Retinal flavoprotein autofluorescence as a measure of retinal health.

PubMed

Elner, Susan G; Elner, Victor M; Field, Matthew G; Park, Seung; Heckenlively, John R; Petty, Howard R

2008-01-01

To establish that increased autofluorescence of mitochondrial flavoproteins, an indicator of mitochondrial oxidative stress, correlates with retinal cell dysfunction. Retinal flavoprotein autofluorescence (FA) was imaged in humans with a fundus camera modified with 467DF8-nm excitation and 535-nm emission filters and a back-illuminated, electron-multiplying, charge-coupled device camera interfaced with a computer equipped with customized image capture software. Multiple digital images, centered on the fovea, were obtained from each eye. Histograms of pixel intensities in grayscale units were analyzed for average intensity and average curve width. Adults with diabetes mellitus, age-related macular degeneration (ARMD), central serous retinopathy, and retinal dystrophies, as well as healthy control volunteers, were imaged. Monolayers of cultured human retinal pigment epithelial (HRPE) cells, HRPE cells exposed to sublethal doses of H2O2, and HRPE cells exposed to H2O2 in the presence of antioxidants were imaged for FA using fluorescent photomicroscopy. Control patients demonstrated low levels of retinal FA, which increased progressively with age. Diabetics without visible retinopathy demonstrated increased FA levels compared to control volunteers (P < .001). Diabetics with retinopathy demonstrated significantly higher FA values than those without retinopathy (P < .04). Patients with ARMD, central serous retinopathy, or retinal dystrophies also demonstrated significantly increased FA. Compared to control RPE cells, cells oxidatively stressed with H2O2 had significantly elevated FA (P < .05), which was prevented by antioxidants (P < .05). Retinal FA is significantly increased with age and diseases known to be mediated by oxidative stress. Retinal FA imaging may provide a novel, noninvasive method of assessing retinal health and retinal dysfunction prior to retinal cell death.

EGFR blockade enriches for lung cancer stem-like cells through Notch3-dependent signaling

PubMed Central

Arasada, Rajeswara Rao; Amann, Joseph M.; Rahman, Mohammad A; Huppert, Stacey S.; Carbone, David P.

2014-01-01

Mutations in the epidermal growth factor receptor (EGFR) are the most common actionable genetic abnormalities yet discovered in lung cancer. However, targeting these mutations with kinase inhibitors is not curative in advanced disease and has yet to demonstrate an impact on potentially curable, early-stage disease, with some data suggesting adverse outcomes. Here, we report that treatment of EGFR-mutated lung cancer cell lines with erlotinib, while showing robust cell death, enriches the ALDH+ stem-like cells through EGFR-dependent activation of Notch3. Additionally, we demonstrate that erlotinib treatment increases the clonogenicity of lung cancer cells in a sphere-forming assay, suggesting increased stem-like cell potential. We demonstrate that inhibition of EGFR kinase activity leads to activation of Notch transcriptional targets in a gamma secretase inhibitor sensitive manner and causes Notch activation. leading to an increase in ALDH high+ cells. We also find a kinase-dependent physical association between the Notch3 and EGFR receptors and tyrosine phosphorylation of Notch3. This could explain the worsened survival observed in some studies of erlotinib treatment at early-stage disease, and suggests that specific dual targeting might overcome this adverse effect. PMID:25125655

Electric utility acid fuel cell stack technology advancement

NASA Astrophysics Data System (ADS)

Congdon, J. V.; Goller, G. J.; Greising, G. J.; Obrien, J. J.; Randall, S. A.; Sandelli, G. J.; Breault, R. D.; Austin, G. W.; Bopse, S.; Coykendall, R. D.

1984-11-01

The principal effort under this program was directed at the fuel cell stack technology required to accomplish the initial feasibility demonstrations of increased cell stack operating pressures and temperatures, increased cell active area, incorporation of the ribbed substrate cell configuration at the bove conditions, and the introduction of higher performance electrocatalysts. The program results were successful with the primary accomplishments being: (1) fabrication of 10 sq ft ribbed substrate, cell components including higher performing electrocatalysts; (2) assembly of a 10 sq ft, 30-cell short stack; and (3) initial test of this stack at 120 psia and 405 F. These accomplishments demonstrate the feasibility of fabricating and handling large area cells using materials and processes that are oriented to low cost manufacture. An additional accomplishment under the program was the testing of two 3.7 sq ft short stacks at 12 psia/405 F to 5400 and 4500 hours respectively. These tests demonstrate the durability of the components and the cell stack configuration to a nominal 5000 hours at the higher pressure and temperature condition planned for the next electric utility power plant.

Electric utility acid fuel cell stack technology advancement

NASA Technical Reports Server (NTRS)

Congdon, J. V.; Goller, G. J.; Greising, G. J.; Obrien, J. J.; Randall, S. A.; Sandelli, G. J.; Breault, R. D.; Austin, G. W.; Bopse, S.; Coykendall, R. D.

1984-01-01

The principal effort under this program was directed at the fuel cell stack technology required to accomplish the initial feasibility demonstrations of increased cell stack operating pressures and temperatures, increased cell active area, incorporation of the ribbed substrate cell configuration at the bove conditions, and the introduction of higher performance electrocatalysts. The program results were successful with the primary accomplishments being: (1) fabrication of 10 sq ft ribbed substrate, cell components including higher performing electrocatalysts; (2) assembly of a 10 sq ft, 30-cell short stack; and (3) initial test of this stack at 120 psia and 405 F. These accomplishments demonstrate the feasibility of fabricating and handling large area cells using materials and processes that are oriented to low cost manufacture. An additional accomplishment under the program was the testing of two 3.7 sq ft short stacks at 12 psia/405 F to 5400 and 4500 hours respectively. These tests demonstrate the durability of the components and the cell stack configuration to a nominal 5000 hours at the higher pressure and temperature condition planned for the next electric utility power plant.

B cell activation. III. B cell plasma membrane depolarization and hyper- Ia antigen expression induced by receptor immunoglobulin cross-linking are coupled

PubMed Central

1983-01-01

We report investigation of the relationship between ligand-induced B cell plasma membrane depolarization and increased expression of membrane-associated, I-A subregion encoded (mI-A) antigens. Results demonstrate that equal frequencies of B cells are stimulated to undergo membrane depolarization and to increase mI-A expression in response to mitogen, anti-Ig, and thymus-independent (TI) or thymus-dependent (TD) antigens. Further, a cause-and-effect relationship between these two events is suggested by results that demonstrate that inhibition of anti- Fab--induced depolarization by valinomycin also inhibits the subsequent increase in mI-A antigen expression and "passive" (non-ligand-mediated) depolarization of murine B cells by K+ results in hyper-mI-A antigen expression. Based upon these results we hypothesize that antigen- mediated receptor cross-linking results in signal transduction via membrane depolarization, which is resultant in increased mI-A antigen synthesis and cell surface expression. This increase in mI-A antigen density may render the B cell more receptive to subsequent interaction with I-region-restricted helper T cells. PMID:6415207

In vivo selection of human embryonic stem cell-derived cells expressing methotrexate-resistant dihydrofolate reductase

PubMed Central

Gori, Jennifer L.; Tian, Xinghui; Swanson, Debra; Gunther, Roland; Shultz, Leonard D.; McIvor, R. Scott; Kaufman, Dan S.

2009-01-01

SUMMARY Human embryonic stem cells (hESCs) provide a novel source of hematopoietic and other cell populations suitable for gene therapy applications. Preclinical studies to evaluate engraftment of hESC-derived hematopoietic cells transplanted into immunodeficient mice demonstrate only limited repopulation. Expression of a drug resistance gene, such as Tyr22-dihydrofolate reductase (Tyr22-DHFR), coupled to methotrexate (MTX) chemotherapy has the potential to selectively increase engraftment of gene-modified hESC-derived cells in mouse xenografts. Here, we describe the generation of Tyr22-DHFR – GFP expressing hESCs that maintain pluripotency, produce teratomas and can differentiate into MTXr-hemato-endothelial cells. We demonstrate that MTX administered to nonobese diabetic/severe combined immunodeficient/IL-2Rγcnull (NSG) mice after injection of Tyr22-DHFR-derived cells significantly increases human CD34+ and CD45+ cell engraftment in the bone marrow (BM) and peripheral blood of transplanted MTX-treated mice. These results demonstrate that MTX treatment supports selective, long-term engraftment of Tyr22-DHFR-cells in vivo, and provides a novel approach for combined human cell and gene therapy. PMID:19829316

A Novel Strategy to Increase the Proliferative Potential of Adult Human β-Cells While Maintaining Their Differentiated Phenotype

PubMed Central

Aly, Haytham; Rohatgi, Nidhi; Marshall, Connie A.; Grossenheider, Tiffani C.; Miyoshi, Hiroyuki; Stappenbeck, Thaddeus S.; Matkovich, Scot J.; McDaniel, Michael L.

2013-01-01

Our previous studies demonstrated that Wnt/GSK-3/β-catenin and mTOR signaling are necessary to stimulate proliferative processes in adult human β-cells. Direct inhibition of GSK-3, that engages Wnt signaling downstream of the Wnt receptor, increases β-catenin nuclear translocation and β-cell proliferation but results in lower insulin content. Our current goal was to engage canonical and non-canonical Wnt signaling at the receptor level to significantly increase human β-cell proliferation while maintaining a β-cell phenotype in intact islets. We adopted a system that utilized conditioned medium from L cells that expressed Wnt3a, R-spondin-3 and Noggin (L-WRN conditioned medium). In addition we used a ROCK inhibitor (Y-27632) and SB-431542 (that results in RhoA inhibition) in these cultures. Treatment of intact human islets with L-WRN conditioned medium plus inhibitors significantly increased DNA synthesis ∼6 fold in a rapamycin-sensitive manner. Moreover, this treatment strikingly increased human β-cell proliferation ∼20 fold above glucose alone. Only the combination of L-WRN conditioned medium with RhoA/ROCK inhibitors resulted in substantial proliferation. Transcriptome-wide gene expression profiling demonstrated that L-WRN medium provoked robust changes in several signaling families, including enhanced β-catenin-mediated and β-cell-specific gene expression. This treatment also increased expression of Nr4a2 and Irs2 and resulted in phosphorylation of Akt. Importantly, glucose-stimulated insulin secretion and content were not downregulated by L-WRN medium treatment. Our data demonstrate that engaging Wnt signaling at the receptor level by this method leads to necessary crosstalk between multiple signaling pathways including activation of Akt, mTOR, Wnt/β-catenin, PKA/CREB, and inhibition of RhoA/ROCK that substantially increase human β-cell proliferation while maintaining the β-cell phenotype. PMID:23776620

A novel strategy to increase the proliferative potential of adult human β-cells while maintaining their differentiated phenotype.

PubMed

Aly, Haytham; Rohatgi, Nidhi; Marshall, Connie A; Grossenheider, Tiffani C; Miyoshi, Hiroyuki; Stappenbeck, Thaddeus S; Matkovich, Scot J; McDaniel, Michael L

2013-01-01

Our previous studies demonstrated that Wnt/GSK-3/β-catenin and mTOR signaling are necessary to stimulate proliferative processes in adult human β-cells. Direct inhibition of GSK-3, that engages Wnt signaling downstream of the Wnt receptor, increases β-catenin nuclear translocation and β-cell proliferation but results in lower insulin content. Our current goal was to engage canonical and non-canonical Wnt signaling at the receptor level to significantly increase human β-cell proliferation while maintaining a β-cell phenotype in intact islets. We adopted a system that utilized conditioned medium from L cells that expressed Wnt3a, R-spondin-3 and Noggin (L-WRN conditioned medium). In addition we used a ROCK inhibitor (Y-27632) and SB-431542 (that results in RhoA inhibition) in these cultures. Treatment of intact human islets with L-WRN conditioned medium plus inhibitors significantly increased DNA synthesis ∼6 fold in a rapamycin-sensitive manner. Moreover, this treatment strikingly increased human β-cell proliferation ∼20 fold above glucose alone. Only the combination of L-WRN conditioned medium with RhoA/ROCK inhibitors resulted in substantial proliferation. Transcriptome-wide gene expression profiling demonstrated that L-WRN medium provoked robust changes in several signaling families, including enhanced β-catenin-mediated and β-cell-specific gene expression. This treatment also increased expression of Nr4a2 and Irs2 and resulted in phosphorylation of Akt. Importantly, glucose-stimulated insulin secretion and content were not downregulated by L-WRN medium treatment. Our data demonstrate that engaging Wnt signaling at the receptor level by this method leads to necessary crosstalk between multiple signaling pathways including activation of Akt, mTOR, Wnt/β-catenin, PKA/CREB, and inhibition of RhoA/ROCK that substantially increase human β-cell proliferation while maintaining the β-cell phenotype.

N-acetyl-L-cysteine increases MnSOD activity and enhances the recruitment of quiescent human fibroblasts to the proliferation cycle during wound healing

PubMed Central

Mao, Gaowei; Goswami, Monali; Kalen, Amanda L.; Goswami, Prabhat C.; Sarsour, Ehab H.

2016-01-01

Background The rebuilding of the connective tissue during wound healing requires the recruitment of fibroblasts to the wound area as well as reentry of quiescent fibroblasts to the proliferative cycle. Whether this process can be modulated by a small molecular weight thiol antioxidant N-acetyl-L-cysteine (NAC) was tested in normal human skin fibroblasts (NHFs) in this study. Methods and Results By using a uni-directional wound healing assay, NAC treated cells demonstrated a decreased migration rate but increased number of proliferating cells recruited into the wound area post wounding. Fifteen day quiescent control and NAC treated NHFs were re-plated at a lower density and cell numbers counted at different days post-plating. Interestingly, NAC treated cells exhibited increased cellular proliferation indicated by both decreased cell population doubling time and increased S phase cells. NAC treated cells demonstrated decreased steady state levels of reactive oxygen species as well as increased protein and activity levels of manganese superoxide dismutase (MnSOD). NAC treatment failed to induce proliferation in quiescent cells lacking MnSOD expression. Conclusions These results demonstrate that NAC enhanced the recruitment of quiescent NHFs into proliferation cycle during wound healing. Our results also suggest that the wound healing properties of NAC might be due to its ability to induce and enhance MnSOD expression and activity. Altogether, these findings suggest NAC might be potentially developed as a dietary intervention to improve tissue injury in animals and humans. PMID:26671656

Cord Blood Stem Cell Procurement in Minority Donors

DTIC Science & Technology

2009-03-01

stem cell transplantation. The educational process and expansion of collection sites has given us a steady supply of cord blood for clinical use; and now we have the operational nucleus of several collection sites that is self-perpetuating a continual drive to expand to affiliated institutions. The greatest benefit of this project is the demonstration of how we solved the problem of increasing the overall yield of the cord blood units. We convincingly demonstrate that putting resources into individual patient education and prenatal visits is not likely to increase the cell

Combined pitavastatin and dacarbazine treatment activates apoptosis and autophagy resulting in synergistic cytotoxicity in melanoma cells.

PubMed

Al-Qatati, Abeer; Aliwaini, Saeb

2017-12-01

Melanoma is an aggressive skin cancer and its incidence is increasing faster than any other type of cancer. Whilst dacarbazine (DTIC) is the standard chemotherapy for metastatic melanoma, it has limited success. Statins, including pitavastatin, have been demonstrated to have a range of anti-cancer effects in a number of human cancer cell lines. The present study therefore explored the anti-cancer activity of combined DTIC and pitavastatin in A375 and WM115 human melanoma cells. Cell survival assays demonstrated that combined DTIC and pitavastatin treatment resulted in synergistic cell death. Cell cycle analyses further revealed that this combined treatment resulted in a G1 cell cycle arrest, as well as a sub-G1 population, indicative of apoptosis. Activation of apoptosis was confirmed by Annexin V-fluorescein isothiocyanate/propidium iodide double-staining and an increase in the levels of active caspase 3 and cleaved poly (ADP-ribose) polymerase. Furthermore, it was demonstrated that apoptosis occurs through the intrinsic pathway, evident from the release of cytochrome c. Finally, combined DTIC and pitavastatin treatment was demonstrated to also activate autophagy as part of a cell death mechanism. The present study provides novel evidence to suggest that the combined treatment of DTIC and pitavastatin may be effective in the treatment of melanoma.

Combined pitavastatin and dacarbazine treatment activates apoptosis and autophagy resulting in synergistic cytotoxicity in melanoma cells

PubMed Central

Al-Qatati, Abeer; Aliwaini, Saeb

2017-01-01

Melanoma is an aggressive skin cancer and its incidence is increasing faster than any other type of cancer. Whilst dacarbazine (DTIC) is the standard chemotherapy for metastatic melanoma, it has limited success. Statins, including pitavastatin, have been demonstrated to have a range of anti-cancer effects in a number of human cancer cell lines. The present study therefore explored the anti-cancer activity of combined DTIC and pitavastatin in A375 and WM115 human melanoma cells. Cell survival assays demonstrated that combined DTIC and pitavastatin treatment resulted in synergistic cell death. Cell cycle analyses further revealed that this combined treatment resulted in a G1 cell cycle arrest, as well as a sub-G1 population, indicative of apoptosis. Activation of apoptosis was confirmed by Annexin V-fluorescein isothiocyanate/propidium iodide double-staining and an increase in the levels of active caspase 3 and cleaved poly (ADP-ribose) polymerase. Furthermore, it was demonstrated that apoptosis occurs through the intrinsic pathway, evident from the release of cytochrome c. Finally, combined DTIC and pitavastatin treatment was demonstrated to also activate autophagy as part of a cell death mechanism. The present study provides novel evidence to suggest that the combined treatment of DTIC and pitavastatin may be effective in the treatment of melanoma. PMID:29344241

Optimized flexible cover films for improved conversion efficiency in thin film flexible solar cells

NASA Astrophysics Data System (ADS)

Guterman, Sidney; Wen, Xin; Gudavalli, Ganesh; Rhajbhandari, Pravakar; Dhakal, Tara P.; Wilt, David; Klotzkin, David

2018-05-01

Thin film solar cell technologies are being developed for lower cost and flexible applications. For such technologies, it is desirable to have inexpensive, flexible cover strips. In this paper, we demonstrate that transparent silicone cover glass adhesive can be doped with TiO2 nanoparticles to achieve an optimal refractive index and maximize the performance of the cell. Cells covered with the film doped with nanoparticles at the optimal concentration demonstrated a ∼1% increase in photocurrent over the plain (undoped) film. In addition, fused silica beads can be incorporated into the flexible cover slip to realize a built-in pseudomorphic glass diffuser layer as well. This additional degree of freedom in engineering flexible solar cell covers allows maximal performance from a given cell for minimal increased cost.

Hyperoside protects human kidney‑2 cells against oxidative damage induced by oxalic acid.

PubMed

Chen, Yongliang; Ye, Lihong; Li, Wangjian; Li, Dongzhang; Li, Feng

2018-05-02

The majority of renal calculi (kidney stones) are calcium stones. Oxidative damage to renal tubular epithelial cells induced by reactive oxygen species (ROS) is the predominant cause of calcium oxalate stone formation. Hyperoside (Hyp) is a flavonol glycoside extracted from medicinal plants and appears to exhibit potent antioxidant activity in various cells. The aim of the present study was to investigate the protective effect of Hyp on renal cells exposed to oxidative stress simulated by oxalic acid (OA), and to determine whether the underlying mechanism involves the nuclear factor E2‑related factor2 (Nrf2)‑antioxidative response element signaling pathway. The study determined the indicators of high oxidative stress, including ROS and hydrogen peroxide (H2O2) in human kidney‑2 cells and the results demonstrated that the levels of ROS, as evaluated by flow cytometry, and H2O2 were significantly increased following treatment with OA (5 mmol/l) for 24 h (OA group), compared with those in the untreated control group. The increased activity of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase in these cells explained this observation, as it is a major source of ROS. The results demonstrated that, in the OA group, the adhesion of calcium oxalate crystals and lactate dehydrogenase (LDH) were significantly increased, and MTT assay demonstrated that cell viability was inhibited, compared with the control, which indicated that severe injury of cells was induced by OA. However, when the cells were pre‑treated with Hyp prior to treatment with OA (drug group), the levels of ROS and H2O2, and the activities of NADPH oxidase and LD were increased, and the adhesion of calcium oxalate crystals to cells was reduced, compared with the OA group. Western blot analysis and reverse transcription‑quantitative polymerase chain reaction demonstrated that the protein and mRNA expression levels of Nrf2, heme oxygenase‑1 (HO‑1) and NAD(P)H: quinineoxidoreductase 1 (NQO1) in the Hyp groups were significantly increased, compared with those in the OA group, with the exception of Nrf2 mRNA. These results suggested that Hyp had a marked protective effect on renal cells against the oxidative damage and cytotoxicity simulated by OA. This is the first report, to the best of our knowledge, demonstrating that the ability of Hyp to enhance the endogenous functions of antioxidation and detoxification in cells may involve the Nrf2/HO‑1/NQO1 pathway.

Final Technical Report: Affordable, High-Performance, Intermediate Temperature Solid Oxide Fuel Cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Blackburn, Bryan M.; Bishop, Sean; Gore, Colin

In this project, we improved the power output and voltage efficiency of our intermediate temperature solid oxide fuel cells (IT-SOFCs) with a focus on ~600 °C operation. At these temperatures and with the increased power density (i.e., fewer cells for same power output), the stack cost should be greatly reduced while extending durability. Most SOFC stacks operate at temperatures greater than 800 °C. This can greatly increase the cost of the system (stacks and BOP) as well as maintenance costs since the most common degradation mechanisms are thermally driven. Our approach uses no platinum group metal (PGM) materials and themore » lower operating temperature allows use of simple stainless steel interconnects and commercial off-the-shelf gaskets in the stack. Furthermore, for combined heating and power (CHP) applications the stack exhaust still provides “high quality” waste heat that can be recovered and used in a chiller or boiler. The anticipated performance, durability, and resulting cost improvements (< $700/kWe) will also move us closer to reaching the full potential of this technology for distributed generation (DG) and residential/commercial CHP. This includes eventual extension to cleaner, more efficient portable generators, auxiliary power units (APUs), and range extenders for transportation. The research added to the understanding of the area investigated by exploring various methods for increasing power density (Watts/square centimeter of active area in each cell) and increasing cell efficiency (increasing the open circuit voltage, or cell voltage with zero external electrical current). The results from this work demonstrated an optimized cell that had greater than 1 W/cm2 at 600 °C and greater than 1.6 W/cm2 at 650 °C. This was demonstrated in large format sizes using both 5 cm by 5 cm and 10 cm by 10 cm cells. Furthermore, this work demonstrated that high stability (no degradation over > 500 hours) can be achieved together with high performance in large format cells as large as 10 cm by 10 cm when operated at ~600 °C. The project culminated in the demonstration of a 12-cell stack using the porous anode-based SOFC technology.« less

Alkaline ceramidase 2 and its bioactive product sphingosine are novel regulators of the DNA damage response

PubMed Central

Xu, Ruijuan; Wang, Kai; Mileva, Izolda; Hannun, Yusuf A.; Obeid, Lina M.; Mao, Cungui

2016-01-01

Human cells respond to DNA damage by elevating sphingosine, a bioactive sphingolipid that induces programmed cell death (PCD) in response to various forms of stress, but its regulation and role in the DNA damage response remain obscure. Herein we demonstrate that DNA damage increases sphingosine levels in tumor cells by upregulating alkaline ceramidase 2 (ACER2) and that the upregulation of the ACER2/sphingosine pathway induces PCD in response to DNA damage by increasing the production of reactive oxygen species (ROS). Treatment with the DNA damaging agent doxorubicin increased both ACER2 expression and sphingosine levels in HCT116 cells in a dose-dependent manner. ACER2 overexpression increased sphingosine in HeLa cells whereas knocking down ACER2 inhibited the doxorubicin-induced increase in sphingosine in HCT116 cells, suggesting that DNA damage elevates sphingosine by upregulating ACER2. Knocking down ACER2 inhibited an increase in the apoptotic and necrotic cell population and the cleavage of poly ADP ribose polymerase (PARP) in HCT116 cells in response to doxorubicin as well as doxorubicin-induced release of lactate dehydrogenase (LDH) from these cells. Similar to treatment with doxorubicin, ACER2 overexpression induced an increase in the apoptotic and necrotic cell population and PARP cleavage in HeLa cells and LDH release from cells, suggesting that ACER2 upregulation mediates PCD in response to DNA damage through sphingosine. Mechanistic studies demonstrated that the upregulation of the ACER2/sphingosine pathway induces PCD by increasing ROS levels. Taken together, these results suggest that the ACER2/sphingosine pathway mediates PCD in response to DNA damage through ROS production. PMID:26943039

Correlation of energy disorder and open-circuit voltage in hybrid perovskite solar cells

DOE PAGES

Shao, Yuchuan; Yuan, Yongbo; Huang, Jinsong

2016-01-11

Organometal trihalide perovskites have been demonstrated as excellent light absorbers for high efficiency photovoltaic applications. Previous approaches to increasing the solar cell efficiency have focussed on optimisation of the grain morphology of perovskite thin films. Here, we show that the structural order of the electron-transport layers also has a significant impact on solar cell performance. We demonstrate that the power conversion efficiency of CH 3NH 3PbI 3 planar-heterojunction photovoltaic cells increases from 17.1% to 19.4% when the energy disorder in the fullerene electron-transport layer is reduced by a simple solvent annealing process. The increase in efficiency is the result ofmore » the enhancement in open-circuit voltage from 1.04 V to 1.13 V without sacrificing the short-circuit current and fill factor. Finally, these results shed light on the origin of open-circuit voltage in perovskite solar cells, and provide a new path to further increase their efficiency« less

Ganoderma lucidum polysaccharides protect fibroblasts against UVB-induced photoaging

PubMed Central

Zeng, Qinghai; Zhou, Fang; Lei, Li; Chen, Jing; Lu, Jianyun; Zhou, Jianda; Cao, Ke; Gao, Lihua; Xia, Fang; Ding, Shu; Huang, Lihua; Xiang, Hong; Wang, Jingjing; Xiao, Yangfan; Xiao, Rong; Huang, Jinhua

2017-01-01

Ganoderma lucidum has featured in traditional Chinese medicine for >1,000 years. Ganoderma polysaccharides (GL-PS), a major active ingredient in Ganoderma, confer immune regulation, antitumor effects and significant antioxidant effects. The aim of the present study was to investigate the efficacy and mechanism of GL-PS-associated inhibition of ultraviolet B (UVB)-induced photoaging in human fibroblasts in vitro. Primary human skin fibroblasts were cultured, and a fibroblast photoaging model was built through exposure to UVB. Cell viability was measured by MTT assay. Aged cells were stained using a senescence-associated β-galactosidase staining (SA-β-gal) kit. ELISA kits were used to analyze matrix metalloproteinase (MMP) −1 and C-telopeptides of Type I collagen (CICP) protein levels in cellular supernatant. ROS levels were quantified by flow cytometry. Cells exposed to UVB had decreased cell viability, increased aged cells, decreased CICP protein expression, increased MMP-1 protein expression, and increased cellular ROS levels compared with non-exposed cells. However, cells exposed to UVB and treated with 10, 20 and 40 µg/ml GL-PS demonstrated increased cell viability, decreased aged cells, increased CICP protein expression, decreased MMP-1 protein expression, and decreased cellular ROS levels compared with UVB exposed/GL-PS untreated cells. These results demonstrate that GL-PS protects fibroblasts against photoaging by eliminating UVB-induced ROS. This finding indicates GL-PS treatment may serve as a novel strategy for antiphotoaging. PMID:27959406

10.2% power conversion efficiency polymer tandem solar cells consisting of two identical sub-cells.

PubMed

You, Jingbi; Chen, Chun-Chao; Hong, Ziruo; Yoshimura, Ken; Ohya, Kenichiro; Xu, Run; Ye, Shenglin; Gao, Jing; Li, Gang; Yang, Yang

2013-08-07

Polymer tandem solar cells with 10.2% power conversion efficiency are demonstrated via stacking two PDTP-DFBT:PC₇₁ BM bulk heterojunctions, connected by MoO₃/PEDOT:PSS/ZnO as an interconnecting layer. The tandem solar cells increase the power conversion efficiency of the PDTP-DFBT:PC₇₁ BM system from 8.1% to 10.2%, successfully demonstrating polymer tandem solar cells with identical sub-cells of double-digit efficiency. Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Upregulation of Glucose Uptake and Hexokinase Activity of Primary Human CD4+ T Cells in Response to Infection with HIV-1

PubMed Central

Kavanagh Williamson, Maia; Coombes, Naomi; Juszczak, Florian; Athanasopoulos, Marios; Khan, Mariam B.; Eykyn, Thomas R.; Srenathan, Ushani; Dias Zeidler, Julianna; Huthoff, Hendrik

2018-01-01

Infection of primary CD4+ T cells with HIV-1 coincides with an increase in glycolysis. We investigated the expression of glucose transporters (GLUT) and glycolytic enzymes in human CD4+ T cells in response to infection with HIV-1. We demonstrate the co-expression of GLUT1, GLUT3, GLUT4, and GLUT6 in human CD4+ T cells after activation, and their concerted overexpression in HIV-1 infected cells. The investigation of glycolytic enzymes demonstrated activation-dependent expression of hexokinases HK1 and HK2 in human CD4+ T cells, and a highly significant increase in cellular hexokinase enzyme activity in response to infection with HIV-1. HIV-1 infected CD4+ T cells showed a marked increase in expression of HK1, as well as the functionally related voltage-dependent anion channel (VDAC) protein, but not HK2. The elevation of GLUT, HK1, and VDAC expression in HIV-1 infected cells mirrored replication kinetics and was dependent on virus replication, as evidenced by the use of reverse transcription inhibitors. Finally, we demonstrated that the upregulation of HK1 in HIV-1 infected CD4+ T cells is independent of the viral accessory proteins Vpu, Vif, Nef, and Vpr. Though these data are consistent with HIV-1 dependency on CD4+ T cell glucose metabolism, a cellular response mechanism to infection cannot be ruled out. PMID:29518929

HMG-CoA reductase is negatively associated with PCV2 infection and PCV2-induced apoptotic cell death.

PubMed

Yang, Xin; Ouyang, Hongsheng; Chen, Fuwang; Pang, Daxing; Dong, Meichen; Yang, Susu; Liu, Xiaoyun; Peng, Zhiyuan; Wang, Fei; Zhang, Xiao; Ren, Linzhu

2014-06-01

We examined the role of HMG-CoA reductase (HMGCR) during porcine circovirus 2 (PCV2) infection. The results demonstrated that levels of endogenous HMGCR were not significantly different in PCV2-infected cells and mock-infected cells. However, the level of phosphorylated HMGCR, an inactivated form of HMGCR, was increased in PCV2-infected cells. Furthermore, HMGCR was upregulated by overexpression, silenced by siRNA or inactivated using its dominant-negative form in PK-15 cells. The results showed that PCV2 infection was inhibited by HMGCR overexpression, whereas it was significantly increased in HMGCR-silenced cells and HMGCR inhibitor-treated cells. Moreover, there was a robust apoptotic response at 48 h post-infection (p.i.) in HMGCR-inactivated cells, and this response was significantly greater than that observed in PK-15 cells. A modest apoptotic response was also observed in HMGCR-silenced cells. Caspase-3 activity was also analysed in PCV2-infected cells at 48 h p.i. As expected, caspase-3 activity was significantly increased in HMGCR-inactivated and -silenced cells compared with PK-15 cells. PCV2 replication was dose-dependently increased in HMGCR-inactivated cells when treated with increasing amounts of caspase-3 inhibitor. Altogether, HMGCR was negatively associated with PCV2 infection and PCV2-induced apoptotic cell death. These data demonstrated that HMGCR can be used as a candidate target for PCV2 disease control and antivirus research. Furthermore, the cells generated in this study can be used to evaluate the potential effects of HMGCR on PCV2 replication. © 2014 The Authors.

Impact of calcium signaling during infection of Neisseria meningitidis to human brain microvascular endothelial cells.

PubMed

Asmat, Tauseef M; Tenenbaum, Tobias; Jonsson, Ann-Beth; Schwerk, Christian; Schroten, Horst

2014-01-01

The pili and outer membrane proteins of Neisseria meningitidis (meningococci) facilitate bacterial adhesion and invasion into host cells. In this context expression of meningococcal PilC1 protein has been reported to play a crucial role. Intracellular calcium mobilization has been implicated as an important signaling event during internalization of several bacterial pathogens. Here we employed time lapse calcium-imaging and demonstrated that PilC1 of meningococci triggered a significant increase in cytoplasmic calcium in human brain microvascular endothelial cells, whereas PilC1-deficient meningococci could not initiate this signaling process. The increase in cytosolic calcium in response to PilC1-expressing meningococci was due to efflux of calcium from host intracellular stores as demonstrated by using 2-APB, which inhibits the release of calcium from the endoplasmic reticulum. Moreover, pre-treatment of host cells with U73122 (phospholipase C inhibitor) abolished the cytosolic calcium increase caused by PilC1-expressing meningococci demonstrating that active phospholipase C (PLC) is required to induce calcium transients in host cells. Furthermore, the role of cytosolic calcium on meningococcal adherence and internalization was documented by gentamicin protection assay and double immunofluorescence (DIF) staining. Results indicated that chelation of intracellular calcium by using BAPTA-AM significantly impaired PilC1-mediated meningococcal adherence to and invasion into host endothelial cells. However, buffering of extracellular calcium by BAPTA or EGTA demonstrated no significant effect on meningococcal adherence to and invasion into host cells. Taken together, these results indicate that meningococci induce calcium release from intracellular stores of host endothelial cells via PilC1 and cytoplasmic calcium concentrations play a critical role during PilC1 mediated meningococcal adherence to and subsequent invasion into host endothelial cells.

Role of epithelial-mesenchymal transition (EMT) and fibroblast function in cerium oxide nanoparticles-induced lung fibrosis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ma, Jane

The emission of cerium oxide nanoparticles (CeO{sub 2}) from diesel engines, using cerium compounds as a catalyst to lower the diesel exhaust particles, is a health concern. We have previously shown that CeO{sub 2} induced pulmonary inflammation and lung fibrosis. The objective of the present study was to investigate the modification of fibroblast function and the role of epithelial-mesenchymal transition (EMT) in CeO{sub 2}-induced fibrosis. Male Sprague-Dawley rats were exposed to CeO{sub 2} (0.15 to 7 mg/kg) by a single intratracheal instillation and sacrificed at various times post-exposure. The results show that at 28 days after CeO{sub 2} (3.5 mg/kg)more » exposure, lung fibrosis was evidenced by increased soluble collagen in bronchoalveolar lavage fluid, elevated hydroxyproline content in lung tissues, and enhanced sirius red staining for collagen in the lung tissue. Lung fibroblasts and alveolar type II (ATII) cells isolated from CeO{sub 2}-exposed rats at 28 days post-exposure demonstrated decreasing proliferation rate when compare to the controls. CeO{sub 2} exposure was cytotoxic and altered cell function as demonstrated by fibroblast apoptosis and aggregation, and ATII cell hypertrophy and hyperplasia with increased surfactant. The presence of stress fibers, expressed as α-smooth muscle actin (SMA), in CeO{sub 2}-exposed fibroblasts and ATII cells was significantly increased compared to the control. Immunohistofluorescence analysis demonstrated co-localization of TGF-β or α-SMA with prosurfactant protein C (SPC)-stained ATII cells. These results demonstrate that CeO{sub 2} exposure affects fibroblast function and induces EMT in ATII cells that play a role in lung fibrosis. These findings suggest potential adverse health effects in response to CeO{sub 2} nanoparticle exposure. - Highlights: • CeO{sub 2} exposure induced lung fibrosis. • CeO{sub 2} were detected in lung tissue, alveolar type II (ATII) cells and fibroblasts. • CeO{sub 2} caused ATII cell hypertrophy and hyperplasia and altered fibroblast function. • Increased α-SMA in CeO{sub 2}-exposed lung fibroblasts indicating myofibroblast formation. • CeO{sub 2} induced EMT in ATII cells demonstrated as increased α-SMA expression.« less

Lysophosphatidic acid triggers mast cell-driven atherosclerotic plaque destabilization by increasing vascular inflammation[S

PubMed Central

Bot, Martine; de Jager, Saskia C. A.; MacAleese, Luke; Lagraauw, H. Maxime; van Berkel, Theo J. C.; Quax, Paul H. A.; Kuiper, Johan; Heeren, Ron M. A.; Biessen, Erik A. L.; Bot, Ilze

2013-01-01

Lysophosphatidic acid (LPA), a bioactive lysophospholipid, accumulates in the atherosclerotic plaque. It has the capacity to activate mast cells, which potentially exacerbates plaque progression. In this study, we thus aimed to investigate whether LPA contributes to plaque destabilization by modulating mast cell function. We here show by an imaging mass spectrometry approach that several LPA species are present in atherosclerotic plaques. Subsequently, we demonstrate that LPA is a potent mast cell activator which, unlike other triggers, favors release of tryptase. Local perivascular administration of LPA to an atherosclerotic carotid artery segment increases the activation status of perivascular mast cells and promotes intraplaque hemorrhage and macrophage recruitment without impacting plaque cell apoptosis. The mast cell stabilizer cromolyn could prevent intraplaque hemorrhage elicited by LPA-mediated mast cell activation. Finally, the involvement of mast cells in these events was further emphasized by the lack of effect of perivascular LPA administration in mast cell deficient animals. We demonstrate that increased accumulation of LPA in plaques induces perivascular mast cell activation and in this way contributes to plaque destabilization in vivo. This study points to local LPA availability as an important factor in atherosclerotic plaque stability. PMID:23396975

Lysophosphatidic acid triggers mast cell-driven atherosclerotic plaque destabilization by increasing vascular inflammation.

