melatonin. In addition, melatonin blocked tunicamycin-induced NF-κB transcriptional activity, p65 nuclear translocation, and p38 MAPK activation. Melatonin-mediated p38 MAPK inhibition contributed to decreased COX-2 mRNA stability. Taken together, our results suggest that melatonin enhances antitumor function through up-regulation of Bim expression and down-regulation of COX-2 expression in tunicamycin-treated MDA-MB-231 cells. PMID:25711465

  17. Regulation of pulmonary inflammation by mesenchymal cells

    NARCIS (Netherlands)

    Alkhouri, Hatem; Poppinga, Wilfred Jelco; Tania, Navessa Padma; Ammit, Alaina; Schuliga, Michael

    2014-01-01

    Pulmonary inflammation and tissue remodelling are common elements of chronic respiratory diseases such as asthma, chronic obstructive pulmonary disease (COPD), idiopathic pulmonary fibrosis (IPF), and pulmonary hypertension (PH). In disease, pulmonary mesenchymal cells not only contribute to tissue

  18. Endogenous ligands of the aryl hydrocarbon receptor regulate lung dendritic cell function.

    Science.gov (United States)

    Thatcher, Thomas H; Williams, Marc A; Pollock, Stephen J; McCarthy, Claire E; Lacy, Shannon H; Phipps, Richard P; Sime, Patricia J

    2016-01-01

    The aryl hydrocarbon receptor (AhR) is a transcription factor that has been extensively studied as a regulator of toxicant metabolism. However, recent evidence indicates that the AhR also plays an important role in immunity. We hypothesized that the AhR is a novel, immune regulator of T helper type 2 (Th2) -mediated allergic airway disease. Here, we report that AhR-deficient mice develop increased allergic responses to the model allergen ovalbumin (OVA), which are driven in part by increased dendritic cell (DC) functional activation. AhR knockout (AhR(-/-) ) mice sensitized and challenged with OVA develop an increased inflammatory response in the lung compared with wild-type controls, with greater numbers of inflammatory eosinophils and neutrophils, greater T-cell proliferation, greater production of Th2 cytokines, and higher levels of OVA-specific IgE and IgG1. Lung DCs from AhR(-/-) mice stimulated antigen-specific proliferation and Th2 cytokine production by naive T cells in vitro. Additionally, AhR(-/-) DCs produced higher levels of tumour necrosis factor-α and interleukin-6, which promote Th2 differentiation, and expressed higher cell surface levels of stimulatory MHC Class II and CD86 molecules. Overall, loss of the AhR was associated with enhanced T-cell activation by pulmonary DCs and heightened pro-inflammatory allergic responses. This suggests that endogenous AhR ligands are involved in the normal regulation of Th2-mediated immunity in the lung via a DC-dependent mechanism. Therefore, the AhR may represent an important target for therapeutic intervention in allergic airways inflammation.

  19. Down-regulation of hypothalamic pro-opiomelanocortin (POMC) expression after weaning is associated with hyperphagia-induced obesity in JCR rats overexpressing neuropeptide Y.

    Science.gov (United States)

    Diané, Abdoulaye; Pierce, W David; Russell, James C; Heth, C Donald; Vine, Donna F; Richard, Denis; Proctor, Spencer D

    2014-03-14

    We hypothesised that hypothalamic feeding-related neuropeptides are differentially expressed in obese-prone and lean-prone rats and trigger overeating-induced obesity. To test this hypothesis, in the present study, we measured energy balance and hypothalamic neuropeptide Y (NPY) and pro-opiomelanocortin (POMC) mRNA expressions in male JCR:LA-cp rats. We compared, in independent cohorts, free-feeding obese-prone (Obese-FF) and lean-prone (Lean-FF) rats at pre-weaning (10 d old), weaning (21-25 d old) and early adulthood (8-12 weeks). A group of Obese-pair-feeding (PF) rats pair-fed to the Lean-FF rats was included in the adult cohort. The body weights of 10-d-old Obese-FF and Lean-FF pups were not significantly different. However, when the pups were shifted from dams' milk to solid food (weaning), the obese-prone rats exhibited more energy intake over the days than the lean-prone rats and higher body and fat pad weights and fasting plasma glucose, leptin, insulin and lipid levels. These differences were consistent with higher energy consumption and lower energy expenditure. In the young adult cohort, the differences between the Obese-FF and Lean-FF rats became more pronounced, yielding significant age effects on most of the parameters of the metabolic syndrome, which were reduced in the Obese-PF rats. The obese-prone rats displayed higher NPY expression than the lean-prone rats at pre-weaning and weaning, and the expression levels did not differ by age. In contrast, POMC expression exhibited significant age-by-genotype differences. At pre-weaning, there was no genotype difference in POMC expression, but in the weanling cohort, obese-prone pups exhibited lower POMC expression than the lean-prone rats. This genotype difference became more pronounced at adulthood. Overall, the development of hyperphagia-induced obesity in obese-prone JCR rats is related to POMC expression down-regulation in the presence of established NPY overexpression.

  20. Regulation of Neuronal Cell Death by c-Abl-Hippo/MST2 Signaling Pathway

    Science.gov (United States)

    Xiao, Lei; Bai, Yujie; Qu, Aiqin; Zheng, Zheng; Yuan, Zengqiang

    2012-01-01

    Background Mammalian Ste20-like kinases (MSTs) are the mammalian homologue of Drosophila hippo and play critical roles in regulation of cell death, organ size control, proliferation and tumorigenesis. MSTs exert pro-apoptotic function through cleavage, autophosphorylation and in turn phosphorylation of downstream targets, such as Histone H2B and FOXO (Forkhead box O). Previously we reported that protein kinase c-Abl mediates oxidative stress-induced neuronal cell death through phosphorylating MST1 at Y433, which is not conserved among mammalian MST2, Drosophila Hippo and C.elegans cst-1/2. Methodology/Principal Findings Using immunoblotting, in vitro kinase and cell death assay, we demonstrate that c-Abl kinase phosphorylates MST2 at an evolutionarily conserved site, Y81, within the kinase domain. We further show that the phosphorylation of MST2 by c-Abl leads to the disruption of the interaction with Raf-1 proteins and the enhancement of homodimerization of MST2 proteins. It thereby enhances the MST2 activation and induces neuronal cell death. Conclusions/Significance The identification of the c-Abl tyrosine kinase as a novel upstream activator of MST2 suggests that the conserved c-Abl-MST signaling cascade plays an important role in oxidative stress-induced neuronal cell death. PMID:22590567

  1. c-Myb regulates NOX1/p38 to control survival of colorectal carcinoma cells.

    Science.gov (United States)

    Pekarčíková, Lucie; Knopfová, Lucia; Beneš, Petr; Šmarda, Jan

    2016-08-01

    The c-Myb transcription factor is important for maintenance of immature cells of many tissues including colon epithelium. Overexpression of c-Myb occurring in colorectal carcinomas (CRC) as well as in other cancers often marks poor prognosis. However, the molecular mechanism explaining how c-Myb contributes to progression of CRC has not been fully elucidated. To address this point, we investigated the way how c-Myb affects sensitivity of CRC cells to anticancer drugs. Using CRC cell lines expressing exogenous c-myb we show that c-Myb protects CRC cells from the cisplatin-, oxaliplatin-, and doxorubicin-induced apoptosis, elevates reactive oxygen species via up-regulation of NOX1, and sustains the pro-survival p38 MAPK pathway. Using pharmacological inhibitors and gene silencing of p38 and NOX1 we found that these proteins are essential for the protective effect of c-Myb and that NOX1 acts upstream of p38 activation. In addition, our result suggests that transcription of NOX1 is directly controlled by c-Myb and these genes are strongly co-expressed in human tumor tissue of CRC patients. The novel c-Myb/NOX1/p38 signaling axis that protects CRC cells from chemotherapy described in this study could provide a new base for design of future therapies of CRC. PMID:27107996

  2. Regulation of split anergy in natural killer cells by inhibition of cathepsins C and H and cystatin F.

    Science.gov (United States)

    Magister, Špela; Tseng, Han-Ching; Bui, Vickie T; Kos, Janko; Jewett, Anahid

    2015-09-01

    Freshly isolated human primary NK cells induce preferential lysis of Oral Squamous Carcinoma Stem Cells (OSCSCs) when compared to differentiated Oral Squamous Carcinoma Cells (OSCCs), while anti-CD16 antibody and monocytes induce functional split anergy in primary NK cells by decreasing the cytotoxic function of NK cells and increasing the release of IFN-γ. Since NK92 cells have relatively lower levels of cytotoxicity when compared to primary NK cells, and have the ability to increase secretion of regulatory cytokines IL-10 and IL-6, we used these cells as a model of NK cell anergy to identify and to study the upstream regulators of anergy. We demonstrate in this paper that the levels of truncated monomeric cystatin F, which is known to inhibit the functions of cathepsins C and H, is significantly elevated in NK92 cells and in anergized primary NK cells. Furthermore, cystatin F co-localizes with cathepsins C and H in the lysosomal/endosomal vesicles of NK cells. Accordingly, the mature forms of aminopeptidases cathepsins C and H, which regulate the activation of effector granzymes in NK cells, are significantly decreased, whereas the levels of pro-cathepsin C enzyme is increased in anergized NK cells after triggering of the CD16 receptor. In addition, the levels of granzyme B is significantly decreased in anti-CD16mAb and target cell anergized primary NK cells and NK92 cells. Our study provides the cellular and molecular mechanisms by which target cells may utilize to inhibit the cytotoxic function of NK cells.

  3. Expression of T-cell KV1.3 potassium channel correlates with pro-inflammatory cytokines and disease activity in ulcerative colitis

    DEFF Research Database (Denmark)

    Koch Hansen, Lars; Møller, Linda Maria Sevelsted; Rabjerg, Maj;

    2014-01-01

    BACKGROUND AND AIMS: Potassium channels, KV1.3 and KCa3.1, have been suggested to control T-cell activation, proliferation, and cytokine production and may thus constitute targets for anti-inflammatory therapy. Ulcerative colitis (UC) is a chronic inflammatory bowel disease characterized...... with active UC (n=33) and controls (n=15). Protein and mRNA expression of KV1.3 and KCa3.1, immune cell markers, and pro-inflammatory cytokines were determined by quantitative-real-time-polymerase-chain-reaction (qPCR) and immunofluorescence, and correlated with clinical parameters of inflammation. In......% of CD8(+) T-cells. KV1.3 was virtually absent on infiltrating macrophages. KV1.3 mRNA expression correlated significantly with mRNA expression of pro-inflammatory cytokines TNF-α (R(2)=0.61) and IL-17A (R(2)=0.51), the mayo endoscopic subscore (R(2)=0.13), and histological inflammation (R(2)=0.23). In...

  4. c-Myc regulates cell proliferation during lens development.

    Directory of Open Access Journals (Sweden)

    Gabriel R Cavalheiro

    Full Text Available Myc protooncogenes play important roles in the regulation of cell proliferation, growth, differentiation and survival during development. In various developing organs, c-myc has been shown to control the expression of cell cycle regulators and its misregulated expression is detected in many human tumors. Here, we show that c-myc gene (Myc is highly expressed in developing mouse lens. Targeted deletion of c-myc gene from head surface ectoderm dramatically impaired ocular organogenesis, resulting in severe microphtalmia, defective anterior segment development, formation of a lens stalk and/or aphakia. In particular, lenses lacking c-myc presented thinner epithelial cell layer and growth impairment that was detectable soon after its inactivation. Defective development of c-myc-null lens was not caused by increased cell death of lens progenitor cells. Instead, c-myc loss reduced cell proliferation, what was associated with an ectopic expression of Prox1 and p27(Kip1 proteins within epithelial cells. Interestingly, a sharp decrease in the expression of the forkhead box transcription factor Foxe3 was also observed following c-myc inactivation. These data represent the first description of the physiological roles played by a Myc family member in mouse lens development. Our findings support the conclusion that c-myc regulates the proliferation of lens epithelial cells in vivo and may, directly or indirectly, modulate the expression of classical cell cycle regulators in developing mouse lens.

  5. FXR: a metabolic regulator and cell protector

    Institute of Scientific and Technical Information of China (English)

    Yan-Dong Wang; Wei-Dong Chen; David D Moore; Wendong Huang

    2008-01-01

    Farnesoid X receptor (FXR) is a member of the nuclear receptor superfamily of ligand-activated transcription fac-tors. As a metabolic regulator, FXR plays key roles in bile acid, cholesterol, lipid, and glucose metabolism. Therefore, FXR is a potential drug target for a number of metabolic disorders, especially those related to the metabolic syn-drome. More recently, our group and others have extended the functions of FXR to more than metabolic regulation, which include anti-bacterial growth in intestine, liver regeneration, and hepatocarcinogenesis. These new findings suggest that FXR has much broader roles than previously thought, and also higl light FXR as a drug target for mul-tiple diseases. This review summarizes the basic information of FXR but focuses on its new functions.

  6. Epigenetic regulator Lid maintains germline stem cells through regulating JAK-STAT signaling pathway activity

    Directory of Open Access Journals (Sweden)

    Lama Tarayrah

    2015-11-01

    Full Text Available Signaling pathways and epigenetic mechanisms have both been shown to play essential roles in regulating stem cell activity. While the role of either mechanism in this regulation is well established in multiple stem cell lineages, how the two mechanisms interact to regulate stem cell activity is not as well understood. Here we report that in the Drosophila testis, an H3K4me3-specific histone demethylase encoded by little imaginal discs (lid maintains germline stem cell (GSC mitotic index and prevents GSC premature differentiation. Lid is required in germ cells for proper expression of the Stat92E transcription factor, the downstream effector of the Janus kinase signal transducer and activator of transcription (JAK-STAT signaling pathway. Our findings support a germ cell autonomous role for the JAK-STAT pathway in maintaining GSCs and place Lid as an upstream regulator of this pathway. Our study provides new insights into the biological functions of a histone demethylase in vivo and sheds light on the interaction between epigenetic mechanisms and signaling pathways in regulating stem cell activities.

  7. Methyl jasmonate induces apoptosis and pro-apoptotic autophagy via the ROS pathway in human non-small cell lung cancer

    OpenAIRE

    Zhang, Mutian; Su, Ling; Xiao, Zhenna; Liu, Xianfang; Liu, Xiangguo

    2016-01-01

    Methyl jasmonate (MJ) is a botanical hormone that serves as a signal transduction intermediate and regulates cell death in stressed plants. MJ induces cell cycle arrest, apoptosis and non-apoptotic cell death selectively in cancer cells. However, the underlying mechanism of MJ-induced apoptosis remains unclear. In this study, we examined the molecular mechanism through which MJ induces apoptosis in human non-small cell lung cancer (NSCLC). We found that MJ triggered apoptosis via the DDIT3-TN...

  8. The Role of Janus Kinase (JAK)-3 in Regulating Toll-Like Receptor-Mediated Inflammatory Cytokine Production in Innate Immune Cells

    OpenAIRE

    Wang, Huizhi; Brown, Jonathan; Gao, Shegan; Liang, Shuang; Jotwani, Ravi; Zhou, Huaxin; Suttles, Jill; Scott, David; Lamont, Richard J.

    2013-01-01

    The role of Janus kinase (JAK)-3 in TLR-mediated innate immune responses is poorly understood, although the suppressive function of JAK3 inhibition in adaptive immune response has been well studied. In this study, we found that JAK3 inhibition enhanced TLR-mediated immune responses by differentially regulating pro- and anti- inflammatory cytokine production in innate immune cells. Specifically, JAK3 inhibition by pharmacological inhibitors or specific siRNA, or JAK3 gene knockout resulted in ...