PubMed

Bot, Martine; de Jager, Saskia C A; MacAleese, Luke; Lagraauw, H Maxime; van Berkel, Theo J C; Quax, Paul H A; Kuiper, Johan; Heeren, Ron M A; Biessen, Erik A L; Bot, Ilze

2013-05-01

Lysophosphatidic acid (LPA), a bioactive lysophospholipid, accumulates in the atherosclerotic plaque. It has the capacity to activate mast cells, which potentially exacerbates plaque progression. In this study, we thus aimed to investigate whether LPA contributes to plaque destabilization by modulating mast cell function. We here show by an imaging mass spectrometry approach that several LPA species are present in atherosclerotic plaques. Subsequently, we demonstrate that LPA is a potent mast cell activator which, unlike other triggers, favors release of tryptase. Local perivascular administration of LPA to an atherosclerotic carotid artery segment increases the activation status of perivascular mast cells and promotes intraplaque hemorrhage and macrophage recruitment without impacting plaque cell apoptosis. The mast cell stabilizer cromolyn could prevent intraplaque hemorrhage elicited by LPA-mediated mast cell activation. Finally, the involvement of mast cells in these events was further emphasized by the lack of effect of perivascular LPA administration in mast cell deficient animals. We demonstrate that increased accumulation of LPA in plaques induces perivascular mast cell activation and in this way contributes to plaque destabilization in vivo. This study points to local LPA availability as an important factor in atherosclerotic plaque stability.

Fluoxetine Induces Proliferation and Inhibits Differentiation of Hypothalamic Neuroprogenitor Cells In Vitro

PubMed Central

Sousa-Ferreira, Lígia; Aveleira, Célia; Botelho, Mariana; Álvaro, Ana Rita; Pereira de Almeida, Luís; Cavadas, Cláudia

2014-01-01

A significant number of children undergo maternal exposure to antidepressants and they often present low birth weight. Therefore, it is important to understand how selective serotonin reuptake inhibitors (SSRIs) affect the development of the hypothalamus, the key center for metabolism regulation. In this study we investigated the proliferative actions of fluoxetine in fetal hypothalamic neuroprogenitor cells and demonstrate that fluoxetine induces the proliferation of these cells, as shown by increased neurospheres size and number of proliferative cells (Ki-67+ cells). Moreover, fluoxetine inhibits the differentiation of hypothalamic neuroprogenitor cells, as demonstrated by decreased number of mature neurons (Neu-N+ cells) and increased number of undifferentiated cells (SOX-2+ cells). Additionally, fluoxetine-induced proliferation and maintenance of hypothalamic neuroprogenitor cells leads to changes in the mRNA levels of appetite regulator neuropeptides, including Neuropeptide Y (NPY) and Cocaine-and-Amphetamine-Regulated-Transcript (CART). This study provides the first evidence that SSRIs affect the development of hypothalamic neuroprogenitor cells in vitro with consequent alterations on appetite neuropeptides. PMID:24598761

Cisplatin radiosensitizes radioresistant human mesenchymal stem cells.

PubMed

Rühle, Alexander; Perez, Ramon Lopez; Glowa, Christin; Weber, Klaus-Josef; Ho, Anthony D; Debus, Jürgen; Saffrich, Rainer; Huber, Peter E; Nicolay, Nils H

2017-10-20

Cisplatin-based chemo-radiotherapy is widely used to treat cancers with often severe therapy-associated late toxicities. While mesenchymal stem cells (MSCs) were shown to aid regeneration of cisplatin- or radiation-induced tissue lesions, the effect of the combined treatment on the stem cells remains unknown. Here we demonstrate that cisplatin treatment radiosensitized human bone marrow-derived MSCs in a dose-dependent manner and increased levels of radiation-induced apoptosis. However, the defining stem cell properties of MSCs remained largely intact after cisplatin-based chemo-radiation, and stem cell motility, adhesion, surface marker expression and the characteristic differentiation potential were not significantly influenced. The increased cisplatin-mediated radiosensitivity was associated with a cell cycle shift of MSCs towards the radiosensitive G2/M phase and increased residual DNA double-strand breaks. These data demonstrate for the first time a dose-dependent radiosensitization effect of MSCs by cisplatin. Clinically, the observed increase in radiation sensitivity and subsequent loss of regenerative MSCs may contribute to the often severe late toxicities observed after cisplatin-based chemo-radiotherapy in cancer patients.

CD147 regulates the expression of MCT1 and lactate export in multiple myeloma cells

PubMed Central

Walters, Denise K; Arendt, Bonnie K; Jelinek, Diane F

2013-01-01

Increased use of the glycolytic pathway, even in the presence of oxygen, has recently been recognized as a key characteristic of malignant cells. However, the glycolytic phenotype results in increased lactic acid production and, in order to prevent cellular acidosis, tumor cells must increase proton efflux via upregulation of pH regulators such as proton-pumps, sodium-proton exchangers, and/or monocarboxylate transporters (MCT) (e.g., MCT1, MCT4). Interestingly, expression of MCT1 and MCT4 has been previously shown to be dependent upon expression of the transmembrane glycoprotein CD147. Recently, we demonstrated that primary patient multiple myeloma (MM) cells and human MM cell lines (HMCLs) overexpress CD147. Therefore, the goal of the current study was to specifically determine if MCT1 and MCT4 were also overexpressed in MM cells. RT-PCR analysis demonstrated both primary patient MM cells and HMCLs overexpress MCT1 and MCT4 mRNA. Notably, primary MM cells or HMCLs were found to express variable levels of MCT1 and/or MCT4 at the protein level despite CD147 expression. In those HMCLs positive for MCT1 and/or MCT4 protein expression, MCT1 and/or MCT4 were found to be associated with CD147. Specific siRNA-mediated downregulation of MCT1 but not MCT4 resulted in decreased HMCL proliferation, decreased lactate export, and increased cellular media pH. However, western blot analysis revealed that downregulation of MCT1 also downregulated CD147 and vice versa despite no effect on mRNA levels. Taken together, these data demonstrate the association between MCT1 and CD147 proteins in MM cells and importance of their association for lactate export and proliferation in MM cells. PMID:24013424

CD147 regulates the expression of MCT1 and lactate export in multiple myeloma cells.

PubMed

Walters, Denise K; Arendt, Bonnie K; Jelinek, Diane F

2013-10-01

Increased use of the glycolytic pathway, even in the presence of oxygen, has recently been recognized as a key characteristic of malignant cells. However, the glycolytic phenotype results in increased lactic acid production and, in order to prevent cellular acidosis, tumor cells must increase proton efflux via upregulation of pH regulators such as proton-pumps, sodium-proton exchangers, and/or monocarboxylate transporters (MCT) (e.g., MCT1, MCT4). Interestingly, expression of MCT1 and MCT4 has been previously shown to be dependent upon expression of the transmembrane glycoprotein CD147. Recently, we demonstrated that primary patient multiple myeloma (MM) cells and human MM cell lines (HMCLs) overexpress CD147. Therefore, the goal of the current study was to specifically determine if MCT1 and MCT4 were also overexpressed in MM cells. RT-PCR analysis demonstrated both primary patient MM cells and HMCLs overexpress MCT1 and MCT4 mRNA. Notably, primary MM cells or HMCLs were found to express variable levels of MCT1 and/or MCT4 at the protein level despite CD147 expression. In those HMCLs positive for MCT1 and/or MCT4 protein expression, MCT1 and/or MCT4 were found to be associated with CD147. Specific siRNA-mediated downregulation of MCT1 but not MCT4 resulted in decreased HMCL proliferation, decreased lactate export, and increased cellular media pH. However, western blot analysis revealed that downregulation of MCT1 also downregulated CD147 and vice versa despite no effect on mRNA levels. Taken together, these data demonstrate the association between MCT1 and CD147 proteins in MM cells and importance of their association for lactate export and proliferation in MM cells.

Systemic Inflammation in Progressive Multiple Sclerosis Involves Follicular T-Helper, Th17- and Activated B-Cells and Correlates with Progression

PubMed Central

Christensen, Jeppe Romme; Börnsen, Lars; Ratzer, Rikke; Piehl, Fredrik; Khademi, Mohsen; Olsson, Tomas; Sørensen, Per Soelberg; Sellebjerg, Finn

2013-01-01

Pathology studies of progressive multiple sclerosis (MS) indicate a major role of inflammation including Th17-cells and meningeal inflammation with ectopic lymphoid follicles, B-cells and plasma cells, the latter indicating a possible role of the newly identified subset of follicular T-helper (TFH) cells. Although previous studies reported increased systemic inflammation in progressive MS it remains unclear whether systemic inflammation contributes to disease progression and intrathecal inflammation. This study aimed to investigate systemic inflammation in progressive MS and its relationship with disease progression, using flow cytometry and gene expression analysis of CD4+ and CD8+T-cells, B-cells, monocytes and dendritic cells. Furthermore, gene expression of cerebrospinal fluid cells was studied. Flow cytometry studies revealed increased frequencies of ICOS+TFH-cells in peripheral blood from relapsing-remitting (RRMS) and secondary progressive (SPMS) MS patients. All MS subtypes had decreased frequencies of Th1 TFH-cells, while primary progressive (PPMS) MS patients had increased frequency of Th17 TFH-cells. The Th17-subset, interleukin-23-receptor+CD4+T-cells, was significantly increased in PPMS and SPMS. In the analysis of B-cells, we found a significant increase of plasmablasts and DC-SIGN+ and CD83+B-cells in SPMS. ICOS+TFH-cells and DC-SIGN+B-cells correlated with disease progression in SPMS patients. Gene expression analysis of peripheral blood cell subsets substantiated the flow cytometry findings by demonstrating increased expression of IL21, IL21R and ICOS in CD4+T-cells in progressive MS. Cerebrospinal fluid cells from RRMS and progressive MS (pooled SPMS and PPMS patients) had increased expression of TFH-cell and plasmablast markers. In conclusion, this study is the first to demonstrate the potential involvement of activated TFH-cells in MS. The increased frequencies of Th17-cells, activated TFH- and B-cells parallel findings from pathology studies which, along with the correlation between activated TFH- and B-cells and disease progression, suggest a pathogenic role of systemic inflammation in progressive MS. These observations may have implications for the treatment of progressive MS. PMID:23469245

Beyond Demonstration: The Role of Fuel Cells in DoD’s Energy Strategy

DTIC Science & Technology

2011-10-19

to consider fuel cell power. Increase awareness of unmanned vehicle designers, provid- ers, and operators about fuel cell systems as an option for...UAV to assist in the disaster relief and recovery efforts following the March 2011 earthquake and resulting tsunami off the eastern coast of Japan... Daiichi nuclear plant. The Global 34 CHAPTER 6 BEYOND DEMONSTRATION: THE ROLE OF FUEL CELLS IN DoD’S ENERGY STRATEGY Hawk was also used for

HSP-70 mitigates LPS/SKI-induced cell damage by increasing sphingosine kinase 1 (SK1).

PubMed

Ding, Xuan Z; Feng, Xiao R; Borschel, Richard H; Nikolich, Mikeljon P; Feng, Jie; Li, Yan S; Hoover, David L

2010-06-01

Heat shock proteins (HSPs) are potent protectors of cellular integrity against environmental stresses, including toxic microbial products. To investigate the mechanism of HSP-70 cell protection against bacterial lipopolysaccharide (LPS), we established a stable HSP-70 gene-transfected RAW 264.7 murine macrophage model of LPS-induced cell death. Bacterial LPS increases the activity of sphingosine kinase 1 (SK1), which catalyzes formation of sphingosine-1-phosphate (S1P). S1P functions as a critical signal for initiation and maintenance of diverse aspects of immune cell activation and function. When mouse macrophages were incubated with Escherichia coli LPS (1 microg/ml) and sphingosine kinase inhibitor (SKI, 5 microM), 90% of cells died. Neither LPS nor SKI alone at these doses damaged the cells. The LPS/SKI-induced cell death was partially reversed by overexpression of HSP-70 in gene-transfected macrophages. The specificity of HSP-70 in this reversal was demonstrated by transfection of HSP-70-specific siRNA. Down-regulation of HSP-70 expression after transfection of siRNA specific for HSP-70 was associated with increased LPS/SKI-induced cell damage. Overexpression of human or murine HSP-70 (HSPA1A and Hspa1a, respectively) increased both cellular SK1 mRNA and protein levels. Cellular heat shock also increased SK1 protein. These studies confirm the importance of SK1 as a protective moiety in LPS-induced cell injury and demonstrate that HSP-70-mediated protection from cells treated with LPS/SKI is accompanied by upregulating expression of SK1. HSP-70-mediated increases in SK1 and consequent increased levels of S1P may also play a role in protection of cells from other processes that lead to programmed cell death. Published by Elsevier Inc.

Leiomyoma Cells in 3-Dimensional Cultures Demonstrate an Attenuated Response to Fasudil, a Rho-Kinase Inhibitor, When Compared to 2-Dimensional Cultures

PubMed Central

Malik, Minnie; Britten, Joy; Segars, James

2014-01-01

Uterine leiomyomata are common benign tumors in women of reproductive age and demonstrate an attenuated response to mechanical signaling that involves Rho and integrins. To further characterize the impairment in Rho signaling, we studied the effect of Rho-kinase inhibitor, fasudil, on extracellular matrix production, in 2-dimensional (2D) and 3-dimensional (3D) cultures of leiomyoma and myometrial cells. Leiomyoma 2D cultures demonstrated a rapid decrease in gene transcripts and protein for fibronectin, procollagen 1A, and versican. In 3D cultures, fibronectin and procollagen 1A proteins demonstrated increased levels at lower concentrations of fasudil, followed by a concentration-dependent decrease. Versican protein increased up to 3-fold, whereas fibromodulin demonstrated a significant decrease of 1.92-fold. Myometrial 2D or 3D cultures demonstrated a decrease in all proteins after 72 hours of treatment. The 3D leiomyoma cultures demonstrated a significant increase in active RhoA, followed by a concentration-dependent decrease at higher concentrations. A concentration-dependent increase in phospho-extracellular regulated signal kinase and proapoptotic protein Bax was observed in 3D leiomyoma cultures. Fasudil relaxed the contraction of the 3D collagen gels caused by myometrium and leiomyoma cell growth. These findings indicate that the altered state of Rho signaling in leiomyoma was more clearly observed in 3D cultures. The results also suggest that fasudil may have clinical applicability for treatment of uterine leiomyoma. PMID:25084783

Elevated concentrations of nonesterified fatty acids increase monocyte expression of CD11b and adhesion to endothelial cells.

PubMed

Zhang, Wei-Yang; Schwartz, Eric; Wang, Yingjie; Attrep, Jeanne; Li, Zhi; Reaven, Peter

2006-03-01

Monocyte proinflammatory activity has been demonstrated in obesity, insulin resistance, and type 2 diabetes, metabolic conditions that are frequently associated with elevated levels of nonesterified fatty acids (NEFA). We therefore tested the hypothesis that NEFA may induce monocyte inflammation. Monocytes exposed to NEFA for 2 days demonstrated a dose-related increase in intracellular reactive oxygen species (ROS) formation and adhesion to endothelial cells. All of these effects were inhibited by the coaddition of antioxidants such as glutathione or butylated hydroxytoluene, by inhibition of ROS generation by NADPH oxidase inhibitors, and by inhibition of protein kinase C, a recognized stimulator of NAPDH oxidase. Monocytes exposed to NEFA also demonstrated a significant increase in CD11b message expression. Stimulation of monocyte adhesion to endothelial cells by NEFA was inhibited by addition of neutralizing antibodies to either CD11b or CD18. Finally, surface expression of CD11b increased significantly on monocytes as measured by flow cytometry, after their incubation with NEFA. These studies indicate that elevated concentrations of NEFA may enhance integrin facilitated monocyte adhesion to endothelial cells and these effects appear mediated, in part, through activation of NADPH oxidase and oxidative stress.

Robust Revascularization in Models of Limb Ischemia Using a Clinically Translatable Human Stem Cell-Derived Endothelial Cell Product.

PubMed

MacAskill, Mark G; Saif, Jaimy; Condie, Alison; Jansen, Maurits A; MacGillivray, Thomas J; Tavares, Adriana A S; Fleisinger, Lucija; Spencer, Helen L; Besnier, Marie; Martin, Ernesto; Biglino, Giovanni; Newby, David E; Hadoke, Patrick W F; Mountford, Joanne C; Emanueli, Costanza; Baker, Andrew H

2018-03-28

Pluripotent stem cell-derived differentiated endothelial cells offer high potential in regenerative medicine in the cardiovascular system. With the aim of translating the use of a human stem cell-derived endothelial cell product (hESC-ECP) for treatment of critical limb ischemia (CLI) in man, we report a good manufacturing practice (GMP)-compatible protocol and detailed cell tracking and efficacy data in multiple preclinical models. The clinical-grade cell line RC11 was used to generate hESC-ECP, which was identified as mostly endothelial (60% CD31 + /CD144 + ), with the remainder of the subset expressing various pericyte/mesenchymal stem cell markers. Cell tracking using MRI, PET, and qPCR in a murine model of limb ischemia demonstrated that hESC-ECP was detectable up to day 7 following injection. Efficacy in several murine models of limb ischemia (immunocompromised/immunocompetent mice and mice with either type I/II diabetes mellitus) demonstrated significantly increased blood perfusion and capillary density. Overall, we demonstrate a GMP-compatible hESC-ECP that improved ischemic limb perfusion and increased local angiogenesis without engraftment, paving the way for translation of this therapy. Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.

A novel three-dimensional heterotypic spheroid model for the assessment of the activity of cancer immunotherapy agents.

PubMed

Herter, Sylvia; Morra, Laura; Schlenker, Ramona; Sulcova, Jitka; Fahrni, Linda; Waldhauer, Inja; Lehmann, Steffi; Reisländer, Timo; Agarkova, Irina; Kelm, Jens M; Klein, Christian; Umana, Pablo; Bacac, Marina

2017-01-01

The complexity of the tumor microenvironment is difficult to mimic in vitro, particularly regarding tumor-host interactions. To enable better assessment of cancer immunotherapy agents in vitro, we developed a three-dimensional (3D) heterotypic spheroid model composed of tumor cells, fibroblasts, and immune cells. Drug targeting, efficient stimulation of immune cell infiltration, and specific elimination of tumor or fibroblast spheroid areas were demonstrated following treatment with a novel immunocytokine (interleukin-2 variant; IgG-IL2v) and tumor- or fibroblast-targeted T cell bispecific antibody (TCB). Following treatment with IgG-IL2v, activation of T cells, NK cells, and NKT cells was demonstrated by increased expression of the activation marker CD69 and enhanced cytokine secretion. The combination of TCBs with IgG-IL2v molecules was more effective than monotherapy, as shown by enhanced effects on immune cell infiltration; activation; increased cytokine secretion; and faster, more efficient elimination of targeted cells. This study demonstrates that the 3D heterotypic spheroid model provides a novel and versatile tool for in vitro evaluation of cancer immunotherapy agents and allows for assessment of additional aspects of the activity of cancer immunotherapy agents, including analysis of immune cell infiltration and drug targeting.

In vivo Host Environment Alters Pseudomonas aeruginosa Susceptibility to Aminoglycoside Antibiotics

PubMed Central

Pan, Xiaolei; Dong, Yuanyuan; Fan, Zheng; Liu, Chang; Xia, Bin; Shi, Jing; Bai, Fang; Jin, Yongxin; Cheng, Zhihui; Jin, Shouguang; Wu, Weihui

2017-01-01

During host infection, Pseudomonas aeruginosa coordinately regulates the expression of numerous genes to adapt to the host environment while counteracting host clearance mechanisms. As infected patients take antibiotics, the invading bacteria encounter antibiotics in the host milieu. P. aeruginosa is highly resistant to antibiotics due to multiple chromosomally encoded resistant determinants. And numerous in vitro studies have demonstrated the regulatory mechanisms of antibiotic resistance related genes in response to antibiotics. However, it is not well-known how host environment affects bacterial response to antibiotics. In this study, we found that P. aeruginosa cells directly isolated from mice lungs displayed higher susceptibility to tobramycin than in vitro cultured bacteria. In vitro experiments demonstrated that incubation with A549 and differentiated HL60 (dHL60) cells sensitized P. aeruginosa to tobramycin. Further studies revealed that reactive oxygen species produced by the host cells contributed to the increased bacterial susceptibility. At the same concentration of tobramycin, presence of A549 and dHL60 cells resulted in higher expression of heat shock proteins, which are known inducible by tobramycin. Further analyses revealed decreased membrane potential upon incubation with the host cells and modification of lipopolysaccharide, which contributed to the increased susceptibility to tobramycin. Therefore, our results demonstrate that contact with host cells increased bacterial susceptibility to tobramycin. PMID:28352614

DOE Office of Scientific and Technical Information (OSTI.GOV)

Steiner, Myles A; Perl, Emmett; Simon, John D

We demonstrate dual junction (Al)GaInP/GaAs solar cells that are designed to operate at 400 degrees C and 1000X concentration in a hybrid photovoltaic-solar thermal concentrator system. The cells have a front metallization and anti-reflection coating that are stable under 400 degrees C operation. We show how the cell performance degrades with increasing aluminum compositions in the top cell. Our best cell is a GaInP/GaAs tandem that demonstrated 15+/-1% efficiency at 400 degrees C over a concentration range of 300-1000 suns, with several pathways to improved performance.

Exosomes as mediators of platinum resistance in ovarian cancer.

PubMed

Crow, Jennifer; Atay, Safinur; Banskota, Samagya; Artale, Brittany; Schmitt, Sarah; Godwin, Andrew K

2017-02-14

Exosomes have been implicated in the cell-cell transfer of oncogenic proteins and genetic material. We speculated this may be one mechanism by which an intrinsically platinum-resistant population of epithelial ovarian cancer (EOC) cells imparts its influence on surrounding tumor cells. To explore this possibility we utilized a platinum-sensitive cell line, A2780 and exosomes derived from its resistant subclones, and an unselected, platinum-resistant EOC line, OVCAR10. A2780 cells demonstrate a ~2-fold increase in viability upon treatment with carboplatin when pre-exposed to exosomes from platinum-resistant cells as compared to controls. This coincided with increased epithelial to mesenchymal transition (EMT). DNA sequencing of EOC cell lines revealed previously unreported somatic mutations in the Mothers Against Decapentaplegic Homolog 4 (SMAD4) within platinum-resistant cells. A2780 cells engineered to exogenously express these SMAD4 mutations demonstrate up-regulation of EMT markers following carboplatin treatment, are more resistant to carboplatin, and release exosomes which impart a ~1.7-fold increase in resistance in naive A2780 recipient cells as compared to controls. These studies provide the first evidence that acquired SMAD4 mutations enhance the chemo-resistance profile of EOC and present a novel mechanism in which exchange of tumor-derived exosomes perpetuates an EMT phenotype, leading to the development of subpopulations of platinum-refractory cells.

Ganoderma lucidum polysaccharides protect fibroblasts against UVB-induced photoaging.

PubMed

Zeng, Qinghai; Zhou, Fang; Lei, Li; Chen, Jing; Lu, Jianyun; Zhou, Jianda; Cao, Ke; Gao, Lihua; Xia, Fang; Ding, Shu; Huang, Lihua; Xiang, Hong; Wang, Jingjing; Xiao, Yangfan; Xiao, Rong; Huang, Jinhua

2017-01-01

Ganoderma lucidum has featured in traditional Chinese medicine for >1,000 years. Ganoderma polysaccharides (GL-PS), a major active ingredient in Ganoderma, confer immune regulation, antitumor effects and significant antioxidant effects. The aim of the present study was to investigate the efficacy and mechanism of GL‑PS‑associated inhibition of ultraviolet B (UVB)‑induced photoaging in human fibroblasts in vitro. Primary human skin fibroblasts were cultured, and a fibroblast photoaging model was built through exposure to UVB. Cell viability was measured by MTT assay. Aged cells were stained using a senescence‑associated β-galactosidase staining (SA‑β‑gal) kit. ELISA kits were used to analyze matrix metalloproteinase (MMP) ‑1 and C‑telopeptides of Type I collagen (CICP) protein levels in cellular supernatant. ROS levels were quantified by flow cytometry. Cells exposed to UVB had decreased cell viability, increased aged cells, decreased CICP protein expression, increased MMP‑1 protein expression, and increased cellular ROS levels compared with non‑exposed cells. However, cells exposed to UVB and treated with 10, 20 and 40 µg/ml GL‑PS demonstrated increased cell viability, decreased aged cells, increased CICP protein expression, decreased MMP‑1 protein expression, and decreased cellular ROS levels compared with UVB exposed/GL‑PS untreated cells. These results demonstrate that GL‑PS protects fibroblasts against photoaging by eliminating UVB‑induced ROS. This finding indicates GL‑PS treatment may serve as a novel strategy for antiphotoaging.

Nanoscale analysis of caspofungin-induced cell surface remodelling in Candida albicans

NASA Astrophysics Data System (ADS)

El-Kirat-Chatel, Sofiane; Beaussart, Audrey; Alsteens, David; Jackson, Desmond N.; Lipke, Peter N.; Dufrêne, Yves F.

2013-01-01

The advent of fungal pathogens that are resistant to the classic repertoire of antifungal drugs has increased the need for new therapeutic agents. A prominent example of such a novel compound is caspofungin, known to alter cell wall biogenesis by inhibiting β-1,3-d-glucan synthesis. Although much progress has been made in understanding the mechanism of action of caspofungin, little is known about its influence on the biophysical properties of the fungal cells. Here, we use atomic force microscopy (AFM) to demonstrate that caspofungin induces major remodelling of the cell surface properties of Candida albicans. Caspofungin causes major morphological and structural alterations of the cells, which correlate with a decrease of the cell wall mechanical strength. Moreover, we find that the drug induces the massive exposure of the cell adhesion protein Als1 on the cell surface and leads to increased cell surface hydrophobicity, two features that trigger cell aggregation. This behaviour is not observed in yeast species lacking Als1, demonstrating the key role that the protein plays in determining the aggregation phenotype of C. albicans. The results show that AFM opens up new avenues for understanding the molecular bases of microbe-drug interactions and for developing new therapeutic agents.The advent of fungal pathogens that are resistant to the classic repertoire of antifungal drugs has increased the need for new therapeutic agents. A prominent example of such a novel compound is caspofungin, known to alter cell wall biogenesis by inhibiting β-1,3-d-glucan synthesis. Although much progress has been made in understanding the mechanism of action of caspofungin, little is known about its influence on the biophysical properties of the fungal cells. Here, we use atomic force microscopy (AFM) to demonstrate that caspofungin induces major remodelling of the cell surface properties of Candida albicans. Caspofungin causes major morphological and structural alterations of the cells, which correlate with a decrease of the cell wall mechanical strength. Moreover, we find that the drug induces the massive exposure of the cell adhesion protein Als1 on the cell surface and leads to increased cell surface hydrophobicity, two features that trigger cell aggregation. This behaviour is not observed in yeast species lacking Als1, demonstrating the key role that the protein plays in determining the aggregation phenotype of C. albicans. The results show that AFM opens up new avenues for understanding the molecular bases of microbe-drug interactions and for developing new therapeutic agents. Electronic supplementary information (ESI) available. See DOI: 10.1039/c2nr33215a

RNase L Suppresses Androgen Receptor Signaling, Cell Migration and Matrix Metalloproteinase Activity in Prostate Cancer Cells

PubMed Central

Dayal, Shubham; Zhou, Jun; Manivannan, Praveen; Siddiqui, Mohammad Adnan; Ahmad, Omaima Farid; Clark, Matthew; Awadia, Sahezeel; Garcia-Mata, Rafael; Shemshedini, Lirim; Malathi, Krishnamurthy

2017-01-01

The interferon antiviral pathways and prostate cancer genetics converge on a regulated endoribonuclease, RNase L. Positional cloning and linkage studies mapped Hereditary Prostate Cancer 1 (HPC1) to RNASEL. To date, there is no correlation of viral infections with prostate cancer, suggesting that RNase L may play additional roles in tumor suppression. Here, we demonstrate a role of RNase L as a suppressor of androgen receptor (AR) signaling, cell migration and matrix metalloproteinase activity. Using RNase L mutants, we show that its nucleolytic activity is dispensable for both AR signaling and migration. The most prevalent HPC1-associated mutations in RNase L, R462Q and E265X, enhance AR signaling and cell migration. RNase L negatively regulates cell migration and attachment on various extracellular matrices. We demonstrate that RNase L knockdown cells promote increased cell surface expression of integrin β1 which activates Focal Adhesion Kinase-Sarcoma (FAK-Src) pathway and Ras-related C3 botulinum toxin substrate 1-guanosine triphosphatase (Rac1-GTPase) activity to increase cell migration. Activity of matrix metalloproteinase (MMP)-2 and -9 is significantly increased in cells where RNase L levels are ablated. We show that mutations in RNase L found in HPC patients may promote prostate cancer by increasing expression of AR-responsive genes and cell motility and identify novel roles of RNase L as a prostate cancer susceptibility gene. PMID:28257035

Mangiferin positively regulates osteoblast differentiation and suppresses osteoclast differentiation

PubMed Central

Sekiguchi, Yuusuke; Mano, Hiroshi; Nakatani, Sachie; Shimizu, Jun; Kataoka, Aya; Ogura, Kana; Kimira, Yoshifumi; Ebata, Midori; Wada, Masahiro

2017-01-01

Mangiferin is a polyphenolic compound present in Salacia reticulata. It has been reported to reduce bone destruction and inhibit osteoclastic differentiation. This study aimed to determine whether mangiferin directly affects osteoblast and osteoclast proliferation and differentiation, and gene expression in MC3T3-E1 osteoblastic cells and osteoclast-like cells derived from primary mouse bone marrow macrophage cells. Mangiferin induced significantly greater WST-1 activity, indicating increased cell proliferation. Mangiferin induced significantly increased alkaline phosphatase staining, indicating greater cell differentiation. Reverse transcription-polymerase chain reaction (RT-PCR) demonstrated that mangiferin significantly increased the mRNA level of runt-related transcription factor 2 (RunX2), but did not affect RunX1 mRNA expression. Mangiferin significantly reduced the formation of tartrate-resistant acid phosphatase-positive multinuclear cells. RT-PCR demonstrated that mangiferin significantly increased the mRNA level of estrogen receptor β (ERβ), but did not affect the expression of other osteoclast-associated genes. Mangiferin may inhibit osteoclastic bone resorption by suppressing differentiation of osteoclasts and promoting expression of ERβ mRNA in mouse bone marrow macrophage cells. It also has potential to promote osteoblastic bone formation by promoting cell proliferation and inducing cell differentiation in preosteoblast MC3T3-E1 cells via RunX2. Mangiferin may therefore be useful in improving bone disease outcomes. PMID:28627701

Mangiferin positively regulates osteoblast differentiation and suppresses osteoclast differentiation.

PubMed

Sekiguchi, Yuusuke; Mano, Hiroshi; Nakatani, Sachie; Shimizu, Jun; Kataoka, Aya; Ogura, Kana; Kimira, Yoshifumi; Ebata, Midori; Wada, Masahiro

2017-08-01

Mangiferin is a polyphenolic compound present in Salacia reticulata. It has been reported to reduce bone destruction and inhibit osteoclastic differentiation. This study aimed to determine whether mangiferin directly affects osteoblast and osteoclast proliferation and differentiation, and gene expression in MC3T3‑E1 osteoblastic cells and osteoclast‑like cells derived from primary mouse bone marrow macrophage cells. Mangiferin induced significantly greater WST‑1 activity, indicating increased cell proliferation. Mangiferin induced significantly increased alkaline phosphatase staining, indicating greater cell differentiation. Reverse transcription‑polymerase chain reaction (RT‑PCR) demonstrated that mangiferin significantly increased the mRNA level of runt‑related transcription factor 2 (RunX2), but did not affect RunX1 mRNA expression. Mangiferin significantly reduced the formation of tartrate‑resistant acid phosphatase‑positive multinuclear cells. RT‑PCR demonstrated that mangiferin significantly increased the mRNA level of estrogen receptor β (ERβ), but did not affect the expression of other osteoclast‑associated genes. Mangiferin may inhibit osteoclastic bone resorption by suppressing differentiation of osteoclasts and promoting expression of ERβ mRNA in mouse bone marrow macrophage cells. It also has potential to promote osteoblastic bone formation by promoting cell proliferation and inducing cell differentiation in preosteoblast MC3T3‑E1 cells via RunX2. Mangiferin may therefore be useful in improving bone disease outcomes.

Piracetam ameliorated oxygen and glucose deprivation-induced injury in rat cortical neurons via inhibition of oxidative stress, excitatory amino acids release and P53/Bax.

PubMed

He, Zhi; Hu, Min; Zha, Yun-hong; Li, Zi-cheng; Zhao, Bo; Yu, Ling-ling; Yu, Min; Qian, Ying

2014-05-01

Our previous work has demonstrated that piracetam inhibited the decrease in amino acid content induced by chronic hypoperfusion, ameliorated the dysfunction of learning and memory in a hypoperfusion rat model, down-regulated P53, and BAX protein, facilitated the synaptic plasticity, and may be helpful in the treatment of vascular dementia. To explore the precise mechanism, the present study further evaluated effects of piracetam on Oxygen and glucose deprivation (OGD)-induced neuronal damage in rat primary cortical cells. The addition of piracetam to the cultured cells 12 h before OGD for 4 h significantly reduced neuronal damage as determined by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay and lactate dehydrogenase release experiments. Piracetam also lowered the levels of malondialdehyde, nitrogen monoxidum, and xanthine oxidase which was increased in the OGD cells, and enhanced the activities of superoxide dismutase and glutathione peroxidase, which were decreased in the OGD cells. We also demonstrated that piracetam could decrease glutamate and aspartate release when cortical cells were subjected to OGD. Furthermore, Western blot study demonstrated that piracetam attenuated the increased expression of P53 and BAX protein in OGD cells. These observations demonstrated that piracetam reduced OGD-induced neuronal damage by inhibiting the oxidative stress and decreasing excitatory amino acids release and lowering P53/Bax protein expression in OGD cells.

Enhanced Cellular Adhesion on Titanium by Silk Functionalized with titanium binding and RGD peptides

PubMed Central

Vidal, Guillaume; Blanchi, Thomas; Mieszawska, Aneta J.; Calabrese, Rossella; Rossi, Claire; Vigneron, Pascale; Duval, Jean-Luc; Kaplan, David L.; Egles, Christophe

2012-01-01

Soft tissue adhesion on titanium represents a challenge for implantable materials. In order to improve adhesion at the cell/material interface we used a new approach based on the molecular recognition of titanium by specific peptides. Silk fibroin protein was chemically grafted with titanium binding peptide (TiBP) to increase adsorption of these chimeric proteins to the metal surface. Quartz Crystal Microbalance was used to quantify the specific adsorption of TiBP-functionalized silk and an increase in protein deposition by more than 35% was demonstrated due to the presence of the binding peptide. A silk protein grafted with TiBP and fibronectin-derived RGD peptide was then prepared. The adherence of fibroblasts on the titanium surface modified with the multifunctional silk coating demonstrated an increase in the number of adhering cells by 60%. The improved adhesion was demonstrated by Scanning Electron Microscopy and immunocytochemical staining of focal contact points. Chick embryo organotypic culture also revealed strong adhesion of endothelial cells expanding on the multifunctional silk-peptide coating. These results demonstrated that silk functionalized with TiBP and RGD represents a promising approach to modify cell-biomaterial interfaces, opening new perspectives for implantable medical devices, especially when reendothelialization is required. PMID:22975628

Irradiated fibroblasts promote epithelial–mesenchymal transition and HDGF expression of esophageal squamous cell carcinoma

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bao, Ci-Hang; Wang, Xin-Tong; Ma, Wei

2015-03-06

Recent evidence suggested that nonirradiated cancer-associated fibroblasts (CAFs) promoted aggressive phenotypes of cancer cells through epithelial–mesenchymal transition (EMT). Hepatoma-derived growth factor (HDGF) is a radiosensitive gene of esophageal squamous cell carcinoma (ESCC). This study aimed to investigate the effect of irradiated fibroblasts on EMT and HDGF expression of ESCC. Our study demonstrated that coculture with nonirradiated fibroblasts significantly increased the invasive ability of ESCC cells and the increased invasiveness was further accelerated when they were cocultured with irradiated fibroblasts. Scattering of ESCC cells was also accelerated by the supernatant from irradiated fibroblasts. Exposure of ESCC cells to supernatant from irradiatedmore » fibroblasts resulted in decreased E-cadherin, increased vimentin in vitro and β-catenin was demonstrated to localize to the nucleus in tumor cells with irradiated fibroblasts in vivo models. The expression of HDGF and β-catenin were increased in both fibroblasts and ESCC cells of irradiated group in vitro and in vivo models. Interestingly, the tumor cells adjoining the stromal fibroblasts displayed strong nuclear HDGF immunoreactivity, which suggested the occurrence of a paracrine effect of fibroblasts on HDGF expression. These data suggested that irradiated fibroblasts promoted invasion, growth, EMT and HDGF expression of ESCC. - Highlights: • Irradiated CAFs accelerated invasiveness and scattering of ESCC cell lines. • Irradiated CAFs promoted EMT of ESCC cells. • Irradiated fibroblasts induced nuclear β-catenin relocalization in ESCC cells. • Irradiated fibroblasts increased HDGF expression in vitro and in vivo.« less

RON kinase isoforms demonstrate variable cell motility in normal cells.

PubMed

Greenbaum, Alissa; Rajput, Ashwani; Wan, Guanghua

2016-09-01

Aberrant RON (Recepteur d'Origine Nantais) tyrosine kinase activation causes the epithelial cell to evade normal growth pathways, resulting in unregulated cell proliferation, increased cell motility and decreased apoptosis. Wildtype (wt) RON has been shown to play a role in metastasis of epithelial malignancies. It presents an important potential therapeutic target for colorectal, breast, gastric and pancreatic cancer. Little is known about functional differences amongst RON isoforms RON155, RON160 and RON165. The purpose of this study was to determine the effect of various RON kinase isoforms on cell motility. Cell lines with stable expression of wtRON were generated by inserting the coding region of RON in pTagRFP (tagged red fluorescence protein plasmid). The expression constructs of RON variants (RON155, RON160 and RON165) were generated by creating a mutagenesis-based wtRON-pTag RFP plasmid and stably transfected into HEK 293 cells. The wound closure scratch assay was used to investigate the effect on cell migratory capacity of wild type RON and its variants. RON transfected cells demonstrated increased cell motility compared to HEK293 control cells. RON165 cell motility was significantly increased compared to RON160 (mean percentage of wound covered 37.37% vs. 32.40%; p = 0.03). RON tyrosine kinase isoforms have variable cell motility. This may reflect a difference in the behavior of malignant epithelial cells and their capacity for metastasis.

Sodium citrate inhibits the proliferation of human gastric adenocarcinoma epithelia cells

PubMed Central

Xia, Yuan; Zhang, Xulong; Bo, Agula; Sun, Juan; Li, Minhui

2018-01-01

The objective of the present study was to investigate the cytotoxic effects of sodium citrate on human gastric adenocarcinoma epithelia AGS cells. Numerous cytotoxicity-associated sodium citrate-induced effects were assessed, including cell viability and proliferation, cytokine expression and caspase activity. In vitro studies demonstrated that incubation with sodium citrate (>3.125 mM) inhibited AGS cell viability and proliferation in a dose-dependent manner. Incubation with sodium citrate for 24 h revealed that the levels of interleukin-1β (IL-1β), IL-8 and tumor necrosis factor increased with an increasing of dose of sodium citrate, whereas the IL-6 levels exhibited only a slight alteration. In addition, increases in caspase-3 and −9 activities were associated with increased duration of treatment and dosage of sodium citrate. Collectively, the results of the present study demonstrated that treatment with sodium citrate at higher concentrations or for longer durations exerts a cytotoxic effect on AGS cells via the induction of the intrinsic apoptosis pathway and the alteration in the levels of certain cytokines. PMID:29616124

Tanshinone IIA promotes the proliferation of WB-F344 hepatic oval cells via Wnt/β-catenin signaling

PubMed Central

ZE, XINGYU; JIA, JIDONG; LI, XINMIN; YOU, HONG; ZHAO, XINYAN; ZHANG, DONG; WANG, BAOEN

2016-01-01

Tanshinone IIA (TSA) is a widely used traditional Chinese medicine, which has been demonstrated to protect damaged liver cells and is currently administered in the treatment of liver fibrosis. Liver precursor cells, also termed oval cells, are key in the repair of liver tissues following injury. However, whether TSA improves the function of liver cells and protects the liver from injury by enhancing the growth and proliferation of hepatic oval cells remains to be elucidated. In the present study, low to moderate concentrations of TSA were observed to stimulate proliferation, did not induce apoptosis in WB-F344 rat hepatic oval cells and the increased expression levels of β-catenin. WB-F344 cells were treated with various concentrations of TSA (0–80 µg/ml) for 24, 48, 72 and 96 h. Cell proliferation was measured using a Cell Counting kit-8 (CCK-8) assay, a 5-ethynyl-2′-deoxyuridine assay and a carboxyfluorescein diacetate succinimidyl ester (CFSE) assay. The CCK-8 assay demonstrated that treatment of WB-F344 cells with 20–40 µg/ml TSA for up to 72 h significantly increased proliferation. Similar results were observed in the subsequent EdU and CFSE assays. Furthermore, a terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling assay demonstrated that 20–40 µg/ml TSA treatment for up to 96 h did not induce apoptosis of the WB-F344 cells. Notably, the results of western blot, immunofluorescence and reverse transcription-quantitative polymerase chain reaction analyses demonstrated that treatment of the WB-F344 cells with 20–40 µg/ml TSA for up to 72 h significantly increased the expression levels of β-catenin. These data indicated that TSA at concentrations between 20 and 40 µg/ml may induce WB-F344 cell proliferation by activating the canonical Wnt signaling pathway. The results of the present study suggest that TSA may be a useful natural agent to enhance repair and regeneration of the injured liver, and improve liver regeneration following orthotopic liver transplantation. PMID:26709094

Ataxia telangiectasia mutated (ATM) modulates long interspersed element-1 (L1) retrotransposition in human neural stem cells

PubMed Central

Coufal, Nicole G.; Garcia-Perez, Josè Luis; Peng, Grace E.; Marchetto, Maria C. N.; Muotri, Alysson R.; Mu, Yangling; Carson, Christian T.; Macia, Angela; Moran, John V.; Gage, Fred H.

2011-01-01

Long interspersed element-1 (L1) retrotransposons compose ∼20% of the mammalian genome, and ongoing L1 retrotransposition events can impact genetic diversity by various mechanisms. Previous studies have demonstrated that endogenous L1 retrotransposition can occur in the germ line and during early embryonic development. In addition, recent data indicate that engineered human L1s can undergo somatic retrotransposition in human neural progenitor cells and that an increase in human-specific L1 DNA content can be detected in the brains of normal controls, as well as in Rett syndrome patients. Here, we demonstrate an increase in the retrotransposition efficiency of engineered human L1s in cells that lack or contain severely reduced levels of ataxia telangiectasia mutated, a serine/threonine kinase involved in DNA damage signaling and neurodegenerative disease. We demonstrate that the increase in L1 retrotransposition in ataxia telangiectasia mutated-deficient cells most likely occurs by conventional target-site primed reverse transcription and generate either longer, or perhaps more, L1 retrotransposition events per cell. Finally, we provide evidence suggesting an increase in human-specific L1 DNA copy number in postmortem brain tissue derived from ataxia telangiectasia patients compared with healthy controls. Together, these data suggest that cellular proteins involved in the DNA damage response may modulate L1 retrotransposition. PMID:22159035

Activation of FoxM1 Revitalizes the Replicative Potential of Aged β-Cells in Male Mice and Enhances Insulin Secretion

PubMed Central

Golson, Maria L.; Dunn, Jennifer C.; Maulis, Matthew F.; Dadi, Prasanna K.; Osipovich, Anna B.; Magnuson, Mark A.; Jacobson, David A.

2015-01-01

Type 2 diabetes incidence increases with age, while β-cell replication declines. The transcription factor FoxM1 is required for β-cell replication in various situations, and its expression declines with age. We hypothesized that increased FoxM1 activity in aged β-cells would rejuvenate proliferation. Induction of an activated form of FoxM1 was sufficient to increase β-cell mass and proliferation in 12-month-old male mice after just 2 weeks. Unexpectedly, at 2 months of age, induction of activated FoxM1 in male mice improved glucose homeostasis with unchanged β-cell mass. Cells expressing activated FoxM1 demonstrated enhanced glucose-stimulated Ca2+ influx, which resulted in improved glucose tolerance through enhanced β-cell function. Conversely, our laboratory has previously demonstrated that mice lacking FoxM1 in the pancreas display glucose intolerance or diabetes with only a 60% reduction in β-cell mass, suggesting that the loss of FoxM1 is detrimental to β-cell function. Ex vivo insulin secretion was therefore examined in size-matched islets from young mice lacking FoxM1 in β-cells. Foxm1-deficient islets indeed displayed reduced insulin secretion. Our studies reveal that activated FoxM1 increases β-cell replication while simultaneously enhancing insulin secretion and improving glucose homeostasis, making FoxM1 an attractive therapeutic target for diabetes. PMID:26251404

Calcium-mediated shaping of naive CD4 T-cell phenotype and function

PubMed Central

Guichard, Vincent; Bonilla, Nelly; Durand, Aurélie; Audemard-Verger, Alexandra; Guilbert, Thomas; Martin, Bruno

2017-01-01

Continuous contact with self-major histocompatibility complex ligands is essential for the survival of naive CD4 T cells. We have previously shown that the resulting tonic TCR signaling also influences their fate upon activation by increasing their ability to differentiate into induced/peripheral regulatory T cells. To decipher the molecular mechanisms governing this process, we here focus on the TCR signaling cascade and demonstrate that a rise in intracellular calcium levels is sufficient to modulate the phenotype of mouse naive CD4 T cells and to increase their sensitivity to regulatory T-cell polarization signals, both processes relying on calcineurin activation. Accordingly, in vivo calcineurin inhibition leads the most self-reactive naive CD4 T cells to adopt the phenotype of their less self-reactive cell-counterparts. Collectively, our findings demonstrate that calcium-mediated activation of the calcineurin pathway acts as a rheostat to shape both the phenotype and effector potential of naive CD4 T cells in the steady-state. PMID:29239722

Catabolic Effects of Endothelial Cell-Derived Microparticles on Disc Cells: Implications in Intervertebral Disc Neovascularization and Degeneration

PubMed Central

Pohl, Pedro H. I.; Lozito, Thomas P.; Cuperman, Thais; Yurube, Takashi; Moon, Hong J.; Ngo, Kevin; Tuan, Rocky S.; Croix, Claudette St.; Sowa, Gwendolyn A.; Rodrigues, Luciano M. R.; Kang, James D.; Vo, Nam V.

2017-01-01

Neovascularization of intervertebral discs, a phenomenon considered pathological since normal discs are primarily avascular structures, occurs most frequently in annulus fibrosus (AF) of degenerated discs. Endothelial cells (ECs) are involved in this process, but the mechanism of the interaction between AF and endothelial cells is unclear. In this study we evaluated the effects on matrix catabolic activity of AF cells by the extracellular endothelial microparticles (EMPs) and soluble protein factors (SUP fraction) produced from ECs. Passage 1 human AF cells grown in monolayer cultures were treated for 72 hours with 250μg of EMPs or SUP fraction isolated from culture of the microvascular endothelial cell line, HEMC-I. Live-cell imaging revealed uptake of EMPs by AF cells. RT-PCR analysis demonstrated increased mRNA expression of MMP-1 (50.3 fold), MMP-3 (4.5 fold) and MMP-13 (5.5 fold) in AF cell cultures treated with EMPs compared to untreated control. Western analysis also demonstrated increased MMP protein expression in EMP-treated AF cells. AF cells treated with the SUP fraction also exhibited a dramatic increase in MMP mRNA and protein expression. Increased MMP expression is primarily due to EMP or SUP stimulation of AF cells since EMPs or SUP fraction alone contained negligible amount of MMPs. Interestingly, MMP activity was elevated in AF cell cultures treated with EMPs but not with SUP. This study revealed enhanced matrix catabolism as a molecular consequence of action of ECs on AF cells via EMPs, which might be expected during neo-angiogenesis of degenerating disc. PMID:27246627

Cell-delivered magnetic nanoparticles caused hyperthermia-mediated increased survival in a murine pancreatic cancer model.

PubMed

Basel, Matthew T; Balivada, Sivasai; Wang, Hongwang; Shrestha, Tej B; Seo, Gwi Moon; Pyle, Marla; Abayaweera, Gayani; Dani, Raj; Koper, Olga B; Tamura, Masaaki; Chikan, Viktor; Bossmann, Stefan H; Troyer, Deryl L

2012-01-01

Using magnetic nanoparticles to absorb alternating magnetic field energy as a method of generating localized hyperthermia has been shown to be a potential cancer treatment. This report demonstrates a system that uses tumor homing cells to actively carry iron/iron oxide nanoparticles into tumor tissue for alternating magnetic field treatment. Paramagnetic iron/ iron oxide nanoparticles were synthesized and loaded into RAW264.7 cells (mouse monocyte/ macrophage-like cells), which have been shown to be tumor homing cells. A murine model of disseminated peritoneal pancreatic cancer was then generated by intraperitoneal injection of Pan02 cells. After tumor development, monocyte/macrophage-like cells loaded with iron/ iron oxide nanoparticles were injected intraperitoneally and allowed to migrate into the tumor. Three days after injection, mice were exposed to an alternating magnetic field for 20 minutes to cause the cell-delivered nanoparticles to generate heat. This treatment regimen was repeated three times. A survival study demonstrated that this system can significantly increase survival in a murine pancreatic cancer model, with an average post-tumor insertion life expectancy increase of 31%. This system has the potential to become a useful method for specifically and actively delivering nanoparticles for local hyperthermia treatment of cancer.

Localization of intercellular adhesion molecule-1 (ICAM-1) in the lungs of silica-exposed mice.

PubMed Central

Nario, R C; Hubbard, A K

1997-01-01

Intercellular adhesion molecule-1 (ICAM-1) is expressed on a variety of cells including endothelial cells, alveolar epithelial cells, and alveolar macrophages. Endothelial/epithelial cell ICAM-1 participates in the migration of leukocytes out of the blood in response to pulmonary inflammation, whereas alveolar macrophage ICAM-1 may represent cell activation. Our previous studies have shown that there is increased expression of ICAM-1 in lung tissue during acute inflammation following intratracheal injection with silica particles (2 mg/mouse). This increased expression was shown to play a role, in part, in the migration of neutrophils from the circulation into the tissue parenchyma. The aim of the current work is to localize expression of ICAM-1 during acute inflammation in lungs of mice exposed to either silica or the nuisance dust, titanium dioxide. In silica-exposed mice, a significant increase in ICAM-1 was detected on day-1 and localized by immunohistochemistry to aggregates of pulmonary macrophages and to type II epithelial cells. Areas of the lung with increased ICAM-1 expression also showed increased tumor necrosis factor alpha expression. Immunocytochemical staining of bronchoalveolar lavage (BAL) cells demonstrated increased ICAM-1 expression associated with alveolar macrophages 3, 5, and 7 days following silica exposure. Finally, soluble ICAM-1 levels in the BAL fluid were significantly increased in mice exposed to silica on the same days. Titanium dioxide exposure elicited a minimal increase in expression of ICAM-1 in the lungs. These data demonstrate that exposure to the toxic particle silica specifically increases ICAM-1 expression localized to pulmonary macrophages and type II epithelial cells. Images Figure 2. B Figure 2. A Figure 2. D Figure 2. C Figure 3. A Figure 3. B Figure 5. B Figure 5. A Figure 5. C PMID:9400721

Increased FasL expression correlates with apoptotic changes in granulocytes cultured with oxidized clozapine

DOE Office of Scientific and Technical Information (OSTI.GOV)

Husain, Zaheed; Department of Pathology, Harvard Medical School, Boston, MA; Almeciga, Ingrid

Clozapine has been associated with a 1% incidence of agranulocytosis. The formation of an oxidized intermediate clozapine metabolite has been implicated in direct polymorphonuclear (PMN) toxicity. We utilized two separate systems to analyze the role of oxidized clozapine in inducing apoptosis in treated cells. Human PMN cells incubated with clozapine (0-10 {mu}M) in the presence of 0.1 mM H{sub 2}O{sub 2} demonstrated a progressive decrease of surface CD16 expression along with increased apoptosis. RT-PCR analysis showed decreased CD16 but increased FasL gene expression in clozapine-treated PMN cells. No change in constitutive Fas expression was observed in treated cells. In HL-60more » cells induced to differentiate with retinoic acid (RA), a similar increase in FasL expression, but no associated changes in CD16 gene expression, was observed following clozapine treatments. Our results demonstrate increased FasL gene expression in oxidized clozapine-induced apoptotic neutrophils suggesting that apoptosis in granulocytes treated with clozapine involves Fas/FasL interaction that initiates a cascade of events leading to clozapine-induced agranulocytosis.« less

High Throughput Screening for Compounds That Alter Muscle Cell Glycosylation Identifies New Role for N-Glycans in Regulating Sarcolemmal Protein Abundance and Laminin Binding*

PubMed Central

Cabrera, Paula V.; Pang, Mabel; Marshall, Jamie L.; Kung, Raymond; Nelson, Stanley F.; Stalnaker, Stephanie H.; Wells, Lance; Crosbie-Watson, Rachelle H.; Baum, Linda G.

2012-01-01

Duchenne muscular dystrophy is an X-linked disorder characterized by loss of dystrophin, a cytoskeletal protein that connects the actin cytoskeleton in skeletal muscle cells to extracellular matrix. Dystrophin binds to the cytoplasmic domain of the transmembrane glycoprotein β-dystroglycan (β-DG), which associates with cell surface α-dystroglycan (α-DG) that binds laminin in the extracellular matrix. β-DG can also associate with utrophin, and this differential association correlates with specific glycosylation changes on α-DG. Genetic modification of α-DG glycosylation can promote utrophin binding and rescue dystrophic phenotypes in mouse dystrophy models. We used high throughput screening with the plant lectin Wisteria floribunda agglutinin (WFA) to identify compounds that altered muscle cell surface glycosylation, with the goal of finding compounds that increase abundance of α-DG and associated sarcolemmal glycoproteins, increase utrophin usage, and increase laminin binding. We identified one compound, lobeline, from the Prestwick library of Food and Drug Administration-approved compounds that fulfilled these criteria, increasing WFA binding to C2C12 cells and to primary muscle cells from wild type and mdx mice. WFA binding and enhancement by lobeline required complex N-glycans but not O-mannose glycans that bind laminin. However, inhibiting complex N-glycan processing reduced laminin binding to muscle cell glycoproteins, although O-mannosylation was intact. Glycan analysis demonstrated a general increase in N-glycans on lobeline-treated cells rather than specific alterations in cell surface glycosylation, consistent with increased abundance of multiple sarcolemmal glycoproteins. This demonstrates the feasibility of high throughput screening with plant lectins to identify compounds that alter muscle cell glycosylation and identifies a novel role for N-glycans in regulating muscle cell function. PMID:22570487

Evidence for pleural epithelial-mesenchymal transition in murine compensatory lung growth

PubMed Central

Ysasi, Alexandra B.; Wagner, Willi L.; Valenzuela, Cristian D.; Kienzle, Arne; Servais, Andrew B.; Bennett, Robert D.; Tsuda, Akira; Ackermann, Maximilian; Mentzer, Steven J.

2017-01-01

In many mammals, including rodents and humans, removal of one lung results in the compensatory growth of the remaining lung; however, the mechanism of compensatory lung growth is unknown. Here, we investigated the changes in morphology and phenotype of pleural cells after pneumonectomy. Between days 1 and 3 after pneumonectomy, cells expressing α-smooth muscle actin (SMA), a cytoplasmic marker of myofibroblasts, were significantly increased in the pleura compared to surgical controls (p < .01). Scanning electron microscopy of the pleural surface 3 days post-pneumonectomy demonstrated regions of the pleura with morphologic features consistent with epithelial-mesenchymal transition (EMT); namely, cells with disrupted intercellular junctions and an acquired mesenchymal (rounded and fusiform) morphotype. To detect the migration of the transitional pleural cells into the lung, a biotin tracer was used to label the pleural mesothelial cells at the time of surgery. By post-operative day 3, image cytometry of post-pneumonectomy subpleural alveoli demonstrated a 40-fold increase in biotin+ cells relative to pneumonectomy-plus-plombage controls (p < .01). Suggesting a similar origin in space and time, the distribution of cells expressing biotin, SMA, or vimentin demonstrated a strong spatial autocorrelation in the subpleural lung (p < .001). We conclude that post-pneumonectomy compensatory lung growth involves EMT with the migration of transitional mesothelial cells into subpleural alveoli. PMID:28542402

Sorafenib induces cathepsin B-mediated apoptosis of bladder cancer cells by regulating the Akt/PTEN pathway. The Akt inhibitor, perifosine, enhances the sorafenib-induced cytotoxicity against bladder cancer cells

PubMed Central

Amantini, Consuelo; Morelli, Maria Beatrice; Santoni, Matteo; Soriani, Alessandra; Cardinali, Claudio; Farfariello, Valerio; Eleuteri, Anna Maria; Bonfili, Laura; Mozzicafreddo, Matteo; Nabissi, Massimo; Cascinu, Stefano; Santoni, Giorgio

2015-01-01

Sorafenib, a tyrosine kinase inhibitor, has been demonstrated to exert anti-tumor effects. However, the molecular mechanisms underlying its effects on bladder cancer remain unknown. Here, we evaluated the mechanisms responsible for the sorafenib-induced anti-tumor effects on 5637 and T24 bladder cancer cells. We demonstrated that sorafenib reduces cell viability, stimulates lysosome permeabilization and induces apoptosis of bladder cancer cells. These effects are dependent by the activation of cathepsin B released from lysosomes. The sorafenib-increased cathepsin B activity induced the proteolysis of Bid into tBid that stimulates the intrinsic pathway of apoptosis characterized by mitochondrial membrane depolarization, oxygen radical generation and cytochrome c release. Moreover, we found that cathepsin B enzymatic activity, induced by sorafenib, is dependent on its dephosphorylation via PTEN activation and Akt inactivation. Pretreatment with orthovanadate rescued bladder cancer cells from apoptosis. In addition, the Akt inhibitor perifosine increased the sensitivity of bladder cancer cells to sorafenib-induced cytotoxicity. Overall, our results show that apoptotic cell death induced by sorafenib in bladder cancer cells is dependent on cathepsin B activity and involved PTEN and Akt signaling pathways. The Akt inhibitor perifosine increased the cytotoxic effects of sorafenib in bladder cancer cells. PMID:26097873

Learning Increases the Survival of Newborn Neurons Provided that Learning Is Difficult to Achieve and Successful

ERIC Educational Resources Information Center

Curlik, Daniel M., II; Shors, Tracey J.

2011-01-01

Learning increases neurogenesis by increasing the survival of new cells generated in the adult hippocampal formation [Shors, T. J. Saving new brain cells. "Scientific American," 300, 46-52, 2009]. However, only some types of learning are effective. Recent studies demonstrate that animals that learn the conditioned response (CR) but require more…

DOE Office of Scientific and Technical Information (OSTI.GOV)

Park, Serk In, E-mail: serkin@korea.edu; The BK21 Plus Program for Biomedical Sciences, Korea University College of Medicine, Seoul; Department of Medicine and Center for Bone Biology, Vanderbilt University School of Medicine, Nashville, TN

The radiation stress induces cytotoxic responses of cell death as well as cytoprotective responses of cell survival. Understanding exact cellular mechanism and signal transduction pathways is important in improving cancer radiotherapy. Increasing evidence suggests that cyclic AMP response element binding protein (CREB)/activating transcription factor (ATF) family proteins act as a survival factor and a signaling molecule in response to stress. We postulated that CREB inhibition via CRE decoy oligonucleotide increases tumor cell sensitization to γ-irradiation-induced cytotoxic stress. In the present study, we demonstrate that CREB phosphorylation and CREB DNA-protein complex formation increased in time- and radiation dose-dependent manners, while theremore » was no significant change in total protein level of CREB. In addition, CREB was phosphorylated in response to γ-irradiation through p38 MAPK pathway. Further investigation revealed that CREB blockade by decoy oligonucleotides functionally inhibited transactivation of CREB, and significantly increased radiosensitivity of multiple human cancer cell lines including TP53- and/or RB-mutated cells with minimal effects on normal cells. We also demonstrate that tumor cells ectopically expressing dominant negative mutant CREB (KCREB) and the cells treated with p38 MAPK inhibitors were more sensitive to γ-irradiation than wild type parental cells or control-treated cells. Taken together, we conclude that CREB protects tumor cells from γ-irradiation, and combination of CREB inhibition plus ionizing radiation will be a promising radiotherapeutic approach. - Highlights: • γ-Irradiation induced CREB phosphorylation and CRE-directed transcription in tumor. • γ-Irradiation-induced transcriptional activation of CREB was via p38 MAPK pathway. • CRE blockade increased radiosensitivity of tumor cells but not of normal cells. • CRE decoy oligonucleotides or p38 MAPK inhibitors can be used as radiosensitizers.« less

Epigenetic modulation of dental pulp stem cells: implications for regenerative endodontics.

PubMed

Duncan, H F; Smith, A J; Fleming, G J P; Cooper, P R

2016-05-01

Dental pulp stem cells (DPSCs) offer significant potential for use in regenerative endodontics, and therefore, identifying cellular regulators that control stem cell fate is critical to devising novel treatment strategies. Stem cell lineage commitment and differentiation are regulated by an intricate range of host and environmental factors of which epigenetic influence is considered vital. Epigenetic modification of DNA and DNA-associated histone proteins has been demonstrated to control cell phenotype and regulate the renewal and pluripotency of stem cell populations. The activities of the nuclear enzymes, histone deacetylases, are increasingly being recognized as potential targets for pharmacologically inducing stem cell differentiation and dedifferentiation. Depending on cell maturity and niche in vitro, low concentration histone deacetylase inhibitor (HDACi) application can promote dedifferentiation of several post-natal and mouse embryonic stem cell populations and conversely increase differentiation and accelerate mineralization in DPSC populations, whilst animal studies have shown an HDACi-induced increase in stem cell marker expression during organ regeneration. Notably, both HDAC and DNA methyltransferase inhibitors have also been demonstrated to dramatically increase the reprogramming of somatic cells to induced pluripotent stem cells (iPSCs) for use in regenerative therapeutic procedures. As the regulation of cell fate will likely remain the subject of intense future research activity, this review aims to describe the current knowledge relating to stem cell epigenetic modification, focusing on the role of HDACi on alteration of DPSC phenotype, whilst presenting the potential for therapeutic application as part of regenerative endodontic regimens. © 2015 International Endodontic Journal. Published by John Wiley & Sons Ltd.

Protein oxidation and degradation during proliferative senescence of human MRC-5 fibroblasts.

PubMed

Sitte, N; Merker, K; von Zglinicki, T; Grune, T

2000-03-01

One of the highlights of age-related changes of cellular metabolism is the accumulation of oxidized proteins. The aging process on a cellular level can be treated either as the ongoing proliferation until a certain number of cell divisions is reached (the Hayflick limit) or as the aging of nondividing cells, that is, the age-related changes in cells without proliferation. The present investigation was undertaken to reveal the changes in protein turnover, proteasome activity, and protein oxidation status during proliferative senescence. We were able to demonstrate that the activity of the cytosolic proteasomal system declines dramatically during the proliferative senescence of human MRC-5 fibroblasts. Regardless of the loss in activity, it could be demonstrated that there are no changes in the transcription and translation of proteasomal subunits. This decline in proteasome activity was accompanied by an increased concentration of oxidized proteins. Cells at higher proliferation stages were no longer able to respond with increased degradation of endogenous [(35)S]-Met-radiolabeled proteins after hydrogen peroxide- or quinone-induced oxidative stress. It could be demonstrated that oxidized proteins in senescent human MRC-5 fibroblasts are not as quickly removed as they are in young cells. Therefore, our study demonstrates that the accumulation of oxidized proteins and decline in protein turnover and activity of the proteasomal system are not only a process of postmitotic aging but also occur during proliferative senescence and result in an increased half-life of oxidized proteins.

Non-Saccharomyces yeasts protect against epithelial cell barrier disruption induced by Salmonella enterica subsp. enterica serovar Typhimurium.

PubMed

Smith, I M; Baker, A; Arneborg, N; Jespersen, L

2015-11-01

The human gastrointestinal epithelium makes up the largest barrier separating the body from the external environment. Whereas invasive pathogens cause epithelial barrier disruption, probiotic micro-organisms modulate tight junction regulation and improve epithelial barrier function. In addition, probiotic strains may be able to reduce epithelial barrier disruption caused by pathogenic species. The aim of this study was to explore non-Saccharomyces yeast modulation of epithelial cell barrier function in vitro. Benchmarking against established probiotic strains, we evaluated the ability of four nonpathogenic yeast species to modulate transepithelial electrical resistance (TER) across a monolayer of differentiated human colonocytes (Caco-2 cells). Further, we assessed yeast modulation of a Salmonella Typhimurium-induced epithelial cell barrier function insult. Our findings demonstrate distinct patterns of non-Saccharomyces yeast modulation of epithelial cell barrier function. While the established probiotic yeast Saccharomyces boulardii increased TER across a Caco-2 monolayer by 30%, Kluyveromyces marxianus exhibited significantly stronger properties of TER enhancement (50% TER increase). In addition, our data demonstrate significant yeast-mediated modulation of Salmonella-induced epithelial cell barrier disruption and identify K. marxianus and Metschnikowia gruessii as two non-Saccharomyces yeasts capable of protecting human epithelial cells from pathogen invasion. This study demonstrates distinct patterns of non-Saccharomyces yeast modulation of epithelial cell barrier function in vitro. Further, our data demonstrate significant yeast-mediated modulation of Salmonella Typhimurium-induced epithelial cell barrier disruption and identify Kluyveromyces marxianus and Metschnikowia gruessii as two non-Saccharomyces yeasts capable of protecting human epithelial cells from pathogen invasion. This study is the first to demonstrate significant non-Saccharomyces yeast-mediated epithelial cell barrier protection from Salmonella invasion, thus encouraging future efforts aimed at confirming the observed effects in vivo and driving further strain development towards novel yeast probiotics. © 2015 The Society for Applied Microbiology.

Insulin-Like Growth Factor Binding Proteins Increase Intracellular Calcium Levels in Two Different Cell Lines

PubMed Central

Seurin, Danielle; Lombet, Alain; Babajko, Sylvie; Godeau, François; Ricort, Jean-Marc

2013-01-01

Background Insulin-like growth factor binding proteins (IGFBPs) are six related secreted proteins that share IGF-dependent and -independent functions. If the former functions begin to be well described, the latter are somewhat more difficult to investigate and to characterize. At the cellular level, IGFBPs were shown to modulate numerous processes including cell growth, differentiation and apoptosis. However, the molecular mechanisms implicated remain largely unknown. We previously demonstrated that IGFBP-3, but not IGFBP-1 or IGFBP-5, increase intracellular calcium concentration in MCF-7 cells (Ricort J-M et al. (2002) FEBS lett 527: 293–297). Methodology/Principal Findings We perform a global analysis in which we studied, by two different approaches, the binding of each IGFBP isoform (i.e., IGFBP-1 to -6) to the surface of two different cellular models, MCF-7 breast adenocarcinoma cells and C2 myoblast proliferative cells, as well as the IGFBP-induced increase of intracellular calcium concentration. Using both confocal fluorescence microscopy and flow cytometry analysis, we showed that all IGFBPs bind to MCF-7 cell surface. By contrast, only four IGFBPs can bind to C2 cell surface since neither IGFBP-2 nor IGFBP-4 were detected. Among the six IGFBPs tested, only IGFBP-1 did not increased intracellular calcium concentration whatever the cellular model studied. By contrast, IGFBP-2, -3, -4 and -6, in MCF-7 cells, and IGFBP-3, -5 and -6, in C2 proliferative cells, induce a rapid and transient increase in intracellular free calcium concentration. Moreover, IGFBP-2 and -3 (in MCF-7 cells) and IGFBP-5 (in C2 cells) increase intracellular free calcium concentration by a pertussis toxin sensitive signaling pathway. Conclusions Our results demonstrate that IGFBPs are able to bind to cell surface and increase intracellular calcium concentration. By characterizing the IGFBPs-induced cell responses and intracellular couplings, we highlight the cellular specificity and complexity of the IGF-independent actions of these IGF binding proteins. PMID:23527161

Somatostatin inhibits intestinal mucosal mast cell degranulation in normal conditions and during mast cell hyperplasia.

PubMed

Saavedra, Y; Vergara, P

2003-03-28

Several studies demonstrate that intestinal mucosal mast cells (IMMC) are modulated by nervous reflexes as well as by intraluminal content. We recently demonstrated that peptones, such as ovalbumin hydrolysate (OVH), induce the release of rat mast cell protease II (RMCP II), indicating IMMC degranulation. The response is due to complex neuroendocrine reflexes. Somatostatin (SS) and its analogues have been used as potential treatments for inflammation in other body systems with contradictory results. The aim of this study was to evaluate if somatostatin could contribute to the reduction of intestinal mucosal mast cell degranulation. Anesthetized rats were prepared for duodenal perfusion and mast cell activation was measured by analysis of RMCP II concentration in the duodenal perfusate. Somatostatin significantly decreased RMCP II concentration in both nonstimulated conditions and after ovalbumin hydrolysate perfusion. However, when somatostatin was given previously to OVH, the peptone still induced a slight increase of RMCP II. Similar effects were observed in animals previously treated with capsaicin. These protocols were repeated in animals infected with Trichinella spiralis, which induces mucosal mast cell hyperplasia. In these cases, somatostatin blocked the effect of OVH, thus, preventing an increase in RMCP II concentration. Fresh frozen tissue sections from the duodenum were processed in an attempt to demonstrate the presence of SS receptors in mast cells using immunofluorescence and Fluo-peptide labeling techniques. Confocal images from duodenum specimens demonstrate the existence of SS receptors in positive cells for RMCP II. Taken together, these results indicate that somatostatin diminishes mast cell activity and in consequence could prevent the intestinal responses to mast cell hyperplasia. Copyright 2002 Elsevier Science B.V.

Non-Invasive Radiofrequency Field Treatment of 4T1 Breast Tumors Induces T-cell Dependent Inflammatory Response.

PubMed

Newton, Jared M; Flores-Arredondo, Jose H; Suki, Sarah; Ware, Matthew J; Krzykawska-Serda, Martyna; Agha, Mahdi; Law, Justin J; Sikora, Andrew G; Curley, Steven A; Corr, Stuart J

2018-02-22

Previous work using non-invasive radiofrequency field treatment (RFT) in cancer has demonstrated its therapeutic potential as it can increase intratumoral blood perfusion, localization of intravenously delivered drugs, and promote a hyperthermic intratumoral state. Despite the well-known immunologic benefits that febrile hyperthermia can induce, an investigation of how RFT could modulate the intra-tumoral immune microenvironment had not been studied. Thus, using an established 4T1 breast cancer model in immune competent mice, we demonstrate that RFT induces a transient, localized, and T-cell dependent intratumoral inflammatory response. More specifically we show that multi- and singlet-dose RFT promote an increase in tumor volume in immune competent Balb/c mice, which does not occur in athymic nude models. Further leukocyte subset analysis at 24, 48, and 120 hours after a single RFT show a rapid increase in tumoral trafficking of CD4+ and CD8+ T-cells 24 hours post-treatment. Additional serum cytokine analysis reveals an increase in numerous pro-inflammatory cytokines and chemokines associated with enhanced T-cell trafficking. Overall, these data demonstrate that non-invasive RFT could be an effective immunomodulatory strategy in solid tumors, especially for enhancing the tumoral trafficking of lymphocytes, which is currently a major hindrance of numerous cancer immunotherapeutic strategies.

Placenta-derived mesenchymal stromal cells and their exosomes exert therapeutic effects in Duchenne muscular dystrophy.

PubMed

Bier, Ariel; Berenstein, Peter; Kronfeld, Noam; Morgoulis, Daria; Ziv-Av, Amotz; Goldstein, Hodaya; Kazimirsky, Gila; Cazacu, Simona; Meir, Rinat; Popovtzer, Rachela; Dori, Amir; Brodie, Chaya

2018-05-03

Duchenne muscular dystrophy (DMD) is a degenerative lethal, X-linked disease of skeletal and cardiac muscles caused by mutations in the dystrophin gene. Cell therapy using different cell types, including mesenchymal stromal cells (MSCs), has been considered as a potential approach for the treatment of DMD. MSCs can be obtained from autologous sources such as bone marrow and adipose tissues or from allogeneic placenta and umbilical cord. The safety and therapeutic impact of these cells has been demonstrated in pre-clinical and clinical studies and their functions are attributed to paracrine effects that are mediated by secreted cytokines and extracellular vesicles. Here, we studied the therapeutic effects of placenta-derived MSCs (PL-MSCs) and their secreted exosomes using mouse and human myoblasts from healthy controls, Duchenne patients and mdx mice. Treatment of myoblasts with conditioned medium or exosomes secreted by PL-MSCs increased the differentiation of these cells and decreased the expression of fibrogenic genes in DMD patient myoblasts. In addition, these treatments also increased the expression of utrophin in these cells. Using a quantitative miR-29c reporter, we demonstrated that the PL-MSC effects were partly mediated by the transfer of exosomal miR-29c. Intramuscular transplantation of PL-MSCs in mdx mice resulted in decreased creatine kinase levels. PL-MSCs significantly decreased the expression of TGF-β and the level of fibrosis in the diaphragm and cardiac muscles, inhibited inflammation and increased utrophin expression. In vivo imaging analyses using MSCs labeled with gold nanoparticles or fluorescent dyes demonstrated localization of the cells in the muscle tissues up to 3 weeks post treatment. Altogether, these results demonstrate that PL-MSCs and their secreted exosomes have important clinical applications in cell therapy of DMD partly via the targeted delivery of exosomal miR-29c. Copyright © 2018. Published by Elsevier Ltd.

TMPRSS4 induces cancer cell invasion through pro-uPA processing.