  9. Regulated genes in mesenchymal stem cells and gastriccancer

    Institute of Scientific and Technical Information of China (English)

    Shihori Tanabe; Kazuhiko Aoyagi; Hiroshi Yokozaki; Hiroki Sasaki

    2015-01-01

    AIM To investigate the genes regulated in mesenchymalstem cells (MSCs) and diffuse-type gastric cancer (GC),gene expression was analyzed.METHODS: Gene expression of MSCs and diffuse-typeGC cells were analyzed by microarray. Genes relatedto stem cells, cancer and the epithelial-mesenchymaltransition (EMT) were extracted from human genelists using Gene Ontology and reference information.Gene panels were generated, and messenger RNAgene expression in MSCs and diffuse-type GC cells wasanalyzed. Cluster analysis was performed using the NCSSsoftware.RESULTS: The gene expression of regulator of G-proteinsignaling 1 (RGS1) was up-regulated in diffuse-type GCcells compared with MSCs. A panel of stem-cell relatedgenes and genes involved in cancer or the EMT wereexamined. Stem-cell related genes, such as growtharrest-specific 6, musashi RNA-binding protein 2 andhairy and enhancer of split 1 (Drosophila), NOTCHfamily genes and Notch ligands, such as delta-like 1(Drosophila) and Jagged 2, were regulated.CONCLUSION: Expression of RGS1 is up-regulated,and genes related to stem cells and NOTCH signalingare altered in diffuse-type GC compared with MSCs.

  10. Study of PHI on regulating apoptosis of Burkitt lymphoma Daudi cell line%PHI对Burkitt淋巴瘤Daudi细胞株凋亡调控的实验研究

    Institute of Scientific and Technical Information of China (English)

    洪苓苓; 黄轶群; 马旭东

    2011-01-01

    Objective:To investigate the effect of PHI on Burkitt lymphoma Daudi cell line in vitro and discuss its potential mechanism.Method:The viability of Daudi cells was observed by MTT method.Apoptotic rate was measured by flow cytometry.The expressions of apoptosis associated protein Bcl-2, proCaspase-9, proCaspase-3,McL-1, XIAP and Cyt-C were detected by Western Blot.Result:Compared with untreated cells, PHI inhibited the proliferation and induced the apoptosis of Daudi cells.It inhibited the expression of Bcl-2, McL-1, XIAP, Cyt-C,proCaspase-9, and proCaspase-3.Conclusion: In vitro, PHI could down-regulate the expression of Bcl-2, McL-1,XIAP, Cyt-C, proCaspase-9, and proCaspase-3.PHI might induce the apoptosis through mitochondrion pathway and inhibite the cells growth in Burkitt lymphoma Daudi cell line.PH1 might be a potential new drug for anti Burkitt lymphoma.%目的:研究PHI在体外对伯基特淋巴瘤淋巴瘤Daudi细胞株的作用,观察PHI对诱导淋巴瘤细胞凋亡的影响,初步探讨其可能的机制.方法:用MTT比色法检测体外Daudi细胞在PHI作用后增殖率的变化.用流式细胞术观察PHI诱导Daudi细胞凋亡.用蛋白免疫印迹法(Western Blot)观察在PHI作用下凋亡相关蛋白Bcl-2、proCaspase-9、proCaspase-3、McL-1、XIAP、Cyt-C表达的变化.结果:经PHI处理的Daudi细胞增殖受到明显抑制;PHI可下调Daudi细胞凋亡相关蛋白Bcl-2、McL-1、XIAP、Cyt-C,proCaspase-9、proCaspase-3的表达,诱导Daudi细胞凋亡.结论:PHI抑制伯基特淋巴瘤淋巴瘤细胞凋亡相关蛋白Bcl-2、McL-1、XIAP、Cyt-C、proCaspase-9、proCaspase-3的表达,可能通过影响线粒体凋亡途径促进Daudi细胞凋亡,抑制其细胞增殖,可能是治疗淋巴瘤的潜在新药.

  11. Regulation of Hematopoietic Stem Cells by Bone Marrow Stromal Cells

    OpenAIRE

    Anthony, Bryan; Link, Daniel C.

    2013-01-01

    Hematopoietic stem cells (HSCs) reside in specialized microenvironments (niches) in the bone marrow. The stem cell niche is thought to provide signals that support key HSC properties, including self-renewal capacity and long-term multilineage repopulation ability. The stromal cells that comprise the stem cell niche and the signals that they generate that support HSC function are the subjects of intense investigation. Here we review the complex and diverse stromal cell populations that reside ...

  12. Regulation of catalase expression in healthy and cancerous cells.

    Science.gov (United States)

    Glorieux, Christophe; Zamocky, Marcel; Sandoval, Juan Marcelo; Verrax, Julien; Calderon, Pedro Buc

    2015-10-01

    Catalase is an important antioxidant enzyme that dismutates hydrogen peroxide into water and molecular oxygen. The catalase gene has all the characteristics of a housekeeping gene (no TATA box, no initiator element sequence, high GC content in promoter) and a core promoter that is highly conserved among species. We demonstrate in this review that within this core promoter, the presence of DNA binding sites for transcription factors, such as NF-Y and Sp1, plays an essential role in the positive regulation of catalase expression. Additional transcription factors, such as FoxO3a, are also involved in this regulatory process. There is strong evidence that the protein Akt/PKB in the PI3K signaling pathway plays a major role in the expression of catalase by modulating the activity of FoxO3a. Over the past decade, other transcription factors (PPARγ, Oct-1, etc.), as well as genetic, epigenetic, and posttranscriptional processes, have emerged as crucial contributors to the regulation of catalase expression. Altered expression levels of catalase have been reported in cancer tissues compared to their normal counterparts. Deciphering the molecular mechanisms that regulate catalase expression could, therefore, be of crucial importance for the future development of pro-oxidant cancer chemotherapy.

  13. miR-148 regulates Mitf in melanoma cells.

    Directory of Open Access Journals (Sweden)

    Benedikta S Haflidadóttir

    Full Text Available The Microphthalmia associated transcription factor (Mitf is an important regulator in melanocyte development and has been shown to be involved in melanoma progression. The current model for the role of Mitf in melanoma assumes that the total activity of the protein is tightly regulated in order to secure cell proliferation. Previous research has shown that regulation of Mitf is complex and involves regulation of expression, splicing, protein stability and post-translational modifications. Here we show that microRNAs (miRNAs are also involved in regulating Mitf in melanoma cells. Sequence analysis revealed conserved binding sites for several miRNAs in the Mitf 3'UTR sequence. Furthermore, miR-148 was shown to affect Mitf mRNA expression in melanoma cells through a conserved binding site in the 3'UTR sequence of mouse and human Mitf. In addition we confirm the previously reported effects of miR-137 on Mitf. Other miRNAs, miR-27a, miR-32 and miR-124 which all have conserved binding sites in the Mitf 3'UTR sequence did not have effects on Mitf. Our data show that miR-148 and miR-137 present an additional level of regulating Mitf expression in melanocytes and melanoma cells. Loss of this regulation, either by mutations or by shortening of the 3'UTR sequence, is therefore a likely factor in melanoma formation and/or progression.

  14. MAPK signal pathways in the regulation of cell proliferation in mammalian cells

    Institute of Scientific and Technical Information of China (English)

    2002-01-01

    MAPK families play an important role in complex cellular programs like proliferation, differentiation,development, transformation, and apoptosis. At least three MAPK families have been characterized: extracellular signal-regulated kinase (ERK), Jun kinase (JNK/SAPK) and p38 MAPK. The above effects are fulfilled by regulation of cell cycle engine and other cell proliferation related proteins. In this paper we discussed their functions and cooperation with other signal pathways in regulation of cell proliferation.

  15. Evolution of cell cycle control: same molecular machines, different regulation

    DEFF Research Database (Denmark)

    de Lichtenberg, Ulrik; Jensen, Thomas Skøt; Brunak, Søren;

    2007-01-01

    Decades of research has together with the availability of whole genomes made it clear that many of the core components involved in the cell cycle are conserved across eukaryotes, both functionally and structurally. These proteins are organized in complexes and modules that are activated or...... layers of regulation together control the activity of cell cycle complexes and how this regulation has evolved. The results show surprisingly poor conservation of both the transcriptional and the post-translation regulation of individual genes and proteins; however, the changes in one layer of regulation...... are often mirrored by changes in other layers, implying that independent layers of control coevolve. By taking a bird's eye view of the cell cycle, we demonstrate how the modular organization of cellular systems possesses a built-in flexibility, which allows evolution to find many different solutions...

  16. Cell cycle regulation of human WEE1.

    OpenAIRE

    McGowan, C H; Russell, P.

    1995-01-01

    WEE1 kinase negatively regulates entry into mitosis by catalyzing the inhibitory tyrosine phosphorylation of CDC2/cyclin B kinase. We report here an investigation of human WEE1. Endogenous WEE1 migrates as an approximately 94 kDa protein in SDS-PAGE, substantially larger than the 49 kDa protein encoded by the original human WEE1 cDNA clone that was truncated at the 5'-end. Antibody depletion experiments demonstrate that WEE1 accounts for most of the activity that phosphorylates CDC2 on Tyr15 ...

  17. Pro-bone and antifat effects of green tea and its polyphenol, epigallocatechin, in rat mesenchymal stem cells in vitro.

    Science.gov (United States)

    Ko, Chun Hay; Siu, Wing Sum; Wong, Hing Lok; Shum, Wai Ting; Fung, Kwok Pui; San Lau, Clara Bik; Leung, Ping Chung

    2011-09-28

    Green tea has been demonstrated recently as a potent bone supportive agent. Our previous studies showed that green tea and its polyphenolic constituents can promote bone-forming osteoblast activities and inhibit the bone-resorpting osteoclast formation. The objective of the present study was to investigate whether green tea and its components can regulate the osteogenic and adipogenic differentiation in pluripotent rat mesenchymal stem cells (MSCs). The rat MSCs were isolated from the bone marrow of tibiae and femora. The cells were treated with decaffeinated green tea extract (GTE) and six tea polyphenols under osteogenic induction. The alkaline phosphatase (ALP) activities and matrix calcium (Ca) deposition were assessed after 7 and 14 days of treatment. Our results demonstrated that GTE could significantly increase ALP dose dependently in the concentrations without cytotoxicity (0-100 μg/mL). Among six tested tea polyphenols, epigallocatechin (EGC) was shown to be the most effective in promoting osteogenic differentiation. At 20 μM, EGC increased ALP levels and Ca deposition significantly by 2.3- and 1.7-fold, respectively, when compared with the control group. EGC also increased the mRNA expression of bone formation markers runt-related transcription factor 2, ALP, osteonectin, and osteopontin. Furthermore, EGC demonstrated its antiadipogenicity by decreasing the adipocyte formation and inhibiting the mRNA expression levels of the adipogenic markers peroxisome proliferator-activated receptor γ, ccaat/enhancer-binding protein β, and fatty acid binding protein 4. In conclusion, this is the first report of the dual action of green tea polyphenol EGC in promoting osteogenesis and inhibiting adipocyte formation in MSCs. Our results provide scientific evidence to support the potential use of green tea in supporting the bone against degenerative diseases such as osteoporosis.

  18. Acute progression of BCR-FGFR1 induced murine B-lympho/myeloproliferative disorder suggests involvement of lineages at the pro-B cell stage.

    Directory of Open Access Journals (Sweden)

    Mingqiang Ren

    Full Text Available Constitutive activation of FGFR1, through rearrangement with various dimerization domains, leads to atypical myeloproliferative disorders where, although T cell lymphoma are common, the BCR-FGFR1 chimeric kinase results in CML-like leukemia. As with the human disease, mouse bone marrow transduction/transplantation with BCR-FGFR1 leads to CML-like myeloproliferation as well as B-cell leukemia/lymphoma. The murine disease described in this report is virtually identical to the human disease in that both showed bi-lineage involvement of myeloid and B-cells, splenomegaly, leukocytosis and bone marrow hypercellularity. A CD19(+ IgM(- CD43(+ immunophenotype was seen both in primary tumors and two cell lines derived from these tumors. In all primary tumors, subpopulations of these CD19(+ IgM(- CD43(+ were also either B220(+ or B220(-, suggesting a block in differentiation at the pro-B cell stage. The B220(- phenotype was retained in one of the cell lines while the other was B220(+. When the two cell lines were transplanted into syngeneic mice, all animals developed the same B-lymphoblastic leukemia within 2-weeks. Thus, the murine model described here closely mimics the human disease with bilineage myeloid and B-cell leukemia/lymphoma which provides a representative model to investigate therapeutic intervention and a better understanding of the etiology of the disease.

  19. Tetracycline-Regulated Suppression of Amber Codons in Mammalian Cells

    OpenAIRE

    Park, Ho-Jin; RajBhandary, Uttam L.

    1998-01-01

    As an approach to inducible suppression of nonsense mutations in mammalian cells, we described recently an amber suppression system in mammalian cells dependent on coexpression of Escherichia coli glutaminyl-tRNA synthetase (GlnRS) along with the E. coli glutamine-inserting amber suppressor tRNA. Here, we report on tetracycline-regulated expression of the E. coli GlnRS gene and, thereby, tetracycline-regulated suppression of amber codons in mammalian HeLa and COS-1 cells. The E. coli GlnRS co...

  20. Mechanism of T cell regulation by microRNAs

    International Nuclear Information System (INIS)

    MicroRNAs (miRNAs) are small, non-coding single-stranded RNAs that can modulate target gene expression at post-transcriptional level and participate in cell proliferation, differentiation, and apoptosis. T cells have important functions in acquired immune response; miRNAs regulate this immune response by targeting the mRNAs of genes involved in T cell development, proliferation, differentiation, and function. For instance, miR-181 family members function in progression by targeting Bcl2 and CD69, among others. MiR-17 to miR-92 clusters function by binding to CREB1, PTEN, and Bim. Considering that the suppression of T cell-mediated immune responses against tumor cells is involved in cancer progression, we should investigate the mechanism by which miRNA regulates T cells to develop new approaches for cancer treatment

  1. Metabolic regulation of regulatory T cell development and function

    Directory of Open Access Journals (Sweden)

    David John Coe

    2014-11-01

    Full Text Available It is now well established that the effector T cell (Teff response is regulated by a series of metabolic switches. Quiescent T cells predominantly require ATP-generating processes, whereas proliferating Teff require high metabolic flux through growth-promoting pathways, such as glycolysis. Pathways that control metabolism and immune cell function are intimately linked, and changes in cell metabolism at both the cell and system levels have been shown to enhance or suppress specific T cell effector functions. Furthermore, functionally distinct T cell subsets have been shown to require distinct energetic and biosynthetic pathways to support their specific functional needs. In particular, naturally occurring regulatory T cells (Treg are characterized by a unique metabolic signature distinct to that of conventional Teff cells. We here briefly review the signaling pathways that control Treg metabolism and how this metabolic phenotype integrates their differentiation and function. Ultimately, these metabolic features may provide new opportunities for the therapeutic modulation of unwanted immune responses.

  2. Histone deacetylase inhibitors strongly sensitise neuroblastoma cells to TRAIL-induced apoptosis by a caspases-dependent increase of the pro- to anti-apoptotic proteins ratio

    International Nuclear Information System (INIS)

    Neuroblastoma (NB) is the second most common solid childhood tumour, an aggressive disease for which new therapeutic strategies are strongly needed. Tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) selectively induces apoptosis in most tumour cells, but not in normal tissues and therefore represents a valuable candidate in apoptosis-inducing therapies. Caspase-8 is silenced in a subset of highly malignant NB cells, which results in full TRAIL resistance. In addition, despite constitutive caspase-8 expression, or its possible restoration by different strategies, NB cells remain weakly sensitive to TRAIL indicating a need to develop strategies to sensitise NB cells to TRAIL. Histone deacetylase inhibitors (HDACIs) are a new class of anti-cancer agent inducing apoptosis or cell cycle arrest in tumour cells with very low toxicity toward normal cells. Although HDACIs were recently shown to increase death induced by TRAIL in weakly TRAIL-sensitive tumour cells, the precise involved sensitisation mechanisms have not been fully identified. NB cell lines were treated with various doses of HDACIs and TRAIL, then cytotoxicity was analysed by MTS/PMS proliferation assays, apoptosis was measured by the Propidium staining method, caspases activity by colorimetric protease assays, and (in)activation of apoptotic proteins by immunoblotting. Sub-toxic doses of HDACIs strongly sensitised caspase-8 positive NB cell lines to TRAIL induced apoptosis in a caspases dependent manner. Combined treatments increased the activation of caspases and Bid, and the inactivation of the anti-apoptotic proteins XIAP, Bcl-x, RIP, and survivin, thereby increasing the pro- to anti-apoptotic protein ratio. It also enhanced the activation of the mitochondrial pathway. Interestingly, the kinetics of caspases activation and inactivation of anti-apoptotic proteins is accelerated by combined treatment with TRAIL and HDACIs compared to TRAIL alone. In contrast, cell surface expression of TRAIL

  3. TGF-β Signaling Regulates Pancreatic β-Cell Proliferation through Control of Cell Cycle Regulator p27 Expression

    International Nuclear Information System (INIS)

    Proliferation of pancreatic β-cells is an important mechanism underlying β-cell mass adaptation to metabolic demands. Increasing β-cell mass by regeneration may ameliorate or correct both type 1 and type 2 diabetes, which both result from inadequate production of insulin by β-cells of the pancreatic islet. Transforming growth factor β (TGF-β) signaling is essential for fetal development and growth of pancreatic islets. In this study, we exposed HIT-T15, a clonal pancreatic β-cell line, to TGF-β signaling. We found that inhibition of TGF-β signaling promotes proliferation of the cells significantly, while TGF-β signaling stimulation inhibits proliferation of the cells remarkably. We confirmed that this proliferative regulation by TGF-β signaling is due to the changed expression of the cell cycle regulator p27. Furthermore, we demonstrated that there is no observed effect on transcriptional activity of p27 by TGF-β signaling. Our data show that TGF-β signaling mediates the cell-cycle progression of pancreatic β-cells by regulating the nuclear localization of CDK inhibitor, p27. Inhibition of TGF-β signaling reduces the nuclear accumulation of p27, and as a result this inhibition promotes proliferation of β-cells

  4. The inhibitory activity of cocoa phenolic extract against pro-inflammatory mediators secretion induced by lipopolysaccharide in RAW 264.7 cells.

    Science.gov (United States)

    Ranneh, Yazan; Ali, Faisal; Al-Qubaisi, Mothanna; Esa, Norhaizan Mohd; Ismail, Amin

    2016-01-01

    Cocoa is a rich source of polyphenols that has been traditionally used as the treatment of several types of inflammation related disease. The response to inflammation comprises the consecutive release of mediators and the enlistment of circulating leukocytes, such as macrophages. Currently, Cocoa-derived polyphenolics have shown anti-inflammatory effects in vivo, but the therapeutic benefits in vitro remain unclear. Therefore, in this study, the effect of cocoa polyphenolic extract (CPE) on RAW 264.7 macrophage cells sensitized by lipopolysaccharide as in vitro inflammatory model was investigated. The anti-inflammatory activity of CPE was assessed by measuring its ability to inhibit the pro-inflammatory enzyme 5-lipoxygenase (5-LOX) and the pro-inflammatory mediators prostaglandin E2 (PGE2), reactive oxygen species (ROS), nitric oxide (NO) and tumor necrosis factor-alpha (TNF-α). The results show that CPE significantly inhibits 5-LOX activity (p < 0.01). In addition, CPE dose-dependently suppressed the production of PGE2, ROS, NO and TNF-α in RAW 264.7 cells. These data suggest that CPE may be used for the treatment of inflammation and it's related-diseases. PMID:27190746

  5. Neural progenitor cells regulate microglia functions and activity.

    Science.gov (United States)

    Mosher, Kira I; Andres, Robert H; Fukuhara, Takeshi; Bieri, Gregor; Hasegawa-Moriyama, Maiko; He, Yingbo; Guzman, Raphael; Wyss-Coray, Tony

    2012-11-01

    We found mouse neural progenitor cells (NPCs) to have a secretory protein profile distinct from other brain cells and to modulate microglial activation, proliferation and phagocytosis. NPC-derived vascular endothelial growth factor was necessary and sufficient to exert at least some of these effects in mice. Thus, neural precursor cells may not only be shaped by microglia, but also regulate microglia functions and activity.

  6. Plant guard cell anion channel SLAC1 regulates stomatal closure

    OpenAIRE

    Vahisalu, Triin

    2010-01-01

    Plants are rooted to their growth place; therefore it is important that they react adequately to changes in environmental conditions. Stomatal pores, which are formed of a pair of guard cells in leaf epidermis, regulate plant gas-exchange. Importantly, guard cells protect the plant from desiccation in drought conditions by reducing the aperture of the stomatal pore. They serve also as the first barrier against the major air pollutant ozone, but the behaviour of guard cells during ozone expo...

  7. Insulin and glucagon regulate pancreatic α-cell proliferation.

    Directory of Open Access Journals (Sweden)

    Zhuo Liu

    Full Text Available Type 2 diabetes mellitus (T2DM results from insulin resistance and β-cell dysfunction, in the setting of hyperglucagonemia. Glucagon is a 29 amino acid peptide hormone, which is secreted from pancreatic α cells: excessively high circulating levels of glucagon lead to excessive hepatic glucose output. We investigated if α-cell numbers increase in T2DM and what factor (s regulate α-cell turnover. Lepr(db/Lepr(db (db/db mice were used as a T2DM model and αTC1 cells were used to study potential α-cell trophic factors. Here, we demonstrate that in db/db mice α-cell number and plasma glucagon levels increased as diabetes progressed. Insulin treatment (EC50 = 2 nM of α cells significantly increased α-cell proliferation in a concentration-dependent manner compared to non-insulin-treated α cells. Insulin up-regulated α-cell proliferation through the IR/IRS2/AKT/mTOR signaling pathway, and increased insulin-mediated proliferation was prevented by pretreatment with rapamycin, a specific mTOR inhibitor. GcgR antagonism resulted in reduced rates of cell proliferation in αTC1 cells. In addition, blockade of GcgRs in db/db mice improved glucose homeostasis, lessened α-cell proliferation, and increased intra-islet insulin content in β cells in db/db mice. These studies illustrate that pancreatic α-cell proliferation increases as diabetes develops, resulting in elevated plasma glucagon levels, and both insulin and glucagon are trophic factors to α-cells. Our current findings suggest that new therapeutic strategies for the treatment of T2DM may include targeting α cells and glucagon.

  8. Upregulation of Phagocytic Clearance of Apoptotic Cells by Autoimmune Regulator

    Institute of Scientific and Technical Information of China (English)

    石亮; 胡丽华; 李一荣

    2010-01-01

    To investigate the effect of autoimmune regulator(AIRE) on phagocytic clearance of apoptotic cells,a recombinant expression vector containing full-length human AIRE cDNA was transfected into 16HBE cells.After incubation with transfected 16HBE cells,engulfment of apoptotic HL-60 cells induced by camptothecin was detected by myeloperoxidase(MPO) staining.The change in the expression of Rac 1 in transfected 16HBE cells was determined by RT-PCR and Western blotting.The results showed that the phagocytosis perce...

  9. Regulation of cell cycle by the anaphase spindle midzone

    Directory of Open Access Journals (Sweden)

    Sluder Greenfield

    2004-12-01

    Full Text Available Abstract Background A number of proteins accumulate in the spindle midzone and midbody of dividing animal cells. Besides proteins essential for cytokinesis, there are also components essential for interphase functions, suggesting that the spindle midzone and/or midbody may play a role in regulating the following cell cycle. Results We microsurgically severed NRK epithelial cells during anaphase or telophase, such that the spindle midzone/midbody was associated with only one of the daughter cells. Time-lapse recording of cells severed during early anaphase indicated that the cell with midzone underwent cytokinesis-like cortical contractions and progressed normally through the interphase, whereas the cell without midzone showed no cortical contraction and an arrest or substantial delay in the progression of interphase. Similar microsurgery during telophase showed a normal progression of interphase for both daughter cells with or without the midbody. Microsurgery of anaphase cells treated with cytochalasin D or nocodazole indicated that interphase progression was independent of cortical ingression but dependent on microtubules. Conclusions We conclude that the mitotic spindle is involved in not only the separation of chromosomes but also the regulation of cell cycle. The process may involve activation of components in the spindle midzone that are required for the cell cycle, and/or degradation of components that are required for cytokinesis but may interfere with the cell cycle.

  10. Regulation of embryonic cell adhesion by the prion protein.

    Directory of Open Access Journals (Sweden)

    Edward Málaga-Trillo

    2009-03-01

    Full Text Available Prion proteins (PrPs are key players in fatal neurodegenerative disorders, yet their physiological functions remain unclear, as PrP knockout mice develop rather normally. We report a strong PrP loss-of-function phenotype in zebrafish embryos, characterized by the loss of embryonic cell adhesion and arrested gastrulation. Zebrafish and mouse PrP mRNAs can partially rescue this knockdown phenotype, indicating conserved PrP functions. Using zebrafish, mouse, and Drosophila cells, we show that PrP: (1 mediates Ca(+2-independent homophilic cell adhesion and signaling; and (2 modulates Ca(+2-dependent cell adhesion by regulating the delivery of E-cadherin to the plasma membrane. In vivo time-lapse analyses reveal that the arrested gastrulation in PrP knockdown embryos is due to deficient morphogenetic cell movements, which rely on E-cadherin-based adhesion. Cell-transplantation experiments indicate that the regulation of embryonic cell adhesion by PrP is cell-autonomous. Moreover, we find that the local accumulation of PrP at cell contact sites is concomitant with the activation of Src-related kinases, the recruitment of reggie/flotillin microdomains, and the reorganization of the actin cytoskeleton, consistent with a role of PrP in the modulation of cell adhesion via signaling. Altogether, our data uncover evolutionarily conserved roles of PrP in cell communication, which ultimately impinge on the stability of adherens cell junctions during embryonic development.

  11. Cell volume regulation in goldfish intestinal mucosa.

    Science.gov (United States)

    Groot, J A

    1981-11-01

    1. Ion and water content of goldfish intestinal mucosa, stripped free from muscular layers were measured under various incubation conditions. 2. Ouabain induces an increase in cation content that is electrically compensated for by chloride. The increase in solute content is accompanied by an increase in water content. 3. When extracellular chloride is partially replaced by sulphate, ouabain does induce cell shrinkage. 4. Anoxia induces a rapid increase in cell volume that is restored by oxygenation of the incubation solution. Ouabain prevents the restoration of volume. 5. It is concluded that the classical ouabain-sensitive Na/K pump participates in the maintenance of cellular volume. We suggest that the constancy in volume after ouabain poisoning as is reported for many tissues might be due to a low chloride conductance of its membranes. 6. Anisotonic media (range: 0.6-1.2 isotonicity), made by variation on mannitol concentration, induce changes in cell water content that deviates from the simplified van't Hoff equation by about 10%. No change in water content after the initial increase was found. 7. We conclude that goldfish enterocytes do not possess a mechanism for rapid volume readjustment. PMID:7322833

  12. Induction of interactions between CD44 and hyaluronic acid by a short exposure of human T cells to diverse pro-inflammatory mediators.

    Science.gov (United States)

    Ariel, A; Lider, O; Brill, A; Cahalon, L; Savion, N; Varon, D; Hershkoviz, R

    2000-07-01

    Migration of T cells into extravascular sites of inflammation is mediated by cell-cell and cell-matrix adhesion receptors, including the hyaluronan-binding glycoprotein, CD44. The biochemical nature of CD44 variants and the ligand specificity, function and the regulation of activation of CD44 expressed on various cell types have been extensively studied. However, little is still known about the short-term influence of cytokines and chemokines on the activation of CD44 on human T cells. Therefore, we studied the role of inflammatory mediators in regulating the adhesion of T cells from human peripheral blood to immobilized hyaluronan under static or shear stress conditions. We found that the CD44-dependent adhesion, under static and shear stress (i.e. relative gradual resistance to flow of 150 and 1500 s-1) conditions, of T cells to hyaluronan requires a T-cell activation of 2-3 hr and is regulated by the cross-linking of CD3, cytokines (e.g. interleukin-2 and tumour necrosis factor-alpha), and chemokines (e.g. MIP-1beta, interleukin-8, and RANTES). This T-cell adhesion was manifested by polarization, spreading and co-localization of cell surface CD44 with a rearranged actin cytoskeleton in hyaluronan-bound T cells. Thus, cytokines and chemokines present in the vicinities of blood vessel walls or present intravascularly in tissues where immune reactions take place, can rapidly activate the CD44 molecules expressed on T cells. PMID:10929056

  13. Regulated cell-to-cell variation in a cell-fate decision system.

    Science.gov (United States)

    Colman-Lerner, Alejandro; Gordon, Andrew; Serra, Eduard; Chin, Tina; Resnekov, Orna; Endy, Drew; Pesce, C Gustavo; Brent, Roger

    2005-09-29

    Here we studied the quantitative behaviour and cell-to-cell variability of a prototypical eukaryotic cell-fate decision system, the mating pheromone response pathway in yeast. We dissected and measured sources of variation in system output, analysing thousands of individual, genetically identical cells. Only a small proportion of total cell-to-cell variation is caused by random fluctuations in gene transcription and translation during the response ('expression noise'). Instead, variation is dominated by differences in the capacity of individual cells to transmit signals through the pathway ('pathway capacity') and to express proteins from genes ('expression capacity'). Cells with high expression capacity express proteins at a higher rate and increase in volume more rapidly. Our results identify two mechanisms that regulate cell-to-cell variation in pathway capacity. First, the MAP kinase Fus3 suppresses variation at high pheromone levels, while the MAP kinase Kss1 enhances variation at low pheromone levels. Second, pathway capacity and expression capacity are negatively correlated, suggesting a compensatory mechanism that allows cells to respond more precisely to pheromone in the presence of a large variation in expression capacity. PMID:16170311

  14. Conditional overexpression of Stat3alpha in differentiating myeloid cells results in neutrophil expansion and induces a distinct, antiapoptotic and pro-oncogenic gene expression pattern.

    Science.gov (United States)

    Redell, Michele S; Tsimelzon, Anna; Hilsenbeck, Susan G; Tweardy, David J

    2007-10-01

    Normal neutrophil development requires G-CSF signaling, which includes activation of Stat3. Studies of G-CSF-mediated Stat3 signaling in cell culture and transgenic mice have yielded conflicting data regarding the role of Stat3 in myelopoiesis. The specific functions of Stat3 remain unclear, in part, because two isoforms, Stat3alpha and Stat3beta, are expressed in myeloid cells. To understand the contribution of each Stat3 isoform to myelopoiesis, we conditionally overexpressed Stat3alpha or Stat3beta in the murine myeloid cell line 32Dcl3 (32D) and examined the consequences of overexpression on cell survival and differentiation. 32D cells induced to overexpress Stat3alpha, but not Stat3beta, generated a markedly higher number of neutrophils in response to G-CSF. This effect was a result of decreased apoptosis but not of increased proliferation. Comparison of gene expression profiles of G-CSF-stimulated, Stat3alpha-overexpressing 32D cells with those of cells with normal Stat3alpha expression revealed novel Stat3 gene targets, which may contribute to neutrophil expansion and improved survival, most notably Slc28a2, a purine nucleoside transporter, which is critical for maintenance of intracellular nucleotide levels and prevention of apoptosis, and Gpr65, an acid-sensing, G protein-coupled receptor with pro-oncogenic and antiapoptotic functions. PMID:17634277

  15. AKT activation drives the nuclear localization of CSE1L and a pro-oncogenic transcriptional activation in ovarian cancer cells.