PubMed

Min, Hye-Jin; Lee, Myung Kyu; Lee, Jung Weon; Kim, Semi

2014-03-28

TMPRSS4 is a novel type II transmembrane serine protease that is highly expressed on the cell surface in pancreatic, thyroid, colon, and other cancer tissues. Previously, we demonstrated that TMPRSS4 mediates cancer cell invasion, epithelial-mesenchymal transition, and metastasis and that increased TMPRSS4 expression correlates with colorectal cancer progression. We also demonstrated that TMPRSS4 upregulates urokinase-type plasminogen activator (uPA) gene expression to induce cancer cell invasion. However, it remains unknown how proteolytic activity of TMPRSS4 contributes to invasion. In this study, we report that TMPRSS4 directly converted inactive pro-uPA into the active form through its proteolytic activity. Analysis of conditioned medium from cells overexpressing TMPRSS4 demonstrated that the active TMPRSS4 protease domain is released from the cells and is associated with the plasma membrane. Furthermore, TMPRSS4 could increase pro-uPA-mediated invasion in a serine proteolytic activity-dependent manner. These observations suggest that TMPRSS4 is an upstream regulator of pro-uPA activation. This study provides valuable insights into the proteolytic function of TMPRSS4 as well as mechanisms for the control of invasion. Copyright © 2014 Elsevier Inc. All rights reserved.

HIF-2α mediates a marked increase in migration and stemness characteristics in a subset of glioma cells under hypoxia by activating an Oct-4/Sox-2-Mena (INV) axis.

PubMed

Bhagat, Mohita; Palanichamy, Jayanth Kumar; Ramalingam, Pradeep; Mudassir, Madeeha; Irshad, Khushboo; Chosdol, Kunzang; Sarkar, Chitra; Seth, Pankaj; Goswami, Sumanta; Sinha, Subrata; Chattopadhyay, Parthaprasad

2016-05-01

Hypoxia is a salient feature of most solid tumors and plays a central role in tumor progression owing to its multiple contributions to therapeutic resistance, metastasis, angiogenesis and stemness properties. Reports exist in literature about hypoxia increasing stemness characteristics and invasiveness potential of malignant cells. In order to delineate molecular crosstalk among factors driving glioma progression, we used knockdown and overexpression strategies. We have demonstrated that U87MG and A172 glioma cells inherently have a subset of cells with high migratory potential due to migration-inducing Mena transcripts. These cells also have elevated stemness markers (Sox-2 and Oct-4). There was a significant increase of number in this subset of migratory cells on exposure to hypoxia with corresponding elevation (over 1000 fold) in migration-inducing Mena transcripts. We were able to demonstrate that a HIF-2α-Sox-2/Oct-4-Mena (INV) axis that is strongly activated in hypoxia and markedly increases the migratory potential of the cells. Such cells also formed tumor spheres with greater efficiency. We have correlated our in-vitro results with human glioblastoma samples and found that hypoxia, invasiveness and stemness markers correlated well in native tumor samples. This study identifies a novel signaling mechanism mediated by HIF-2α in regulating invasiveness and stemness characteristics, suggesting that under hypoxic conditions, some tumor cells acquire more migratory potential by increased Pan Mena and Mena INV expression as a consequence of this HIF-2α mediated increase in Oct-4 and Sox-2. These properties would help the cells to form a new nidus after local invasion or metastasis. Copyright © 2016 Elsevier Ltd. All rights reserved.

HIV-1 Tat protein induces glial cell autophagy through enhancement of BAG3 protein levels.

PubMed

Bruno, Anna Paola; De Simone, Francesca Isabella; Iorio, Vittoria; De Marco, Margot; Khalili, Kamel; Sariyer, Ilker Kudret; Capunzo, Mario; Nori, Stefania Lucia; Rosati, Alessandra

2014-01-01

BAG3 protein has been described as an anti-apoptotic and pro-autophagic factor in several neoplastic and normal cells. We previously demonstrated that BAG3 expression is elevated upon HIV-1 infection of glial and T lymphocyte cells. Among HIV-1 proteins, Tat is highly involved in regulating host cell response to viral infection. Therefore, we investigated the possible role of Tat protein in modulating BAG3 protein levels and the autophagic process itself. In this report, we show that transfection with Tat raises BAG3 levels in glioblastoma cells. Moreover, BAG3 silencing results in highly reducing Tat- induced levels of LC3-II and increasing the appearance of sub G0/G1 apoptotic cells, in keeping with the reported role of BAG3 in modulating the autophagy/apoptosis balance. These results demonstrate for the first time that Tat protein is able to stimulate autophagy through increasing BAG3 levels in human glial cells.

Quercetin increased the antiproliferative activity of green tea polyphenol (−)-epigallocatechin gallate in prostate cancer cells

PubMed Central

Wang, Piwen; Heber, David; Henning, Susanne M.

2012-01-01

We previously demonstrated that 50% of (−)-epigallocatechin gallate (EGCG) was present in methylated form (4″-MeEGCG) in human prostate tissue, which is less bioactive. We therefore investigated whether quercetin, a natural inhibitor of catechol-O-methyl transferase (COMT), will inhibit EGCG methylation leading to enhanced antiproliferative activity of EGCG in prostate cancer cells. Incubation with both, quercetin and EGCG, for 2 hr increased the cellular concentrations of EGCG by 4 to 8-fold and 6 to 10-fold in androgen-independent PC-3 cells and androgen-dependent LNCaP cells, respectively. Concurrently, the percent of 4″-MeEGCG in the total EGCG was decreased from 39% to 15% in PC-3 cells and from 61% to 38% in LNCaP cells. Quercetin and EGCG in combination synergistically inhibited cell proliferation, caused cell cycle arrest and induced apoptosis in PC-3 cells. In LNCaP cells EGCG and quercetin exhibited a stronger antiproliferative activity leading to an additive effect. The synergistic effect of these two agents in PC-3 cells could be based on the fact that EGCG primarily inhibited COMT activity while quercetin reduced the amount of COMT protein. In summary, quercetin combined with EGCG in vitro demonstrated enhanced inhibition of cell proliferation by increasing the intracellular concentration of EGCG and decreasing EGCG methylation. PMID:22452782

Exosomes as mediators of platinum resistance in ovarian cancer

PubMed Central

Crow, Jennifer; Atay, Safinur; Banskota, Samagya; Artale, Brittany; Schmitt, Sarah; Godwin, Andrew K

2017-01-01

Exosomes have been implicated in the cell-cell transfer of oncogenic proteins and genetic material. We speculated this may be one mechanism by which an intrinsically platinum-resistant population of epithelial ovarian cancer (EOC) cells imparts its influence on surrounding tumor cells. To explore this possibility we utilized a platinum-sensitive cell line, A2780 and exosomes derived from its resistant subclones, and an unselected, platinum-resistant EOC line, OVCAR10. A2780 cells demonstrate a ~2-fold increase in viability upon treatment with carboplatin when pre-exposed to exosomes from platinum-resistant cells as compared to controls. This coincided with increased epithelial to mesenchymal transition (EMT). DNA sequencing of EOC cell lines revealed previously unreported somatic mutations in the Mothers Against Decapentaplegic Homolog 4 (SMAD4) within platinum-resistant cells. A2780 cells engineered to exogenously express these SMAD4 mutations demonstrate up-regulation of EMT markers following carboplatin treatment, are more resistant to carboplatin, and release exosomes which impart a ~1.7-fold increase in resistance in naive A2780 recipient cells as compared to controls. These studies provide the first evidence that acquired SMAD4 mutations enhance the chemo-resistance profile of EOC and present a novel mechanism in which exchange of tumor-derived exosomes perpetuates an EMT phenotype, leading to the development of subpopulations of platinum-refractory cells. PMID:28060758

Local and Systemic CD4+ T Cell Exhaustion Reverses with Clinical Resolution of Pulmonary Sarcoidosis

PubMed Central

Hawkins, Charlene; Shaginurova, Guzel; Shelton, D. Auriel; Herazo-Maya, Jose D.; Oswald-Richter, Kyra A.; Young, Anjuli; Celada, Lindsay J.; Kaminski, Naftali; Sevin, Carla

2017-01-01

Investigation of the Th1 immune response in sarcoidosis CD4+ T cells has revealed reduced proliferative capacity and cytokine expression upon TCR stimulation. In other disease models, such cellular dysfunction has been associated with a step-wise, progressive loss of T cell function that results from chronic antigenic stimulation. T cell exhaustion is defined by decreased cytokine production upon TCR activation, decreased proliferation, increased expression of inhibitory cell surface receptors, and increased susceptibility to apoptosis. We characterized sarcoidosis CD4+ T cell immune function in systemic and local environments among subjects undergoing disease progression compared to those experiencing disease resolution. Spontaneous and TCR-stimulated Th1 cytokine expression and proliferation assays were performed in 53 sarcoidosis subjects and 30 healthy controls. PD-1 expression and apoptosis were assessed by flow cytometry. Compared to healthy controls, sarcoidosis CD4+ T cells demonstrated reductions in Th1 cytokine expression, proliferative capacity (p < 0.05), enhanced apoptosis (p < 0.01), and increased PD-1 expression (p < 0.001). BAL-derived CD4+ T cells also demonstrated multiple facets of T cell exhaustion (p < 0.05). Reversal of CD4+ T cell exhaustion was observed in subjects undergoing spontaneous resolution (p < 0.05). Sarcoidosis CD4+ T cells exhibit loss of cellular function during progressive disease that follows the archetype of T cell exhaustion. PMID:29234685

Identification of the APC/C co-factor FZR1 as a novel therapeutic target for multiple myeloma.

PubMed

Crawford, Lisa J; Anderson, Gordon; Johnston, Cliona K; Irvine, Alexandra E

2016-10-25

Multiple Myeloma (MM) is a haematological neoplasm characterised by the clonal proliferation of malignant plasma cells in the bone marrow. The success of proteasome inhibitors in the treatment of MM has highlighted the importance of the ubiquitin proteasome system (UPS) in the pathogenesis of this disease. In this study, we analysed gene expression of UPS components to identify novel therapeutic targets within this pathway in MM. Here we demonstrate how this approach identified previously validated and novel therapeutic targets. In addition we show that FZR1 (Fzr), a cofactor of the multi-subunit E3 ligase complex anaphase-promoting complex/cyclosome (APC/C), represents a novel therapeutic target in myeloma. The APC/C associates independently with two cofactors, Fzr and Cdc20, to control cell cycle progression. We found high levels of FZR1 in MM primary cells and cell lines and demonstrate that expression is further increased on adhesion to bone marrow stromal cells (BMSCs). Specific knockdown of either FZR1 or CDC20 reduced viability and induced growth arrest of MM cell lines, and resulted in accumulation of APC/CFzr substrate Topoisomerase IIα (TOPIIα) or APC/CCdc20 substrate Cyclin B. Similar effects were observed following treatment with proTAME, an inhibitor of both APC/CFzr and APC/CCdc20. Combinations of proTAME with topoisomerase inhibitors, etoposide and doxorubicin, significantly increased cell death in MM cell lines and primary cells, particularly if TOPIIα levels were first increased through pre-treatment with proTAME. Similarly, combinations of proTAME with the microtubule inhibitor vincristine resulted in enhanced cell death. This study demonstrates the potential of targeting the APC/C and its cofactors as a therapeutic approach in MM.

Ligation of TLR7 on CD19(+) CD1d(hi) B cells suppresses allergic lung inflammation via regulatory T cells.

PubMed

Khan, Adnan R; Amu, Sylvie; Saunders, Sean P; Hams, Emily; Blackshields, Gordon; Leonard, Martin O; Weaver, Casey T; Sparwasser, Tim; Sheils, Orla; Fallon, Padraic G

2015-06-01

B cells have been described as having the capacity to regulate cellular immune responses and suppress inflammatory processes. One such regulatory B-cell population is defined as IL-10-producing CD19(+) CD1d(hi) cells. Previous work has identified an expansion of these cells in mice infected with the helminth, Schistosoma mansoni. Here, microarray analysis of CD19(+) CD1d(hi) B cells from mice infected with S. mansoni demonstrated significantly increased Tlr7 expression, while CD19(+) CD1d(hi) B cells from uninfected mice also demonstrated elevated Tlr7 expression. Using IL-10 reporter, Il10(-/-) and Tlr7(-/-) mice, we formally demonstrate that TLR7 ligation of CD19(+) CD1d(hi) B cells increases their capacity to produce IL-10. In a mouse model of allergic lung inflammation, the adoptive transfer of TLR7-elicited CD19(+) CD1d(hi) B cells reduced airway inflammation and associated airway hyperresponsiveness. Using DEREG mice to deplete FoxP3(+) T regulatory cells in allergen-sensitized mice, we show that that TLR7-elicited CD19(+) CD1d(hi) B cells suppress airway hyperresponsiveness via a T regulatory cell dependent mechanism. These studies identify that TLR7 stimulation leads to the expansion of IL-10-producing CD19(+) CD1d(hi) B cells, which can suppress allergic lung inflammation via T regulatory cells. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

TALE-mediated epigenetic suppression of CDKN2A increases replication in human fibroblasts.

PubMed

Bernstein, Diana L; Le Lay, John E; Ruano, Elena G; Kaestner, Klaus H

2015-05-01

Current strategies to alter disease-associated epigenetic modifications target ubiquitously expressed epigenetic regulators. This approach does not allow specific genes to be controlled in specific cell types; therefore, tools to selectively target epigenetic modifications in the desired cell type and strategies to more efficiently correct aberrant gene expression in disease are needed. Here, we have developed a method for directing DNA methylation to specific gene loci by conjugating catalytic domains of DNA methyltransferases (DNMTs) to engineered transcription activator-like effectors (TALEs). We demonstrated that these TALE-DNMTs direct DNA methylation specifically to the targeted gene locus in human cells. Further, we determined that minimizing direct nucleotide sequence repeats within the TALE moiety permits efficient lentivirus transduction, allowing easy targeting of primary cell types. Finally, we demonstrated that directed DNA methylation with a TALE-DNMT targeting the CDKN2A locus, which encodes the cyclin-dependent kinase inhibitor p16, decreased CDKN2A expression and increased replication of primary human fibroblasts, as intended. Moreover, overexpression of p16 in these cells reversed the proliferative phenotype, demonstrating the specificity of our epigenetic targeting. Together, our results demonstrate that TALE-DNMTs can selectively target specific genes and suggest that this strategy has potential application for the development of locus-specific epigenetic therapeutics.

TALE-mediated epigenetic suppression of CDKN2A increases replication in human fibroblasts

PubMed Central

Bernstein, Diana L.; Le Lay, John E.; Ruano, Elena G.; Kaestner, Klaus H.

2015-01-01

Current strategies to alter disease-associated epigenetic modifications target ubiquitously expressed epigenetic regulators. This approach does not allow specific genes to be controlled in specific cell types; therefore, tools to selectively target epigenetic modifications in the desired cell type and strategies to more efficiently correct aberrant gene expression in disease are needed. Here, we have developed a method for directing DNA methylation to specific gene loci by conjugating catalytic domains of DNA methyltransferases (DNMTs) to engineered transcription activator–like effectors (TALEs). We demonstrated that these TALE-DNMTs direct DNA methylation specifically to the targeted gene locus in human cells. Further, we determined that minimizing direct nucleotide sequence repeats within the TALE moiety permits efficient lentivirus transduction, allowing easy targeting of primary cell types. Finally, we demonstrated that directed DNA methylation with a TALE-DNMT targeting the CDKN2A locus, which encodes the cyclin-dependent kinase inhibitor p16, decreased CDKN2A expression and increased replication of primary human fibroblasts, as intended. Moreover, overexpression of p16 in these cells reversed the proliferative phenotype, demonstrating the specificity of our epigenetic targeting. Together, our results demonstrate that TALE-DNMTs can selectively target specific genes and suggest that this strategy has potential application for the development of locus-specific epigenetic therapeutics. PMID:25866970

Inhibition of Hsp90 acts synergistically with topoisomerase II poisons to increase the apoptotic killing of cells due to an increase in topoisomerase II mediated DNA damage.

PubMed

Barker, Catherine R; McNamara, Anne V; Rackstraw, Stephen A; Nelson, David E; White, Mike R; Watson, Alastair J M; Jenkins, John R

2006-01-01

Topoisomerase II plays a crucial role during chromosome condensation and segregation in mitosis and meiosis and is a highly attractive target for chemotherapeutic agents. We have identified previously topoisomerase II and heat shock protein 90 (Hsp90) as part of a complex. In this paper we demonstrate that drug combinations targeting these two enzymes cause a synergistic increase in apoptosis. The objective of our study was to identify the mode of cell killing and the mechanism behind the increase in topoisomerase II mediated DNA damage. Importantly we demonstrate that Hsp90 inhibition results in an increased topoiosmerase II activity but not degradation of topoisomerase II and it is this, in the presence of a topoisomerase II poison that causes the increase in cell death. Our results suggest a novel mechanism of action where the inhibition of Hsp90 disrupts the Hsp90-topoisomerase II interaction leading to an increase in and activation of unbound topoisomerase II, which, in the presence of a topoisomerase II poison leads to the formation of an increased number of cleavable complexes ultimately resulting in rise in DNA damage and a subsequent increase cell death.

Inhibition of Hsp90 acts synergistically with topoisomerase II poisons to increase the apoptotic killing of cells due to an increase in topoisomerase II mediated DNA damage

PubMed Central

Barker, Catherine R.; McNamara, Anne V.; Rackstraw, Stephen A.; Nelson, David E.; White, Mike R.; Watson, Alastair J. M.; Jenkins, John R.

2006-01-01

Topoisomerase II plays a crucial role during chromosome condensation and segregation in mitosis and meiosis and is a highly attractive target for chemotherapeutic agents. We have identified previously topoisomerase II and heat shock protein 90 (Hsp90) as part of a complex. In this paper we demonstrate that drug combinations targeting these two enzymes cause a synergistic increase in apoptosis. The objective of our study was to identify the mode of cell killing and the mechanism behind the increase in topoisomerase II mediated DNA damage. Importantly we demonstrate that Hsp90 inhibition results in an increased topoiosmerase II activity but not degradation of topoisomerase II and it is this, in the presence of a topoisomerase II poison that causes the increase in cell death. Our results suggest a novel mechanism of action where the inhibition of Hsp90 disrupts the Hsp90–topoisomerase II interaction leading to an increase in and activation of unbound topoisomerase II, which, in the presence of a topoisomerase II poison leads to the formation of an increased number of cleavable complexes ultimately resulting in rise in DNA damage and a subsequent increase cell death. PMID:16504968

An increase or a decrease in myosin II phosphorylation inhibits macrophage motility

PubMed Central

1991-01-01

Myosin II purified from mammalian non-muscle cells is phosphorylated on the 20-kD light chain subunit (MLC20) by the Ca2+/calmodulin-dependent enzyme myosin light chain kinase (MLCK). The importance of MLC20 phosphorylation in regulating cell motility was investigated by introducing either antibodies to MLCK (MK-Ab) or a Ca2+/calmodulin- independent, constitutively active form of MLCK (MK-) into macrophages. The effects of these proteins on cell motility were then determined using a quantitative chemotaxis assay. Chemotaxis is significantly diminished in macrophages containing MK-Ab compared to macrophages containing control antibodies. Moreover, there is an inverse relationship between the number of cells that migrate and the amount of MK-Ab introduced into cells. Interestingly, there is also an inverse relationship between the number of cells that migrate and the amount of MK- introduced into cells. Other experiments demonstrated that MK-Ab decreased intracellular MLC20 phosphorylation while MK- increased MLC20 phosphorylation. MK- also increased the amount of myosin associated with the cytoskeleton. These data demonstrate that the regulation of MLCK is an important aspect of cell motility and suggest that MLC20 phosphorylation must be maintained within narrow limits during translational motility by mammalian cells. PMID:2071674

Epstein-Barr Virus Latent Membrane Protein 2A (LMP2A)-Mediated changes in Fas Expression and Fas-Dependent Apoptosis: Role of Lyn/Syk activation

PubMed Central

Incrocci, Ryan; Hussain, Samira; Stone, Amanda; Bieging, Kathryn; Alt, Lauren A.C.; Fay, Michael J.; Swanson-Mungerson, Michelle

2015-01-01

Epstein-Barr virus Latent Membrane Protein 2A (LMP2A) is expressed in EBV-infected B cells in the germinal center, a site of significant apoptosis induced by engagement of Fas on activated B cells. Signals from the B cell receptor (BCR) protect germinal center B cells from Fas-mediated apoptosis, and since LMP2A is a BCR mimic, we hypothesized that LMP2A would also protect B cells from Fas-mediated apoptosis. Surprisingly, latently-infected human and murine B cell lines expressing LMP2A were more sensitive to Fas-mediated apoptosis, as determined by increases in Annexin-V staining, and cleavage of caspase-8, −3 and PARP. Additional studies show that LMP2A-expressing B cell lines demonstrate a Lyn- and Syk-dependent increase in sensitivity to Fas-mediated apoptosis, due to an LMP2A-dependent enhancement in Fas expression. These findings demonstrate the ability for LMP2A to directly increase a pro-apoptotic molecule and have implications for EBV latency as well as the treatment of EBV-associated malignancies. PMID:26255694

Adipose‑derived stem cells and hyaluronic acid based gel compatibility, studied in vitro.

PubMed

Guo, Jiayan; Guo, Shu; Wang, Yuxin; Yu, Yanqiu

2017-10-01

Minimally invasive aesthetic and cosmetic procedures have increased in popularity. Injectable dermal fillers provide soft tissue augmentation, improve facial rejuvenation and wrinkles, and correct tissue defects. To investigate the use of adipose‑derived stem cells integrated with a hyaluronic acid based gel as a dermal filler, the present study used cytotoxicity studies, proliferation studies, adipogenic and osteogenic differentiation, apoptosis assays and scanning electron microscopy. Although hyaluronic acid induced low levels of apoptosis in adipose‑derived stem cells, its significantly promoted proliferation of adipose‑derived stem cells. Hyaluronic acid demonstrates little toxicity against adipose‑derived stem cells. Adipose‑derived stem cells were able to differentiate into adipocytes and osteoblasts. Furthermore, scanning electron microscopy revealed that adipose‑derived stem cells maintained intact structures on the surface of hyaluronic acid as well as in it, and demonstrated abundant cell attachments. The present study demonstrated the compatibility of adipose‑derived stem cells and hyaluronic acid based gels in vitro.

Demonstration of NK cell-mediated lysis of varicella-zoster virus (VZV)-infected cells: characterization of the effector cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Tilden, A.B.; Cauda, R.; Grossi, C.E.

1986-06-01

Infection with varicella-zoster virus (VZV) rendered RAJI cells more susceptible to lysis by non-adherent blood lymphocytes. At an effector to target ratio of 80:1 the mean percentage of /sup 51/Cr release of VZV-infected RAJI cells was 41 +/- 12%, whereas that of uninfected RAJI cells was 15 +/- 6%. The increased susceptibility to lysis was associated with increased effector to target conjugate formation in immunofluorescence binding assays. The effector cells cytotoxic for VZV-infected RAJI cells were predominantly Leu-11a/sup +/ Leu-4/sup -/ granular lymphocytes as demonstrated by fluorescence-activated cell sorting. The effector cell active against VZV-infected RAJI cells appeared similar tomore » those active against herpes simplex virus (HSV)-infected cells, because in cold target competition experiments the lysis of /sup 51/Cr-labeled VZV-infected RAJI cells was efficiently inhibited by either unlabeled VZV-infected RAJI cells (mean 71% inhibition, 2:1 ratio unlabeled to labeled target) or HSV-infected RAJI cells (mean 69% inhibition) but not by uninfected RAJI cells (mean 10% inhibition). In contrast, competition experiments revealed donor heterogeneity in the overlap between effector cells for VZV- or HSV-infected RAJI vs K-562 cells.« less

Defective TFH Cell Function and Increased TFR Cells Contribute to Defective Antibody Production in Aging.

PubMed

Sage, Peter T; Tan, Catherine L; Freeman, Gordon J; Haigis, Marcia; Sharpe, Arlene H

2015-07-14

Defective antibody production in aging is broadly attributed to immunosenescence. However, the precise immunological mechanisms remain unclear. Here, we demonstrate an increase in the ratio of inhibitory T follicular regulatory (TFR) cells to stimulatory T follicular helper (TFH) cells in aged mice. Aged TFH and TFR cells are phenotypically distinct from those in young mice, exhibiting increased programmed cell death protein-1 expression but decreased ICOS expression. Aged TFH cells exhibit defective antigen-specific responses, and programmed cell death protein-ligand 1 blockade can partially rescue TFH cell function. In contrast, young and aged TFR cells have similar suppressive capacity on a per-cell basis in vitro and in vivo. Together, these studies reveal mechanisms contributing to defective humoral immunity in aging: an increase in suppressive TFR cells combined with impaired function of aged TFH cells results in reduced T-cell-dependent antibody responses in aged mice. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

Protein and chemotherapy profiling of extracellular vesicles harvested from therapeutic induced senescent triple negative breast cancer cells

PubMed Central

Kavanagh, E L; Lindsay, S; Halasz, M; Gubbins, L C; Weiner-Gorzel, K; Guang, M H Z; McGoldrick, A; Collins, E; Henry, M; Blanco-Fernández, A; Gorman, P O'; Fitzpatrick, P; Higgins, M J; Dowling, P; McCann, A

2017-01-01

Triple negative breast cancer (TNBC) is an aggressive subtype with relatively poor clinical outcomes and limited treatment options. Chemotherapy, while killing cancer cells, can result in the generation of highly chemoresistant therapeutic induced senescent (TIS) cells that potentially form stem cell niches resulting in metastases. Intriguingly, senescent cells release significantly more extracellular vesicles (EVs) than non-senescent cells. Our aim was to profile EVs harvested from TIS TNBC cells compared with control cells to identify a potential mechanism by which TIS TNBC cells maintain survival in the face of chemotherapy. TIS was induced and confirmed in Cal51 TNBC cells using the chemotherapeutic paclitaxel (PTX) (Taxol). Mass spectrometry (MS) analysis of EVs harvested from TIS compared with control Cal51 cells was performed using Ingenuity Pathway Analysis and InnateDB programs. We demonstrate that TIS Cal51 cells treated with 75 nM PTX for 7 days became senescent (senescence-associated β-galactosidase (SA-β-Gal) positive, Ki67-negative, increased p21 and p16, G2/M cell cycle arrest) and released significantly more EVs (P=0.0002) and exosomes (P=0.0007) than non-senescent control cells. Moreover, TIS cells displayed an increased expression of the multidrug resistance protein 1/p-glycoprotein. MS analysis demonstrated that EVs derived from senescent Cal51 cells contained 142 proteins with a significant increased fold change compared with control EVs. Key proteins included ATPases, annexins, tubulins, integrins, Rabs and insoluble senescence-associated secretory phenotype (SASP) factors. A fluorescent analogue of PTX (Flutax-2) allowed appreciation of the removal of chemotherapy in EVs from senescent cells. Treatment of TIS cells with the exosome biogenesis inhibitor GW4869 resulted in reduced SA-β-Gal staining (P=0.04). In summary, this study demonstrates that TIS cells release significantly more EVs compared with control cells, containing chemotherapy and key proteins involved in cell proliferation, ATP depletion, apoptosis and the SASP. These findings may partially explain why cancer senescent cells remain viable despite chemotherapeutic challenge. PMID:28991260

A synchronous increase in hydraulic conductive capacity and mechanical support in conifers with relatively uniform xylem structure.

PubMed

Jagels, Richard; Visscher, George E

2006-02-01

The dual function provided by longitudinal tracheids in conifers has led to a generally held trade-off concept that increasing wall thickness and/or volume of latewood tracheids improves mechanical support, while increasing cell diameter and/or volume of earlywood tracheids enhances conductive potential. Yet, some conifers have either uniform cell structure across the growth ring or, at most, a small amount of latewood. How do these trees accomplish the needs for increasing support and conduction with height growth? We examined Metasequoia glyptostroboides, a species that we previously demonstrated improves its mechanical properties with increasing age without a change in specific gravity or secondary wall microfibril angle. In this paper, we showed that lignin and extractive contents are not contributing factors, and through composite structure analysis, we eliminated a role for tracheid length. Using micromorphometric analysis, we demonstrated that as cell diameter increases, total primary wall decreases, secondary wall increases, and strength and conductive capacity increase with no change in specific gravity. Meta-analysis using other species of Cupressaceae, Podocarpaceae, and Araucariaceae provided strong corroborative evidence for this design strategy.

Semaphorin 4C Protects against Allergic Inflammation: Requirement of Regulatory CD138+ Plasma Cells.

PubMed

Xue, Di; Kaufman, Gabriel N; Dembele, Marieme; Beland, Marianne; Massoud, Amir H; Mindt, Barbara C; Fiter, Ryan; Fixman, Elizabeth D; Martin, James G; Friedel, Roland H; Divangahi, Maziar; Fritz, Jörg H; Mazer, Bruce D

2017-01-01

The regulatory properties of B cells have been studied in autoimmune diseases; however, their role in allergic diseases is poorly understood. We demonstrate that Semaphorin 4C (Sema4C), an axonal guidance molecule, plays a crucial role in B cell regulatory function. Mice deficient in Sema4C exhibited increased airway inflammation after allergen exposure, with massive eosinophilic lung infiltrates and increased Th2 cytokines. This phenotype was reproduced by mixed bone marrow chimeric mice with Sema4C deficient only in B cells, indicating that B lymphocytes were the key cells affected by the absence of Sema4C expression in allergic inflammation. We determined that Sema4C-deficient CD19 + CD138 + cells exhibited decreased IL-10 and increased IL-4 expression in vivo and in vitro. Adoptive transfer of Sema4c -/- CD19 + CD138 + cells induced marked pulmonary inflammation, eosinophilia, and increased bronchoalveolar lavage fluid IL-4 and IL-5, whereas adoptive transfer of wild-type CD19 + CD138 + IL-10 + cells dramatically decreased allergic airway inflammation in wild-type and Sema4c -/- mice. This study identifies a novel pathway by which Th2-mediated immune responses are regulated. It highlights the importance of plasma cells as regulatory cells in allergic inflammation and suggests that CD138 + B cells contribute to cytokine balance and are important for maintenance of immune homeostasis in allergic airways disease. Furthermore, we demonstrate that Sema4C is critical for optimal regulatory cytokine production in CD138 + B cells. Copyright © 2016 by The American Association of Immunologists, Inc.

Norepinephrine regulates hepatic innate immune system in leptin-deficient mice with nonalcoholic steatohepatitis.

PubMed

Li, Zhiping; Oben, Jude A; Yang, Shiqi; Lin, Huizhi; Stafford, Elizabeth A; Soloski, Mark J; Thomas, Steven A; Diehl, Anna Mae

2004-08-01

It is not known why natural killer T (NKT) cells, which modulate liver injury by regulating local cytokine production, are reduced in leptin-deficient ob/ob mice. NKT cells express adrenoceptors. Thus, we hypothesize that the low norepinephrine (NE) activity of ob/ob mice promotes depletion of liver NKT cells, thereby sensitizing ob/ob livers to lipopolysaccharide (LPS) toxicity. To evaluate this hypothesis, hepatic NKT cells were quantified in wild-type mice before and after treatment with NE inhibitors, and in dopamine beta-hydroxylase knockout mice (which cannot synthesize NE) and ob/ob mice before and after 4 weeks of NE supplementation. Decreasing NE activity consistently reduces liver NKT cells, while increasing NE has the opposite effect. Analysis of hepatic and thymic NKT cells in mice of different ages demonstrate an age-related accumulation of hepatic NKT cells in normal mice, while liver NKT cells become depleted after birth in ob/ob mice, which have increased apoptosis of hepatic NKT cells. NE treatment inhibits apoptosis and restores hepatic NKT cells. In ob/ob mice with reduced hepatic NKT cells, hepatic T and NKT cells produce excessive T helper (Th)-1 proinflammatory cytokines and the liver is sensitized to LPS toxicity. NE treatment decreases Th-1 cytokines, increases production of Th-2 cytokines, and reduces hepatotoxicity. Studies of CD1d-deficient mice, which lack the receptor required for NKT cell development, demonstrate that they are also unusually sensitive to LPS hepatotoxicity. In conclusion, low NE activity increases hepatic NKT cell apoptosis and depletes liver NKT cells, promoting proinflammatory polarization of hepatic cytokine production that sensitizes the liver to LPS toxicity. Copyright 2004 American Association for the Study of Liver Diseases

Catabolic effects of endothelial cell-derived microparticles on disc cells: Implications in intervertebral disc neovascularization and degeneration.

PubMed

Pohl, Pedro H I; Lozito, Thomas P; Cuperman, Thais; Yurube, Takashi; Moon, Hong J; Ngo, Kevin; Tuan, Rocky S; St Croix, Claudette; Sowa, Gwendolyn A; Rodrigues, Luciano M R; Kang, James D; Vo, Nam V

2016-08-01

Neovascularization of intervertebral discs, a phenomenon considered pathological since normal discs are primarily avascular structures, occurs most frequently in annulus fibrosus (AF) of degenerated discs. Endothelial cells (ECs) are involved in this process, but the mechanism of the interaction between AF and endothelial cells is unclear. In this study, we evaluated the effects on matrix catabolic activity of AF cells by the extracellular endothelial microparticles (EMPs) and soluble protein factors (SUP fraction) produced from ECs. Passage 1 human AF cells grown in monolayer cultures were treated for 72 h with 250 µg of EMPs or SUP fraction isolated from culture of the microvascular endothelial cell line, HEMC-I. Live-cell imaging revealed uptake of EMPs by AF cells. RT-PCR analysis demonstrated increased mRNA expression of MMP-1 (50.3-fold), MMP-3 (4.5-fold) and MMP-13 (5.5-fold) in AF cell cultures treated with EMPs compared to untreated control. Western analysis also demonstrated increased MMP protein expression in EMP-treated AF cells. AF cells treated with the SUP fraction also exhibited a dramatic increase in MMP mRNA and protein expression. Increased MMP expression is primarily due to EMP or SUP stimulation of AF cells since EMPs or SUP fraction alone contained negligible amount of MMPs. Interestingly, MMP activity was elevated in AF cell cultures treated with EMPs but not with SUP. This study revealed enhanced matrix catabolism as a molecular consequence of action of ECs on AF cells via EMPs, which might be expected during neo-angiogenesis of degenerating disc. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 34:1466-1474, 2016. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc.

Dual-functioning peptides discovered by phage display increase the magnitude and specificity of BMSC attachment to mineralized biomaterials.

PubMed

Ramaraju, Harsha; Miller, Sharon J; Kohn, David H

2017-07-01

Design of biomaterials for cell-based therapies requires presentation of specific physical and chemical cues to cells, analogous to cues provided by native extracellular matrices (ECM). We previously identified a peptide sequence with high affinity towards apatite (VTKHLNQISQSY, VTK) using phage display. The aims of this study were to identify a human MSC-specific peptide sequence through phage display, combine it with the apatite-specific sequence, and verify the specificity of the combined dual-functioning peptide to both apatite and human bone marrow stromal cells. In this study, a combinatorial phage display identified the cell binding sequence (DPIYALSWSGMA, DPI) which was combined with the mineral binding sequence to generate the dual peptide DPI-VTK. DPI-VTK demonstrated significantly greater binding affinity (1/K D ) to apatite surfaces compared to VTK, phosphorylated VTK (VTK phos ), DPI-VTK phos , RGD-VTK, and peptide-free apatite surfaces (p < 0.01), while significantly increasing hBMSC adhesion strength (τ 50 , p < 0.01). MSCs demonstrated significantly greater adhesion strength to DPI-VTK compared to other cell types, while attachment of MC3T3 pre-osteoblasts and murine fibroblasts was limited (p < 0.01). MSCs on DPI-VTK coated surfaces also demonstrated increased spreading compared to pre-osteoblasts and fibroblasts. MSCs cultured on DPI-VTK coated apatite films exhibited significantly greater proliferation compared to controls (p < 0.001). Moreover, early and late stage osteogenic differentiation markers were elevated on DPI-VTK coated apatite films compared to controls. Taken together, phage display can identify non-obvious cell and material specific peptides to increase human MSC adhesion strength to specific biomaterial surfaces and subsequently increase cell proliferation and differentiation. These new peptides expand biomaterial design methodology for cell-based regeneration of bone defects. This strategy of combining cell and material binding phage display derived peptides is broadly applicable to a variety of systems requiring targeted adhesion of specific cell populations, and may be generalized to the engineering of any adhesion surface. Copyright © 2017 Elsevier Ltd. All rights reserved.

Allosteric Regulation of E-Cadherin Adhesion*

PubMed Central

Shashikanth, Nitesh; Petrova, Yuliya I.; Park, Seongjin; Chekan, Jillian; Maiden, Stephanie; Spano, Martha; Ha, Taekjip; Gumbiner, Barry M.; Leckband, Deborah E.

2015-01-01

Cadherins are transmembrane adhesion proteins that maintain intercellular cohesion in all tissues, and their rapid regulation is essential for organized tissue remodeling. Despite some evidence that cadherin adhesion might be allosterically regulated, testing of this has been hindered by the difficulty of quantifying altered E-cadherin binding affinity caused by perturbations outside the ectodomain binding site. Here, measured kinetics of cadherin-mediated intercellular adhesion demonstrated quantitatively that treatment with activating, anti-E-cadherin antibodies or the dephosphorylation of a cytoplasmic binding partner, p120ctn, increased the homophilic binding affinity of E-cadherin. Results obtained with Colo 205 cells, which express inactive E-cadherin and do not aggregate, demonstrated that four treatments, which induced Colo 205 aggregation and p120ctn dephosphorylation, triggered quantitatively similar increases in E-cadherin affinity. Several processes can alter cell aggregation, but these results directly demonstrated the allosteric regulation of cell surface E-cadherin by p120ctn dephosphorylation. PMID:26175155

Probing cellular mechanics with Acoustic Force Spectroscopy.

PubMed

Sorkin, Raya; Bergamaschi, Giulia; Kamsma, Douwe; Brand, Guy; Dekel, Elya; Ofir-Birin, Yifat; Rudik, Ariel; Gironella, Marta; Ritort, Felix; Regev-Rudzki, Neta; Roos, Wouter; Wuite, Gijs J L

2018-06-21

A large number of studies demonstrate that cell mechanics and pathology are intimately linked. In particular, Red Blood Cell (RBC) deformability is key to their function, and is dramatically altered in the time course of diseases such as anemia and malaria. Due to the physiological importance of cell mechanics, many methods for cell mechanical probing have been developed. While single cell methods provide very valuable information, they are often technically challenging and lack high data throughput needed to distinguish differences in heterogeneous populations, while fluid flow high throughput methods miss the accuracy to detect subtle differences. Here, we present a new method for multiplexed single-cell mechanical probing using Acoustic Force Spectroscopy (AFS). We demonstrate that mechanical differences induced by chemical treatments of known effect can be measured and quantified. Furthermore, we explore the effect of extracellular-vesicles (EVs) uptake on RBC mechanics and demonstrate that EVs uptake increases RBC deformability. Our findings demonstrate the ability of AFS to manipulate cells with high stability and precision, and pave the way to further new insights into cellular mechanics and mechanobiology in health and disease, as well as potential biomedical applications.

Xin, an actin binding protein, is expressed within muscle satellite cells and newly regenerated skeletal muscle fibers.

PubMed

Hawke, Thomas J; Atkinson, Daniel J; Kanatous, Shane B; Van der Ven, Peter F M; Goetsch, Sean C; Garry, Daniel J

2007-11-01

Xin is a muscle-specific actin binding protein of which its role and regulation within skeletal muscle is not well understood. Here we demonstrate that Xin mRNA is robustly upregulated (>16-fold) within 12 h of skeletal muscle injury and is localized to the muscle satellite cell population. RT-PCR confirmed the expression pattern of Xin during regeneration, as well as within primary muscle myoblast cultures, but not other known stem cell populations. Immunohistochemical staining of single myofibers demonstrate Xin expression colocalized with the satellite cell marker Syndecan-4 further supporting the mRNA expression of Xin in satellite cells. In situ hybridization of regenerating muscle 5-7 days postinjury illustrates Xin expression within newly regenerated myofibers. Promoter-reporter assays demonstrate that known myogenic transcription factors [myocyte enhancer factor-2 (MEF2), myogenic differentiation-1 (MyoD), and myogenic factor-5 (Myf-5)] transactivate Xin promoter constructs supporting the muscle-specific expression of Xin. To determine the role of Xin within muscle precursor cells, proliferation, migration, and differentiation analysis using Xin, short hairpin RNA (shRNA) were undertaken in C2C12 myoblasts. Reducing endogenous Xin expression resulted in a 26% increase (P < 0.05) in cell proliferation and a 20% increase (P < 0.05) in myoblast migratory capacity. Skeletal muscle myosin heavy chain protein levels were increased (P < 0.05) with Xin shRNA administration; however, this was not accompanied by changes in myoglobin protein (another marker of differentiation) nor overt morphological differences relative to differentiating control cells. Taken together, the present findings support the hypothesis that Xin is expressed within muscle satellite cells during skeletal muscle regeneration and is involved in the regulation of myoblast function.

HIV integration sites in latently infected cell lines: evidence of ongoing replication.

PubMed

Symons, Jori; Chopra, Abha; Malatinkova, Eva; De Spiegelaere, Ward; Leary, Shay; Cooper, Don; Abana, Chike O; Rhodes, Ajantha; Rezaei, Simin D; Vandekerckhove, Linos; Mallal, Simon; Lewin, Sharon R; Cameron, Paul U

2017-01-13

Assessing the location and frequency of HIV integration sites in latently infected cells can potentially inform our understanding of how HIV persists during combination antiretroviral therapy. We developed a novel high throughput sequencing method to evaluate HIV integration sites in latently infected cell lines to determine whether there was virus replication or clonal expansion in these cell lines observed as multiple integration events at the same position. We modified a previously reported method using random DNA shearing and PCR to allow for high throughput robotic processing to identify the site and frequency of HIV integration in latently infected cell lines. Latently infected cell lines infected with intact virus demonstrated multiple distinct HIV integration sites (28 different sites in U1, 110 in ACH-2 and 117 in J1.1 per 150,000 cells). In contrast, cell lines infected with replication-incompetent viruses (J-Lat cells) demonstrated single integration sites. Following in vitro passaging of the ACH-2 cell line, we observed a significant increase in the frequency of unique HIV integration sites and there were multiple mutations and large deletions in the proviral DNA. When the ACH-2 cell line was cultured with the integrase inhibitor raltegravir, there was a significant decrease in the number of unique HIV integration sites and a transient increase in the frequency of 2-LTR circles consistent with virus replication in these cells. Cell lines latently infected with intact HIV demonstrated multiple unique HIV integration sites indicating that these cell lines are not clonal and in the ACH-2 cell line there was evidence of low level virus replication. These findings have implications for the use of latently infected cell lines as models of HIV latency and for the use of these cells as standards.

Tumor suppressors Sav/Scrib and oncogene Ras regulate stem cell transformation in adult Drosophila Malpighian Tubules

PubMed Central

Zeng, Xiankun; Singh, Shree Ram; Hou, David; Hou, Steven X.

2012-01-01

An increasing body of evidence suggests that tumors might originate from a few transformed cells that share many properties with normal stem cells. However, it remains unclear how normal stem cells are transformed into cancer stem cells. Here, we demonstrated that mutations causing the loss of tumor suppressor Sav or Scrib or activation of the oncogene Ras transform normal stem cells into cancer stem cells through a multistep process in the adult Drosophila Malpighian Tubules (MTs). In wild-type MTs, each stem cell generates one self-renewing and one differentiating daughter cell. However, in flies with loss-of-function sav or scrib or gain-of-function Ras mutations, both daughter cells grew and behaved like stem cells, leading to the formation of tumors in MTs. Ras functioned downstream of Sav and Scrib in regulating the stem cell transformation. The Ras-transformed stem cells exhibited many of the hallmarks of cancer, such as increased proliferation, reduced cell death, and failure to differentiate. We further demonstrated that several signal transduction pathways (including MEK/MAPK, RhoA, PKA, and TOR) mediate Rasṕ function in the stem cell transformation. Therefore, we have identified a molecular mechanism that regulates stem cell transformation, and this finding may lead to strategies for preventing tumor formation in certain organs. PMID:20432470

Acidosis increases the susceptibility of respiratory epithelial cells to Pseudomonas aeruginosa-induced cytotoxicity.

PubMed

Torres, Iviana M; Demirdjian, Sally; Vargas, Jennifer; Goodale, Britton C; Berwin, Brent

2017-07-01

Bacterial infection can lead to acidosis of the local microenvironment, which is believed to exacerbate disease pathogenesis; however, the mechanisms by which changes in pH alter disease progression are poorly understood. We test the hypothesis that acidosis enhances respiratory epithelial cell death in response to infection with Pseudomonas aeruginosa Our findings support the idea that acidosis in the context of P. aeruginosa infection results in increased epithelial cell cytotoxicity due to ExoU intoxication. Importantly, enforced maintenance of neutral pH during P. aeruginosa infection demonstrates that cytotoxicity is dependent on the acidosis. Investigation of the underlying mechanisms revealed that host cell cytotoxicity correlated with increased bacterial survival during an acidic infection that was due to reduced bactericidal activity of host-derived antimicrobial peptides. These findings extend previous reports that the activities of antimicrobial peptides are pH-dependent and provide novel insights into the consequences of acidosis on infection-derived pathology. Therefore, this report provides the first evidence that physiological levels of acidosis increase the susceptibility of epithelial cells to acute Pseudomonas infection and demonstrates the benefit of maintaining pH homeostasis during a bacterial infection. Copyright © 2017 the American Physiological Society.

Involvement of PUMA in pericyte migration induced by methamphetamine.

PubMed

Zhang, Yanhong; Zhang, Yuan; Bai, Ying; Chao, Jie; Hu, Gang; Chen, Xufeng; Yao, Honghong

2017-07-01

Mounting evidence indicates that methamphetamine causes blood-brain barrier damage, with emphasis on endothelial cells. The role of pericytes in methamphetamine-induced BBB damage remains unknown. Our study demonstrated that methamphetamine increased the migration of pericytes from the endothelial basement membrane. However, the detailed mechanisms underlying this process remain poorly understood. Thus, we examined the molecular mechanisms involved in methamphetamine-induced pericyte migration. The results showed that exposure of C3H/10T1/2 cells and HBVPs to methamphetamine increased PUMA expression via activation of the sigma-1 receptor, MAPK and Akt/PI3K pathways. Moreover, methamphetamine treatment resulted in the increased migration of C3H/10T1/2 cells and HBVPs. Knockdown of PUMA in pericytes transduced with PUMA siRNA attenuated the methamphetamine-induced increase in cell migration through attenuation of integrin and tyrosine kinase mechanisms, implicating a role of PUMA in the migration of C3H/10T1/2 cells and HBVPs. This study has demonstrated that methamphetamine-mediated pericytes migration involves PUMA up-regulation. Thus, targeted studies of PUMA could provide insights to facilitate the development of a potential therapeutic approach for alleviation of methamphetamine-induced pericyte migration. Copyright © 2017. Published by Elsevier Inc.

Quercetin induced apoptosis of human oral cancer SAS cells through mitochondria and endoplasmic reticulum mediated signaling pathways.

PubMed

Ma, Yi-Shih; Yao, Chien-Ning; Liu, Hsin-Chung; Yu, Fu-Shun; Lin, Jen-Jyh; Lu, Kung-Wen; Liao, Ching-Lung; Chueh, Fu-Shin; Chung, Jing-Gung

2018-06-01

Oral cancer is a cause of cancer-associated mortality worldwide and the treatment of oral cancer includes radiation, surgery and chemotherapy. Quercetin is a component from natural plant products and it has been demonstrated that quercetin is able to induce cytotoxic effects through induction of cell apoptosis in a number of human cancer cell lines. However, there is no available information to demonstrate that quercetin is able to induce apoptosis in human oral cancer cells. In the present study, the effect of quercetin on the cell death via the induction of apoptosis in human oral cancer SAS cells was investigated using flow cytometry, Annexin V/propidium iodide (PI) double staining, western blotting and confocal laser microscopy examination, to test for cytotoxic effects at 6-48 h after treatment with quercetin. The rate of cell death increased with the duration of quercetin treatment based on the results of a cell viability assay, increased Annexin V/PI staining, increased reactive oxygen species and Ca 2+ production, decreased the levels of mitochondrial membrane potential (ΔΨ m ), increased proportion of apoptotic cells and altered levels of apoptosis-associated protein expression in SAS cells. The results from western blotting revealed that quercetin increased Fas, Fas-Ligand, fas-associated protein with death domain and caspase-8, all of which associated with cell surface death receptor. Furthermore, quercetin increased the levels of activating transcription factor (ATF)-6α, ATF-6β and gastrin-releasing peptide-78 which indicated an increase in endoplasm reticulum stress, increased levels of the pro-apoptotic protein BH3 interacting-domain death antagonist, and decreased levels of anti-apoptotic proteins B-cell lymphoma (Bcl) 2 and Bcl-extra large which may have led to the decreases of ΔΨ m . Additionally, confocal microscopy suggested that quercetin was able to increase the expression levels of cytochrome c , apoptosis-inducing factor and endonuclease G, which are associated with apoptotic pathways. Therefore, it is hypothesized that quercetin may potentially be used as a novel anti-cancer agent for the treatment of oral cancer in future.

Bmi1 regulates auditory hair cell survival by maintaining redox balance.

PubMed

Chen, Y; Li, L; Ni, W; Zhang, Y; Sun, S; Miao, D; Chai, R; Li, H

2015-01-22

Reactive oxygen species (ROS) accumulation are involved in noise- and ototoxic drug-induced hair cell loss, which is the major cause of hearing loss. Bmi1 is a member of the Polycomb protein family and has been reported to regulate mitochondrial function and ROS level in thymocytes and neurons. In this study, we reported the expression of Bmi1 in mouse cochlea and investigated the role of Bmi1 in hair cell survival. Bmi1 expressed in hair cells and supporting cells in mouse cochlea. Bmi1(-/-) mice displayed severe hearing loss and patched outer hair cell loss from postnatal day 22. Ototoxic drug-induced hair cells loss dramatically increased in Bmi1(-/-) mice compared with that in wild-type controls both in vivo and in vitro, indicating Bmi1(-/-) hair cells were significantly more sensitive to ototoxic drug-induced damage. Cleaved caspase-3 and TUNEL staining demonstrated that apoptosis was involved in the increased hair cell loss of Bmi1(-/-) mice. Aminophenyl fluorescein and MitoSOX Red staining showed the level of free radicals and mitochondrial ROS increased in Bmi1(-/-) hair cells due to the aggravated disequilibrium of antioxidant-prooxidant balance. Furthermore, the antioxidant N-acetylcysteine rescued Bmi1(-/-) hair cells from neomycin injury both in vitro and in vivo, suggesting that ROS accumulation was mainly responsible for the increased aminoglycosides sensitivity in Bmi1(-/-) hair cells. Our findings demonstrate that Bmi1 has an important role in hair cell survival by controlling redox balance and ROS level, thus suggesting that Bmi1 may work as a new therapeutic target for the prevention of hair cell death.

Cell phones change the way we walk.

PubMed

Lamberg, Eric M; Muratori, Lisa M

2012-04-01

Cell phone use among pedestrians leads to increased cognitive distraction, reduced situation awareness and increases in unsafe behavior. Performing a dual-task, such as talking or texting with a cell phone while walking, may interfere with working memory and result in walking errors. At baseline, thirty-three participants visually located a target 8m ahead; then vision was occluded and they were instructed to walk to the remembered target. One week later participants were assigned to either walk, walk while talking on a cell phone, or walk while texting on a cell phone toward the target with vision occluded. Duration and final location of the heel were noted. Linear distance traveled, lateral angular deviation from the start line, and gait velocity were derived. Changes from baseline to testing were analyzed with paired t-tests. Participants engaged in cell phone use presented with significant reductions in gait velocity (texting: 33% reduction, p=0.01; talking: 16% reduction, p=0.02). Moreover, participants who were texting while walking demonstrated a 61% increase in lateral deviation (p=0.04) and 13% increase in linear distance traveled (p=0.03). These results suggest that the dual-task of walking while using a cell phone impacts executive function and working memory and influences gait to such a degree that it may compromise safety. Importantly, comparison of the two cell phone conditions demonstrates texting creates a significantly greater interference effect on walking than talking on a cell phone. Copyright © 2011 Elsevier B.V. All rights reserved.

Inhibition of SIRT1 by a small molecule induces apoptosis in breast cancer cells.

PubMed

Kalle, Arunasree M; Mallika, A; Badiger, Jayasree; Alinakhi; Talukdar, Pinaki; Sachchidanand

2010-10-08

Overexpression of SIRT1, a NAD+-dependent class III histone deacetylases (HDACs), is implicated in many cancers and therefore could become a promising antitumor target. Here we demonstrate a small molecule SIRT1 inhibitor, ILS-JGB-1741(JGB1741) with potent inhibitory effects on the proliferation of human metastatic breast cancer cells, MDA-MB 231. The molecule has been designed using medicinal chemistry approach based on known SIRT1 inhibitor, sirtinol. The molecule showed a significant inhibition of SIRT1 activity compared to sirtinol. Studies on the antitumor effects of JGB on three different cancer cell lines, K562, HepG2 and MDA-MB 231 showed an IC₅₀ of 1, 10 and 0.5 μM, respectively. Further studies on MDA-MB 231 cells showed a dose-dependent increase in K9 and K382 acetylation of H3 and p53, respectively. Results also demonstrated that JGB1741-induced apoptosis is associated with increase in cytochrome c release, modulation in Bax/Bcl2 ratio and cleavage of PARP. Flowcytometric analysis showed increased percentage of apoptotic cells, decrease in mitochondrial membrane potential and increase in multicaspase activation. In conclusion, the present study indicates the potent apoptotic effects of JGB1741 in MDA-MB 231 cells. Copyright © 2010 Elsevier Inc. All rights reserved.

Poly(ADP-ribose) polymerase-1 (Parp-1)-deficient mice demonstrate abnormal antibody responses

PubMed Central

Ambrose, Helen E; Willimott, Shaun; Beswick, Richard W; Dantzer, Françoise; de Murcia, Josiane Ménissier; Yelamos, José; Wagner, Simon D

2009-01-01

Poly(ADP-ribosylation) of acceptor proteins is an epigenetic modification involved in DNA strand break repair, recombination and transcription. Here we provide evidence for the involvement of poly(ADP-ribose) polymerase-1 (Parp-1) in antibody responses. Parp-1−/− mice had increased numbers of T cells and normal numbers of total B cells. Marginal zone B cells were mildly reduced in number, and numbers of follicular B cells were preserved. There were abnormal levels of basal immunoglobulins, with reduced levels of immunoglobulin G2a (IgG2a) and increased levels of IgA and IgG2b. Analysis of specific antibody responses showed that T cell-independent responses were normal but T cell-dependent responses were markedly reduced. Germinal centres were normal in size and number. In vitro purified B cells from Parp-1−/− mice proliferated normally and showed normal IgM secretion, decreased switching to IgG2a but increased IgA secretion. Collectively our results demonstrate that Parp-1 has essential roles in normal T cell-dependent antibody responses and the regulation of isotype expression. We speculate that Parp-1 forms a component of the protein complex involved in resolving the DNA double-strand breaks that occur during class switch recombination. PMID:18778284

Cell-delivered magnetic nanoparticles caused hyperthermia-mediated increased survival in a murine pancreatic cancer model

PubMed Central

Basel, Matthew T; Balivada, Sivasai; Wang, Hongwang; Shrestha, Tej B; Seo, Gwi Moon; Pyle, Marla; Abayaweera, Gayani; Dani, Raj; Koper, Olga B; Tamura, Masaaki; Chikan, Viktor; Bossmann, Stefan H; Troyer, Deryl L

2012-01-01

Using magnetic nanoparticles to absorb alternating magnetic field energy as a method of generating localized hyperthermia has been shown to be a potential cancer treatment. This report demonstrates a system that uses tumor homing cells to actively carry iron/iron oxide nanoparticles into tumor tissue for alternating magnetic field treatment. Paramagnetic iron/ iron oxide nanoparticles were synthesized and loaded into RAW264.7 cells (mouse monocyte/ macrophage-like cells), which have been shown to be tumor homing cells. A murine model of disseminated peritoneal pancreatic cancer was then generated by intraperitoneal injection of Pan02 cells. After tumor development, monocyte/macrophage-like cells loaded with iron/ iron oxide nanoparticles were injected intraperitoneally and allowed to migrate into the tumor. Three days after injection, mice were exposed to an alternating magnetic field for 20 minutes to cause the cell-delivered nanoparticles to generate heat. This treatment regimen was repeated three times. A survival study demonstrated that this system can significantly increase survival in a murine pancreatic cancer model, with an average post-tumor insertion life expectancy increase of 31%. This system has the potential to become a useful method for specifically and actively delivering nanoparticles for local hyperthermia treatment of cancer. PMID:22287840

Cytotoxic effect of Erythroxylum daphnites extract is associated with G1 cell cycle arrest and apoptosis in oral squamous cell carcinoma.

PubMed

Elias, Silvia T; Macedo, Carolina C S; Simeoni, Luiz A; Silveira, Dâmaris; Magalhães, Pérola O; Lofrano-Porto, Adriana; Coletta, Ricardo D; Neves, Francisco A R; Guerra, Eliete N S

2016-01-01

Plant-derived molecules showing antineoplastic effects have recently gained increased attention as potential adjuvants to traditional therapies for various cancers. Cerrado biome in Brazil contains high floral biodiversity, but knowledge about the potential therapeutic effects of compounds derived from that flora is still limited. The present study investigated the antineoplastic activity of Erythroxylum daphnites Mart., a Brazilian native plant from Cerrado biome, in the SCC-9 oral squamous cell carcinoma cell line. Cells were treated with various concentrations of hexane extract of Erythroxylum daphnites leaves (EDH) and assessed for cytotoxicity, proliferation, and apoptosis. Thin layer chromatography was conducted to characterize the substances present in EDH. Our results showed that EDH exerted anti-proliferative effects in SCC-9 cells by stabilizing the cell cycle at G1 phase in association with reduced intracellular levels of cyclins D and E and increased level of p21. EDH also demonstrated pro-apoptotic properties, as shown by an increased expression of caspase-3. Triterpenes were the major constituents of EDH. Our findings demonstrated a cytotoxic effect of EDH against SCC-9 cells in vitro mediated by the restraint of cellular proliferation and induction of apoptosis. Taken together, these findings support EDH constituents as potential therapeutic adjuvants for oral cancer.

Suspension state increases reattachment of breast cancer cells by up-regulating lamin A/C.

PubMed

Zhang, Xiaomei; Lv, Yonggang

2017-12-01

Extravasation is a rate-limiting step of tumor metastasis, for which adhesion to endothelium of circulating tumor cells (CTCs) is the prerequisite. The suspension state of CTCs undergoing detachment from primary tumor is a persistent biomechanical cue, which potentially regulates the biophysical characteristics and cellular behaviors of tumor cells. In this study, breast tumor cells MDA-MB-231 in suspension culture condition were used to investigate the effect of suspension state on reattachment of CTCs. Our study demonstrated that suspension state significantly increased the adhesion ability of breast tumor cells. In addition, suspension state markedly promoted the formation of stress fibers and focal adhesions and reduced the motility in reattached breast cancer cells. Moreover, lamin A/C was reversibly accumulated at posttranscriptional level under suspension state, improving the cell stiffness of reattached breast cancer cells. Disruption of actin cytoskeleton by cytochalasin D caused lamin A/C accumulation. Conversely, decreasing actomyosin contraction by ROCK inhibitor Y27632 reduced lamin A/C level. Knocking down lamin A/C weakened the suspension-induced increase of adhesion, and also abolished the suspension-induced decrease of motility and increase of stress fibers and focal adhesion in reattaching tumor cells, suggesting a crucial role of lamin A/C. In conclusion, it was demonstrated that suspension state promoted the reattachment of breast tumor cells by up-regulating lamin A/C via cytoskeleton disruption. These findings highlight the important role of suspension state for tumor cells in tumor metastasis. Copyright © 2017 Elsevier B.V. All rights reserved.

Long term exposure to environmental concentrations of diesel exhaust particles does not impact the phenotype of human bronchial epithelial cells.

PubMed

Savary, Camille C; Bellamri, Nessrine; Morzadec, Claudie; Langouët, Sophie; Lecureur, Valérie; Vernhet, Laurent

2018-06-19

Chronic exposure to diesel engine exhausts is associated with an increased risk of pulmonary diseases including lung cancer. Diesel engine exhausts contain large amounts of diesel exhaust particles (DEP) on which are adsorbed several carcinogenic compounds such as polycyclic aromatic hydrocarbons. Acute toxicity of high concentrations of DEP has been largely demonstrated in various in vitro cellular models. In contrast, the cellular and molecular impacts of low environmental concentrations of DEP on the phenotype of chronically exposed lung epithelial cells remain to be investigated. In the present study, we show that long term exposure (6 months) to 2 μg/ml (0.4 μg/cm 2 ) DEP (standard reference material 1650b) increased cytochrome P4501A mRNA levels in the human bronchial epithelial BEAS-2B cell line. However, chronic exposure to DEP did not change cell morphology, trigger epithelial-mesenchymal transition or increase anchorage-independent cell growth. Moreover, DEP increase neither the levels of reactive oxygen species or those of γ-histone H2AX, nor the expression of interleukin-6 and interleukin-8. Our results thus demonstrate that the chronic exposure to low DEP concentrations could increase cytochrome P501A gene expression in BEAS-2B cells but did not induce molecular effects related to genotoxicity, oxidative stress or inflammation. Copyright © 2018 Elsevier Ltd. All rights reserved.

Human mesenchymal stem cell differentiation to NP-like cells in chitosan-glycerophosphate hydrogels.

PubMed

Richardson, Stephen M; Hughes, Nesta; Hunt, John A; Freemont, Anthony J; Hoyland, Judith A

2008-01-01

Intervertebral disc (IVD) degeneration is one of the major causes of low back pain. As current clinical treatments are aimed at restoring biomechanical function and providing symptomatic relief, interest in methods focused on biological repair has increased. Several tissue engineering approaches using different cell types and hydrogels/scaffolds have been proposed. Owing to the unsuitable nature of degenerate cells for tissue engineering attention has focused on the use of mesenchymal stem cells (MSCs). Additionally, while rigid scaffolds have been demonstrated to allow MSC differentiation to the chondrocyte-like cells of the IVD, hydrogels are being increasingly studied as they allow minimally invasive implantation without extensive damage to the IVD. Here, we have studied the temperature-sensitive hydrogel chitosan-glycerophosphate (C/Gp), seeded with human MSCs and cultured for 4 weeks in standard medium. We have analysed the gene and protein expression profile of the MSCs and compared it to that of both nucleus pulposus (NP) cells and articular chondrocytes cultured in C/Gp. Gene expression analysis for chondrocytic-cell marker genes demonstrated differentiation of MSCs to a phenotype which showed similarities to both articular chondrocytes and NP cells. Conventional PCR demonstrated a lack of expression of osteogenic marker genes and the hypertrophic marker gene type X collagen. MSCs also secreted both proteoglycans and collagens in a ratio, which more closely resembled that of NP cells than articular chondrocytes. These results therefore suggest that MSC-seeded C/Gp gels could be used clinically for the regeneration of the degenerate human IVD.

AlGaInP/GaAs tandem solar cells for power conversion at 400°C and high concentration

NASA Astrophysics Data System (ADS)

Steiner, Myles A.; Perl, Emmett E.; Simon, John; Friedman, Daniel J.; Jain, Nikhil; Sharps, Paul; McPheeters, Claiborne; Lee, Minjoo Larry

2017-09-01

We demonstrate dual junction (Al)GaInP/GaAs solar cells that are designed to operate at 400°C and 1000X concentration in a hybrid photovoltaic-solar thermal concentrator system. The cells have a front metallization and anti-reflection coating that are stable under 400°C operation. We show how the cell performance degrades with increasing aluminum compositions in the top cell. Our best cell is a GaInP/GaAs tandem that demonstrated 15±1% efficiency at 400°C over a concentration range of 300-1000 suns, with several pathways to improved performance.

[Inhibition effects of black rice pericarp extracts on cell proliferation of PC-3 cells].

PubMed

Jiang, Weiwei; Yu, Xudong; Ren, Guofeng

2013-05-01

To observe the inhibitive effects of black rice pericarp extracts on cell proliferation of human prostate cancer cell PC-3 and to explore its effecting mechanism. The black rice pericarp extract was used to treat the PC-3 cells. The inhibitory effect of black rice pericarp extract on cells proliferation of PC-3 was tested by MTT method. Cell apoptosis rates and cell cycle were measured by flow cytometric assay (FCM). Western blot was used to study the protein expression levels of p38, p-p38, JNK, p-JNK. A dose-dependent and time-dependent proliferation inhibition of black rice pericarp extract was demonstrated in PC-3. The most prominent experiment condition was inhibitory concentration with 300microg/ml and treated for 72 h. The experiment result of flow cytometry analysis demonstrates that the apoptosis rate of PC-3 cells increased along with the increasing of black rice pericarp extract concentration, and a G1-S cell cycle arrest was induced in a dose-dependent manner. After PC-3 cell was treated with black rice pericarp extract for 72 h, the expressions of p-p38, p-JNK protein increased. Black rice pericarp extract could inhibit proliferation, change the cell cycle distributions and induce apoptosis in human prostatic cancer cell PC-3. Its inhibitory effect may be through promoting activation of the JNK, p38 signaling pathway. These results suggest that black rice pericarp extract maybe has an inhibitory effect on prostatic cancer.

Natural Killer Cell Recruitment to the Lung During Influenza A Virus Infection Is Dependent on CXCR3, CCR5, and Virus Exposure Dose

PubMed Central

Carlin, Lindsey E.; Hemann, Emily A.; Zacharias, Zeb R.; Heusel, Jonathan W.; Legge, Kevin L.

2018-01-01

Natural killer (NK) cells are vital components of the antiviral immune response, but their contributions in defense against influenza A virus (IAV) are not well understood. To better understand NK cell responses during IAV infections, we examined the magnitude, kinetics, and contribution of NK cells to immunity and protection during high- and low-dose IAV infections. Herein, we demonstrate an increased accumulation of NK cells in the lung in high-dose vs. low-dose infections. In part, this increase is due to the local proliferation of pulmonary NK cells. However, the majority of NK cell accumulation within the lungs and airways during an IAV infection is due to recruitment that is partially dependent upon CXCR3 and CCR5, respectively. Therefore, altogether, our results demonstrate that NK cells are actively recruited to the lungs and airways during IAV infection and that the magnitude of the recruitment may relate to the inflammatory environment found within the tissues during high- and low-dose IAV infections. PMID:29719539

Glabridin induces apoptosis and cell cycle arrest in oral cancer cells through the JNK1/2 signaling pathway.

PubMed

Chen, Chang-Tai; Chen, Yi-Tzu; Hsieh, Yi-Hsien; Weng, Chia-Jui; Yeh, Jung-Chun; Yang, Shun-Fa; Lin, Chiao-Wen; Yang, Jia-Sin

2018-06-01

Glabridin, a flavonoid extracted from licorice (Glycyrrhiza glabra), possesses various biological properties, including anticancer activities. However, the effect of glabridin on oral cancer cell apoptosis and the underlying molecular mechanisms has not been elucidated. In this study, we demonstrated that glabridin treatment significantly inhibits cell proliferation in human oral cancer SCC-9 and SAS cell lines. Flow cytometric assays demonstrated that glabridin induced several features of apoptosis, such as sub-G1 phase cell increase and phosphatidylserine externalization. Furthermore, glabridin induced apoptosis dose-dependently in SCC-9 cells through caspase-3, -8, and -9 activation and poly (ADP-ribose) polymerase cleavage. Moreover, glabridin increased the phosphorylation of the extracellular signal-regulated kinase, p38, and c-Jun N-terminal kinase (JNK) pathways in a dose-dependent manner. Moreover, the inhibition of the JNK1/2 inhibitor significantly reversed the glabridin-induced activation of the caspase pathway. In conclusion, our findings suggest that glabridin induces oral cancer cell apoptosis through the JNK1/2 pathway and is a potential therapeutic agent for oral cancer. © 2018 Wiley Periodicals, Inc.

xCT (SLC7A11)-mediated metabolic reprogramming promotes non-small cell lung cancer progression.

PubMed

Ji, Xiangming; Qian, Jun; Rahman, S M Jamshedur; Siska, Peter J; Zou, Yong; Harris, Bradford K; Hoeksema, Megan D; Trenary, Irina A; Heidi, Chen; Eisenberg, Rosana; Rathmell, Jeffrey C; Young, Jamey D; Massion, Pierre P

2018-05-23

Many tumors increase uptake and dependence on glucose, cystine or glutamine. These basic observations on cancer cell metabolism have opened multiple new diagnostic and therapeutic avenues in cancer research. Recent studies demonstrated that smoking could induce the expression of xCT (SLC7A11) in oral cancer cells, suggesting that overexpression of xCT may support lung tumor progression. We hypothesized that overexpression of xCT occurs in lung cancer cells to satisfy the metabolic requirements for growth and survival. Our results demonstrated that 1) xCT was highly expressed at the cytoplasmic membrane in non-small cell lung cancer (NSCLC), 2) the expression of xCT was correlated with advanced stage and predicted a worse 5-year survival, 3) targeting xCT transport activity in xCT overexpressing NSCLC cells with sulfasalazine decreased cell proliferation and invasion in vitro and in vivo and 4) increased dependence on glutamine was observed in xCT overexpressed normal airway epithelial cells. These results suggested that xCT regulate metabolic requirements during lung cancer progression and be a potential therapeutic target in NSCLC.

Oligomerization of Clostridium perfringens Epsilon Toxin Is Dependent upon Caveolins 1 and 2

PubMed Central

Fennessey, Christine M.; Sheng, Jinsong; Rubin, Donald H.; McClain, Mark S.

2012-01-01

Evidence from multiple studies suggests that Clostridium perfringens ε-toxin is a pore-forming toxin, assembling into oligomeric complexes in the plasma membrane of sensitive cells. In a previous study, we used gene-trap mutagenesis to identify mammalian factors contributing to toxin activity, including caveolin-2 (CAV2). In this study, we demonstrate the importance of caveolin-2 and its interaction partner, caveolin-1 (CAV1), in ε-toxin-induced cytotoxicity. Using CAV2-specific shRNA in a toxin-sensitive human kidney cell line, ACHN, we confirmed that cells deficient in CAV2 exhibit increased resistance to ε-toxin. Similarly, using CAV1-specific shRNA, we demonstrate that cells deficient in CAV1 also exhibit increased resistance to the toxin. Immunoprecipitation of CAV1 and CAV2 from ε-toxin-treated ACHN cells demonstrated interaction of both CAV1 and -2 with the toxin. Furthermore, blue-native PAGE indicated that the toxin and caveolins were components of a 670 kDa protein complex. Although ε-toxin binding was only slightly perturbed in caveolin-deficient cells, oligomerization of the toxin was dramatically reduced in both CAV1- and CAV2-deficient cells. These results indicate that CAV1 and -2 potentiate ε-toxin induced cytotoxicity by promoting toxin oligomerization – an event which is requisite for pore formation and, by extension, cell death. PMID:23056496

Oligomerization of Clostridium perfringens epsilon toxin is dependent upon caveolins 1 and 2.

PubMed

Fennessey, Christine M; Sheng, Jinsong; Rubin, Donald H; McClain, Mark S

2012-01-01

Evidence from multiple studies suggests that Clostridium perfringens ε-toxin is a pore-forming toxin, assembling into oligomeric complexes in the plasma membrane of sensitive cells. In a previous study, we used gene-trap mutagenesis to identify mammalian factors contributing to toxin activity, including caveolin-2 (CAV2). In this study, we demonstrate the importance of caveolin-2 and its interaction partner, caveolin-1 (CAV1), in ε-toxin-induced cytotoxicity. Using CAV2-specific shRNA in a toxin-sensitive human kidney cell line, ACHN, we confirmed that cells deficient in CAV2 exhibit increased resistance to ε-toxin. Similarly, using CAV1-specific shRNA, we demonstrate that cells deficient in CAV1 also exhibit increased resistance to the toxin. Immunoprecipitation of CAV1 and CAV2 from ε-toxin-treated ACHN cells demonstrated interaction of both CAV1 and -2 with the toxin. Furthermore, blue-native PAGE indicated that the toxin and caveolins were components of a 670 kDa protein complex. Although ε-toxin binding was only slightly perturbed in caveolin-deficient cells, oligomerization of the toxin was dramatically reduced in both CAV1- and CAV2-deficient cells. These results indicate that CAV1 and -2 potentiate ε-toxin induced cytotoxicity by promoting toxin oligomerization - an event which is requisite for pore formation and, by extension, cell death.

Transcription factor CREB is involved in CaSR-mediated cytoskeleton gene expression.

PubMed

Huang, Shuaishuai; Ren, Yu; Wang, Ping; Li, Yanyuan; Wang, Xue; Zhuang, Haihui; Fang, Rong; Wang, Yuduo; Liu, Ningsheng; Hehir, Michael; Zhou, Jeff X

2015-03-01

Our previous studies illustrated that a steady increase of intracellular calcium concentration ([Ca2+]i) was important for maintaining microtubules (MTs) rearrangement in apoptotic cells. However, little is known about the effect of calcium sensing receptor (CaSR)-mediated increase in [Ca2+]i on cytoskeleton gene expression. We examined the impact of taxol or CaSR agonist/antagonist on the regulation of [Ca2+]i concentration, cytoskeleton arrangement, phosphorylated CREB and cytoskeleton gene expressions in HeLa cells with dominant negative plasmid of CREB (PM). This study demonstrated that Gdcl3 (a specific CaSR agonist) evoked a rapid increase of [Ca2+]i, formed a rigid bundle of MTs which surrounded the nucleus and decreased the cytoskeleton gene expressions in HeLa cells. These effects were rescued by addition of NPS2390 (a specific CaSR antagonist). Moreover, CaSR activity affected cytoskeleton gene expression through transcription factor CREB. Histoscores of pCREB immunoreactivity in tissues of cervical adenocarcinoma, renal clear cell carcinoma, and diffuse large B-cell lymphoma were markedly increased compared with non malignant tissue. These data demonstrate, for the first time, that CaSR-mediated increase in [Ca2+]i probably modulate cytoskeleton organization and gene expression via transcription factor. © 2014 Wiley Periodicals, Inc.

Towards ultra-thin plasmonic silicon wafer solar cells with minimized efficiency loss.