    Science.gov (United States)

    Lorenzato, Annalisa; Biolatti, Marta; Delogu, Giuseppe; Capobianco, Giampiero; Farace, Cristiano; Dessole, Salvatore; Cossu, Antonio; Tanda, Francesco; Madeddu, Roberto; Olivero, Martina; Di Renzo, Maria Flavia

    2013-10-15

    The human homolog of the yeast cse1 gene (CSE1L) is over-expressed in ovarian cancer. CSE1L forms complex with Ran and importin-α and has roles in nucleocytoplasmic traffic and gene expression. CSE1L accumulated in the nucleus of ovarian cancer cell lines, while it was localized also in the cytoplasm of other cancer cell lines. Nuclear localization depended on AKT, which was constitutively active in ovarian cancer cells, as the CSE1L protein translocated to the cytoplasm when AKT was inactivated. Moreover, the expression of a constitutively active AKT forced the translocation of CSE1L from the cytoplasm to the nucleus in other cancer cells. Nuclear accrual of CSE1L was associated to the nuclear accumulation of the phosphorylated Ran Binding protein 3 (RanBP3), which depended on AKT as well. Also in samples of human ovarian cancer, AKT activation was associated to nuclear accumulation of CSE1L and phosphorylation of RanBP3. Expression profiling of ovarian cancer cells after CSE1L silencing showed that CSE1L was required for the expression of genes promoting invasion and metastasis. In agreement, CSE1L silencing impaired motility and invasiveness of ovarian cancer cells. Altogether these data show that in ovarian cancer cells activated AKT by affecting RanBP3 phosphorylation determines the nuclear accumulation of CSE1L and likely the nuclear concentration of transcription factors conveying pro-oncogenic signals. PMID:23948303

  16. Dietary gluten alters the balance of pro-inflammatory and anti-inflammatory cytokines in T cells of BALB/c mice

    DEFF Research Database (Denmark)

    Antvorskov, Julie C.; Fundova, Petra; Buschard, Karsten;

    2013-01-01

    in the cytokine pattern of Foxp3(+) regulatory T cells, as well as Foxp3(-) T cells, isolated from intestinal mucosal lymphoid tissue and non-mucosal lymphoid compartments in BALB/c mice. The gluten-containing standard diet markedly changed the cytokine expression within Foxp3(-) T cells, in all lymphoid organs......-inflammatory cytokine profile with higher proportion of transforming growth factor-β (TGF-β)(+) Foxp3(-) T cells in all tested lymphoid tissues and higher IL-10 expression within non-T cells in spleen, and a tendency towards a mucosal increase in TGF-β(+) Foxp3(+) regulatory T cells. Our data shows that the gluten...... tested, towards a higher expression of pro-inflammatory interferon-γ (IFN-γ), interleukin-17 (IL-17) and IL-2. In Foxp3(+) regulatory T cells, gluten ingestion resulted in a mucosal increase in IL-17 and IL-2 and an overall increase in IFN-γ and IL-4. The gluten-free diet induced an anti...

  17. Heparan sulfate regulates ADAM12 through a molecular switch mechanism

    DEFF Research Database (Denmark)

    Sørensen, Hans P; Vives, Romain R; Manetopoulos, Christina;

    2008-01-01

    tumor progression and chondrocyte proliferation in osteoarthritic cartilage, is shown to possess a pro/catalytic domain cationic molecular switch, regulated by exogenous heparan sulfate and heparin but also endogenous cell surface proteoglycans and the polyanion, calcium pentosan polysulfate. Sheddase...

  18. Viral infections and cell cycle G2/M regulation

    Institute of Scientific and Technical Information of China (English)

    Richard Y.ZHAO; Robert T.ELDER

    2005-01-01

    Progression of cells from G2 phase of the cell cycle to mitosis is a tightly regulated cellular process that requires activation of the Cdc2 kinase, which determines onset of mitosis in all eukaryotic cells. In both human and fission yeast(Schizosaccharomyces pombe) cells, the activity of Cdc2 is regulated in part by the phosphorylation status of tyrosine 15(Tyr15) on Cdc2, which is phosphorylated by Wee1 kinase during late G2 and is rapidly dephosphorylated by the Cdc25 tyrosine phosphatase to trigger entry into mitosis. These Cdc2 regulators are the downstream targets of two well-characterized G2/M checkpoint pathways which prevent cells from entering mitosis when cellular DNA is damaged or when DNA replication is inhibited. Increasing evidence suggests that Cdc2 is also commonly targeted by viral proteins,which modulate host cell cycle machinery to benefit viral survival or replication. In this review, we describe the effect of viral protein R (Vpr) encoded by human immunodeficiency virus type 1 (HIV-1) on cell cycle G2/M regulation. Based on our current knowledge about this viral effect, we hypothesize that Vpr induces cell cycle G2 arrest through a mechanism that is to some extent different from the classic G2/M checkpoints. One the unique features distinguishing Vpr-induced G2 arrest from the classic checkpoints is the role of phosphatase 2A (PP2A) in Vpr-induced G2 arrest.Interestingly, PP2A is targeted by a number of other viral proteins including SV40 small T antigen, polyomavirus T antigen, HTLV Tax and adenovirus E4orf4. Thus an in-depth understanding of the molecular mechanisms underlying Vpr-induced G2 arrest will provide additional insights into the basic biology of cell cycle G2/M regulation and into the biological significance of this effect during host-pathogen interactions.

  19. Pro Tools HD

    CERN Document Server

    Camou, Edouard

    2013-01-01

    An easy-to-follow guide for using Pro Tools HD 11 effectively.This book is ideal for anyone who already uses ProTools and wants to learn more, or is new to Pro Tools HD and wants to use it effectively in their own audio workstations.

  20. Mesenchymal stem cell printing and process regulated cell properties.

    Science.gov (United States)

    Snyder, Jessica; Rin Son, Ae; Hamid, Qudus; Wang, Chengyang; Lui, Yigong; Sun, Wei

    2015-01-01

    This topical review with original analysis and empirical results compares cell sensitivity to physical stress during printing. The objective is to frame a reproducible causation between printing environment and printed cell morphology, viability and phenotype stability. Content includes: (1) a topical review classifies the overlap between physical stress vectors during printing and mesenchymal stem cell sensitivities. (2) Original flow analysis frames the feasible range of stress duration and intensity during manufacturing. (3) Preliminary empirical results define cell properties as a function of minimum, mean and maximum stress conditions. The review and analytical characterization serve as an essential precursor to interpret surprising empirical results. Results identify key cell properties are stress-dependent and controllable based on printing process parameter selection. Printing's minimum stress condition preserves cell viability. The maximum stress increases heterogeneity of cell response, induces inelastic ultra-structural distortion of the cell membrane and chromatin, and increases necrotic subpopulations post-printing. The review, analysis and preliminary results support the feasibility of modulating cell properties during fabrication by prescriptively tuning the stress environment. The process control over cell morphology, health and the rate of differentiation is both a direct result of strain during printing and an in-direct result of increased distress signaling from necrotic sub-populations. PMID:26696405

  1. Glucocorticoid-induced leucine zipper (GILZ regulates testicular FOXO1 activity and spermatogonial stem cell (SSC function.

    Directory of Open Access Journals (Sweden)

    Devi Ngo

    Full Text Available Spermatogonia stem cell (SSC self-renewal and differentiation are tightly regulated processes that ensure a continued production of mature sperm throughout male adulthood. In the present study, we investigated the role of glucocorticoid-induced leucine zipper (GILZ in maintenance of the male germline and spermatogenesis. GILZ was detectable in germ cells of wild type mice on the day of birth, suggesting a role for GILZ in prospermatogonia and SSC pool formation. Gilz KO mice were generated and adult males were azoospermic and sterile. During the first wave of spermatogenesis in Gilz KO mice, spermatogenesis arrested part way through pachytene of meiosis I. Subsequent waves resulted in a progressive depletion of germ cells through apoptosis to ultimately produce a Sertoli cell-only phenotype. Further, in contrast to wild type littermates, PLZF(+ cells were detected in the peri-luminal region of Gilz KO mice at day 6 post-natal, suggesting a defect in prospermatogonia migration in the absence of GILZ. At age 30 days, transient accumulation of PLZF(+ cells in a subset of tubules and severely compromised spermatogenesis were observed in Gilz KO mice, consistent with defective SSC differentiation. GILZ deficiency was associated with an increase in FOXO1 transcriptional activity, which leads to activation of a selective set of FOXO1 target genes, including a pro-apoptotic protein, BIM. On the other hand, no evidence of a heightened immune response was observed. Together, these results suggest that GILZ suppresses FOXO1 nuclear translocation, promotes SSC differentiation over self-renewal, and favours germ cell survival through inhibition of BIM-dependent pro-apoptotic signals. These findings provide a mechanism for the effects of GILZ on spermatogenesis and strengthen the case for GILZ being a critical molecule in the regulation of male fertility.

  2. Glutamate Receptor Agonists and Glutamate Transporter Antagonists Regulate Differentiation of Osteoblast Lineage Cells.

    Science.gov (United States)

    Xie, Wenjie; Dolder, Silvia; Siegrist, Mark; Wetterwald, Antoinette; Hofstetter, Willy

    2016-08-01

    Development and function of osteoblast lineage cells are regulated by a complex microenvironment consisting of the bone extracellular matrix, cells, systemic hormones and cytokines, autocrine and paracrine factors, and mechanical load. Apart from receptors that transduce extracellular signals into the cell, molecular transporters play a crucial role in the cellular response to the microenvironment. Transporter molecules are responsible for cellular uptake of nutritional components, elimination of metabolites, ion transport, and cell-cell communication. In this report, the expression of molecular transporters in osteoblast lineage cells was investigated to assess their roles in cell development and activity. Low-density arrays, covering membrane and vesicular transport molecules, were used to assess gene expression in osteoblasts representing early and late differentiation states. Receptors and transporters for the amino acid glutamate were found to be differentially expressed during osteoblast development. Glutamate is a neurotransmitter in the central nervous system, and the mechanisms of its release, signal transduction, and cellular reabsorption in the synaptic cleft are well understood. Less clear, however, is the control of equivalent processes in peripheral tissues. In primary osteoblasts, inhibition of glutamate transporters with nonselective inhibitors leads to an increase in the concentration of extracellular glutamate. This change was accompanied by a decrease in osteoblast proliferation, stimulation of alkaline phosphatase, and the expression of transcripts encoding osteocalcin. Enzymatic removal of extracellular glutamate abolished these pro-differentiation effects, as did the inhibition of PKC- and Erk1/2-signaling pathways. These findings demonstrate that glutamate signaling promotes differentiation and activation of osteoblast lineage cells. Consequently, the glutamate system may represent a putative therapeutic target to induce an anabolic response

  3. Stem Cells in the Trabecular Meshwork for Regulating Intraocular Pressure.

    Science.gov (United States)

    Yun, Hongmin; Zhou, Yi; Wills, Andrew; Du, Yiqin

    2016-06-01

    Intraocular pressure (IOP) is still the main treatment target for glaucoma. Outflow resistance mainly exists at the trabecular meshwork (TM) outflow pathway, which is responsible for IOP regulation. Changes of TM cellularity and TM extracellular matrix turnover may play important roles in IOP regulation. In this article, we review basic anatomy and physiology of the outflow pathway and TM stem cell characteristics regarding the location, isolation, identification and function. TM stem cells are localized at the insert region of the TM and are label-retaining in vivo. They can be isolated by side-population cell sorting, cloning culture, or sphere culture. TM stem cells are multipotent with the ability to home to the TM region and differentiate into TM cells in vivo. Other stem cell types, such as adipose-derived stem cells, mesenchymal stem cells and induced pluripotent stem cells have been discovered for TM cell differentiation and TM regeneration. We also review glaucomatous animal models, which are suitable to study stem cell-based therapies for TM regeneration. PMID:27183473

  4. Molecular biological mechanism II. Molecular mechanisms of cell cycle regulation

    International Nuclear Information System (INIS)

    The cell cycle in eukaryotes is regulated by central cell cycle controlling protein kinase complexes. These protein kinase complexes consist of a catalytic subunit from the cyclin-dependent protein kinase family (CDK), and a regulatory subunit from the cyclin family. Cyclins are characterised by their periodic cell cycle related synthesis and destruction. Each cell cycle phase is characterised by a specific set of CDKs and cyclins. The activity of CDK/cyclin complexes is mainly regulated on four levels. It is controlled by specific phosphorylation steps, the synthesis and destruction of cyclins, the binding of specific inhibitor proteins, and by active control of their intracellular localisation. At several critical points within the cell cycle, named checkpoints, the integrity of the cellular genome is monitored. If damage to the genome or an unfinished prior cell cycle phase is detected, the cell cycle progression is stopped. These cell cycle blocks are of great importance to secure survival of cells. Their primary importance is to prevent the manifestation and heritable passage of a mutated genome to daughter cells. Damage sensing, DNA repair, cell cycle control and apoptosis are closely linked cellular defence mechanisms to secure genome integrity. Disregulation in one of these defence mechanisms are potentially correlated with an increased cancer risk and therefore in at least some cases with an increased radiation sensitivity. (orig.)

  5. MHC class II molecules regulate growth in human T cells

    DEFF Research Database (Denmark)

    Nielsen, M; Odum, Niels; Bendtzen, K;

    1994-01-01

    modulate several T cell responses. Here, we studied further the role of class II molecules in the regulation of T cell growth. Costimulation of class II molecules by immobilized HLA-DR mAb significantly enhanced interleukin (IL)-2-supported T cell growth of the majority of CD4+, CD45RAlow, ROhigh T cell......-like) as well as T cells producing both cytokines (THO-like) responded to class II mAb. The costimulatory effect was not restricted to IL-2-driven T cell growth, since TCR/CD3-induced T cell activation was also enhanced by HLA-DR mAb. Moreover, class II costimulation potentiated CD28-mAb-induced T cell...

  6. Transcriptional regulation of dendritic cell diversity.

    Science.gov (United States)

    Chopin, Michaël; Allan, Rhys S; Belz, Gabrielle T

    2012-01-01

    Dendritic cells (DCs) are specialized antigen presenting cells that are exquisitely adapted to sense pathogens and induce the development of adaptive immune responses. They form a complex network of phenotypically and functionally distinct subsets. Within this network, individual DC subsets display highly specific roles in local immunosurveillance, migration, and antigen presentation. This division of labor amongst DCs offers great potential to tune the immune response by harnessing subset-specific attributes of DCs in the clinical setting. Until recently, our understanding of DC subsets has been limited and paralleled by poor clinical translation and efficacy. We have now begun to unravel how different DC subsets develop within a complex multilayered system. These findings open up exciting possibilities for targeted manipulation of DC subsets. Furthermore, ground-breaking developments overcoming a major translational obstacle - identification of similar DC populations in mouse and man - now sets the stage for significant advances in the field. Here we explore the determinants that underpin cellular and transcriptional heterogeneity within the DC network, how these influence DC distribution and localization at steady-state, and the capacity of DCs to present antigens via direct or cross-presentation during pathogen infection.