PubMed

Zhang, Yinan; Stokes, Nicholas; Jia, Baohua; Fan, Shanhui; Gu, Min

2014-05-13

The cost-effectiveness of market-dominating silicon wafer solar cells plays a key role in determining the competiveness of solar energy with other exhaustible energy sources. Reducing the silicon wafer thickness at a minimized efficiency loss represents a mainstream trend in increasing the cost-effectiveness of wafer-based solar cells. In this paper we demonstrate that, using the advanced light trapping strategy with a properly designed nanoparticle architecture, the wafer thickness can be dramatically reduced to only around 1/10 of the current thickness (180 μm) without any solar cell efficiency loss at 18.2%. Nanoparticle integrated ultra-thin solar cells with only 3% of the current wafer thickness can potentially achieve 15.3% efficiency combining the absorption enhancement with the benefit of thinner wafer induced open circuit voltage increase. This represents a 97% material saving with only 15% relative efficiency loss. These results demonstrate the feasibility and prospect of achieving high-efficiency ultra-thin silicon wafer cells with plasmonic light trapping.

Metuzumab enhanced chemosensitivity and apoptosis in non-small cell lung carcinoma

PubMed Central

Feng, Fei; Wang, Bin; Sun, Xiuxuan; Zhu, Yumeng; Tang, Hao; Nan, Gang; Wang, Lijuan; Wu, Bo; Huhe, Muren; Liu, Shuangshuang; Diao, Tengyue; Hou, Rong; Zhang, Yang; Zhang, Zheng

2017-01-01

ABSTRACT Targeted therapeutics is used as an alternative treatment of non-small cell lung cancer (NSCLC); however, treatment effect is far from being satisfactory, and therefore identification of new targets is needed. We have previously shown that metuzumab inhibit tumor growth in vivo. The present study was performed to investigate the anti-tumor efficacy of metuzumab combined with gemcitabine and cisplatin (GP), paclitaxel and cisplatin (TP) or navelbine and cisplatin (NP) regimens in multiple NSCLC cell lines. Our results demonstrate that, in comparison to single agent metuzumab or GP treated cells, metuzumab combined with GP display inhibitory effects on tumor growth. Furthermore, we found that metuzumab elevated the sensitivity of cell lines to gemcitabine, which was identified by MTT assay. Flow cytometric analysis showed that metuzumab combined with gemcitabine (GEM) treatment led to an obvious G1 arrest and an elevated apoptosis in A549, NCI-H460 and NCI-H520 cells. Western blot analysis also demonstrated a significantly reduced level of cyclin D1, Bcl-2, and an obviously increase level of Bax and full-length caspase-3 in A549, NCI-H460 and NCI-H520 cells treated with metuzumab/gemcitabine combination in comparison with single agent treated cells. In addition, metuzumab/gemcitabine treated A549, NCI-H460 and NCI-H520 cells also demonstrated a significantly increase in deoxycytidine kinase (dCK) protein level compared with single agent metuzumab or gemcitabine treated cells. Xenograft models also demonstrated that this metuzumab/gemcitabine combination led to upregulation of dCK. Taken together, the mechanisms of metuzumab combined with GP repress tumor growth were that the combined treatment significantly inhibited the tumor cell proliferation, apoptosis and cell cycle in vitro and in vivo and at least partially by induction of dCK expression. Our results suggested that metuzumab could significantly enhance chemosensitivity of human NSCLC cells to gemcitabine. Metuzumab/gemcitabine combination treatment may be a potentially useful therapeutic regimen for NSCLC patients. PMID:28055291

Propionibacterium acnes induces an adjuvant effect in B-1 cells and affects their phagocyte differentiation via a TLR2-mediated mechanism.

PubMed

Gambero, Monica; Teixeira, Daniela; Butin, Liane; Ishimura, Mayari Eika; Mariano, Mario; Popi, Ana Flavia; Longo-Maugéri, Ieda Maria

2016-09-01

B-1 lymphocytes are present in large numbers in the mouse peritoneal cavity, as are macrophages, and are responsible for natural IgM production. These lymphocytes migrate to inflammatory foci and are also involved in innate immunity. It was also demonstrated that B-1 cells are able to differentiated into phagocytes (B-1CDP), which is characterized by expression of F4/80 and increased phagocytic activity. B-1 cell responses to antigens and adjuvants are poorly characterized. It has been shown that Propionibacterium acnes suspensions induce immunomodulatory effects in both macrophages and B-2 lymphocytes. We recently demonstrated that this bacterium has the ability to increase B-1 cell populations both in vitro and in vivo. P. acnes induces B-1CDP differentiation, increases the expression of TLR2, TLR4 and TLR9 and augments the expression of CD80, CD86 and CD40 in B-1 and B-1CDP cells. Because P. acnes has been shown to modulate TLR expression, in this study, we investigated the role of TLR2 and TLR4 in B-1 cell population, including B-1CDP differentiation and phagocytic activity in vitro and in vivo. Interestingly, we have demonstrated that TLR2 signaling could be involved in the increase in the B-1 cell population induced by P. acnes. Furthermore, the early differentiation of B-1CDP is also dependent of TLR2. It was also observed that TLR signals also interfere in the phagocytic ability of B-1 cells and their phagocytes. According to these data, it is clear that P. acnes promotes an important adjuvant effect in B-1 cells by inducing them to differentiate into B-1CDP cells and modulates their phagocytic functions both in vivo and in vitro. Moreover, most of these effects are mediated primarily via TLR2. These data reinforce the findings that such bacterial suspensions have powerful adjuvant properties. The responses of B-1 cells to exogenous stimulation indicate that these cells are important to the innate immune response. Copyright © 2016 Elsevier GmbH. All rights reserved.

Low-Level Light Therapy Potentiates NPe6-mediated Photodynamic Therapy in a Human Osteosarcoma Cell Line via Increased ATP

PubMed Central

Tsai, Shang-Ru; Yin, Rui; Huang, Ying-Ying; Sheu, Bor-Ching; Lee, Si-Chen; Hamblin, Michael R.

2015-01-01

Background Low-Level Light Therapy (LLLT) is used to stimulate healing, reduce pain and inflammation, and preserve tissue from dying. LLLT has been shown to protect cells in culture from dying after various cytotoxic insults, and LLLT is known to increase the cellular ATP content. Previous studies have demonstrated that maintaining a sufficiently high ATP level is necessary for the efficient induction and execution of apoptosis steps after photodynamic therapy (PDT). Methods We asked whether LLLT would protect cells from cytotoxicity due to PDT, or conversely whether LLLT would enhance the efficacy of PDT mediated by mono-L-aspartyl chlorin(e6) (NPe6). Increased ATP could lead to enhanced cell uptake of NPe6 by the energy dependent process of endocytosis, and also to more efficient apoptosis. In this study, human osteosarcoma cell line MG-63 was subjected to 1.5 J/cm2 of 810 nm near infrared radiation (NIR) followed by addition of 10 μM NPe6 and after 2 h incubation by 1.5 J/cm2 of 652 nm red light for PDT. Results PDT combined with LLLT led to higher cell death and increased intracellular reactive oxygen species compared to PDT alone. The uptake of NPe6 was moderately increased by LLLT, and cellular ATP was increased. The mitochondrial respiratory chain inhibitor antimycin A abrogated the LLLT-induced increase in cytotoxicity. Conclusions Taken together, these results demonstrate that LLLT potentiates NPe6-mediated PDT via increased ATP synthesis and is a potentially promising strategy that could be applied in clinical PDT. PMID:25462575

Enhancement of committed hematopoietic stem cell colony formation by nandrolone decanoate after sublethal whole body irradiation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Gallicchio, V.S.; Chen, M.G.; Watts, T.D.

1984-11-01

The ability of an anabolic steroid, nandrolone decanoate, to increase committed topoietic stem cell (CFU-gm, CFU-e, and BFU-e) colony formation after sublethal irradiation was evaluated. Immediately after receiving whole body irradiation and on the next two days, each mouse was injected intraperitoneally with nandrolone decanoate (1.25 mg) in propylene glycol. Irradiated control mice received only propylene glycol. Compared to controls, drug-treated mice showed marked peripheral blood leukocytosis and more stable packed red cell volume. Drug-treated mice also demonstrated increased erythropoiesis, as CFU-e/BFU-e concentrations from both marrow (9% to 581%) and spleen (15% to 797%) were elevated. Granulopoiesis was increased similarly,more » as CFU-gm concentrations from marrow (38% to 685%) and spleen (9% to 373%) were elevated. These results demonstrate that nandrolone decanoate enhances hematopoietic stem cell recovery after sublethal whole body irradiation. This suggests that following hematopoietic suppression, nandrolone decanoate may stimulate the recovery of hematopoiesis at the stem cell level and in peripheral blood.« less

Narcolepsy Type 1 Is Associated with a Systemic Increase and Activation of Regulatory T Cells and with a Systemic Activation of Global T Cells.

PubMed

Lecendreux, Michel; Churlaud, Guillaume; Pitoiset, Fabien; Regnault, Armelle; Tran, Tu Anh; Liblau, Roland; Klatzmann, David; Rosenzwajg, Michelle

2017-01-01

Narcolepsy is a rare neurologic disorder characterized by excessive daytime sleepiness, cataplexy and disturbed nocturnal sleep patterns. Narcolepsy type 1 (NT1) has been shown to result from a selective loss of hypothalamic hypocretin-secreting neurons with patients typically showing low CSF-hypocretin levels (<110 pg/ml). This specific loss of hypocretin and the strong association with the HLA-DQB1*06:02 allele led to the hypothesis that NT1 could be an immune-mediated pathology. Moreover, susceptibility to NT1 has recently been associated with several pathogens, particularly with influenza A H1N1 virus either through infection or vaccination. The goal of this study was to compare peripheral blood immune cell populations in recent onset pediatric NT1 subjects (post or non-post 2009-influenza A H1N1 vaccination) to healthy donors. We demonstrated an increased number of central memory CD4+ T cells (CD62L+ CD45RA-) associated to an activated phenotype (increase in CD69 and CD25 expression) in NT1 patients. Percentage and absolute count of regulatory T cells (Tregs) in NT1 patients were increased associated with an activated phenotype (increase in GITR and LAP expression), and of activated memory phenotype. Cytokine production by CD4+ and CD8+ T cells after activation was not modified in NT1 patients. In H1N1 vaccinated NT1 patients, absolute counts of CD3+, CD8+ T cells, and B cells were increased compared to non-vaccinated NT1 patients. These results support a global T cell activation in NT1 patients and thus support a T cell-mediated autoimmune origin of NT1, but do not demonstrate the pathological role of H1N1 prophylactic vaccination. They should prompt further studies of T cells, particularly of Tregs (such as suppression and proliferation antigen specific assays, and also T-cell receptor sequencing), in NT1.

Membrane Cholesterol Modulates Superwarfarin Toxicity

DOE Office of Scientific and Technical Information (OSTI.GOV)

Marangoni, M. Natalia; Martynowycz, Michael W.; Kuzmenko, Ivan

Superwarfarins are modified analogs of warfarin with additional lipophilic aromatic rings, up to 100-fold greater potency, and longer biological half-lives. We hypothesized that increased hydrophobicity allowed interactions with amphiphilic membranes and modulation of biological responses. We find that superwarfarins brodifacoum and difenacoum increase lactate production and cell death in neuroblastoma cells. In contrast, neither causes changes in glioma cells that have higher cholesterol content. After choleterol depletion, lactate production was increased and cell viability was reduced. Drug-membrane interactions were examined by surface X-ray scattering using Langmuir monolayers of dipalmitoylphosphatidylcholine and/or cholesterol. Specular X-ray reflectivity data revealed that superwarfarins, but notmore » warfarin, intercalate between dipalmitoylphosphatidylcholine molecules, whereas grazing incidence X-ray diffraction demonstrated changes in lateral crystalline order of the film. Neither agent showed significant interactions with monolayers containing >20% cholesterol. These findings demonstrate an affinity of superwarfarins to biomembranes and suggest that cellular responses to these agents are regulated by cholesterol content.« less

Salinomycin sensitizes antimitotic drugs-treated cancer cells by increasing apoptosis via the prevention of G2 arrest

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kim, Ju-Hwa; Yoo, Hye-In; Kang, Han Sung

Highlights: Black-Right-Pointing-Pointer Sal sensitizes antimitotic drugs-treated cancer cells. Black-Right-Pointing-Pointer Sal sensitizes them by prevention of G2 arrest and reduced cyclin D1 levels. Black-Right-Pointing-Pointer Sal also sensitizes them by increasing DNA damage and reducing p21 level. Black-Right-Pointing-Pointer A low concentration of Sal effectively sensitized the cancer cells to antimitotic drugs. -- Abstract: Here, we investigated whether Sal could sensitize cancer cells to antimitotic drugs. We demonstrated that Sal sensitized paclitaxcel (PAC)-, docetaxcel (DOC)-, vinblastin (VIN)-, or colchicine (COL)-treated cancer cell lines, suggesting that Sal has the ability to sensitize the cells to any form of microtubule-targeting drugs. Sensitization to the antimitoticmore » drugs could be achieved with very low concentrations of Sal, suggesting that there is a possibility to minimize Sal toxicity associated with human cancer patient treatments. Sensitization by Sal increased apoptosis, which was observed by C-PARP production. Sal sensitized the cancer cells to antimitotic drugs by preventing G2 arrest, suggesting that Sal contributes to the induction of mitotic catastrophe. Sal generally reduced cyclin D1 levels in PAC-, DOC-, and VIN-treated cells. In addition, Sal treatment increased pH2AX levels and reduced p21 levels in antimitotic drugs-treated cells. These observations suggest that the mechanisms underlying Sal sensitization to DNA-damaging compounds, radiation, and microtubule-targeting drugs are similar. Our data demonstrated that Sal sensitizes cancer cells to antimitotic drugs by increasing apoptosis through the prevention of G2 arrest via conserved Sal-sensitization mechanisms. These results may contribute to the development of Sal-based chemotherapy for cancer patients treated with antimitotic drugs.« less

Influence and timing of arrival of murine neural crest on pancreatic beta cell development and maturation.

PubMed

Plank, Jennifer L; Mundell, Nathan A; Frist, Audrey Y; LeGrone, Alison W; Kim, Thomas; Musser, Melissa A; Walter, Teagan J; Labosky, Patricia A

2011-01-15

Interactions between cells from the ectoderm and mesoderm influence development of the endodermally-derived pancreas. While much is known about how mesoderm regulates pancreatic development, relatively little is understood about how and when the ectodermally-derived neural crest regulates pancreatic development and specifically, beta cell maturation. A previous study demonstrated that signals from the neural crest regulate beta cell proliferation and ultimately, beta cell mass. Here, we expand on that work to describe timing of neural crest arrival at the developing pancreatic bud and extend our knowledge of the non-cell autonomous role for neural crest derivatives in the process of beta cell maturation. We demonstrated that murine neural crest entered the pancreatic mesenchyme between the 26 and 27 somite stages (approximately 10.0 dpc) and became intermingled with pancreatic progenitors as the epithelium branched into the surrounding mesenchyme. Using a neural crest-specific deletion of the Forkhead transcription factor Foxd3, we ablated neural crest cells that migrate to the pancreatic primordium. Consistent with previous data, in the absence of Foxd3, and therefore the absence of neural crest cells, proliferation of insulin-expressing cells and insulin-positive area are increased. Analysis of endocrine cell gene expression in the absence of neural crest demonstrated that, although the number of insulin-expressing cells was increased, beta cell maturation was significantly impaired. Decreased MafA and Pdx1 expression illustrated the defect in beta cell maturation; we discovered that without neural crest, there was a reduction in the percentage of insulin-positive cells that co-expressed Glut2 and Pdx1 compared to controls. In addition, transmission electron microscopy analyses revealed decreased numbers of characteristic insulin granules and the presence of abnormal granules in insulin-expressing cells from mutant embryos. Together, these data demonstrate that the neural crest is a critical regulator of beta cell development on two levels: by negatively regulating beta cell proliferation and by promoting beta cell maturation. Copyright © 2010 Elsevier Inc. All rights reserved.

Young Investigator Challenge: A novel, simple method for cell block preparation, implementation, and use over 2 years.

PubMed

Lindsey, Kathryn G; Houser, Patricia M; Shotsberger-Gray, Wanda; Chajewski, Olga S; Yang, Jack

2016-12-01

The cell block is an essential adjunct to conventional cytopreparatory techniques. The need for molecular analysis and immunostains will increase the need for successful cell block preparation. Even with this need, to the authors' knowledge very little has changed regarding the way in which cell blocks are produced. The authors developed A Formalin-Fixed, paraffin Embedded Cytology cell block Technique (AFFECT) that uses a cytospin centrifuge and funnel to deposit a cell pellet into a well on a piece of open-cell, absorbent foam. The foam and the pellet are then sent through normal processing. Herein, the authors present the implementation of this method and some of their experience with its performance over the course of 2 years. Although a comparison of the methods indicated good correlation for the production of a cell block between AFFECT and the agarose method, the AFFECT blocks demonstrated markedly improved cellular morphology. Over the first 6 months of use, AFFECT produced a successful cell block in 74% of cases overall, and in 65% of cases with a cell pellet measuring ≤0.1 mL. The year preceding the implementation of AFFECT and its first year of use were compared for endoscopic and bronchoscopic ultrasound-guided fine-needle aspiration specimens, and demonstrated an improved success rate. The authors developed a novel method of cell block preparation that demonstrates improved histology and has increased the success rate of cell block production compared with the agarose method. Cancer Cytopathol 2016;124:885-892. © 2016 American Cancer Society. © 2016 American Cancer Society.

Pediatric brain tumor cancer stem cells: cell cycle dynamics, DNA repair, and etoposide extrusion

PubMed Central

Hussein, Deema; Punjaruk, Wiyada; Storer, Lisa C.D.; Shaw, Lucy; Ottoman, Ramadan; Peet, Andrew; Miller, Suzanne; Bandopadhyay, Gagori; Heath, Rachel; Kumari, Rajendra; Bowman, Karen J.; Braker, Paul; Rahman, Ruman; Jones, George D.D.; Watson, Susan; Lowe, James; Kerr, Ian D.; Grundy, Richard G.; Coyle, Beth

2011-01-01

Reliable model systems are needed to elucidate the role cancer stem cells (CSCs) play in pediatric brain tumor drug resistance. The majority of studies to date have focused on clinically distinct adult tumors and restricted tumor types. Here, the CSC component of 7 newly established primary pediatric cell lines (2 ependymomas, 2 medulloblastomas, 2 gliomas, and a CNS primitive neuroectodermal tumor) was thoroughly characterized. Comparison of DNA copy number with the original corresponding tumor demonstrated that genomic changes present in the original tumor, typical of that particular tumor type, were retained in culture. In each case, the CSC component was approximately 3–4-fold enriched in neurosphere culture compared with monolayer culture, and a higher capacity for multilineage differentiation was observed for neurosphere-derived cells. DNA content profiles of neurosphere-derived cells expressing the CSC marker nestin demonstrated the presence of cells in all phases of the cell cycle, indicating that not all CSCs are quiescent. Furthermore, neurosphere-derived cells demonstrated an increased resistance to etoposide compared with monolayer-derived cells, having lower initial DNA damage, potentially due to a combination of increased drug extrusion by ATP-binding cassette multidrug transporters and enhanced rates of DNA repair. Finally, orthotopic xenograft models reflecting the tumor of origin were established from these cell lines. In summary, these cell lines and the approach taken provide a robust model system that can be used to develop our understanding of the biology of CSCs in pediatric brain tumors and other cancer types and to preclinically test therapeutic agents. PMID:20978004

Pediatric brain tumor cancer stem cells: cell cycle dynamics, DNA repair, and etoposide extrusion.

PubMed

Hussein, Deema; Punjaruk, Wiyada; Storer, Lisa C D; Shaw, Lucy; Othman, Ramadhan; Ottoman, Ramadan; Peet, Andrew; Miller, Suzanne; Bandopadhyay, Gagori; Heath, Rachel; Kumari, Rajendra; Bowman, Karen J; Braker, Paul; Rahman, Ruman; Jones, George D D; Watson, Susan; Lowe, James; Kerr, Ian D; Grundy, Richard G; Coyle, Beth

2011-01-01

Reliable model systems are needed to elucidate the role cancer stem cells (CSCs) play in pediatric brain tumor drug resistance. The majority of studies to date have focused on clinically distinct adult tumors and restricted tumor types. Here, the CSC component of 7 newly established primary pediatric cell lines (2 ependymomas, 2 medulloblastomas, 2 gliomas, and a CNS primitive neuroectodermal tumor) was thoroughly characterized. Comparison of DNA copy number with the original corresponding tumor demonstrated that genomic changes present in the original tumor, typical of that particular tumor type, were retained in culture. In each case, the CSC component was approximately 3-4-fold enriched in neurosphere culture compared with monolayer culture, and a higher capacity for multilineage differentiation was observed for neurosphere-derived cells. DNA content profiles of neurosphere-derived cells expressing the CSC marker nestin demonstrated the presence of cells in all phases of the cell cycle, indicating that not all CSCs are quiescent. Furthermore, neurosphere-derived cells demonstrated an increased resistance to etoposide compared with monolayer-derived cells, having lower initial DNA damage, potentially due to a combination of increased drug extrusion by ATP-binding cassette multidrug transporters and enhanced rates of DNA repair. Finally, orthotopic xenograft models reflecting the tumor of origin were established from these cell lines. In summary, these cell lines and the approach taken provide a robust model system that can be used to develop our understanding of the biology of CSCs in pediatric brain tumors and other cancer types and to preclinically test therapeutic agents.

Development of mRuby2-Transfected C3H10T1/2 Fibroblasts for Musculoskeletal Tissue Engineering

PubMed Central

Yang, Yunzhi Peter

2015-01-01

Mouse C3H10T1/2 fibroblasts are multipotent, mesenchymal stem cell (MSC)-like progenitor cells that are widely used in musculoskeletal research. In this study, we have established a clonal population of C3H10T1/2 cells stably-transfected with mRuby2, an orange-red fluorescence reporter gene. Flow cytometry analysis and fluorescence imaging confirmed successful transfection of these cells. Cell counting studies showed that untransfected C3H10T1/2 cells and mRuby2-transfected C3H10T1/2 cells proliferated at similar rates. Adipogenic differentiation experiments demonstrated that untransfected C3H10T1/2 cells and mRuby2-transfected C3H10T1/2 cells stained positive for Oil Red O and showed increased expression of adipogenic genes including adiponectin and lipoprotein lipase. Chondrogenic differentiation experiments demonstrated that untransfected C3H10T1/2 cells and mRuby2-transfected C3H10T1/2 cells stained positive for Alcian Blue and showed increased expression of chondrogenic genes including aggrecan. Osteogenic differentiation experiments demonstrated that untransfected C3H10T1/2 cells and mRuby2-transfected C3H10T1/2 cells stained positive for alkaline phosphatase (ALP) as well as Alizarin Red and showed increased expression of osteogenic genes including alp, ocn and osf-1. When seeded on calcium phosphate-based ceramic scaffolds, mRuby2-transfected C3H10T1/2 cells maintained even fluorescence labeling and osteogenic differentiation. In summary, mRuby2-transfected C3H10T1/2 cells exhibit mRuby2 fluorescence and showed little-to-no difference in terms of cell proliferation and differentiation as untransfected C3H10T1/2 cells. These cells will be available from American Type Culture Collection (ATCC; CRL-3268™) and may be a valuable tool for preclinical studies. PMID:26407291

Development of mRuby2-Transfected C3H10T1/2 Fibroblasts for Musculoskeletal Tissue Engineering.

PubMed

Ker, Dai Fei Elmer; Sharma, Rashmi; Wang, Evelyna Tsi Hsin; Yang, Yunzhi Peter

2015-01-01

Mouse C3H10T1/2 fibroblasts are multipotent, mesenchymal stem cell (MSC)-like progenitor cells that are widely used in musculoskeletal research. In this study, we have established a clonal population of C3H10T1/2 cells stably-transfected with mRuby2, an orange-red fluorescence reporter gene. Flow cytometry analysis and fluorescence imaging confirmed successful transfection of these cells. Cell counting studies showed that untransfected C3H10T1/2 cells and mRuby2-transfected C3H10T1/2 cells proliferated at similar rates. Adipogenic differentiation experiments demonstrated that untransfected C3H10T1/2 cells and mRuby2-transfected C3H10T1/2 cells stained positive for Oil Red O and showed increased expression of adipogenic genes including adiponectin and lipoprotein lipase. Chondrogenic differentiation experiments demonstrated that untransfected C3H10T1/2 cells and mRuby2-transfected C3H10T1/2 cells stained positive for Alcian Blue and showed increased expression of chondrogenic genes including aggrecan. Osteogenic differentiation experiments demonstrated that untransfected C3H10T1/2 cells and mRuby2-transfected C3H10T1/2 cells stained positive for alkaline phosphatase (ALP) as well as Alizarin Red and showed increased expression of osteogenic genes including alp, ocn and osf-1. When seeded on calcium phosphate-based ceramic scaffolds, mRuby2-transfected C3H10T1/2 cells maintained even fluorescence labeling and osteogenic differentiation. In summary, mRuby2-transfected C3H10T1/2 cells exhibit mRuby2 fluorescence and showed little-to-no difference in terms of cell proliferation and differentiation as untransfected C3H10T1/2 cells. These cells will be available from American Type Culture Collection (ATCC; CRL-3268™) and may be a valuable tool for preclinical studies.

Intestinal double-positive CD4+CD8+ T cells are highly activated memory cells with an increased capacity to produce cytokines.

PubMed

Pahar, Bapi; Lackner, Andrew A; Veazey, Ronald S

2006-03-01

Peripheral blood and intestinal CD4+CD8+ double-positive (DP) T cells have been described in several species including humans, but their function and immunophenotypic characteristics are still not clearly understood. Here we demonstrate that DP T cells are abundant in the intestinal lamina propria of normal rhesus macaques (Macaca mulatta). Moreover, DP T cells have a memory phenotype and are capable of producing different and/or higher levels of cytokines and chemokines in response to mitogen stimulation compared to CD4+ single-positive T cells. Intestinal DP T cells are also highly activated and have higher expression of CCR5, which makes them preferred targets for simian immunodeficiency virus/HIV infection. Increased levels of CD69, CD25 and HLA-DR, and lower CD62L expression were found on intestinal DP T cells populations compared to CD4+ single-positive T cells. Collectively, these findings demonstrate that intestinal and peripheral blood DP T cells are effector cells and may be important in regulating immune responses, which distinguishes them from the immature DP cells found in the thymus. Finally, these intestinal DP T cells may be important target cells for HIV infection and replication due to their activation, memory phenotype and high expression of CCR5.

Tunneling nanotubes promote intercellular mitochondria transfer followed by increased invasiveness in bladder cancer cells

PubMed Central

Lu, Jinjin; Zheng, Xiufen; Li, Fan; Yu, Yang; Chen, Zhong; Liu, Zheng; Wang, Zhihua; Xu, Hua; Yang, Weimin

2017-01-01

Intercellular transfer of organelles via tunneling nanotubes (TNTs) is a novel means of cell-to-cell communication. Here we demonstrate the existence of TNTs between co-cultured RT4 and T24 bladder cancer cells using light microscopy, fluorescence imaging, and scanning electron microscopy (SEM). Spontaneous unidirectional transfer of mitochondria from T24 to RT4 cells was detected using fluorescence imaging and flow cytometry. The distribution of mitochondria migrated from T24 cells was in good agreement with the original mitochondria in RT4 cells, which may imply mitochondrial fusion. We detected cytoskeleton reconstruction in RT4-Mito-T24 cells by observing F-actin redistribution. Akt, mTOR, and their downstream mediators were activated and increased. The resultant increase in the invasiveness of bladder cancer cells was detected in vitro and in vivo. These data indicate that TNTs promote intercellular mitochondrial transfer between heterogeneous cells, followed by an increase in the invasiveness of bladder cancer cells. PMID:28107184

Functional Involvement of Carbonic Anhydrase in the Lysosomal Response to Cadmium Exposure in Mytilus galloprovincialis Digestive Gland

PubMed Central

Caricato, Roberto; Giordano, M. Elena; Schettino, Trifone; Lionetto, M. Giulia

2018-01-01

Carbonic anhydrase (CA) is a ubiquitous metalloenzyme, whose functions in animals span from respiration to pH homeostasis, electrolyte transport, calcification, and biosynthetic reactions. CA is sensitive to trace metals in a number of species. In mussels, a previous study demonstrated CA activity and protein expression to be enhanced in digestive gland by cadmium exposure. The aim of the present work was to investigate the functional meaning, if any, of this response. To this end the study addressed the possible involvement of CA in the lysosomal system response of digestive gland cells to metal exposure. The in vivo exposure to acetazolamide, specific CA inhibitor, significantly inhibited the acidification of the lysosomal compartment in the digestive gland cells charged with the acidotropic probe LysoSensor Green D-189, demonstrating in vivo the physiological contribution of CA to the acidification of the lysosomes. Under CdCl2 exposure, CA activity significantly increased in parallel to the increase of the fluorescence of LysoSensor Green charged cells, which is in turn indicative of proliferation and/or increase in size of lysosomes. Acetazolamide exposure was able to completely inhibit the cadmium induced Lysosensor fluorescence increase in digestive gland cells. In conclusion, our results demonstrated the functional role of CA in the lysosomal acidification of Mytilus galloprovincialis digestive gland and its involvement in the lysosomal activation following cadmium exposure. CA induction could physiologically respond to a prolonged increased requirement of H+ for supporting lysosomal acidification during lysosomal activation. PMID:29670538

Transplantation of polarized type 2 donor T cells reduces mortality caused by experimental graft-versus-host disease.

PubMed

Krenger, W; Cooke, K R; Crawford, J M; Sonis, S T; Simmons, R; Pan, L; Delmonte, J; Karandikar, M; Ferrara, J L

1996-11-15

Acute graft-versus-host disease (GVHD) is thought to be initiated by alloreactive type 1 T cells that secrete gamma-interferon (IFN-gamma). IFN-gamma induces the production of inflammatory cytokines, e.g., tumor necrosis factor-alpha and interleukin (IL)-1, which are the distal mediators of GVHD. We demonstrate that the transplantation of polarized type 2 murine T cells (i.e., cells secreting IL-4 but not IFN-gamma) together with T-cell-depleted bone marrow results in a significant increase in survival (P<0.001) after bone marrow transplantation across minor histocompatibility barriers (B10.BR-->CBA/J). Further analysis demonstrated that increased survival in recipients of polarized type 2 T cells correlated with diminished production of both IFN-gamma and tumor necrosis factor-alpha but with increases in IL-4 2 weeks after transplantation. Despite improved survival, histologic changes of GVHD were evident in oral mucosal and hepatic tissues at 7 weeks after bone marrow transplantation. These data provide further evidence that inflammatory cytokines in the immediate posttransplant period are pivotal to the development of mortality but that they do not correlate with individual target organ damage.

HGF and c-Met Interaction Promotes Migration in Human Chondrosarcoma Cells

PubMed Central

Tsou, Hsi-Kai; Chen, Hsien-Te; Hung, Ya-Huey; Chang, Chia-Hao; Li, Te-Mao; Fong, Yi-Chin; Tang, Chih-Hsin

2013-01-01

Chondrosarcoma is a type of highly malignant tumor with a potent capacity for local invasion and causing distant metastasis. Chondrosarcoma shows a predilection for metastasis to the lungs. Hepatocyte growth factor (HGF) has been demonstrated to stimulate cancer proliferation, migration, and metastasis. However, the effect of HGF on migration activity of human chondrosarcoma cells is not well known. Here, we found that human chondrosarcoma tissues demonstrated significant expression of HGF, which was higher than that in normal cartilage. We also found that HGF increased the migration and expression of matrix metalloproteinase (MMP)-2 in human chondrosarcoma cells. c-Met inhibitor and siRNA reduced HGF-increased cell migration and MMP-2 expression. HGF treatment resulted in activation of the phosphatidylinositol 3′-kinase (PI3K)/Akt/PKCδ/NF-κB pathway, and HGF-induced expression of MMP-2 and cell migration was inhibited by specific inhibitors or siRNA-knockdown of PI3K, Akt, PKCδ, and NF-κB cascades. Taken together, our results indicated that HGF enhances migration of chondrosarcoma cells by increasing MMP-2 expression through the c-Met receptor/PI3K/Akt/PKCδ/NF-κB signal transduction pathway. PMID:23320110

Allergen-induced Increases in Sputum Levels of Group 2 Innate Lymphoid Cells in Subjects with Asthma.

PubMed

Chen, Ruchong; Smith, Steven G; Salter, Brittany; El-Gammal, Amani; Oliveria, John Paul; Obminski, Caitlin; Watson, Rick; O'Byrne, Paul M; Gauvreau, Gail M; Sehmi, Roma

2017-09-15

Group 2 innate lymphoid cells (ILC2), a major source of type 2 cytokines, initiate eosinophilic inflammatory responses in murine models of asthma. To investigate the role of ILC2 in allergen-induced airway eosinophilic responses in subjects with atopy and asthma. Using a diluent-controlled allergen challenge crossover study, where all subjects (n = 10) developed allergen-induced early and late responses, airway eosinophilia, and increased methacholine airway responsiveness, bone marrow, blood, and sputum samples were collected before and after inhalation challenge. ILC2 (lin - FcεRI - CD45 + CD127 + ST2 + ) and CD4 + T lymphocytes were enumerated by flow cytometry, as well as intracellular IL-5 and IL-13 expression. Steroid sensitivity of ILC2 and CD4 + T cells was investigated in vitro. A significant increase in total, IL-5 + , IL-13 + , and CRTH2 + ILC2 was found in sputum, 24 hours after allergen, coincident with a significant decrease in blood ILC2. Total, IL-5 + , and IL-13 + , but not CRTH2 + , CD4 + T cells significantly increased at 24 and 48 hours after allergen in sputum. In blood and bone marrow, only CD4 + cells demonstrated increased activation after allergen. Airway eosinophilia correlated with IL-5 + ILC2 at all time points and allergen-induced changes in IL-5 + CD4 + cells at 48 hours after allergen. Dexamethasone significantly attenuated IL-2- and IL-33-stimulated IL-5 and IL-13 production by both cell types. Innate and adaptive immune cells are increased in the airways associated with allergic asthmatic responses. Total and type 2 cytokine-positive ILC2 are increased only within the airways, whereas CD4 + T lymphocytes demonstrated local and systemic increases. Steroid sensitivity of both cells may explain effectiveness of this therapy in those with mild asthma.

FGF-1-induced matrix metalloproteinase-9 expression in breast cancer cells is mediated by increased activities of NF-kappaB and activating protein-1.

PubMed

Lungu, Gina; Covaleda, Lina; Mendes, Odete; Martini-Stoica, Heidi; Stoica, George

2008-06-01

Matrix metalloproteinase-9 (MMP-9) plays a critical role in tumor invasion and metastasis. Here, we investigate the effect of fibroblast growth factor-1 (FGF-1) on the expression of MMP-9 in ENU1564, an ethyl-N-nitrosourea-induced rat mammary adenocarcinoma cell line. We observed that FGF-1 induces a dose-dependent increase in MMP-9 mRNA, protein, and activity in ENU1564 cells. To gain insight into the molecular mechanism of MMP-9 regulation by FGF-1, we investigated the role of components of PI3K-Akt and MEK1/2-ERK signaling pathways in our system since NF-kappaB and AP-1 transcription factor binding sites have been characterized in the upstream region of the MMP-9 gene. We demonstrated that FGF-1 increases Akt phosphorylation, triggers nuclear translocation of NF-kappaBp65, and enhances degradation of cytoplasmic IkappaBalpha. Pretreatment of cells with LY294002, a PI3K inhibitor, significantly inhibited MMP-9 protein expression in FGF-1-treated cells. Conversely, our data show that FGF-1 increases ERK phosphorylation in ENU1564 cells, increases c-jun and c-fos mRNA expression in a time-dependent manner, and triggers nuclear translocation of c-jun. Pretreatment of cells with PD98059, a MEK1/2 inhibitor significantly inhibited MMP-9 protein expression in FGF-1 treated cells. Finally, we observed increased DNA binding of NF-kappaB and AP-1 in FGF-1-treated cells and that mutation of either NF-kappaB or AP-1 response elements prevented MMP-9 promoter activation by FGF-1. Taken together, these results demonstrated that FGF-1-induced MMP-9 expression in ENU1564 cells is associated with increasing DNA binding activities of NF-kappaB and AP-1 and involve activation of a dual signaling pathway, PI3K-Akt and MEK1/2-ERK. (c) 2007 Wiley-Liss, Inc.

JAG1-Mediated Notch Signaling Regulates Secretory Cell Differentiation of the Human Airway Epithelium.

PubMed

Gomi, Kazunori; Staudt, Michelle R; Salit, Jacqueline; Kaner, Robert J; Heldrich, Jonna; Rogalski, Allison M; Arbelaez, Vanessa; Crystal, Ronald G; Walters, Matthew S

2016-08-01

Basal cells (BC) are the stem/progenitor cells of the human airway epithelium capable of differentiating into secretory and ciliated cells. Notch signaling activation increases BC differentiation into secretory cells, but the role of individual Notch ligands in regulating this process in the human airway epithelium is largely unknown. The objective of this study was to define the role of the Notch ligand JAG1 in regulating human BC differentiation. JAG1 over-expression in BC increased secretory cell differentiation, with no effect on ciliated cell differentiation. Conversely, knockdown of JAG1 decreased expression of secretory cell genes. These data demonstrate JAG1-mediated Notch signaling regulates differentiation of BC into secretory cells.