  7. The Role of Cyclo(His-Pro) in Neurodegeneration

    Science.gov (United States)

    Grottelli, Silvia; Ferrari, Ilaria; Pietrini, Grazia; Peirce, Matthew J.; Minelli, Alba; Bellezza, Ilaria

    2016-01-01

    Neurodegenerative diseases may have distinct genetic etiologies and pathological manifestations, yet share common cellular mechanisms underpinning neuronal damage and dysfunction. These cellular mechanisms include excitotoxicity, calcium dysregulation, oxidative damage, ER stress and neuroinflammation. Recent data have identified a dual role in these events for glial cells, such as microglia and astrocytes, which are able both to induce and to protect against damage induced by diverse stresses. Cyclo(His-Pro), a cyclic dipeptide derived from the hydrolytic removal of the amino-terminal pyroglutamic acid residue of the hypothalamic thyrotropin-releasing hormone, may be important in regulating the nature of the glial cell contribution. Cyclo(His-Pro) is ubiquitous in the central nervous system and is a key substrate of organic cation transporters, which are strongly linked to neuroprotection. The cyclic dipeptide can also cross the brain-blood-barrier and, once in the brain, can affect diverse inflammatory and stress responses by modifying the Nrf2-NF-κB signaling axis. For these reasons, cyclo(His-Pro) has striking potential for therapeutic application by both parenteral and oral administration routes and may represent an important new tool in counteracting neuroinflammation-based degenerative pathologies. In this review, we discuss the chemistry and biology of cyclo(His-Pro), how it may interact with the biological mechanisms driving neurodegenerative disease, such as amyotrophic lateral sclerosis, and thereby act to preserve or restore neuronal function. PMID:27529240

  8. The Role of Cyclo(His-Pro) in Neurodegeneration.

    Science.gov (United States)

    Grottelli, Silvia; Ferrari, Ilaria; Pietrini, Grazia; Peirce, Matthew J; Minelli, Alba; Bellezza, Ilaria

    2016-01-01

    Neurodegenerative diseases may have distinct genetic etiologies and pathological manifestations, yet share common cellular mechanisms underpinning neuronal damage and dysfunction. These cellular mechanisms include excitotoxicity, calcium dysregulation, oxidative damage, ER stress and neuroinflammation. Recent data have identified a dual role in these events for glial cells, such as microglia and astrocytes, which are able both to induce and to protect against damage induced by diverse stresses. Cyclo(His-Pro), a cyclic dipeptide derived from the hydrolytic removal of the amino-terminal pyroglutamic acid residue of the hypothalamic thyrotropin-releasing hormone, may be important in regulating the nature of the glial cell contribution. Cyclo(His-Pro) is ubiquitous in the central nervous system and is a key substrate of organic cation transporters, which are strongly linked to neuroprotection. The cyclic dipeptide can also cross the brain-blood-barrier and, once in the brain, can affect diverse inflammatory and stress responses by modifying the Nrf2-NF-κB signaling axis. For these reasons, cyclo(His-Pro) has striking potential for therapeutic application by both parenteral and oral administration routes and may represent an important new tool in counteracting neuroinflammation-based degenerative pathologies. In this review, we discuss the chemistry and biology of cyclo(His-Pro), how it may interact with the biological mechanisms driving neurodegenerative disease, such as amyotrophic lateral sclerosis, and thereby act to preserve or restore neuronal function. PMID:27529240

  9. Novel role of KCNQ2/3 channels in regulating neuronal cell viability.

    Science.gov (United States)

    Zhou, X; Wei, J; Song, M; Francis, K; Yu, S P

    2011-03-01

    Overactivation of certain K(+) channels can mediate excessive K(+) efflux and intracellular K(+) depletion, which are early ionic events in apoptotic cascade. The present investigation examined a possible role of the KCNQ2/3 channel or M-channel (also named Kv7.2/7.3 channels) in the pro-apoptotic process. Whole-cell recordings detected much larger M-currents (212 ± 31 pA or 10.5 ± 1.5 pA/pF) in cultured hippocampal neurons than that in cultured cortical neurons (47 ± 21 pA or 2.4 ± 0.8 pA/pF). KCNQ2/3 channel openers N-ethylmaleimide (NEM) and flupirtine caused dose-dependent K(+) efflux, intracellular K(+) depletion, and cell death in hippocampal cultures, whereas little cell death was induced by NEM in cortical cultures. The NEM-induced cell death was antagonized by co-applied KCNQ channel inhibitor XE991 (10 μM), or by elevated extracellular K(+) concentration. Supporting a mediating role of KCNQ2/3 channels in apoptosis, expression of KCNQ2 or KCNQ2/3 channels in Chinese hamster ovary (CHO) cells initiated caspase-3 activation. Consistently, application of NEM (20 μM, 8 h) in hippocampal cultures similarly caused caspase-3 activation assessed by immunocytochemical staining and western blotting. NEM increased the expression of extracellular signal-regulated protein kinases 1 and 2 (ERK1/2), induced mitochondria membrane depolarization, cytochrome c release, formation of apoptosome complex, and apoptosis-inducing factor (AIF) translocation into nuclear. All these events were attenuated by blocking KCNQ2/3 channels. These findings provide novel evidence that KCNQ2/3 channels could be an important regulator in neuronal apoptosis. PMID:20885443

  10. Both common and specialty mushrooms inhibit adhesion molecule expression and in vitro binding of monocytes to human aortic endothelial cells in a pro-inflammatory environment

    Directory of Open Access Journals (Sweden)

    Martin Keith R

    2010-07-01

    Full Text Available Abstract Background Cardiovascular disease (CVD is a leading cause of mortality in the United States as well as globally. Epidemiological studies show that regular fruit and vegetable consumption reduces CVD risk, in part, due to antioxidant activity and immunomodulation since oxidative stress and inflammation are features of atherogenesis. Accumulating evidence also shows that dietary fungi, viz., mushrooms, can protect against chronic disease by altering inflammatory environments such as those associated with CVD although most research has focused on specialty mushrooms. In this study, we tested the ability of both common and specialty mushrooms to inhibit cellular processes associated with CVD. Methods Human aortic endothelial cells (HAEC were incubated overnight with control media with dimethylsulfoxide (DMSO vehicle (1% v/v or containing DMSO extracts of whole dehydrated mushrooms (0.1 mg/mL, which included Agaricus bisporus (white button and crimini, Lentinula edodes (shiitake, Pleurotus ostreatus (oyster, and Grifola frondosa (maitake. Monolayers were subsequently washed and incubated with medium alone or containing the pro-inflammatory cytokine IL-1β (5 ng/mL for 6 h to upregulate pro-atherosclerotic adhesion molecules (AM. AM expression was assayed by ELISA and binding of U937 human monocytes pre-loaded with fluorescent dye was determined. Results White button mushrooms consistently reduced (p Conclusion These data provide evidence that dietary mushrooms can inhibit cellular processes such as adhesion molecule expression and ultimate binding of monocytes to the endothelium under pro-inflammatory conditions, which are associated with CVD. As a result, these findings support the notion that dietary mushrooms can be protective against CVD.

  11. [Physiological regulation of hematopoietic stem cell and its molecular basis].

    Science.gov (United States)

    Dong, Fang; Hao, Sha; Cheng, Hui; Cheng, Tao

    2016-08-25

    As a classical type of tissue stem cells, hematopoietic stem cell (HSC) is the earliest discovered and has been widely applied in the clinic as a great successful example for stem cell therapy. Thus, HSC research represents a leading field in stem cell biology and regenerative medicine. Self-renewal, differentiation, quiescence, apoptosis and trafficking constitute major characteristics of functional HSCs. These characteristics also signify different dynamic states of HSC through physiological interactions with the microenvironment cues in vivo. This review covers our current knowledge on the physiological regulation of HSC and its underlying molecular mechanisms. It is our hope that this review will not only help our colleagues to understand how HSC is physiologically regulated but also serve as a good reference for the studies on stem cell and regenerative medicine in general. PMID:27546503

  12. Naegleria fowleri induces MUC5AC and pro-inflammatory cytokines in human epithelial cells via ROS production and EGFR activation.

    Science.gov (United States)

    Cervantes-Sandoval, Isaac; Serrano-Luna, José de Jesús; Meza-Cervantez, Patricia; Arroyo, Rossana; Tsutsumi, Víctor; Shibayama, Mineko

    2009-11-01

    Naegleria fowleri is an amoeboflagellate responsible for the fatal central nervous system (CNS) disease primary amoebic meningoencephalitis (PAM). This amoeba gains access to the CNS by invading the olfactory mucosa and crossing the cribriform plate. Studies using a mouse model of infection have shown that the host secretes mucus during the very early stages of infection, and this event is followed by an infiltration of neutrophils into the nasal cavity. In this study, we investigated the role of N. fowleri trophozoites in inducing the expression and secretion of airway mucin and pro-inflammatory mediators. Using the human mucoepidermal cell line NCI-H292, we demonstrated that N. fowleri induced the expression of the MUC5AC gene and protein and the pro-inflammatory mediators interleukin-8 (IL-8) and interleukin-1 beta (IL-1 beta), but not tumour necrosis factor-alpha or chemokine c-c motif ligand 11 (eotaxin). Since the production of reactive oxygen species (ROS) is a common phenomenon involved in the signalling pathways of these molecules, we analysed if trophozoites were capable of causing ROS production in NCI-H292 cells by detecting oxidation of the fluorescent probe 2,7-dichlorofluorescein diacetate. NCI-H292 cells generated ROS after 15-30 min of trophozoite stimulation. Furthermore, the expression of MUC5AC, IL-8 and IL-1 beta was inhibited in the presence of the ROS scavenger DMSO. In addition, the use of an epidermal growth factor receptor inhibitor decreased the expression of MUC5AC and IL-8, but not IL-1 beta. We conclude that N. fowleri induces the expression of some host innate defence mechanisms, such as mucin secretion (MUC5AC) and local inflammation (IL-8 and IL-1 beta) in respiratory epithelial cells via ROS production and suggest that these innate immune mechanisms probably prevent most PAM infections.

  13. Changes in some pro-and anti-inflammatory cytokines produced by bovine peripheral blood mononuclear cells following foot and mouth disease vaccination

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    N. Delirezh

    2016-09-01

    Full Text Available Interleukin (IL-17 is exclusively produced by CD4 helper T-cells upon activation. It most often acts as a pro-inflammatory cytokine, which stimulates the release of pro-inflammatory cytokines IL-6, IL-8, TNF-α, and granulocyte-macrophage colony-stimulating factor (GM-CSF. In this study, we studied the in-vitro IL-17 response to specific antigens and a variety of mitogens and compared the IL-17 response to IL-2, IL-4, IL-5, IL-6, IL-10, and IFN-γ responses. We used a foot and mouth disease (FMD vaccine as specific antigens and mitogens (phytohemagglutinin [PHA], pokeweed mitogen [PWM], and concanavalin A [Con A] to stimulate peripheral blood mononuclear cells (PBMCs of vaccinated calves. Cell culture supernatant was harvested and analyzed for cytokines, using commercially available bovine ELISA kits. The mitogens induced a significant increase in IL-17 production. IL-17 was produced at high levels in response to the T cell-stimulated mitogens, PHA, and Con A, and at low levels in response to PWM mitogens. In contrast, level of the produced IL-17 cytokines in response to the FMDV antigens was lower as compared to those produced by mitogens. The FMDV antigens and mitogens significantly increased IL-17 production. There was not a correlation between IL-17 production and type-1 cytokine, IFN-γ, and IL-2, while there was a correlation between type-2 cytokine, IL-4, and IL-5 at either cytokine level produced by PBMCs stimulated by FMDV antigens. Moreover, there was an interaction between IL-17 and IL-6, that is, as IL-6 cytokine level elevated or diminished, IL-17 cytokine level increased or decreased, as well.

  14. Induction and regulation of matrix metalloproteinase-12in human airway smooth muscle cells

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    Papi Alberto

    2005-12-01

    Full Text Available Abstract Background The elastolytic enzyme matrix metalloproteinase (MMP-12 has been implicated in the development of airway inflammation and remodeling. We investigated whether human airway smooth muscle cells could express and secrete MMP-12, thereby participating in the pathogenesis of airway inflammatory diseases. Methods Laser capture microdissection was used to collect smooth muscle cells from human bronchial biopsy sections. MMP-12 mRNA expression was analysed by quantitative real-time RT-PCR. MMP-12 protein expression and secretion from cultured primary airway smooth muscle cells was further analysed by Western blot. MMP-12 protein localization in bronchial tissue sections was detected by immunohistochemistry. MMP-12 activity was determined by zymography. The TransAM AP-1 family kit was used to measure c-Jun activation and nuclear binding. Analysis of variance was used to determine statistical significance. Results We provide evidence that MMP-12 mRNA and protein are expressed by in-situ human airway smooth muscle cells obtained from bronchial biopsies of normal volunteers, and of patients with asthma, COPD and chronic cough. The pro-inflammatory cytokine, interleukin (IL-1β, induced a >100-fold increase in MMP-12 gene expression and a >10-fold enhancement in MMP-12 activity of primary airway smooth muscle cell cultures. Selective inhibitors of extracellular signal-regulated kinase, c-Jun N-terminal kinase and phosphatidylinositol 3-kinase reduced the activity of IL-1β on MMP-12, indicating a role for these kinases in IL-1β-induced induction and release of MMP-12. IL-1β-induced MMP-12 activity and gene expression was down-regulated by the corticosteroid dexamethasone but up-regulated by the inflammatory cytokine tumour necrosis factor (TNF-α through enhancing activator protein-1 activation by IL-1β. Transforming growth factor-β had no significant effect on MMP-12 induction. Conclusion Our findings indicate that human airway smooth

  15. Diallyl disulfide enhances carbon ion beams-induced apoptotic cell death in cervical cancer cells through regulating Tap73 /ΔNp73.

    Science.gov (United States)

    Di, Cuixia; Sun, Chao; Li, Hongyan; Si, Jing; Zhang, Hong; Han, Lu; Zhao, Qiuyue; Liu, Yang; Liu, Bin; Miao, Guoying; Gan, Lu; Liu, Yuanyuan

    2015-01-01

    Diallyl disulfide (DADS), extracted from crushed garlic by steam-distillation, has been reported to provide the anticancer activity in several cancer types. However, the effect of DADS on high-LET carbon beams - induced cell death remains unknown. Therefore, we used human cervical cancer cells to elucidate the molecular effects of this diallyl sulfide. Radiotherapy remains the mainstay of treatment, especially in advanced cervical cancer and there is still space to improve the radiosensitivity to reduce radiation dosage. In this study, we found that radiation effects evoked by high-LET carbon beam was marked by inhibition of cell viability, cell cycle arrest, significant rise of apoptotic cells, regulation of transcription factor, such as p73, as well as alterations of crucial mediator of the apoptosis pathway. We further demonstrated that pretreatment of 10 µM DADS in HeLa cells exposed to radiation resulted in decrease in cell viability and increased radiosensitivity. Additionally, cells pretreated with DADS obviously inhibited the radiation-induced G2/M phase arrest, but promoted radiation-induced apoptosis. Moreover, combination DADS and the radiation exacerbated the activation of apoptosis pathways through up-regulated ration of pro-apoptotic Tap73 to anti-apoptotic ΔNp73, and its downstream proteins, such as FASLG, and APAF1. Taken together, these results suggest that DADS is a potential candidate as radio sensitive agent for cervical cancer. PMID:26505313

  16. Variability in tuberculosis granuloma T cell responses exists, but a balance of pro- and anti-inflammatory cytokines is associated with sterilization.

    Directory of Open Access Journals (Sweden)

    Hannah Priyadarshini Gideon

    2015-01-01

    Full Text Available Lung granulomas are the pathologic hallmark of tuberculosis (TB. T cells are a major cellular component of TB lung granulomas and are known to play an important role in containment of Mycobacterium tuberculosis (Mtb infection. We used cynomolgus macaques, a non-human primate model that recapitulates human TB with clinically active disease, latent infection or early infection, to understand functional characteristics and dynamics of T cells in individual granulomas. We sought to correlate T cell cytokine response and bacterial burden of each granuloma, as well as granuloma and systemic responses in individual animals. Our results support that each granuloma within an individual host is independent with respect to total cell numbers, proportion of T cells, pattern of cytokine response, and bacterial burden. The spectrum of these components overlaps greatly amongst animals with different clinical status, indicating that a diversity of granulomas exists within an individual host. On average only about 8% of T cells from granulomas respond with cytokine production after stimulation with Mtb specific antigens, and few "multi-functional" T cells were observed. However, granulomas were found to be "multi-functional" with respect to the combinations of functional T cells that were identified among lesions from individual animals. Although the responses generally overlapped, sterile granulomas had modestly higher frequencies of T cells making IL-17, TNF and any of T-1 (IFN-γ, IL-2, or TNF and/or T-17 (IL-17 cytokines than non-sterile granulomas. An inverse correlation was observed between bacterial burden with TNF and T-1/T-17 responses in individual granulomas, and a combinatorial analysis of pair-wise cytokine responses indicated that granulomas with T cells producing both pro- and anti-inflammatory cytokines (e.g. IL-10 and IL-17 were associated with clearance of Mtb. Preliminary evaluation suggests that systemic responses in the blood do not

  17. MicroRNA-34a Induces Vascular Smooth Muscle Cells Senescence by SIRT1 Downregulation and Promotes the Expression of Age-Associated Pro-inflammatory Secretory Factors.