Hematopoietic stem cell loss and hematopoietic failure in severe aplastic anemia is driven by macrophages and aberrant podoplanin expression.

PubMed

McCabe, Amanda; Smith, Julianne N P; Costello, Angelica; Maloney, Jackson; Katikaneni, Divya; MacNamara, Katherine C

2018-05-17

Severe aplastic anemia results from profound hematopoietic stem cell loss. T cells and interferon gamma have long been associated with severe aplastic anemia, yet the underlying mechanisms driving hematopoietic stem cell loss remain unknown. Using a mouse model of severe aplastic anemia, we demonstrate that interferon gamma-dependent hematopoietic stem cell loss required macrophages. Interferon gamma was necessary for bone marrow macrophage persistence, despite loss of other myeloid cells and hematopoietic stem cells. Depleting macrophages or abrogating interferon gamma signaling specifically in macrophages did not impair T cell activation or interferon gamma production in the bone marrow but rescued hematopoietic stem cells and reduced mortality. Thus, macrophages are not required for induction of interferon gamma in severe aplastic anemia and rather act as sensors of interferon gamma. Macrophage depletion rescued thrombocytopenia, increased bone marrow megakaryocytes, preserved platelet-primed stem cells, and increased the platelet-repopulating capacity of transplanted hematopoietic stem cells. In addition to the hematopoietic effects, severe aplastic anemia induced loss of non-hematopoietic stromal populations, including podoplanin-positive stromal cells. However, a subset of podoplanin-positive macrophages was increased during disease, and blockade of podoplanin in mice was sufficient to rescue disease. Our data further our understanding of disease pathogenesis demonstrating a novel role for macrophages as sensors of interferon gamma, thus illustrating an important role for the microenvironment in pathogenesis of severe aplastic anemia. Copyright © 2018, Ferrata Storti Foundation.

Bisphenol A induces corticotropin-releasing hormone expression in the placental cells JEG-3.

PubMed

Huang, Hui; Tan, Wenjuan; Wang, C C; Leung, Lai K

2012-11-01

Bisphenol A is utilized to make polycarbonate plastics and is an environmental pollutant. Recent research has indicated that it is an endocrine disruptor and may interfere with reproduction. Placental corticotrophin-releasing hormone (CRH) is a peptide hormone which is involved in fetal development. Increased plasma CRH is associated with elevated risk of premature delivery. In the present study, we demonstrated that bisphenol A increased CRH mRNA expression in the placental JEG-3 cells at or above 25μM. Reporter gene assay also demonstrated that bisphenol A could induce CRH gene transactivity. Since cyclic AMP response element (CRE) is a major regulatory element located in CRH promoter, the sequence-specific binding activity was investigated by using electrophoretic mobility shift assay. Our data indicated that bisphenol A increased the CRE binding activity. Western analysis further illustrated that PKA could be the signal triggering the CRE binding and CRH gene transactivation. In summary, the present study demonstrated that bisphenol A could induce CRH expression in placental cells and the underlying signal transduction pathway was also described. Copyright © 2012 Elsevier Inc. All rights reserved.

Slug promoted vasculogenic mimicry in hepatocellular carcinoma

PubMed Central

Sun, Dan; Sun, Baocun; Liu, Tieju; Zhao, Xiulan; Che, Na; Gu, Qiang; Dong, Xueyi; Yao, Zhi; Li, Rui; Li, Jing; Chi, Jiadong; Sun, Ran

2013-01-01

Vasculogenic mimicry (VM) refers to the unique capability of aggressive tumour cells to mimic the pattern of embryonic vasculogenic networks. Epithelial–mesenchymal transition (EMT) regulator slug have been implicated in the tumour invasion and metastasis of human hepatocellular carcinoma (HCC). However, the relationship between slug and VM formation is not clear. In the study, we demonstrated that slug expression was associated with EMT and cancer stem cell (CSCs) phenotype in HCC patients. Importantly, slug showed statistically correlation with VM formation. We consistently demonstrated that an overexpression of slug in HCC cells significantly increased CSCs subpopulation that was obvious by the increased clone forming efficiency in soft agar and by flowcytometry analysis. Meantime, the VM formation and VM mediator overexpression were also induced by slug induction. Finally, slug overexpression lead to the maintenance of CSCs phenotype and VM formation was demonstrated in vivo. Therefore, the results of this study indicate that slug induced the increase and maintenance of CSCs subpopulation and contributed to VM formation eventually. The related molecular pathways may be used as novel therapeutic targets for the inhibition of HCC angiogenesis and metastasis. PMID:23815612

DOE Office of Scientific and Technical Information (OSTI.GOV)

Woodford, William

This document is the final technical report from 24M Technologies on the project titled: Low Cost, Structurally Advanced Novel Electrode and Cell Manufacturing. All of the program milestones and deliverables were completed during the performance of the award. Specific accomplishments are 1) 24M demonstrated the processability and electrochemical performance of semi-solid electrodes with active volume contents increased by 10% relative to the program baseline; 2) electrode-level metrics, quality, and yield were demonstrated at an 80 cm 2 electrode footprint; 3) these electrodes were integrated into cells with consistent capacities and impedances, including cells delivered to Argonne National Laboratory for independentmore » testing; 4) those processes were scaled to a large-format (> 260 cm 2) electrode footprint and quality and yield were demonstrated; 5) a high-volume manufacturing approach for large-format electrode fabrication was demonstrated; and 6) large-format cells (> 100 Ah capacity) were prototyped with consistent capacity and impedance, including cells which were delivered to Argonne National Laboratory for independent testing.« less

A stochastic step model of replicative senescence explains ROS production rate in ageing cell populations.

PubMed

Lawless, Conor; Jurk, Diana; Gillespie, Colin S; Shanley, Daryl; Saretzki, Gabriele; von Zglinicki, Thomas; Passos, João F

2012-01-01

Increases in cellular Reactive Oxygen Species (ROS) concentration with age have been observed repeatedly in mammalian tissues. Concomitant increases in the proportion of replicatively senescent cells in ageing mammalian tissues have also been observed. Populations of mitotic human fibroblasts cultured in vitro, undergoing transition from proliferation competence to replicative senescence are useful models of ageing human tissues. Similar exponential increases in ROS with age have been observed in this model system. Tracking individual cells in dividing populations is difficult, and so the vast majority of observations have been cross-sectional, at the population level, rather than longitudinal observations of individual cells.One possible explanation for these observations is an exponential increase in ROS in individual fibroblasts with time (e.g. resulting from a vicious cycle between cellular ROS and damage). However, we demonstrate an alternative, simple hypothesis, equally consistent with these observations which does not depend on any gradual increase in ROS concentration: the Stochastic Step Model of Replicative Senescence (SSMRS). We also demonstrate that, consistent with the SSMRS, neither proliferation-competent human fibroblasts of any age, nor populations of hTERT overexpressing human fibroblasts passaged beyond the Hayflick limit, display high ROS concentrations. We conclude that longitudinal studies of single cells and their lineages are now required for testing hypotheses about roles and mechanisms of ROS increase during replicative senescence.

A Stochastic Step Model of Replicative Senescence Explains ROS Production Rate in Ageing Cell Populations

PubMed Central

Lawless, Conor; Jurk, Diana; Gillespie, Colin S.; Shanley, Daryl; Saretzki, Gabriele; von Zglinicki, Thomas; Passos, João F.

2012-01-01

Increases in cellular Reactive Oxygen Species (ROS) concentration with age have been observed repeatedly in mammalian tissues. Concomitant increases in the proportion of replicatively senescent cells in ageing mammalian tissues have also been observed. Populations of mitotic human fibroblasts cultured in vitro, undergoing transition from proliferation competence to replicative senescence are useful models of ageing human tissues. Similar exponential increases in ROS with age have been observed in this model system. Tracking individual cells in dividing populations is difficult, and so the vast majority of observations have been cross-sectional, at the population level, rather than longitudinal observations of individual cells. One possible explanation for these observations is an exponential increase in ROS in individual fibroblasts with time (e.g. resulting from a vicious cycle between cellular ROS and damage). However, we demonstrate an alternative, simple hypothesis, equally consistent with these observations which does not depend on any gradual increase in ROS concentration: the Stochastic Step Model of Replicative Senescence (SSMRS). We also demonstrate that, consistent with the SSMRS, neither proliferation-competent human fibroblasts of any age, nor populations of hTERT overexpressing human fibroblasts passaged beyond the Hayflick limit, display high ROS concentrations. We conclude that longitudinal studies of single cells and their lineages are now required for testing hypotheses about roles and mechanisms of ROS increase during replicative senescence. PMID:22359661

The emerging role of mast cells in liver disease.

PubMed

Jarido, Veronica; Kennedy, Lindsey; Hargrove, Laura; Demieville, Jennifer; Thomson, Joanne; Stephenson, Kristen; Francis, Heather

2017-08-01

The depth of our knowledge regarding mast cells has widened exponentially in the last 20 years. Once thought to be only important for allergy-mediated events, mast cells are now recognized to be important regulators of a number of pathological processes. The revelation that mast cells can influence organs, tissues, and cells has increased interest in mast cell research during liver disease. The purpose of this review is to refresh the reader's knowledge of the development, type, and location of mast cells and to review recent work that demonstrates the role of hepatic mast cells during diseased states. This review focuses primarily on liver diseases and mast cells during autoimmune disease, hepatitis, fatty liver disease, liver cancer, and aging in the liver. Overall, these studies demonstrate the potential role of mast cells in disease progression.

Surveillance on the status of immune cells after Echinnococcus granulosus protoscoleces infection in Balb/c mice.

PubMed

Pan, Wei; Zhou, He-Jun; Shen, Yu-Juan; Wang, Ying; Xu, Yu-Xin; Hu, Yuan; Jiang, Yan-Yan; Yuan, Zhong-Ying; Ugwu, Chidiebere E; Cao, Jian-Ping

2013-01-01

Cystic echinococcosis is a global parasitic disease caused by infection with Echinococcus granulosus larvae with potentially life-threatening complications in humans. To date, the status of the immune cells believed to be associated with the pathogenicity of E. granulosus infection has not been demonstrated clearly. In this study, we developed a multiplex flow cytometry assay to investigate the systemic immune status of innate and adaptive immunity at 30, 180, 360 days post-infection (dpi) in mice infected with E. granulousus. At 30 dpi, an increase in the number of CD11b(+) and CD11c(+) antigen-presenting cells (APCs) was observed. This was accompanied by the slight down-regulated expression of the co-stimulatory molecule MHC-II, indicating the impairment of APCs in early infection through the release of secretory-excretory products. In all infected groups, we observed a significant increase in innate immune cells, including APCs and GR-1(+) cells, and a dramatic increase in the myeloid-derived suppressor cells (MDSC) expressing CD11b(+)/GR-1(+). Moreover, the upregulation of the activated markers CD69, CD44, CD40L, and the downregulation of CD62L were observed in the CD4(+) and CD8(+) T cells following infection. Regulatory T cells expressing CD4(+)/CD25(+)/FoxP3 (+) increased significantly over the course of infection. Our findings demonstrate that the microenvironment in the peripheral immune system after E. granulosus infection changes in subtle but detectably ways, especially during the persistent period of infection. We found that T cells were activated following infection, but observed that the significant increase of immunosuppressive cells such as MDSC and Treg cells could inhibit T cell response to E. granulosus antigens. We suggest these cells may play a neglected but key role in the downregulation of the immune response in long-term parasitic infection. Understanding the basic functions and temporal interactions of these immunosuppressive cells will pave the way for new strategies of parasite vaccine design.

Making Epidermal Bladder Cells Bigger: Developmental- and Salinity-Induced Endopolyploidy in a Model Halophyte.

PubMed

Barkla, Bronwyn J; Rhodes, Timothy; Tran, Kieu-Nga T; Wijesinghege, Chathura; Larkin, John C; Dassanayake, Maheshi

2018-06-01

Endopolyploidy occurs when DNA replication takes place without subsequent mitotic nuclear division, resulting in cell-specific ploidy levels within tissues. In plants, endopolyploidy plays an important role in sustaining growth and development, but only a few studies have demonstrated a role in abiotic stress response. In this study, we investigated the function of ploidy level and nuclear and cell size in leaf expansion throughout development and tracked cell type-specific ploidy in the halophyte Mesembryanthemum crystallinum In addition to developmental endopolyploidy, we examined the effects of salinity stress on ploidy level. We focused specifically on epidermal bladder cells (EBC), which are modified balloon-like trichomes, due to their large size and role in salt accumulation. Our results demonstrate that ploidy increases as the leaves expand in a similar manner for each leaf type, and ploidy levels up to 512C were recorded for nuclei in EBC of leaves of adult plants. Salt treatment led to a significant increase in ploidy levels in the EBC, and these cells showed spatially related differences in their ploidy and nuclear and cell size depending on the positions on the leaf and stem surface. Transcriptome analysis highlighted salinity-induced changes in genes involved in DNA replication, cell cycle, endoreduplication, and trichome development in EBC. The increase in cell size and ploidy observed in M. crystallinum under salinity stress may contribute to salt tolerance by increasing the storage capacity for sodium sequestration brought about by higher metabolic activity driving rapid cell enlargement in the leaf tissue and EBC. © 2018 American Society of Plant Biologists. All rights reserved.

Taraxacum coreanum protects against glutamate-induced neurotoxicity through heme oxygenase-1 expression in mouse hippocampal HT22 cells.

PubMed

Yoon, Chi-Su; Ko, Wonmin; Lee, Dong-Sung; Kim, Dong-Cheol; Kim, Jongsu; Choi, Moonbum; Beom, Jin Seon; An, Ren-Bo; Oh, Hyuncheol; Kim, Youn-Chul

2017-04-01

Taraxacum coreanum Nakai is a dandelion that is native to Korea, and is widely used as an edible and medicinal herb. The present study revealed the neuroprotective effect of this plant against glutamate-induced oxidative stress in HT22 murine hippocampal neuronal cells. Ethanolic extracts from the aerial (TCAE) and the root parts (TCRE) of T. coreanum were prepared. Both extracts were demonstrated, by high performance liquid chromatography, to contain caffeic acid and ferulic acid as representative constituents. TCAE and TCRE significantly increased cell viability against glutamate-induced oxidative stress in mouse hippocampal HT22 cells. Western blot analysis revealed that treatment of HT22 cells with the extracts induced increased expression of the enzyme heme oxygenase-1 (HO-1), compared with untreated cells, in a concentration-dependent manner. Increased HO-1 enzymatic activity, compared with untreated cells, was also demonstrated following treatment with TCAE and TCRE. In addition, western blot analysis of the nuclear fractions of both TCAE and TCRE-treated HT22 cells revealed increased levels of nuclear factor erythroid 2 like 2 (Nrf2) compared with untreated cells, and decreased Nrf2 levels in the cytoplasmic fraction compared with untreated cells. The present study suggested that the neuroprotective effect of T. coreanum is associated with induction of HO-1 expression and Nrf2 translocation to the nucleus. Therefore, T. coreanum exhibits a promising function in prevention of neurodegeneration. Further studies will be required for the isolation and the full characterization of its active substances.

Changes of buoyant density during the S-phase of the cell cycle. Direct evidence demonstrated in acute myeloid leukemia by flowcytometry.

PubMed

Daenen, S; Huiges, W; Modderman, E; Halie, M R

1993-01-01

Studies with synchronized or exponentially growing bacteria and mammalian cell lines are not able to demonstrate small changes in buoyant density during the cell cycle. Flowcytometric analysis of density separated acute myeloid leukemia cells, a system not dependent on time-related variables, shows that the cellular buoyant density increases slightly with up to 0.008 g/ml during the S-phase, at least in cryo-preserved cells used in this study. This contrasts with the generally accepted belief that S-phase cells have a lower or constant buoyant density. A practical implication is that separation of cell (sub)populations based on differences in buoyant density could be flawed to the extent that these populations contain S-phase cells.

The insulin-like growth factor-1 receptor inhibitor PPP produces only very limited resistance in tumor cells exposed to long-term selection.

PubMed

Vasilcanu, D; Weng, W-H; Girnita, A; Lui, W-O; Vasilcanu, R; Axelson, M; Larsson, O; Larsson, C; Girnita, L

2006-05-25

The cyclolignan PPP was recently demonstrated to inhibit the activity of insulin-like growth factor-1 receptor (IGF-1R), without affecting the highly homologous insulin receptor. In addition, PPP caused complete regression of xenografts derived from various types of cancer. These data highlight the use of this compound in cancer treatment. However, a general concern with antitumor agents is development of resistance. In light of this problem, we aimed to investigate whether malignant cells may develop serious resistance to PPP. After trying to select 10 malignant cell lines, with documented IGF-1R expression and apoptotic responsiveness to PPP treatment (IC50s less than 0.1 microM), only two survived an 80-week selection but could only tolerate maximal PPP doses of 0.2 and 0.5 microM, respectively. Any further increase in the PPP dose resulted in massive cell death. These two cell lines were demonstrated not to acquire any essential alteration in responsiveness to PPP regarding IGF-1-induced IGF-1R phosphorylation. Neither did they exhibit any increase in expression of the multidrug resistance proteins MDR1 or MRP1. Consistently, they did not exhibit decreased sensitivity to conventional cytostatic drugs. Rather, the sensitivity was increased. During the first half of the selection period, both cell lines responded with a temporary and moderate increase in IGF-1R expression, which appeared to be because of an increased transcription of the IGF-1R gene. This increase in IGF-1R might be necessary to make cells competent for further selection but only up to a PPP concentration of 0.2 and 0.5 microM. In conclusion, malignant cells develop no or remarkably weak resistance to the IGF-1R inhibitor PPP.

Kir 4.1 inward rectifier potassium channel is upregulated in astrocytes in a murine multiple sclerosis model.

PubMed

Mercado, Francisco; Almanza, Angélica; Rubio, Nazario; Soto, Enrique

2018-06-11

Multiple sclerosis (MS) is a high prevalence degenerative disease characterized at the cellular level by glial and neuronal cell death. The causes of cell death during the disease course are not fully understood. In this work we demonstrate that in a MS model induced by Theiler's murine encephalomyelitis virus (TMEV) infection, the inward rectifier (Kir) 4.1 potassium channel subunit is overexpressed in astrocytes. In voltage clamp experiments the inward current density from TMEV-infected astrocytes was significantly larger than in mock-infected ones. The cRNA hybridization analysis from mock- and TMEV-infected cells showed an upregulation of a potassium transport channel coding sequence. We validated this mRNA increase by RT-PCR and quantitative PCR using Kir 4.1 specific primers. Western blotting experiments confirmed the upregulation of Kir 4.1, and alignment between sequences provided the demonstration that the over-expressed gene encodes for a Kir family member. Flow cytometry showed that the Kir 4.1 protein is located mainly in the cell membrane in mock and TMEV-infected astrocytes. Our results demonstrate an increase in K + inward current in TMEV-infected glial cells, this increment may reduce the neuronal depolarization, contributing to cell resilience mechanisms. Copyright © 2018 Elsevier B.V. All rights reserved.

Engineered cell-cell communication via DNA messaging

PubMed Central

2012-01-01

Background Evolution has selected for organisms that benefit from genetically encoded cell-cell communication. Engineers have begun to repurpose elements of natural communication systems to realize programmed pattern formation and coordinate other population-level behaviors. However, existing engineered systems rely on system-specific small molecules to send molecular messages among cells. Thus, the information transmission capacity of current engineered biological communication systems is physically limited by specific biomolecules that are capable of sending only a single message, typically “regulate transcription.” Results We have engineered a cell-cell communication platform using bacteriophage M13 gene products to autonomously package and deliver heterologous DNA messages of varying lengths and encoded functions. We demonstrate the decoupling of messages from a common communication channel via the autonomous transmission of various arbitrary genetic messages. Further, we increase the range of engineered DNA messaging across semisolid media by linking message transmission or receipt to active cellular chemotaxis. Conclusions We demonstrate decoupling of a communication channel from message transmission within engineered biological systems via the autonomous targeted transduction of user-specified heterologous DNA messages. We also demonstrate that bacteriophage M13 particle production and message transduction occurs among chemotactic bacteria. We use chemotaxis to improve the range of DNA messaging, increasing both transmission distance and communication bit rates relative to existing small molecule-based communication systems. We postulate that integration of different engineered cell-cell communication platforms will allow for more complex spatial programming of dynamic cellular consortia. PMID:22958599

Endothelial-monocyte activating polypeptide II disrupts alveolar epithelial type II to type I cell transdifferentiation

PubMed Central

2012-01-01

Background Distal alveolar morphogenesis is marked by differentiation of alveolar type (AT)-II to AT-I cells that give rise to the primary site of gas exchange, the alveolar/vascular interface. Endothelial-Monocyte Activating Polypeptide (EMAP) II, an endogenous protein with anti-angiogenic properties, profoundly disrupts distal lung neovascularization and alveolar formation during lung morphogenesis, and is robustly expressed in the dysplastic alveolar regions of infants with Bronchopulmonary dysplasia. Determination as to whether EMAP II has a direct or indirect affect on ATII→ATI trans-differentiation has not been explored. Method In a controlled nonvascular environment, an in vitro model of ATII→ATI cell trans-differentiation was utilized to demonstrate the contribution that one vascular mediator has on distal epithelial cell differentiation. Results Here, we show that EMAP II significantly blocked ATII→ATI cell transdifferentiation by increasing cellular apoptosis and inhibiting expression of ATI markers. Moreover, EMAP II-treated ATII cells displayed myofibroblast characteristics, including elevated cellular proliferation, increased actin cytoskeleton stress fibers and Rho-GTPase activity, and increased nuclear:cytoplasmic volume. However, EMAP II-treated cells did not express the myofibroblast markers desmin or αSMA. Conclusion Our findings demonstrate that EMAP II interferes with ATII → ATI transdifferentiation resulting in a proliferating non-myofibroblast cell. These data identify the transdifferentiating alveolar cell as a possible target for EMAP II's induction of alveolar dysplasia. PMID:22214516

Conjugated Oligoelectrolytes Increase Current Response and Organic Containment Removal in Wastewater Microbial Fuel Cells

DTIC Science & Technology

2012-01-01

certain COEs have been demonstrated to sponta- neously insert into liposomes and within the membranes of yeast .14 The driving force for this...ofDSSN+ and a cartoon illustration of its incorporation into a cell membrane. Introduction of yeast modified withDSSN+ into microbial fuel cells (MFCs...leads to an increase of generated current, compared to unmodified yeast , which suggests improved interaction with the charge-collecting electrode.14

Noninvasive pulsed focused ultrasound allows spatiotemporal control of targeted homing for multiple stem cell types in murine skeletal muscle and the magnitude of cell homing can be increased through repeated applications.

PubMed

Burks, Scott R; Ziadloo, Ali; Kim, Saejeong J; Nguyen, Ben A; Frank, Joseph A

2013-11-01

Stem cells are promising therapeutics for cardiovascular diseases, and i.v. injection is the most desirable route of administration clinically. Subsequent homing of exogenous stem cells to pathological loci is frequently required for therapeutic efficacy and is mediated by chemoattractants (cell adhesion molecules, cytokines, and growth factors). Homing processes are inefficient and depend on short-lived pathological inflammation that limits the window of opportunity for cell injections. Noninvasive pulsed focused ultrasound (pFUS), which emphasizes mechanical ultrasound-tissue interactions, can be precisely targeted in the body and is a promising approach to target and maximize stem cell delivery by stimulating chemoattractant expression in pFUS-treated tissue prior to cell infusions. We demonstrate that pFUS is nondestructive to murine skeletal muscle tissue (no necrosis, hemorrhage, or muscle stem cell activation) and initiates a largely M2-type macrophage response. We also demonstrate that local upregulation of chemoattractants in pFUS-treated skeletal muscle leads to enhance homing, permeability, and retention of human mesenchymal stem cells (MSC) and human endothelial precursor cells (EPC). Furthermore, the magnitude of MSC or EPC homing was increased when pFUS treatments and cell infusions were repeated daily. This study demonstrates that pFUS defines transient "molecular zip codes" of elevated chemoattractants in targeted muscle tissue, which effectively provides spatiotemporal control and tunability of the homing process for multiple stem cell types. pFUS is a clinically translatable modality that may ultimately improve homing efficiency and flexibility of cell therapies for cardiovascular diseases. © AlphaMed Press.

Demonstration of a Nano-Enabled Space Power System

NASA Technical Reports Server (NTRS)

Raffaelle, Ryne; Hunter, Roger C.; Baker, Christopher

2017-01-01

The Nano-Enabled Space Power System will demonstrate power systems with nanomaterial-enhanced components as are placement for CubeSat power generation, transmission, and storage. Successful flights of these nano-power systems will accelerate the use of this revolutionary technology in the aerospace industry. The use of nano materials in solar cells, wire harnesses,and lithium ion batteries can increase the device performance without significantly altering the devices physical dimensions or the devices operating range (temperature,voltage, current). In many cases, the use of nanomaterials widens the viable range of operating conditions, such as increased depth of discharge of lithium ion batteries, tunable bandgaps in solar cells, and increased flexure tolerance of wire harnesses.

Frequencies and role of regulatory T cells in patients with (pre)malignant cervical neoplasia

PubMed Central

Visser, J; Nijman, H W; Hoogenboom, B-N; Jager, P; van Baarle, D; Schuuring, E; Abdulahad, W; Miedema, F; van der Zee, A G; Daemen, T

2007-01-01

Oncogenic human papillomavirus (HPV)-infection is crucial for developing cervical cancer and its precursor lesions [cervical intraepithelial neoplasia (CIN)]. Regulatory T cells (Tregs) might be involved in the failure of the immune system to control the development of HPV-induced cancer. We investigated frequencies, phenotype and activity of Tregs in patients with cervical neoplasia. CIN and cervical cancer patients showed increased CD4+/CD25high T cell frequencies in peripheral blood and CD4+ T cell fraction. These CD4+/CD25high T cells represent Tregs as demonstrated by their low proliferation rate, low interferon (IFN)-γ/interleukin (IL)-10 ratio, high expression of CD45RO, GITR, CTLA-4, forkhead box P3 (FoxP3) and low CD45RA expression. Moreover, in HPV16+ cervical cancer patients, in-vitro depletion of CD25+ T cells resulted in increased IFN-γ T cell responses against HPV16 E6- and E7 peptides. Thus, increased frequencies of Tregs in cervical cancer patients may indeed suppress HPV-specific immunity. Longitudinal analysis of CD4+/CD25high T cell frequencies in patients showed a modest decline 1 year after curative surgery or chemoradiation. This study demonstrates increased frequencies and suppressive activity of Tregs in cervical cancer. These results imply that Tregs may suppress the immune control of cervical neoplasia and furthermore that suppression of immunity by Tregs will be another hurdle to overcome in therapeutic immunization strategies against cervical neoplasia. PMID:17937675

Study of wettability and cell viability of H implanted stainless steel

NASA Astrophysics Data System (ADS)

Shafique, Muhammad Ahsan; Ahmad, Riaz; Rehman, Ihtesham Ur

2018-03-01

In the present work, the effect of hydrogen ion implantation on surface wettability and biocompatibility of stainless steel is investigated. Hydrogen ions are implanted in the near-surface of stainless steel to facilitate hydrogen bonding at different doses with constant energy of 500 KeV, which consequently improve the surface wettability. Treated and untreated sample are characterized for surface wettability, incubation of hydroxyapatite and cell viability. Contact angle (CA) study reveals that surface wettability increases with increasing H-ion dose. Raman spectroscopy shows that precipitation of hydroxyapatite over the surface increase with increasing dose of H-ions. Cell viability study using MTT assay describes improved cell viability in treated samples as compared to the untreated sample. It is found that low dose of H-ions is more effective for cell proliferation and the cell count decreases with increasing ion dose. Our study demonstrates that H ion implantation improves the surface wettability and biocompatibility of stainless steel.

Omega-3 Eicosapentaenoic Acid Decreases CD133 Colon Cancer Stem-Like Cell Marker Expression While Increasing Sensitivity to Chemotherapy

PubMed Central

De Carlo, Flavia; Witte, Theodore R.; Hardman, W. Elaine; Claudio, Pier Paolo

2013-01-01

Colorectal cancer is the third leading cause of cancer-related death in the western world. In vitro and in vivo experiments showed that omega-3 polyunsaturated fatty acids (n-3 PUFAs) can attenuate the proliferation of cancer cells, including colon cancer, and increase the efficacy of various anticancer drugs. However, these studies address the effects of n-3 PUFAs on the bulk of the tumor cells and not on the undifferentiated colon cancer stem-like cells (CSLCs) that are responsible for tumor formation and maintenance. CSLCs have also been linked to the acquisition of chemotherapy resistance and to tumor relapse. Colon CSLCs have been immunophenotyped using several antibodies against cellular markers including CD133, CD44, EpCAM, and ALDH. Anti-CD133 has been used to isolate a population of colon cancer cells that retains stem cells properties (CSLCs) from both established cell lines and primary cell cultures. We demonstrated that the n-3 PUFA, eicosapentaenoic acid (EPA), was actively incorporated into the membrane lipids of COLO 320 DM cells. 25 uM EPA decreased the cell number of the overall population of cancer cells, but not of the CD133 (+) CSLCs. Also, we observed that EPA induced down-regulation of CD133 expression and up-regulation of colonic epithelium differentiation markers, Cytokeratin 20 (CK20) and Mucin 2 (MUC2). Finally, we demonstrated that EPA increased the sensitivity of COLO 320 DM cells (total population) to both standard-of-care chemotherapies (5-Fluorouracil and oxaliplatin), whereas EPA increased the sensitivity of the CD133 (+) CSLCs to only 5-Fluorouracil. PMID:23874993

Nitinol-based Nanotubular and Nanowell Coatings for the Modulation of Human Vascular Cell Functions

NASA Astrophysics Data System (ADS)

Lee, Phin Peng

Current approaches to reducing restenosis do not balance the reduction of vascular smooth muscle cell proliferation with the increase in the healing of the endothelium. Here, I present my study on the synthesis and characterization of a nanotubular coating on Nitinol substrates. I found that the coating demonstrated 'pro-healing' properties by increasing primary human aortic endothelial cell spreading, migration and collagen and elastin production. Certain cellular functions such as collagen and elastin production were also found to be affected by changes in nanotube diameter. The coating also reduced the proliferation and mRNA expression of collagen I and MMP2 for primary human aortic smooth muscle cells. I will also demonstrate the synthesis of a nanowell coating on Nitinol stents as well as an additional poly(lactic-co-glycolic acid) coating on top of the nanowells that has the potential for controlling drug release. These findings demonstrate the potential for the coatings to aid in the prevention of restenosis and sets up future explorations of ex vivo and in vivo studies.

Simultaneous Increases in Proliferation and Apoptosis of Vascular Smooth Muscle Cells Accelerate Diabetic Mouse Venous Atherosclerosis

PubMed Central

Liu, Shuying; Zhang, Zhengyu; Wang, Jingjing; Zhou, Yuhuan; Liu, Kefeng; Huang, Jintao; Chen, Dadi; Wang, Junmei; Li, Chaohong

2015-01-01

Aims This study was designed to demonstrate simultaneous increases in proliferation and apoptosis of vascular smooth muscle cells (VSMCs) leading to accelerated vein graft remodeling and to explore the underlying mechanisms. Methods Vein grafts were performed in non-diabetic and diabetic mice. The cultured quiescent VSMCs were subjected to mechanical stretch stress (SS) and/or advanced glycosylation end products (AGEs). Harvested vein grafts and treated VSMCs were used to detect cell proliferation, apoptosis, mitogen-activated protein kinases (MAPKs) activation and SM-α-actin expression. Results Significantly thicker vessel walls and greater increases in proliferation and apoptosis were observed in diabetic vein grafts than those in non-diabetic. Both SS and AGEs were found to induce different activation of three members of MAPKs and simultaneous increases in proliferation and apoptosis of VSMCs, and combined treatment with both had a synergistic effect. VSMCs with strong SM-α-actin expression represented more activated JNKs or p38MAPK, and cell apoptosis, while the cells with weak SM-α-actin expression demonstrated preferential activation of ERKs and cell proliferation. In contrast, inhibition of MAPKs signals triggered significant decreases in VSMC proliferation, and apoptosis. Treatment of the cells with RNA interference of receptor of AGEs (RAGE) also resulted in significant decreases in both proliferation and apoptosis. Conclusions Increased pressure-induced SS triggers simultaneous increases in proliferation and apoptosis of VSMCs in the vein grafts leading to vein arterializations, which can be synergistically accelerated by high glucose-induced AGEs resulting in vein graft atherosclerosis. Either SS or AGEs and their combination induce simultaneous increases in proliferation and apoptosis of VSMCs via different activation of three members of MAPKs resulting from different VSMC subtypes classified by SM-α-actin expression levels. PMID:26488175

Nε-(carboxymethyl) lysine-induced mitochondrial fission and mitophagy cause decreased insulin secretion from β-cells.

PubMed

Lo, Mei-Chen; Chen, Ming-Hong; Lee, Wen-Sen; Lu, Chin-I; Chang, Chuang-Rung; Kao, Shu-Huei; Lee, Horng-Mo

2015-11-15

Nε-(carboxymethyl) lysine-conjugated bovine serum albumin (CML-BSA) is a major component of advanced glycation end products (AGEs). We hypothesised that AGEs reduce insulin secretion from pancreatic β-cells by damaging mitochondrial functions and inducing mitophagy. Mitochondrial morphology and the occurrence of autophagy were examined in pancreatic islets of diabetic db/db mice and in the cultured CML-BSA-treated insulinoma cell line RIN-m5F. In addition, the effects of α-lipoic acid (ALA) on mitochondria in AGE-damaged tissues were evaluated. The diabetic db/db mouse exhibited an increase in the number of autophagosomes in damaged mitochondria and receptor for AGEs (RAGE). Treatment of db/db mice with ALA for 12 wk increased the number of mitochondria with well-organized cristae and fewer autophagosomes. Treatment of RIN-m5F cells with CML-BSA increased the level of RAGE protein and autophagosome formation, caused mitochondrial dysfunction, and decreased insulin secretion. CML-BSA also reduced mitochondrial membrane potential and ATP production, increased ROS and lipid peroxide production, and caused mitochondrial DNA deletions. Elevated fission protein dynamin-related protein 1 (Drp1) level and mitochondrial fragmentation demonstrated the unbalance of mitochondrial fusion and fission in CML-BSA-treated cells. Additionally, increased levels of Parkin and PTEN-induced putative kinase 1 protein suggest that fragmented mitochondria were associated with increased mitophagic activity, and ALA attenuated the CML-BSA-induced mitophage formation. Our study demonstrated that CML-BSA induced mitochondrial dysfunction and mitophagy in pancreatic β-cells. The findings from this study suggest that increased concentration of AGEs may damage β-cells and reduce insulin secretion. Copyright © 2015 the American Physiological Society.

The autophagic- lysosomal pathway determines the fate of glial cells under manganese- induced oxidative stress conditions.

PubMed

Gorojod, R M; Alaimo, A; Porte Alcon, S; Pomilio, C; Saravia, F; Kotler, M L

2015-10-01

Manganese (Mn) overexposure is frequently associated with the development of a neurodegenerative disorder known as Manganism. The Mn-mediated generation of reactive oxygen species (ROS) promotes cellular damage, finally leading to apoptotic cell death in rat astrocytoma C6 cells. In this scenario, the autophagic pathway could play an important role in preventing cytotoxicity. In the present study, we found that Mn induced an increase in the amount and total volume of acidic vesicular organelles (AVOs), a process usually related to the activation of the autophagic pathway. Particularly, the generation of enlarged AVOs was a ROS- dependent event. In this report we demonstrated for the first time that Mn induces autophagy in glial cells. This conclusion emerged from the results obtained employing a battery of autophagy markers: a) the increase in LC3-II expression levels, b) the formation of autophagic vesicles labeled with monodansylcadaverine (MDC) or LC3 and, c) the increase in Beclin 1/ Bcl-2 and Beclin 1/ Bcl-X(L) ratio. Autophagy inhibition employing 3-MA and mAtg5(K130R) resulted in decreased cell viability indicating that this event plays a protective role in Mn- induced cell death. In addition, mitophagy was demonstrated by an increase in LC3 and TOM-20 colocalization. On the other hand, we proposed the occurrence of lysosomal membrane permeabilization (LMP) based in the fact that cathepsins B and D activities are essential for cell death. Both cathepsin B inhibitor (Ca-074 Me) or cathepsin D inhibitor (Pepstatin A) completely prevented Mn- induced cytotoxicity. In addition, low dose of Bafilomycin A1 showed a similar effect, a finding that adds evidence about the lysosomal role in Mn cytotoxicity. Finally, in vivo experiments demonstrated that Mn induces injury and alters LC3 expression levels in rat striatal astrocytes. In summary, our results demonstrated that autophagy is activated to counteract the harmful effect caused by Mn. These data is valuable to be considered in future research concerning Manganism therapies. Copyright © 2015. Published by Elsevier Inc.

Osthole inhibits the tumorigenesis of hepatocellular carcinoma cells.