    Science.gov (United States)

    Badi, Ileana; Burba, Ilaria; Ruggeri, Clarissa; Zeni, Filippo; Bertolotti, Matteo; Scopece, Alessandro; Pompilio, Giulio; Raucci, Angela

    2015-11-01

    Arterial aging is a major risk factor for the occurrence of cardiovascular diseases. The aged artery is characterized by endothelial dysfunction and vascular smooth muscle cells altered physiology together with low-grade chronic inflammation. MicroRNA-34a (miR-34a) has been recently implicated in cardiac, endothelial, and endothelial progenitor cell senescence; however, its contribution to aging-associated vascular smooth muscle cells phenotype has not been explored so far. We found that miR-34a was highly expressed in aortas isolated from old mice. Moreover, its well-known target, the longevity-associated protein SIRT1, was significantly downregulated during aging in both endothelial cells and vascular smooth muscle cells. Increased miR-34a as well as decreased SIRT1 expression was also observed in replicative-senescent human aortic smooth muscle cells. miR-34a overexpression in proliferative human aortic smooth muscle cells caused cell cycle arrest along with enhanced p21 protein levels and evidence of cell senescence. Furthermore, miR-34a ectopic expression induced pro-inflammatory senescence-associated secretory phenotype molecules. Finally, SIRT1 protein significantly decreased upon miR-34a overexpression and restoration of its levels rescued miR-34a-dependent human aortic smooth muscle cells senescence, but not senescence-associated secretory phenotype factors upregulation. Taken together, our findings suggest that aging-associated increase of miR-34a expression levels, by promoting vascular smooth muscle cells senescence and inflammation through SIRT1 downregulation and senescence-associated secretory phenotype factors induction, respectively, may lead to arterial dysfunctions.

  18. The pro-inflammatory cytokine tumor necrosis factor α stimulates expression of the carnitine transporter OCTN2 (novel organic cation transporter 2) and carnitine uptake via nuclear factor-κB in Madin-Darby bovine kidney cells.

    Science.gov (United States)

    Zhou, X; Ringseis, R; Wen, G; Eder, K

    2015-06-01

    Carnitine uptake into tissues is mediated mainly by the novel organic cation transporter 2 (OCTN2), whose expression is upregulated in the liver of early-lactating dairy cows. It has been shown recently that pro-inflammatory cytokines, including tumor necrosis factor α (TNFα), stimulate OCTN2 expression and carnitine uptake in intestinal cells and inflamed intestinal mucosa. Given that many early-lactating dairy cows show typical signs of hepatic and systemic inflammation, such as elevated concentrations of circulating TNFα and activation of the key regulator of inflammation, nuclear factor κB (NF-κB), in tissues, it is possible that upregulation of OCTN2 and increase of carnitine uptake by TNFα is mediated by NF-κB, a mechanism that might contribute to the upregulation of OCNT2 in the liver of early-lactating dairy cows. Thus, in the present study, we tested the hypothesis that TNFα stimulates OCTN2 gene expression and carnitine uptake via NF-κB in the bovine Madin-Darby bovine kidney (MDBK) cell line. Treatment with TNFα caused activation of NF-κB, increased the mRNA and protein concentration of OCTN2, and stimulated the uptake of carnitine in MDBK cells. In contrast, combined treatment of MDBK cells with TNFα and the NF-κB inhibitor BAY 11-7085 completely blocked the effect of TNFα on OCTN2 mRNA and protein concentration and uptake of carnitine. These findings suggest that the bovine OCTN2 gene and carnitine uptake are regulated by NF-κB. Future studies are required to show the in vivo relevance of this regulatory mechanism in cattle.

  19. Regulation of Hedgehog Signalling Inside and Outside the Cell

    Science.gov (United States)

    Ramsbottom, Simon A.; Pownall, Mary E.

    2016-01-01

    The hedgehog (Hh) signalling pathway is conserved throughout metazoans and plays an important regulatory role in both embryonic development and adult homeostasis. Many levels of regulation exist that control the release, reception, and interpretation of the hedgehog signal. The fatty nature of the Shh ligand means that it tends to associate tightly with the cell membrane, and yet it is known to act as a morphogen that diffuses to elicit pattern formation. Heparan sulfate proteoglycans (HSPGs) play a major role in the regulation of Hh distribution outside the cell. Inside the cell, the primary cilium provides an important hub for processing the Hh signal in vertebrates. This review will summarise the current understanding of how the Hh pathway is regulated from ligand production, release, and diffusion, through to signal reception and intracellular transduction.

  20. Huntingtin regulates mammary stem cell division and differentiation.

    Science.gov (United States)

    Elias, Salah; Thion, Morgane S; Yu, Hua; Sousa, Cristovao Marques; Lasgi, Charlène; Morin, Xavier; Humbert, Sandrine

    2014-04-01

    Little is known about the mechanisms of mitotic spindle orientation during mammary gland morphogenesis. Here, we report the presence of huntingtin, the protein mutated in Huntington's disease, in mouse mammary basal and luminal cells throughout mammogenesis. Keratin 5-driven depletion of huntingtin results in a decreased pool and specification of basal and luminal progenitors, and altered mammary morphogenesis. Analysis of mitosis in huntingtin-depleted basal progenitors reveals mitotic spindle misorientation. In mammary cell culture, huntingtin regulates spindle orientation in a dynein-dependent manner. Huntingtin is targeted to spindle poles through its interaction with dynein and promotes the accumulation of NUMA and LGN. Huntingtin is also essential for the cortical localization of dynein, dynactin, NUMA, and LGN by regulating their kinesin 1-dependent trafficking along astral microtubules. We thus suggest that huntingtin is a component of the pathway regulating the orientation of mammary stem cell division, with potential implications for their self-renewal and differentiation properties. PMID:24749073

  1. Huntingtin Regulates Mammary Stem Cell Division and Differentiation

    Directory of Open Access Journals (Sweden)

    Salah Elias

    2014-04-01

    Full Text Available Little is known about the mechanisms of mitotic spindle orientation during mammary gland morphogenesis. Here, we report the presence of huntingtin, the protein mutated in Huntington’s disease, in mouse mammary basal and luminal cells throughout mammogenesis. Keratin 5-driven depletion of huntingtin results in a decreased pool and specification of basal and luminal progenitors, and altered mammary morphogenesis. Analysis of mitosis in huntingtin-depleted basal progenitors reveals mitotic spindle misorientation. In mammary cell culture, huntingtin regulates spindle orientation in a dynein-dependent manner. Huntingtin is targeted to spindle poles through its interaction with dynein and promotes the accumulation of NUMA and LGN. Huntingtin is also essential for the cortical localization of dynein, dynactin, NUMA, and LGN by regulating their kinesin 1-dependent trafficking along astral microtubules. We thus suggest that huntingtin is a component of the pathway regulating the orientation of mammary stem cell division, with potential implications for their self-renewal and differentiation properties.

  2. Regulation of Cell Wall Biogenesis in Saccharomyces cerevisiae: The Cell Wall Integrity Signaling Pathway

    OpenAIRE

    Levin, David E.

    2011-01-01

    The yeast cell wall is a strong, but elastic, structure that is essential not only for the maintenance of cell shape and integrity, but also for progression through the cell cycle. During growth and morphogenesis, and in response to environmental challenges, the cell wall is remodeled in a highly regulated and polarized manner, a process that is principally under the control of the cell wall integrity (CWI) signaling pathway. This pathway transmits wall stress signals from the cell surface to...

  3. Phosphorylation of actopaxin regulates cell spreading and migration

    Science.gov (United States)

    Clarke, Dominic M.; Brown, Michael C.; LaLonde, David P.; Turner, Christopher E.

    2004-01-01

    Actopaxin is an actin and paxillin binding protein that localizes to focal adhesions. It regulates cell spreading and is phosphorylated during mitosis. Herein, we identify a role for actopaxin phosphorylation in cell spreading and migration. Stable clones of U2OS cells expressing actopaxin wild-type (WT), nonphosphorylatable, and phosphomimetic mutants were developed to evaluate actopaxin function. All proteins targeted to focal adhesions, however the nonphosphorylatable mutant inhibited spreading whereas the phosphomimetic mutant cells spread more efficiently than WT cells. Endogenous and WT actopaxin, but not the nonphosphorylatable mutant, were phosphorylated in vivo during cell adhesion/spreading. Expression of the nonphosphorylatable actopaxin mutant significantly reduced cell migration, whereas expression of the phosphomimetic increased cell migration in scrape wound and Boyden chamber migration assays. In vitro kinase assays demonstrate that extracellular signal-regulated protein kinase phosphorylates actopaxin, and treatment of U2OS cells with the MEK1 inhibitor UO126 inhibited adhesion-induced phosphorylation of actopaxin and also inhibited cell migration. PMID:15353548

  4. Mast Cells and Th17 Cells Contribute to the Lymphoma-Associated Pro-Inflammatory Microenvironment of Angioimmunoblastic T-Cell Lymphoma

    OpenAIRE

    Tripodo, Claudio; Gri, Giorgia; Piccaluga, Pier Paolo; Frossi, Barbara; Guarnotta, Carla; Piconese, Silvia; Franco, Giovanni; Vetri, Valeria; Pucillo, Carlo Ennio; Florena, Ada Maria; Colombo, Mario Paolo; Pileri, Stefano Aldo

    2010-01-01

    Reports focusing on the immunological microenvironment of peripheral T-cell lymphomas (PTCL) are rare. Here we studied the reciprocal contribution of regulatory (Treg) and interleukin-17-producing (Th17) T-cells to the composition of the lymphoma-associated microenvironment of angioimmunoblastic T-cell lymphoma (AITL) and PTCL not otherwise specified on tissue microarrays from 30 PTCLs not otherwise specified and 37 AITLs. We found that Th17 but not Treg cells were differently represented in ...

  5. p53 regulation and activity in mouse embryonic stem cells

    OpenAIRE

    Solozobova, Valeriya

    2010-01-01

    P53 is a tumour development p53. The aim of this work was to study the regulation of p53 in embryonic stem cells and its activation in response to DNA damage. p53 was found that p53 becomes transcriptionally active in ES cells after DNA damage. Embryonic stem cells contain a relatively high amount of p53 protein and p53 RNA. After differentiation p53 level is rapidly downregulated. The high abundance of p53 in undifferentiated ES cells is a result of enhanced translation.

  6. Cell Adhesion and Its Endocytic Regulation in Cell Migration during Neural Development and Cancer Metastasis

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    Takeshi Kawauchi

    2012-04-01

    Full Text Available Cell migration is a crucial event for tissue organization during development, and its dysregulation leads to several diseases, including cancer. Cells exhibit various types of migration, such as single mesenchymal or amoeboid migration, collective migration and scaffold cell-dependent migration. The migration properties are partly dictated by cell adhesion and its endocytic regulation. While an epithelial-mesenchymal transition (EMT-mediated mesenchymal cell migration requires the endocytic recycling of integrin-mediated adhesions after the disruption of cell-cell adhesions, an amoeboid migration is not dependent on any adhesions to extracellular matrix (ECM or neighboring cells. In contrast, a collective migration is mediated by both cell-cell and cell-ECM adhesions, and a scaffold cell-dependent migration is regulated by the endocytosis and recycling of cell-cell adhesion molecules. Although some invasive carcinoma cells exhibit an EMT-mediated mesenchymal or amoeboid migration, other cancer cells are known to maintain cadherin-based cell-cell adhesions and epithelial morphology during metastasis. On the other hand, a scaffold cell-dependent migration is mainly utilized by migrating neurons in normal developing brains. This review will summarize the structures of cell adhesions, including adherens junctions and focal adhesions, and discuss the regulatory mechanisms for the dynamic behavior of cell adhesions by endocytic pathways in cell migration in physiological and pathological conditions, focusing particularly on neural development and cancer metastasis.

  7. Mast Cells Regulate Wound Healing in Diabetes.

    Science.gov (United States)

    Tellechea, Ana; Leal, Ermelindo C; Kafanas, Antonios; Auster, Michael E; Kuchibhotla, Sarada; Ostrovsky, Yana; Tecilazich, Francesco; Baltzis, Dimitrios; Zheng, Yongjun; Carvalho, Eugénia; Zabolotny, Janice M; Weng, Zuyi; Petra, Anastasia; Patel, Arti; Panagiotidou, Smaro; Pradhan-Nabzdyk, Leena; Theoharides, Theoharis C; Veves, Aristidis

    2016-07-01

    Diabetic foot ulceration is a severe complication of diabetes that lacks effective treatment. Mast cells (MCs) contribute to wound healing, but their role in diabetes skin complications is poorly understood. Here we show that the number of degranulated MCs is increased in unwounded forearm and foot skin of patients with diabetes and in unwounded dorsal skin of diabetic mice (P diabetic mice. Pretreatment with the MC degranulation inhibitor disodium cromoglycate rescues diabetes-associated wound-healing impairment in mice and shifts macrophages to the regenerative M2 phenotype (P diabetic mice deficient in MCs have delayed wound healing compared with their wild-type (WT) controls, implying that some MC mediator is needed for proper healing. MCs are a major source of vascular endothelial growth factor (VEGF) in mouse skin, but the level of VEGF is reduced in diabetic mouse skin, and its release from human MCs is reduced in hyperglycemic conditions. Topical treatment with the MC trigger substance P does not affect wound healing in MC-deficient mice, but improves it in WT mice. In conclusion, the presence of nondegranulated MCs in unwounded skin is required for proper wound healing, and therapies inhibiting MC degranulation could improve wound healing in diabetes. PMID:27207516

  8. Physiology and Regulation of Calcium Channels in Stomatal Guard Cells

    Energy Technology Data Exchange (ETDEWEB)

    Schroeder, Julian I.

    2007-05-02

    Stomatal pores in the epidermis of leaves regulate the diffusion of CO2 into leaves for photosynthetic carbon fixation and control water loss of plants during drought periods. Guard cells sense CO2, water status, light and other environmental conditions to regulate stomatal apertures for optimization of CO2 intake and plant growth under drought stress. The cytosolic second messenger calcium contributes to stomatal movements by transducing signals and regulating ion channels in guard cells. Studies suggest that both plasma membrane Ca2+ influx channels and vacuolar/organellar Ca2+ release channels contribute to ABA-induced Ca2+ elevations in guard cells. Recent research in the P.I.'s laboratory has led to identification of a novel major cation-selective Ca2+-permeable influx channel (Ica) in the plasma membrane of Arabidopsis guard cells. These advances will allow detailed characterization of Ica plasma membrane Ca2+ influx channels in guard cells. The long term goal of this research project is to gain a first detailed characterization of these novel plasma membrane Ca2+-permeable channel currents in Arabidopsis guard cells. The proposed research will investigate the hypothesis that Ica represents an important Ca2+ influx pathway for ABA and CO2 signal transduction in Arabidopsis guard cells. These studies will lead to elucidation of key signal transduction mechanisms by which plants balance CO2 influx into leaves and transpirational water loss and may contribute to future strategies for manipulating gas exchange for improved growth of crop plants and for biomass production.

  9. Id2 regulates hyporesponsive invariant natural killer T cells

    Science.gov (United States)

    Stradner, Martin H; Cheung, Kitty P; Lasorella, Anna; Goldrath, Ananda W; D’Cruz, Louise M

    2016-01-01

    While the invariant natural killer T (iNKT)-cell response to primary stimulation with the glycolipid, α-galactosylceramide (αGalCer), is robust, the secondary response to this stimulus is muted resulting in a hyporesponsive state characterized by anti-inflammatory interleukin-10 (IL-10) production and high expression of programmed cell death 1 (PD1) and neuropilin 1 (NRP1). The E protein transcription factors and their negative regulators, the Id proteins, have previously been shown to regulate iNKT cell thymic development, subset differentiation and peripheral survival. Here, we provide evidence that the expression of the transcriptional regulator Id2 is downregulated upon stimulation of iNKT cells with their cognate antigen. Moreover, loss of Id2 expression by iNKT cells resulted in a hyporesponsive state, with splenic Id2-deficient iNKT cells expressing low levels of TBET, high levels of PD1 and NRP1 and production of IL-10 upon stimulation. We propose that downregulation of Id2 expression is an essential component of induction of the anti-inflammatory, hyporesponsive state in iNKT cells. PMID:26880074

  10. Difference in Pro-Inflammatory Cytokine Responses Induced in THP1 Cells by Particulate Matter Collected on Days with and without ASIAN Dust Storms

    Directory of Open Access Journals (Sweden)

    Masanari Watanabe

    2015-07-01

    Full Text Available The associations between particulate matter from Asian dust storms (ADS and health disorders differ among studies, and the underlying mechanisms remain unclear. In this study, ADS and non-ADS particles were tested for their potential to induce pro-inflammatory cytokines associated with adverse respiratory effects. Particulate matter was collected in Japan during four periods in 2013 (2 × ADS periods; 2 × non-ADS. THP1 cells were exposed to this particulate matter, and the levels of various interleukins (ILs, and tumor necrosis factor (TNF-α were measured. Levels of IL-2 increased significantly following exposure to all particulate matter samples (compared to levels in a solvent control. Increased levels of IL-10 and TNF-α were also observed following exposure to particles collected during three (one ADS and two non-ADS and two (one ADS and one non-ADS collection periods, respectively. Thus, the effects of particulate matter on cytokine responses differed according to collection period, and the effects of ADS particles differed for each ADS event. Additionally, the levels of pro-inflammatory cytokines induced by ADS particles were not always higher than those induced by non-ADS particles.

  11. MK615 attenuates Porphyromonas gingivalis lipopolysaccharide-induced pro-inflammatory cytokine release via MAPK inactivation in murine macrophage-like RAW264.7 cells.

    Science.gov (United States)

    Morimoto, Yoko; Kikuchi, Kiyoshi; Ito, Takashi; Tokuda, Masayuki; Matsuyama, Takashi; Noma, Satoshi; Hashiguchi, Teruto; Torii, Mitsuo; Maruyama, Ikuro; Kawahara, Ko-Ichi

    2009-11-01

    The Japanese apricot, known as Ume in Japanese, has been a traditional Japanese medicine for centuries, and is a familiar and commonly consumed food. The health benefits of Ume are now being widely recognized and have been strengthened by recent studies showing that MK615, an extract of compounds from Ume, has strong anticancer and anti-inflammatory effects. However, the potential role of MK615 in the periodontal field remains unknown. Here, we found that MK615 significantly reduced the production of pro-inflammatory mediators (tumor necrosis factor-alpha and interleukin-6) induced by Porphyromonas gingivalis lipopolysaccharide (LPS), a major etiological agent in localized chronic periodontitis, in murine macrophage-like RAW264.7 cells. MK615 markedly inhibited the phosphorylation of ERK1/2, p38MAPK, and JNK, which is associated with pro-inflammatory mediator release pathways. Moreover, MK615 completely blocked LPS-triggered NF-kappaB activation. The present results suggest that MK615 has potential as a therapeutic agent for treating inflammatory diseases such as periodontitis. PMID:19706286

  12. Difference in Pro-Inflammatory Cytokine Responses Induced in THP1 Cells by Particulate Matter Collected on Days with and without ASIAN Dust Storms.

    Science.gov (United States)

    Watanabe, Masanari; Kurai, Jun; Sano, Hiroyuki; Yamasaki, Akira; Shimizu, Eiji

    2015-07-01

    The associations between particulate matter from Asian dust storms (ADS) and health disorders differ among studies, and the underlying mechanisms remain unclear. In this study, ADS and non-ADS particles were tested for their potential to induce pro-inflammatory cytokines associated with adverse respiratory effects. Particulate matter was collected in Japan during four periods in 2013 (2 × ADS periods; 2 × non-ADS). THP1 cells were exposed to this particulate matter, and the levels of various interleukins (ILs), and tumor necrosis factor (TNF)-α were measured. Levels of IL-2 increased significantly following exposure to all particulate matter samples (compared to levels in a solvent control). Increased levels of IL-10 and TNF-α were also observed following exposure to particles collected during three (one ADS and two non-ADS) and two (one ADS and one non-ADS) collection periods, respectively. Thus, the effects of particulate matter on cytokine responses differed according to collection period, and the effects of ADS particles differed for each ADS event. Additionally, the levels of pro-inflammatory cytokines induced by ADS particles were not always higher than those induced by non-ADS particles. PMID:26184251

  13. Peptide-modified PELCL electrospun membranes for regulation of vascular endothelial cells.

    Science.gov (United States)

    Zhou, Fang; Jia, Xiaoling; Yang, Yang; Yang, Qingmao; Gao, Chao; Zhao, Yunhui; Fan, Yubo; Yuan, Xiaoyan

    2016-11-01

    The efficiency of biomaterials used in small vascular repair depends greatly on their ability to interact with vascular endothelial cells (VECs). Rapid endothelialization of the vascular grafts is a promising way to prevent thrombosis and intimal hyperplasia. In this work, modification of electrospun membranes of poly(ethylene glycol)-b-poly(l-lactide-co-ε-caprolactone) (PELCL) by three different peptides for regulation of VECs were studied in order to obtain ideal bioactive biomaterials as small diameter vascular grafts. QK (a mimetic peptide to vascular endothelial growth factor), Arg-Glu-Asp-Val (REDV, a specific adhesive peptide to VECs) and Val-Ala-Pro-Gly (VAPG, a specific adhesive peptide to vascular smooth muscle cells) were investigated. Surface properties of the modified membranes and the response of VECs were verified. It was found that protein adsorption and platelet adhesion were effectively suppressed with the introduction of QK, REDV or VAPG peptides on the PELCL electrospun membranes. Both QK- and REDV-modified electrospun membranes could accelerate the proliferation of VECs in the first 9days, and the QK-modified electrospun membrane promoted cell proliferation more significantly than the REDV-modified one. The REDV-modified PELCL membrane was the most favorable for VECs adhesion than QK- and VAPG-modified membranes. It was suggested that QK- or REDV-modified PELCL electrospun membranes may have great potential applications in cardiovascular biomaterials for rapid endothelialization in situ. PMID:27524062

  14. The Nlrp3 Inflammasome Does Not Regulate Alloimmunization to Transfused Red Blood Cells in Mice

    Directory of Open Access Journals (Sweden)

    David R. Gibb

    2016-07-01

    Full Text Available Red blood cell (RBC transfusions are essential for patients with hematological disorders and bone marrow failure syndromes. Despite ABO matching, RBC transfusions can lead to production of alloantibodies against “minor” blood group antigens. Non-ABO alloimmunization is a leading cause of transfusion-associated mortality in the U.S. Despite its clinical importance, little is known about the immunological factors that promote alloimmunization. Prior studies indicate that inflammatory conditions place patients at higher risk for alloimmunization. Additionally, co-exposure to pro-inflammatory pathogen associated molecular patterns (PAMPs promotes alloimmunization in animal models, suggesting that RBC alloimmunization depends on innate immune cell activation. However, the specific innate immune stimuli and sensors that induce a T cell-dependent alloantibody response to transfused RBCs have not been identified. The NLRP3 inflammasome senses chemically diverse PAMPs and damage associated molecular patterns (DAMPs, including extracellular ATP and iron-containing heme. We hypothesized that activation of the NLRP3 inflammasome by endogenous DAMPs from RBCs promotes the alloimmune response to a sterile RBC transfusion. Using genetically modified mice lacking either NLRP3 or multiple downstream inflammasome response elements, we ruled out a role for the NLRP3 inflammasome or any Caspase-1 or -11 dependent inflammasome in regulating RBC alloantibody production to a model antigen.

  15. Lactobacilli, bifidobacteria and E. coli nissle induce pro- and anti-infiammatory cytokines in peripheral blood mononuclear cells

    Institute of Scientific and Technical Information of China (English)

    Ulf Helwig; Stefan Schreiber; Massimo Campieri; Karen M Lammers; Fernando Rizzello; Patricia Brigidi; Verena Rohleder; Elisabetta Caramelli; Paolo Gionchetti; Juergen Schrezenmeir; Ulrich R Foelsch

    2006-01-01

    AIM: To investigate whether the stimulation of peripheral blood mononuclear cells (PBMNC) with the cell debris and cell extraction of different probiotic strains is similar or Species specific.METHODS: Three strains of bifidobacteria, 4 strains of lactobacilli, and E. colinissle were sonicated and centrifuged in order to divide them into cell extract and cell debris. PBMNC were separated by density gradient and incubated for 36 h with either the cell debris or the cell extract of single strains of probiotic bacteria in doses from 102 to 108 CFU/mL. Cell supernatants were taken and interleukin (IL)-10, IL-1β, and tumor necosis factor (TNF)-α were determined by El ISA.RESULTS: Depending on the species super-family, the strains had different stimulation patterns. Except for both L. casei strains, the cell extract of bifidobacteria and lactobacilli had less stimulating capacity than cell debris, whereas the cell extract of E. coli nissle had similar stimulating properties to that of the cell debris of the strain and significantly more stimulating capacity than that of bifidobacteria and lactobacilli. The cell debris of bifidobacteria stimulated more cytokine release than the cell debris of lactobacilli. The cell debris of lactobacilli did not have a stimulating capacity when lower concentrations were used. Neither cell extraction nor cell debris had an inhibitory effect on the production of the tested cytokines by stimulated PBMNC.CONCLUSION: The incubation of probiotic strains,which have been used in clinical trials for inflammatory diseases, with immunocompetent cells leads to different species specific reactions. High IL-10 response to cell debris of bifidobacteria and E. coli nissle can be found. This corresponds to positive effects of bifidobacteria and E.coli nissle in clinical trials for inflammatory bowel disease compared to negative outcomes obtained with lactobacilli.

  16. Cell-specific Regulation of APOBEC3F by Interferons

    Institute of Scientific and Technical Information of China (English)

    Songcheng YING; Xuzhao ZHANG; Phuong Thi Nguyen SARKIS; Rongzhen XU; Xiaofang YU

    2007-01-01

    Human cytidine deaminase APOBEC3F (A3F) has broad anti-viral activity against hepatitis B virus and retroviruses including human immunodeficiency virus type 1. However, its regulation in viral natural target cells such CD4+ T lymphocytes, macrophages, and primary liver cells has not been well studied. Here we showed that A3F was up-regulated by interferon (IFN)-α in primary hepatocytes and multiple liver cell lines as well as macrophages. Although the IFN-α signaling pathway was active in T lymphoid cells and induction of other IFN stimulated genes such as PKR was detected, A3F and APOBEC3G (A3G) were not induced by IFN-o in these cells. Thus, additional factors other than known IFN-stimulated genes also regulated IFN-α-induced A3F expression distinctly. A3F and A3G expression levels in primary hepatocytes, especially after IFN-α stimulation, were comparable to those in CD4+ T lymphocytes in some individuals. Significant variations of A3F and A3G expression in primary hepatocytes from various subjects were observed. Individual variations in A3F and/or A3G regulation and expression might influence the clinical outcomes of hepatitis B infection.

  17. A novel, non-apoptotic role for Scythe/BAT3: a functional switch between the pro- and anti-proliferative roles of p21 during the cell cycle.

    Directory of Open Access Journals (Sweden)

    Sheila T Yong

    Full Text Available BACKGROUND: Scythe/BAT3 is a member of the BAG protein family whose role in apoptosis has been extensively studied. However, since the developmental defects observed in Bat3-null mouse embryos cannot be explained solely by defects in apoptosis, we investigated whether BAT3 is also involved in cell-cycle progression. METHODS/PRINCIPAL FINDINGS: Using a stable-inducible Bat3-knockdown cellular system, we demonstrated that reduced BAT3 protein level causes a delay in both G1/S transition and G2/M progression. Concurrent with these changes in cell-cycle progression, we observed a reduction in the turnover and phosphorylation of the CDK inhibitor p21, which is best known as an inhibitor of DNA replication; however, phosphorylated p21 has also been shown to promote G2/M progression. Our findings indicate that in Bat3-knockdown cells, p21 continues to be synthesized during cell-cycle phases that do not normally require p21, resulting in p21 protein accumulation and a subsequent delay in cell-cycle progression. Finally, we showed that BAT3 co-localizes with p21 during the cell cycle and is required for the translocation of p21 from the cytoplasm to the nucleus during the G1/S transition and G2/M progression. CONCLUSION: Our study reveals a novel, non-apoptotic role for BAT3 in cell-cycle regulation. By maintaining a low p21 protein level during the G1/S transition, BAT3 counteracts the inhibitory effect of p21 on DNA replication and thus enables the cells to progress from G1 to S phase. Conversely, during G2/M progression, BAT3 facilitates p21 phosphorylation by cyclin A/Cdk2, an event required for G2/M progression. BAT3 modulates these pro- and anti-proliferative roles of p21 at least in part by regulating cyclin A abundance, as well as p21 translocation between the cytoplasm and the nucleus to ensure that it functions in the appropriate intracellular compartment during each phase of the cell cycle.

  18. Cell volume regulation in epithelial physiology and cancer

    DEFF Research Database (Denmark)

    Pedersen, Stine Helene Falsig; Hoffmann, Else Kay; Novak, Ivana

    2013-01-01

    regulation both rely on the spatially and temporally coordinated function of ion channels and transporters. In healthy epithelia, specific ion channels/transporters localize to the luminal and basolateral membranes, contributing to functional epithelial polarity. In pathophysiological processes...... such as cancer, transepithelial and cell volume regulatory ion transport are dys-regulated. Furthermore, epithelial architecture and coordinated ion transport function are lost, cell survival/death balance is altered, and new interactions with the stroma arise, all contributing to drug resistance. Since altered......The physiological function of epithelia is transport of ions, nutrients, and fluid either in secretory or absorptive direction. All of these processes are closely related to cell volume changes, which are thus an integrated part of epithelial function. Transepithelial transport and cell volume...

  19. Cell fate determination by ubiquitin-dependent regulation of translation

    Science.gov (United States)

    Werner, Achim; Iwasaki, Shintaro; McGourty, Colleen; Medina-Ruiz, Sofia; Teerikorpi, Nia; Fedrigo, Indro; Ingolia, Nicholas T.; Rape, Michael

    2015-01-01

    Metazoan development depends on accurate execution of differentiation programs that allow pluripotent stem cells to adopt specific fates 1. Differentiation requires changes to chromatin architecture and transcriptional networks, yet whether other regulatory events support cell fate determination is less well understood. Here, we have identified the vertebrate-specific ubiquitin ligase CUL3KBTBD8 as an essential regulator of neural crest specification. CUL3KBTBD8 monoubiquitylates NOLC1 and its paralog TCOF1, whose mutation underlies the neurocristopathy Treacher Collins Syndrome 2,3. Ubiquitylation drives formation of a TCOF1-NOLC1 platform that connects RNA polymerase I with ribosome modification enzymes and remodels the translational program of differentiating cells in favor of neural crest specification. We conclude that ubiquitin-dependent regulation of translation is an important feature of cell fate determination. PMID:26399832

  20. Anti-Proliferative and Pro-Apoptotic Activities of 4-Methyl-2,6-bis(1-phenylethyl)phenol in Cancer Cells.

    Science.gov (United States)

    Sung, Nak Yoon; Kim, Seung Cheol; Kim, Yun Hwan; Kim, Gihyeon; Lee, Yunmi; Sung, Gi-Ho; Kim, Ji Hye; Yang, Woo Seok; Kim, Mi Seon; Baek, Kwang-Soo; Kim, Jong-Hoon; Cho, Jae Youl

    2016-07-01

    It has been found that 4-isopropyl-2,6-bis(1-phenylethyl)phenol (KTH-13), a novel compound isolated from Cordyceps bassiana, is able to suppress tumor cell proliferation by inducing apoptosis. To mass-produce this compound, we established a total synthesis method. Using those conditions, we further synthesized various analogs with structural similarity to KTH-13. In this study, we aimed to test their anti-cancer activity by measuring anti-proliferative and pro-apoptotic activities. Of 8 compounds tested, 4-methyl-2,6-bis(1-phenylethyl)phenol (KTH-13-Me) exhibited the strongest anti-proliferative activity toward MDA-MB 231 cells. KTH-13-Me also similarly suppressed the survival of various cancer cell lines, including C6 glioma, HCT-15, and LoVo cells. Treatment of KTH-13-Me induced several apoptotic signs in C6 glioma cells, such as morphological changes, induction of apoptotic bodies, and nuclear fragmentation and chromatin condensation. Concordantly, early-apoptotic cells were also identified by staining with FITC-Annexin V/PI. Moreover, KTH-13-Me highly enhanced the activation of caspase-3 and caspase-9, and decreased the protein level of Bcl-2. In addition, the phosphorylation levels of Src and STAT3 were diminished in KTH-13-Me-treated C6 cells. Therefore, these results suggest that KTH-13-Me can be developed as a novel anti-cancer drug capable of blocking proliferation, inducing apoptosis, and blocking cell survival signaling in cancer cells. PMID:27068261

  1. Anti-Proliferative and Pro-Apoptotic Activities of 4-Methyl-2,6-bis(1-phenylethyl)phenol in Cancer Cells

    Science.gov (United States)

    Sung, Nak Yoon; Kim, Seung Cheol; Kim, Yun Hwan; Kim, Gihyeon; Lee, Yunmi; Sung, Gi-Ho; Kim, Ji Hye; Yang, Woo Seok; Kim, Mi Seon; Baek, Kwang-Soo; Kim, Jong-Hoon; Cho, Jae Youl

    2016-01-01

    It has been found that 4-isopropyl-2,6-bis(1-phenylethyl)phenol (KTH-13), a novel compound isolated from Cordyceps bassiana, is able to suppress tumor cell proliferation by inducing apoptosis. To mass-produce this compound, we established a total synthesis method. Using those conditions, we further synthesized various analogs with structural similarity to KTH-13. In this study, we aimed to test their anti-cancer activity by measuring anti-proliferative and pro-apoptotic activities. Of 8 compounds tested, 4-methyl-2,6-bis(1-phenylethyl)phenol (KTH-13-Me) exhibited the strongest anti-proliferative activity toward MDA-MB 231 cells. KTH-13-Me also similarly suppressed the survival of various cancer cell lines, including C6 glioma, HCT-15, and LoVo cells. Treatment of KTH-13-Me induced several apoptotic signs in C6 glioma cells, such as morphological changes, induction of apoptotic bodies, and nuclear fragmentation and chromatin condensation. Concordantly, early-apoptotic cells were also identified by staining with FITC-Annexin V/PI. Moreover, KTH-13-Me highly enhanced the activation of caspase-3 and caspase-9, and decreased the protein level of Bcl-2. In addition, the phosphorylation levels of Src and STAT3 were diminished in KTH-13-Me-treated C6 cells. Therefore, these results suggest that KTH-13-Me can be developed as a novel anti-cancer drug capable of blocking proliferation, inducing apoptosis, and blocking cell survival signaling in cancer cells. PMID:27068261

  2. Store-operated calcium channels and pro-inflammatory signals

    Institute of Scientific and Technical Information of China (English)

    Wei-chiao CHANG

    2006-01-01

    In non-excitable cells such as T lymphocytes,hepatocytes,mast cells,endothelia and epithelia,the major pathway for calcium(Ca2+)entry is through store-operated Ca2+ channels in the plasma membrane.These channels are activated by the emptying of intracellular Ca2+ stores,however,neither the gating mechanism nor the downstream targets of these channels has been clear established.Here,I review some of the proposed gating mechanisms of store-operated Ca2+ channels and the functional implications in regulating pro-inflammatory signals.

  3. Transcriptional Enhancers In The Regulation Of T Cell Differentiation

    OpenAIRE

    Nguyen, Michelle L. T.; Sarah A. Jones; Prier, Julia E.; Brendan Edward Russ

    2015-01-01

    The changes in phenotype and function that characterise the differentiation of naïve T cells to effector and memory states are underscored by large-scale, coordinated, and stable changes in gene expression. In turn, these changes are choreographed by the interplay between transcription factors and epigenetic regulators that act to restructure the genome, ultimately ensuring lineage-appropriate gene expression. Here, we focus on the mechanisms that control T cell differentiation, with a partic...

  4. Bone Cell Autophagy Is Regulated by Environmental Factors

    OpenAIRE

    Zahm, Adam M.; Bohensky, Jolene; Adams, Christopher S.; Shapiro, Irving M.; Srinivas, Vickram

    2011-01-01

    The goal of this investigation was to ascertain whether bone cells undergo autophagy and to determine if this process is regulated by environmental factors. We showed that osteocytes in both murine and human cortical bone display a punctuate distribution of microtubule-associated protein light chain 3, indicative of autophagy. In addition, we noted a basal level of autophagy in preosteocyte-like murine long bone-derived osteocytic (MLO)-A5 cells. Autophagy was upregulated following nutrient d...

  5. Sef Regulates Epithelial-Mesenchymal Transition in Breast Cancer Cells.

    Science.gov (United States)

    He, Qing; Gong, Yan; Gower, Lindsey; Yang, Xuehui; Friesel, Robert E

    2016-10-01

    Sef (similar expression to fgf), also know as IL17RD, is a transmembrane protein shown to inhibit fibroblast growth factor signaling in developmental and cancer contexts; however, its role as a tumor suppressor remains to be fully elucidated. Here, we show that Sef regulates epithelial-mesenchymal transition (EMT) in breast cancer cell lines. Sef expression was highest in the normal breast epithelial cell line MCF10A, intermediate expression in MCF-7 cells and lowest in MDA-MB-231 cells. Knockdown of Sef increased the expression of genes associated with EMT, and promoted cell migration, invasion, and a fibroblastic morphology of MCF-7 cells. Overexpression of Sef inhibited the expression of EMT marker genes and inhibited cell migration and invasion in MCF-7 cells. Induction of EMT in MCF10A cells by TGF-β and TNF-α resulted in downregulation of Sef expression concomitant with upregulation of EMT gene expression and loss of epithelial morphology. Overexpression of Sef in MCF10A cells partially blocked cytokine-induced EMT. Sef was shown to block β-catenin mediated luciferase reporter activity and to cause a decrease in the nuclear localization of active β-catenin. Furthermore, Sef was shown to co-immunoprecipitate with β-catenin. In a mouse orthotopic xenograft model, Sef overexpression in MDA-MB-231 cells slowed tumor growth and reduced expression of EMT marker genes. Together, these data indicate that Sef plays a role in the negative regulation of EMT in a β-catenin dependent manner and that reduced expression of Sef in breast tumor cells may be permissive for EMT and the acquisition of a more metastatic phenotype. J. Cell. Biochem. 117: 2346-2356, 2016. © 2016 Wiley Periodicals, Inc. PMID:26950413

  6. Host epithelial geometry regulates breast cancer cell invasiveness

    Science.gov (United States)

    Boghaert, Eline; Gleghorn, Jason P.; Lee, KangAe; Gjorevski, Nikolce; Radisky, Derek C.; Nelson, Celeste M.

    2012-01-01

    Breast tumor development is regulated in part by cues from the local microenvironment, including interactions with neighboring nontumor cells as well as the ECM. Studies using homogeneous populations of breast cancer cell lines cultured in 3D ECM have shown that increased ECM stiffness stimulates tumor cell invasion. However, at early stages of breast cancer development, malignant cells are surrounded by normal epithelial cells, which have been shown to exert a tumor-suppressive effect on cocultured cancer cells. Here we explored how the biophysical characteristics of the host microenvironment affect the proliferative and invasive tumor phenotype of the earliest stages of tumor development, by using a 3D microfabrication-based approach to engineer ducts composed of normal mammary epithelial cells that contained a single tumor cell. We found that the phenotype of the tumor cell was dictated by its position in the duct: proliferation and invasion were enhanced at the ends and blocked when the tumor cell was located elsewhere within the tissue. Regions of invasion correlated with high endogenous mechanical stress, as shown by finite element modeling and bead displacement experiments, and modulating the contractility of the host epithelium controlled the subsequent invasion of tumor cells. Combining microcomputed tomographic analysis with finite element modeling suggested that predicted regions of high mechanical stress correspond to regions of tumor formation in vivo. This work suggests that the mechanical tone of nontumorigenic host epithelium directs the phenotype of tumor cells and provides additional insight into the instructive role of the mechanical tumor microenvironment. PMID:23150585

  7. NK Cell Subtypes as Regulators of Autoimmune Liver Disease

    Science.gov (United States)

    2016-01-01

    As major components of innate immunity, NK cells not only exert cell-mediated cytotoxicity to destroy tumors or infected cells, but also act to regulate the functions of other cells in the immune system by secreting cytokines and chemokines. Thus, NK cells provide surveillance in the early defense against viruses, intracellular bacteria, and cancer cells. However, the effecter function of NK cells must be exquisitely controlled to prevent inadvertent attack against normal “self” cells. In an organ such as the liver, where the distinction between immunotolerance and immune defense against routinely processed pathogens is critical, the plethora of NK cells has a unique role in the maintenance of homeostasis. Once self-tolerance is broken, autoimmune liver disease resulted. NK cells act as a “two-edged weapon” and even play opposite roles with both regulatory and inducer activities in the hepatic environment. That is, NK cells act not only to produce inflammatory cytokines and chemokines, but also to alter the proliferation and activation of associated lymphocytes. However, the precise regulatory mechanisms at work in autoimmune liver diseases remain to be identified. In this review, we focus on recent research with NK cells and their potential role in the development of autoimmune liver disease. PMID:27462349

  8. Lung tumor exosomes induce a pro-inflammatory phenotype in mesenchymal stem cells via NFκB-TLR signaling pathway

    OpenAIRE

    Li, Xiaoxia; Wang, Shihua; Zhu, Rongjia; Li, Hongling; Han, Qin; Zhao, Robert Chunhua

    2016-01-01

    Background In tumor microenvironment, a continuous cross-talk between cancer cells and other cellular components is required to sustain tumor progression. Accumulating evidence suggests that exosomes, a novel way of cell communication, play an important role in such cross-talk. Exosomes could facilitate the direct intercellular transfer of proteins, lipids, and miRNA/mRNA/DNAs between cells. Since mesenchymal stem cells (MSCs) can be attracted to tumor sites and become an important component ...

  9. Controlling the switches: Rho GTPase regulation during animal cell mitosis.

    Science.gov (United States)

    Zuo, Yan; Oh, Wonkyung; Frost, Jeffrey A

    2014-12-01

    Animal cell division is a fundamental process that requires complex changes in cytoskeletal organization and function. Aberrant cell division often has disastrous consequences for the cell and can lead to cell senescence, neoplastic transformation or death. As important regulators of the actin cytoskeleton, Rho GTPases play major roles in regulating many aspects of mitosis and cytokinesis. These include centrosome duplication and separation, generation of cortical rigidity, microtubule-kinetochore stabilization, cleavage furrow formation, contractile ring formation and constriction, and abscission. The ability of Rho proteins to function as regulators of cell division depends on their ability to cycle between their active, GTP-bound and inactive, GDP-bound states. However, Rho proteins are inherently inefficient at fulfilling this cycle and require the actions of regulatory proteins that enhance GTP binding (RhoGEFs), stimulate GTPase activity (RhoGAPs), and sequester inactive Rho proteins in the cytosol (RhoGDIs). The roles of these regulatory proteins in controlling cell division are an area of active investigation. In this review we will delineate the current state of knowledge of how specific RhoGEFs, RhoGAPs and RhoGDIs control mitosis and cytokinesis, and highlight the mechanisms by which their functions are controlled.

  10. Phosphorylation of Large T Antigen Regulates Merkel Cell Polyomavirus Replication

    Energy Technology Data Exchange (ETDEWEB)

    Diaz, Jason; Wang, Xin; Tsang, Sabrina H. [Department of Microbiology, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA 19104 (United States); Jiao, Jing [Department of Pathology and Laboratory Medicine, Children’s Hospital of Philadelphia, Philadelphia, PA 19104 (United States); You, Jianxin, E-mail: jianyou@mail.med.upenn.edu [Department of Microbiology, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA 19104 (United States)

    2014-07-08

    Merkel Cell Polyomavirus (MCPyV) was recently discovered as a novel human polyomavirus that is associated with ~80% of Merkel Cell Carcinomas. The Large Tumor antigen (LT) is an early viral protein which has a variety of functions, including manipulation of the cell cycle and initiating viral DNA replication. Phosphorylation plays a critical regulatory role for polyomavirus LT proteins, but no investigation of MCPyV LT phosphorylation has been performed to date. In this report mass spectrometry analysis reveals three unique phosphorylation sites: T271, T297 and T299. In vivo replication assays confirm that phosphorylation of T271 does not play a role in viral replication, while modification at T297 and T299 have dramatic and opposing effects on LT’s ability to initiate replication from the viral origin. We test these mutants for their ability to bind, unwind, and act as a functional helicase at the viral origin. These studies provide a framework for understanding how phosphorylation of LT may dynamically regulate viral replication. Although the natural host cell of MCPyV has not yet been established, this work provides a foundation for understanding how LT activity is regulated and provides tools for better exploring this regulation in both natural host cells and Merkel cells.

  11. Regulation of cell division in higher plants. Progress report

    Energy Technology Data Exchange (ETDEWEB)

    Jacobs, T.W.

    1992-07-01

    Cell division is arguably the most fundamental of all developmental processes. In higher plants, mitotic activity is largely confined to foci of patterned cell divisions called meristems. From these perpetually embryonic tissues arise the plant`s essential organs of light capture, support, protection and reproduction. Once an adequate understanding of plant cell mitotic regulation is attained, unprecedented opportunities will ensue for analyzing and genetically controlling diverse aspects of development, including plant architecture, leaf shape, plant height, and root depth. The mitotic cycle in a variety of model eukaryotic systems in under the control of a regulatory network of striking evolutionary conservation. Homologues of the yeast cdc2 gene, its catalytic product, p34, and the cyclin regulatory subunits of the MPF complex have emerged as ubiquitous mitotic regulators. We have cloned cdc2-like and cyclin genes from pea. As in other eukaryotic model systems, p34 of Pisum sativum is a subunit of a high molecular weight complex which binds the fission yeast p13 protein and displays histone H1 kinase activity in vitro. Our primary objective in this study is to gain baseline information about the regulation of this higher plant cell division control complex in non-dividing, differentiated cells as well as in synchronous and asynchronous mitotic cells. We are investigating cdc2 and cyclin expression at the levels of protein abundance, protein phosphorylation and quaternary associations.

  12. NSA2, a novel nucleolus protein regulates cell proliferation and cell cycle

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Heyu [Department of Immunology, School of Basic Medical Sciences, Peking University, No. 38 Xueyuan Road, Beijing 100191 (China); Human Disease Genomics Center, Peking University, No. 38 Xueyuan Road, Beijing 100191 (China); Ma, Xi [Department of Immunology, School of Basic Medical Sciences, Peking University, No. 38 Xueyuan Road, Beijing 100191 (China); Human Disease Genomics Center, Peking University, No. 38 Xueyuan Road, Beij