PubMed

Lin, Zhi-Kun; Liu, Jia; Jiang, Guo-Qiang; Tan, Guang; Gong, Peng; Luo, Hai-Feng; Li, Hui-Min; Du, Jian; Ning, Zhen; Xin, Yi; Wang, Zhong-Yu

2017-03-01

Hepatocellular carcinoma (HCC) accounts for approximately 90% of all cases of primary liver cancer, and the majority of patients with HCC are deprived of effective curative methods. Osthole is a Chinese herbal medicine which has been reported to possess various pharmacological functions, including hepatocellular protection. In the present study, we investigated the anticancer activity of osthole using HCC cell lines. We found that osthole inhibited HCC cell proliferation, induced cell cycle arrest, triggered DNA damage and suppressed migration in HCC cell lines. Furthermore, we demonstrated that osthole not only contributed to cell cycle G2/M phase arrest via downregulation of Cdc2 and cyclin B1 levels, but also induced DNA damage via an increase in ERCC1 expression. In addition, osthole inhibited the migration of HCC cell lines by significantly downregulating MMP-2 and MMP-9 levels. Finally, we demonstrated that osthole inhibited epithelial-mesenchymal transition (EMT) via increasing the expression of epithelial biomarkers E-cadherin and β-catenin, and significantly decreasing mesenchymal N-cadherin and vimentin protein expression. These results suggest that osthole may have potential chemotherapeutic activity against HCC.

Chromosome nondisjunction during bipolar mitoses of binucleated intermediates promote aneuploidy formation along with multipolar mitoses rather than chromosome loss in micronuclei induced by asbestos

PubMed Central

Zhang, Tianwei; Lv, Lei; Huang, Yun; Ren, Xiaohui; Shi, Qinghua

2017-01-01

Asbestos is a well-known occupational carcinogen that can cause aneuploidy during the early stages of neoplastic development. To explore the origins of asbestos-induced aneuploidy, we performed long-term live-cell imaging followed by fluorescence in situ hybridization of chromosomes 8 and 12 in human bronchial epithelial (HBEC) and mesothelial (MeT5A) cells. We demonstrate that asbestos induces aneuploidy via binucleated intermediates resulting from cytokinesis failure. On the one hand, asbestos increases chromosome nondisjunction during bipolar divisions of binucleated intermediates and produces near-tetraploidy. On the other hand, asbestos increases multipolar divisions of binucleated intermediates to produce aneuploidy. Surprisingly, chromosomes in asbestos-induced micronucleated cells are not truly lost by the cells, and do not contribute to aneuploid cell formation in either cell type. These results clarify the cellular source of asbestos-induced aneuploidy. In particular, they show the asbestos-induced disruption of bipolar chromosomal segregation in tetraploid cells, thereby demonstrating the causality between binucleated intermediates and aneuploidy evolution, rather than chromosome loss in micronuclei. PMID:28038458

Chromosome nondisjunction during bipolar mitoses of binucleated intermediates promote aneuploidy formation along with multipolar mitoses rather than chromosome loss in micronuclei induced by asbestos.

PubMed

Zhang, Tianwei; Lv, Lei; Huang, Yun; Ren, Xiaohui; Shi, Qinghua

2017-02-14

Asbestos is a well-known occupational carcinogen that can cause aneuploidy during the early stages of neoplastic development. To explore the origins of asbestos-induced aneuploidy, we performed long-term live-cell imaging followed by fluorescence in situ hybridization of chromosomes 8 and 12 in human bronchial epithelial (HBEC) and mesothelial (MeT5A) cells. We demonstrate that asbestos induces aneuploidy via binucleated intermediates resulting from cytokinesis failure. On the one hand, asbestos increases chromosome nondisjunction during bipolar divisions of binucleated intermediates and produces near-tetraploidy. On the other hand, asbestos increases multipolar divisions of binucleated intermediates to produce aneuploidy. Surprisingly, chromosomes in asbestos-induced micronucleated cells are not truly lost by the cells, and do not contribute to aneuploid cell formation in either cell type. These results clarify the cellular source of asbestos-induced aneuploidy. In particular, they show the asbestos-induced disruption of bipolar chromosomal segregation in tetraploid cells, thereby demonstrating the causality between binucleated intermediates and aneuploidy evolution, rather than chromosome loss in micronuclei.

PEG-chitosan hydrogel with tunable stiffness for study of drug response of breast cancer cells

PubMed Central

Chang, Fei-Chien; Tsao, Ching-Ting; Lin, Anqi; Zhang, Mengying; Levengood, Sheeny Lan; Zhang, Miqin

2016-01-01

Mechanical properties of the extracellular matrix have a profound effect on the behavior of anchorage-dependent cells. However, the mechanisms that define the effects of matrix stiffness on cell behavior remains unclear. Therefore, the development and fabrication of synthetic matrices with well-defined stiffness is invaluable for studying the interactions of cells with their biophysical microenvironment in vitro. We demonstrate a methoxypolyethylene glycol (mPEG)-modified chitosan hydrogel network where hydrogel stiffness can be easily modulated under physiological conditions by adjusting the degree of mPEG grafting onto chitosan (PEGylation). We show that the storage modulus of the hydrogel increases as PEGylation decreases and the gels exhibit instant self-recovery after deformation. Breast cancer cells cultured on the stiffest hydrogels adopt a more malignant phenotype with increased resistance to doxorubicin as compared with cells cultured on tissue culture polystyrene or Matrigel. This work demonstrates the utility of mPEG-modified chitosan hydrogel, with tunable mechanical properties, as an improved replacement of conventional culture system for in vitro characterization of breast cancer cell phenotype and evaluation of cancer therapies. PMID:27595012

Modeling the efficiency of a magnetic needle for collecting magnetic cells

NASA Astrophysics Data System (ADS)

Butler, Kimberly S.; Adolphi, Natalie L.; Bryant, H. C.; Lovato, Debbie M.; Larson, Richard S.; Flynn, Edward R.

2014-07-01

As new magnetic nanoparticle-based technologies are developed and new target cells are identified, there is a critical need to understand the features important for magnetic isolation of specific cells in fluids, an increasingly important tool in disease research and diagnosis. To investigate magnetic cell collection, cell-sized spherical microparticles, coated with superparamagnetic nanoparticles, were suspended in (1) glycerine-water solutions, chosen to approximate the range of viscosities of bone marrow, and (2) water in which 3, 5, 10 and 100% of the total suspended microspheres are coated with magnetic nanoparticles, to model collection of rare magnetic nanoparticle-coated cells from a mixture of cells in a fluid. The magnetic microspheres were collected on a magnetic needle, and we demonstrate that the collection efficiency versus time can be modeled using a simple, heuristically-derived function, with three physically-significant parameters. The function enables experimentally-obtained collection efficiencies to be scaled to extract the effective drag of the suspending medium. The results of this analysis demonstrate that the effective drag scales linearly with fluid viscosity, as expected. Surprisingly, increasing the number of non-magnetic microspheres in the suspending fluid results increases the collection of magnetic microspheres, corresponding to a decrease in the effective drag of the medium.

Modeling the Efficiency of a Magnetic Needle for Collecting Magnetic Cells

PubMed Central

Butler, Kimberly S; Adolphi, Natalie L.; Bryant, H C; Lovato, Debbie M; Larson, Richard S; Flynn, Edward R

2014-01-01

As new magnetic nanoparticle-based technologies are developed and new target cells are identified, there is a critical need to understand the features important for magnetic isolation of specific cells in fluids, an increasingly important tool in disease research and diagnosis. To investigate magnetic cell collection, cell-sized spherical microparticles, coated with superparamagnetic nanoparticles, were suspended in 1) glycerine-water solutions, chosen to approximate the range of viscosities of bone marrow, and 2) water in which 3, 5, 10 and 100 % of the total suspended microspheres are coated with magnetic nanoparticles, to model collection of rare magnetic nanoparticle-coated cells from a mixture of cells in a fluid. The magnetic microspheres were collected on a magnetic needle, and we demonstrate that the collection efficiency vs. time can be modeled using a simple, heuristically-derived function, with three physically-significant parameters. The function enables experimentally-obtained collection efficiencies to be scaled to extract the effective drag of the suspending medium. The results of this analysis demonstrate that the effective drag scales linearly with fluid viscosity, as expected. Surprisingly, increasing the number of non-magnetic microspheres in the suspending fluid results increases the collection of magnetic microspheres, corresponding to a decrease in the effective drag of the medium. PMID:24874577

Caspase-mediated pro-apoptotic interaction of panaxadiol and irinotecan in human colorectal cancer cells.

PubMed

Du, Guang-Jian; Wang, Chong-Zhi; Zhang, Zhi-Yu; Wen, Xiao-Dong; Somogyi, Jacqueline; Calway, Tyler; He, Tong-Chuan; Du, Wei; Yuan, Chun-Su

2012-05-01

Panaxadiol is a purified sapogenin of ginseng saponins that exhibits anticancer activity. Irinotecan is a second-line anticancer drug, but clinical treatment with irinotecan is limited due to its side effects. In this study, we have investigated the possible synergistic anticancer effects of panaxadiol and irinotecan on human colorectal cancer cells and explored the potential role of apoptosis in their synergistic activity. The combination of panaxadiol and irinotecan significantly enhanced antiproliferative effects in HCT-116 cells (P< 0.05). Cell cycle analysis demonstrated that combining irinotecan treatment with panaxadiol significantly increased the G1-phase fractions of cells, compared with irinotecan treatment alone. In apoptotic assays, the combination of panaxadiol and irinotecan significantly increased the percentage of apoptotic cells compared with irinotecan alone (P<0.01). Increased activity of caspase-3 and caspase-9 was observed after treating with panaxadiol and irinotecan. The synergistic apoptotic effects were supported by docking analysis, which demonstrated that panaxadiol and irinotecan bound two different chains of the caspase-3 protein. Data from this study suggested that caspase-3- and caspase-9-mediated apoptosis may play an important role in the panaxadiol enhanced antiproliferative effects of irinotecan on human colorectal cancer cells. © 2012 The Authors. JPP © 2012 Royal Pharmaceutical Society.

The Cellular and Molecular Mechanisms for Neutropenia in Barth Syndrome

PubMed Central

Makaryan, Vahagn; Kulik, Willem; Vaz, Frederic M.; Allen, Christopher; Dror, Yigal; Dale, David C.; Aprikyan, Andranik A.

2015-01-01

Barth syndrome (BTHS), a rare, X-linked, recessive disease characterized by neutropenia and cardiomyopathy. BTHS is caused by loss-of-function mutations of the tafazzin (TAZ) gene. We developed a model of BTHS by transfecting human HL60 myeloid progenitor cells with TAZ-specific shRNAs. Results demonstrate a significant down-regulation in TAZ expression, mimicking the effects of naturally-occurring truncation mutations in TAZ. Flow cytometry analyses of cells with TAZ-specific, but not scrambled, shRNAs demonstrate nearly two-fold increase in proportion of annexin-V positive cells and significantly increased dissipation of mitochondrial membrane potential as determined by DIOC6-staining. Transfection of TAZ specific shRNA had similar effects in U937 myeloid cells but not in lymphoid cell lines. Further studies in HL60 myeloid progenitor cells revealed aberrant release of cytochrome c from mitochondria and significantly elevated levels of activated caspase-3 in response to TAZ knock-down. Treatment with caspase-specific inhibitor zVAD-fmk resulted in substantially reduced apoptosis to near-normal levels. These data suggest that neutropenia in BTHS is attributable to increased dissipation of mitochondrial membrane potential, aberrant release of cytochrome c, activation of caspase-3 and accelerated apoptosis of myeloid progenitor cells, and that this defect can be partially restored in vitro by treatment with caspase-specific inhibitors. PMID:22023389

Effect of hyaluronic acid on morphological changes to dentin surfaces and subsequent effect on periodontal ligament cell survival, attachment, and spreading.

PubMed

Mueller, Andrea; Fujioka-Kobayashi, Masako; Mueller, Heinz-Dieter; Lussi, Adrian; Sculean, Anton; Schmidlin, Patrick R; Miron, Richard J

2017-05-01

Hyaluronic acid (HA) is a natural constituent of connective tissues and plays an important role in their development, maintenance, and regeneration. Recently, HA has been shown to improve wound healing. However, no basic in vitro study to date has investigated its mode of action. Therefore, the purpose of this study was to examine morphological changes of dentin surfaces following HA coating and thereafter investigate the influence of periodontal ligament (PDL) cell survival, attachment, and spreading to dentin discs. HA was coated onto dentin discs utilizing either non-cross-linked (HA) or cross-linked (HA cl) delivery systems. Morphological changes to dentin discs were then assessed using scanning electron microscopy (SEM). Thereafter, human PDL cells were seeded under three in vitro conditions including (1) dilution of HA (1:100), (2) dilution of HA (1:10), and (3) HA coated directly to dentin discs. Samples were then investigated for PDL cell survival, attachment, and spreading using a live/dead assay, cell adhesion assay, and SEM imaging, respectively. While control dentin discs demonstrated smooth surfaces both at low and high magnification, the coating of HA altered surface texture of dentin discs by increasing surface roughness. HA cl further revealed greater surface texture/roughness likely due to the cross-linking carrier system. Thereafter, PDL cells were seeded on control and HA coated dentin discs and demonstrated a near 100 % survival rate for all samples demonstrating high biocompatibility of HA at dilutions of both 1:100 and 1:10. Interestingly, non-cross-linked HA significantly increased cell numbers at 8 h, whereas cross-linked HA improved cell spreading as qualitatively assessed by SEM. The results from the present study demonstrate that both carrier systems for HA were extremely biocompatible and demonstrated either improved cell numbers or cell spreading onto dentin discs. Future in vitro and animal research is necessary to further characterize the optimal delivery system of HA for improved clinical use. HA is a highly biocompatible material that may improve PDL cell attachment or spreading on dentin.

Multi-walled carbon nanotubes increase antibody-producing B cells in mice immunized with a tetravalent vaccine candidate for dengue virus.

PubMed

Calegari, Luan P; Dias, Roberto S; de Oliveira, Michelle D; Pessoa, Carine Ribeiro; de Oliveira, André S; Oliveira, Ana F C S; da Silva, Cynthia C; Fonseca, Flavio G; Versiani, Alice F; De Paula, Sérgio O

2016-07-27

In recent times, studies have demonstrated that carbon nanotubes are good candidates for use as vehicles for transfection of exogenous material into the cells. However, there are few studies evaluating the behavior of carbon nanotubes as DNA vectors and few of these studies have used multi-walled carbon nanotubes (MWCNTs) or carboxylated MWCNTs. Thus, this study aims to assess the MWCNTs' (carboxylated or not) efficiency in the increase in expression of the tetravalent vaccine candidate (TVC) plasmid vector for dengue virus in vitro using Vero cells, and in vivo, through the intramuscular route, to evaluate the immunological response profile. Multi-walled carbon nanotubes internalized by Vero cells, have been found in the cytoplasm and nucleus associated with the plasmid. However, it was not efficient to increase the messenger ribonucleic acid (mRNA) compared to the pure vaccine candidate associated with Lipofectamine(®) 2000. The in vivo experiments showed that the use of intramuscular injection of the TVC in combination with MWCNTs reduced the immune response compared to pure TVC, in a general way, although an increase was observed in the population of the antibody-producing B cells, as compared to pure TVC. The results confirm the data found by other authors, which demonstrate the ability of nanotubes to penetrate target cells and reach both the cytoplasm and the cell nucleus. The cytotoxicity values are also in accordance with the literature, which range from 5 to 20 µg/mL. This has been found to be 10 µg/mL in this study. Although the expression levels are higher in cells that receive the pure TVC transfected using Lipofectamine(®) 2000, the nanotubes show an increase in B-cells producing antibodies.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Gao, Hui; Lin, Lu; Haq, Ihtesham Ul

The transcription factor nuclear factor-κB (NF-κB) plays an important role in diverse processes, including cell proliferation and differentiation, apoptosis and inflammation. However, the role of NF-κB in porcine follicle development is not clearly elucidated. In this study, we demonstrated that follicle stimulating hormone (FSH) increased the level of inhibitor of NF-κB (IκB) protein and promoted the cytoplasmic localization of p65, indicating that FSH inhibits the activation of NF-κB in porcine granulosa cells. Moreover, inhibition of NF-κB by FSH or another specific inhibitor of NF-κB, pyrrolidine dithiocarbamate (PDTC), could activate JNK signaling and enhance autophagic activity in porcine granulosa cells. Knockdownmore » of RelA (p65) Subunit of NF-κB by RNA interference abrogated the activation of JNK signaling pathway and the increase of autophagic protein expression by FSH. Meanwhile, the functional significance of FSH or PDTC-mediated autophagy were further investigated. Our results demonstrated that the increased autophagy promoted progesterone secretion in porcine granulosa cells. Blockage of autophagy by chloroquine obviated the FSH or PDTC-induced progesterone production. Taken together, these results indicate that inhibition of NF-κB increased autophagy via JNK signaling, and promote steroidogenesis in porcine granulosa cells. Our results provide new insights into the regulation and function of autophagy in mammalian follicle development. - Highlights: • FSH inhibits the activation of NF-κB in porcine primary granulosa cells. • Inhibition of NF-κB by FSH promotes autophagy via JNK signaling in granulosa cells. • Increased autophagy contributes to progesterone production in granulosa cells. • This is the first report against beclin1 regulation in porcine granulosa cells.« less

Elevated O-GlcNAcylation promotes gastric cancer cells proliferation by modulating cell cycle related proteins and ERK 1/2 signaling.

PubMed

Jiang, Mingzuo; Qiu, Zhaoyan; Zhang, Song; Fan, Xing; Cai, Xiqiang; Xu, Bing; Li, Xiaowei; Zhou, Jinfeng; Zhang, Xiangyuan; Chu, Yi; Wang, Weijie; Liang, Jie; Horvath, Tamas; Yang, Xiaoyong; Wu, Kaichun; Nie, Yongzhan; Fan, Daiming

2016-09-20

O-GlcNAc transferase (OGT) is the only enzyme in mammals that catalyzes the attachment of β-D-N-acetylglucosamine (GlcNAc) to serine or threonine residues of target proteins. Hyper-O-GlcNAcylation is becoming increasingly realized as a general feature of cancer and contributes to rapid proliferation of cancer cells. In this study, we demonstrated that O-GlcNAc and OGT levels were increased in all six gastric cancer (GC) cell lines as compared with immortal gastric epithelial cells. Downregulation of the O-GlcNAcylation level by silencing OGT inhibited cell viability and growth rate via the cdk-2, cyclin D1 and ERK 1/2 pathways. In vivo xenograft assays also demonstrated that the hyper-O-GlcNAc level markedly promoted the proliferation of tumors. Moreover, compared with noncancerous tissues, the O-GlcNAcylation level was increased in cancerous tissues. GC patients with higher levels of O-GlcNAcylation exhibited large tumor sizes (≥5 cm), deep tumor invasion (T3 and T4), high AJCC stages (stage III and IV), more lymph node metastases and lower overall survival. Notably, the phosphorylation level of ERK 1/2 was increased progressively with the increase of O-GlcNAcylation in both SGC 7901 and AGS cells. Consistently, human GC tissue arrays also revealed that ERK 1/2 signaling was positively correlated to O-GlcNAcylation (r = 0.348; P = 0.015). Taken together, here we reported that hyper-O-GlcNAcylation significantly promotes GC cells proliferation by modulating cell cycle related proteins and ERK 1/2 signaling, suggesting that inhibition of OGT may be a potential novel therapeutic target of GC.

Monomeric cocoa catechins enhance β-cell function by increasing mitochondrial respiration.

PubMed

Rowley, Thomas J; Bitner, Benjamin F; Ray, Jason D; Lathen, Daniel R; Smithson, Andrew T; Dallon, Blake W; Plowman, Chase J; Bikman, Benjamin T; Hansen, Jason M; Dorenkott, Melanie R; Goodrich, Katheryn M; Ye, Liyun; O'Keefe, Sean F; Neilson, Andrew P; Tessem, Jeffery S

2017-11-01

A hallmark of type 2 diabetes (T2D) is β-cell dysfunction and the eventual loss of functional β-cell mass. Therefore, mechanisms that improve or preserve β-cell function could be used to improve the quality of life of individuals with T2D. Studies have shown that monomeric, oligomeric and polymeric cocoa flavanols have different effects on obesity, insulin resistance and glucose tolerance. We hypothesized that these cocoa flavanols may have beneficial effects on β-cell function. INS-1 832/13-derived β-cells and primary rat islets cultured with a monomeric catechin-rich cocoa flavanol fraction demonstrated enhanced glucose-stimulated insulin secretion, while cells cultured with total cocoa extract and with oligomeric or polymeric procyanidin-rich fraction demonstrated no improvement. The increased glucose-stimulated insulin secretion in the presence of the monomeric catechin-rich fraction corresponded with enhanced mitochondrial respiration, suggesting improvements in β-cell fuel utilization. Mitochondrial complex III, IV and V components are up-regulated after culture with the monomer-rich fraction, corresponding with increased cellular ATP production. The monomer-rich fraction improved cellular redox state and increased glutathione concentration, which corresponds with nuclear factor, erythroid 2 like 2 (Nrf2) nuclear localization and expression of Nrf2 target genes including nuclear respiratory factor 1 (Nrf1) and GA binding protein transcription factor alpha subunit (GABPA), essential genes for increasing mitochondrial function. We propose a model by which monomeric cocoa catechins improve the cellular redox state, resulting in Nrf2 nuclear migration and up-regulation of genes critical for mitochondrial respiration, glucose-stimulated insulin secretion and ultimately improved β-cell function. These results suggest a mechanism by which monomeric cocoa catechins exert their effects as an effective complementary strategy to benefit T2D patients. Copyright © 2017 Elsevier Inc. All rights reserved.

MEF2‑activated long non‑coding RNA PCGEM1 promotes cell proliferation in hormone‑refractory prostate cancer through downregulation of miR‑148a.

PubMed

Zhang, Shibao; Li, Zongwu; Zhang, Longyang; Xu, Zhonghua

2018-05-04

Prostate cancer gene expression marker 1 (PCGEM1) is a prostate‑specific gene overexpressed in prostate cancer cells that promotes cell proliferation. To study the molecular mechanism of PCGEM1 function in hormone‑refractory prostate cancer, the interaction between myocyte enhancer factor 2 (MEF2) and PCGEM1 was assessed by a luciferase reporter assay and chromatin immunoprecipitation (ChIP) assay. In addition, the underlying mechanism of PCGEM1 regulating expression of microRNA (miR)‑148a in PC3 prostate cancer cells was evaluated. Relative expression levels were measured by reverse transcription‑quantitative polymerase chain reaction, and early apoptosis was measured by flow cytometry. PCGEM1 was demonstrated to be overexpressed in prostate cancer tissues compared with noncancerous tissues. Expression levels of PCGEM1 in PC3 cancer cells were demonstrated to be regulated by MEF2, as PCGME1 mRNA was increased by MEF2 overexpression but decreased by MEF2 silencing. MEF2 was also demonstrated to enhance the activity of PCGEM1 promoter and thus promote PCGEM1 transcription. In addition, downregulation of PCGEM1 expression in PC3 cells increased expression of miR‑148a. By contrast, overexpression of PCGEM1 decreased miR‑148a expression. Finally, PCGME1 silencing by small interfering RNA significantly induced early cell apoptosis but this effect was reduced by a miR‑148a inhibitor. In conclusion, the present study demonstrated a positive regulatory association between MEF2 and PCGEM1, and a reciprocal negative regulatory association between PCGEM1 and miR‑148a that controls cell apoptosis. The present study, therefore, provides new insights into the mechanism of PCGEM1 function in prostate cancer development.

Cancer cells recovering from damage exhibit mitochondrial restructuring and increased aerobic glycolysis.

PubMed

Akakura, Shin; Ostrakhovitch, Elena; Sanokawa-Akakura, Reiko; Tabibzadeh, Siamak

2014-06-13

Instead of relying on mitochondrial oxidative phosphorylation, most cancer cells rely heavily on aerobic glycolysis, a phenomenon termed as "the Warburg effect". We considered that this effect is a direct consequence of damage which persists in cancer cells that recover from damage. To this end, we studied glycolysis and rate of cell proliferation in cancer cells that recovered from severe damage. We show that in vitro Damage-Recovered (DR) cells exhibit mitochondrial structural remodeling, display Warburg effect, and show increased in vitro and in vivo proliferation and tolerance to damage. To test whether cancer cells derived from tumor microenvironment can show similar properties, we isolated Damage-Recovered (T(DR)) cells from tumors. We demonstrate that T(DR) cells also show increased aerobic glycolysis and a high proliferation rate. These findings show that Warburg effect and its consequences are induced in cancer cells that survive severe damage. Copyright © 2014 Elsevier Inc. All rights reserved.

Indoleamine 2,3-dioxygenase and iron are required for Mycobacterium leprae survival.

PubMed

de Mattos Barbosa, Mayara Garcia; da Silva Prata, Rhana Berto; Andrade, Priscila Ribeiro; Ferreira, Helen; de Andrade Silva, Bruno Jorge; da Paixão de Oliveira, Jéssica Araújo; Assis, Tayná Quintella; de Toledo-Pinto, Thiago Gomes; de Lima Bezerra, Ohanna Cavalcanti; da Costa Nery, José Augusto; Rosa, Patricia Sammarco; Bozza, Marcelo Torres; Lara, Flávio Alves; Moraes, Milton Ozório; Schmitz, Veronica; Sarno, Euzenir Nunes; Pinheiro, Roberta Olmo

2017-11-01

Our previous study has demonstrated that IL-10 may modulate both indoleamine 2,3-dioxygenase (IDO) and CD163 expression in lepromatous leprosy (LL) cells, favoring Mycobacterium leprae persistence through induction of regulatory pathways and iron storage. Here, we observed that in LL lesion cells there is an increase in the expression of proteins involved in iron metabolism such as hemoglobin (Hb), haptoglobin, heme oxygenase 1 and transferrin receptor 1 (TfR1) when compared to tuberculoid leprosy (BT) cells. We also found increased iron deposits and diminished expression of the iron exporter ferroportin 1 in LL lesion cells. Hemin, but not FeSO 4 stimulation, was able to enhance M. leprae viability by a mechanism that involves IDO. Analysis of cell phenotype in lesions demonstrated a predominance of M2 markers in LL when compared with BT lesion cells. A positive correlation between CD163 and PPARG with the bacillary index (BI) was observed. In contrast, TNF, STAT1 and CSF2 presented a negative correlation with the BI. In summary, this study demonstrates that iron may regulate IDO expression by a mechanism that involves IL-10, which may contribute for the predominance of M2-like phenotype in LL lesions that favors the phagocytosis and maintenance of M. leprae in host cells. Copyright © 2017 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.

The Role of Epithelial-Mesenchymal Transition in the Formation of Normal and Neoplastic Mammary Epithelial Stem Cells

DTIC Science & Technology

2011-09-01

separating stem cell and non- stem cell populations of normal and breast cancer cells and identified EMT transcription factors most likely involved in... stem cell biology. Preliminary results directly demonstrate that transient induction of EMT increases the number of mammary epithelial stem cells...EMT and entrance into a stem - cell state. The outcome of these experiments holds important implications for the mechanisms controlling the formation of

Tumor necrosis factor-{alpha} enhances IL-15-induced natural killer cell differentiation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lee, Jiwon; Lee, Suk Hyung; Korea University of Science and Technology, Yusong, Daejeon 305-333

2009-09-04

The differentiation of natural killer (NK) cells is regulated by various factors including soluble growth factors and transcription factors. Here, we have demonstrated that tumor necrosis factor-{alpha} (TNF-{alpha}) is a positive regulator of NK cell differentiation. TNF-{alpha} augmented the IL-15-induced expression of NK1.1 and CD122 in mature NK cells, and TNF-{alpha} alone also induced NK cell maturation as well as IL-15. TNF-{alpha} also increased IFN-{gamma} production in NK cells in the presence of IL-15. Meanwhile, mRNA expression of several transcription factors, including T-bet and GATA-3, was increased by the addition of TNF-{alpha} and IL-15. In addition, TNF-{alpha} increased nuclear factor-kappamore » B (NF-{kappa}B) activity in NK cells and inhibition of NF-{kappa}B impeded TNF-{alpha}-enhanced NK cell maturation. Overall, these data suggest that TNF-{alpha} significantly increased IL-15-driven NK cell differentiation by increasing the expression of transcription factors that play crucial roles in NK cell maturation and inducing the NF-{kappa}B activity.« less

A Preliminary Study: Human Fibroid Stro-1+/CD44+ Stem Cells Isolated From Uterine Fibroids Demonstrate Decreased DNA Repair and Genomic Integrity Compared to Adjacent Myometrial Stro-1+/CD44+ Cells.

PubMed

Prusinski Fernung, Lauren E; Al-Hendy, Ayman; Yang, Qiwei

2018-01-01

Although uterine fibroids (UFs) continue to place a major burden on female reproductive health, the mechanisms behind their origin remain undetermined. Normal myometrial stem cells may be transformed into tumor-initiating stem cells, causing UFs, due to unknown causes of somatic mutations in MED12, found in up to 85% of sporadically formed UFs. It is well established in other tumor types that defective DNA repair increases the risk of such tumorigenic somatic mutations, mechanisms not yet studied in UFs. To examine the putative cause(s) of this stem cell transformation, we analyzed DNA repair within stem cells from human UFs compared to those from adjacent myometrium to determine whether DNA repair in fibroid stem cells is compromised. Human fibroid (F) and adjacent myometrial (Myo) stem cells were isolated from fresh tissues, and gene expression relating to DNA repair was analyzed. Fibroid stem cells differentially expressed DNA repair genes related to DNA double- (DSBs) and single-strand breaks. DNA damage was measured using alkaline comet assay. Additionally, DNA DSBs were induced in these stem cells and DNA DSB repair evaluated (1) by determining changes in phosphorylation of DNA DSB-related proteins and (2) by determining differences in γ-H2AX foci formation and relative DNA repair protein RAD50 expression. Overall, F stem cells demonstrated increased DNA damage and altered DNA repair gene expression and signaling, suggesting that human F stem cells demonstrate impaired DNA repair. Compromised F stem cell DNA repair may contribute to further mutagenesis and, consequently, further growth and propagation of UF tumors.

Simvastatin rises reactive oxygen species levels and induces senescence in human melanoma cells by activation of p53/p21 pathway

DOE Office of Scientific and Technical Information (OSTI.GOV)

Guterres, Fernanda Augusta de Lima Barbosa; Martinez, Glaucia Regina; Rocha, Maria Eliane Merlin

2013-11-15

Recent studies demonstrated that simvastatin has antitumor properties in several types of cancer cells, mainly by inducing apoptosis and inhibiting growth. The arrest of proliferation is a feature of cellular senescence; however, the occurrence of senescence in melanoma cells upon simvastatin treatment has not been investigated until now. Our results demonstrated that exposure of human metastatic melanoma cells (WM9) to simvastatin induces a senescent phenotype, characterized by G1 arrest, positive staining for senescence-associated β-galactosidase assay, and morphological changes. Also, the main pathways leading to cell senescence were examined in simvastatin-treated human melanoma cells, and the expression levels of phospho-p53 andmore » p21 were upregulated by simvastatin, suggesting that cell cycle regulators and DNA damage pathways are involved in the onset of senescence. Since simvastatin can act as a pro-oxidant agent, and oxidative stress may be related to senescence, we measured the intracellular ROS levels in WM9 cells upon simvastatin treatment. Interestingly, we found an increased amount of intracellular ROS in these cells, which was accompanied by elevated expression of catalase and peroxiredoxin-1. Collectively, our results demonstrated that simvastatin can induce senescence in human melanoma cells by activation of p53/p21 pathway, and that oxidative stress may be related to this process. - Highlights: • Lower concentrations of simvastatin can induce senescent phenotype in melanoma cells. • Simvastatin induces senescence in human melanoma cells via p53/p21 pathway. • Senescent phenotype is related with increased intracellular ROS. • Partial detoxification of ROS by catalase/peroxiredoxin-1 could lead cells to senescence rather than apoptosis.« less

SLAP deficiency increases TCR avidity leading to altered repertoire and negative selection of cognate antigen-specific CD8+ T cells

PubMed Central

Friend, Samantha F.; Peterson, Lisa K.; Kedl, Ross M.; Dragone, Leonard L.

2014-01-01

How T cell receptor (TCR) avidity influences CD8+ T cell development and repertoire selection is not yet fully understood. To fill this gap, we utilized Src-like adaptor protein (SLAP)-deficient mice as a tool to increase TCR avidity on double positive (DP) thymocytes. We generated SLAP−/− mice with the transgenic MHC class I-restricted TCR (OT-1) and SLAP−/− Vβ5 mice, expressing only the β-chain of the TCR OT-1 transgene, to examine the effects of increased TCR surface levels on CD8+ T cell development and repertoire selection. In comparing SLAP−/− OT-1 and Vβ5 mice with wild-type controls, we performed compositional analysis and assessed thymocyte signaling by measuring CD5 levels. In addition, we performed tetramer and compositional staining to measure affinity for the cognate antigen, ovalbumin (OVA) peptide, presented by MHC. Furthermore, we quantified differences in α-chain repertoire in SLAP−/− Vβ5 mice. We have found that SLAP−/− OT-1 mice have fewer CD8+ thymocytes but have increased CD5 expression. SLAP−/− OT-1 mice have fewer DP thymocytes expressing Vα2, signifying increased endogenous α-chain rearrangement, and more non-OVA-specific CD8+ splenocytes upon tetramer staining. Our data demonstrate that SLAP−/− Vβ5 mice also have fewer OVA-specific cells and increased Vα2 usage in the peripheral Vβ5 CD8+ T cells that were non-OVA-specific, demonstrating differences in α-chain repertoire. These studies provide direct evidence that increased TCR avidity in DP thymocytes enhances CD8+ T cell negative selection deleting thymocytes with specificity for cognate antigen, an antigen the mature T cells may never encounter. Collectively, these studies provide new insights into how TCR avidity during CD8+ T cell development influences repertoire selection. PMID:22956467

SLAP deficiency increases TCR avidity leading to altered repertoire and negative selection of cognate antigen-specific CD8+ T cells.

PubMed

Friend, Samantha F; Peterson, Lisa K; Kedl, Ross M; Dragone, Leonard L

2013-03-01

How T cell receptor (TCR) avidity influences CD8(+) T cell development and repertoire selection is not yet fully understood. To fill this gap, we utilized Src-like adaptor protein (SLAP)-deficient mice as a tool to increase TCR avidity on double positive (DP) thymocytes. We generated SLAP(-/-) mice with the transgenic MHC class I-restricted TCR (OT-1) and SLAP(-/-) Vβ5 mice, expressing only the β-chain of the TCR OT-1 transgene, to examine the effects of increased TCR surface levels on CD8(+) T cell development and repertoire selection. In comparing SLAP(-/-) OT-1 and Vβ5 mice with wild-type controls, we performed compositional analysis and assessed thymocyte signaling by measuring CD5 levels. In addition, we performed tetramer and compositional staining to measure affinity for the cognate antigen, ovalbumin (OVA) peptide, presented by MHC. Furthermore, we quantified differences in α-chain repertoire in SLAP(-/-) Vβ5 mice. We have found that SLAP(-/-) OT-1 mice have fewer CD8(+) thymocytes but have increased CD5 expression. SLAP(-/-) OT-1 mice have fewer DP thymocytes expressing Vα2, signifying increased endogenous α-chain rearrangement, and more non-OVA-specific CD8(+) splenocytes upon tetramer staining. Our data demonstrate that SLAP(-/-) Vβ5 mice also have fewer OVA-specific cells and increased Vα2 usage in the peripheral Vβ5 CD8(+) T cells that were non-OVA-specific, demonstrating differences in α-chain repertoire. These studies provide direct evidence that increased TCR avidity in DP thymocytes enhances CD8(+) T cell negative selection deleting thymocytes with specificity for cognate antigen, an antigen the mature T cells may never encounter. Collectively, these studies provide new insights into how TCR avidity during CD8(+) T cell development influences repertoire selection.

[Effect of ectopic expression of NtEXPA5 gene on cell size and growth of organs of transgenic tobacco plants].

PubMed

Kuluev, B R; Safiullina, M G; Kniazev, A V; Chemeris, A V

2013-01-01

We obtained transgenic tobacco plants demonstrating overexpression of NtEXPA5 gene that encodes alpha-expansin of Nicotiana tabacum. The transgenic plants were characterized by increased size of leaves and stems. However, size of flowers remained almost unchanged. The increase of organ sizes was induced by cell stretching only. Moreover, the number of cell divisions was even decreased. The obtained data suggest tight interaction between cell stretching regulation and cell division, which together provide the basic mechanism aimed at the controlling of plant organ sizes.

Sparse grid techniques for particle-in-cell schemes

NASA Astrophysics Data System (ADS)

Ricketson, L. F.; Cerfon, A. J.

2017-02-01

We propose the use of sparse grids to accelerate particle-in-cell (PIC) schemes. By using the so-called ‘combination technique’ from the sparse grids literature, we are able to dramatically increase the size of the spatial cells in multi-dimensional PIC schemes while paying only a slight penalty in grid-based error. The resulting increase in cell size allows us to reduce the statistical noise in the simulation without increasing total particle number. We present initial proof-of-principle results from test cases in two and three dimensions that demonstrate the new scheme’s efficiency, both in terms of computation time and memory usage.

T Lymphocyte Activation Threshold and Membrane Reorganization Perturbations in Unique Culture Model

NASA Technical Reports Server (NTRS)

Adams, C. L.; Sams, C. F.

2000-01-01

Quantitative activation thresholds and cellular membrane reorganization are mechanisms by which resting T cells modulate their response to activating stimuli. Here we demonstrate perturbations of these cellular processes in a unique culture system that non-invasively inhibits T lymphocyte activation. During clinorotation, the T cell activation threshold is increased 5-fold. This increased threshold involves a mechanism independent of TCR triggering. Recruitment of lipid rafts to the activation site is impaired during clinorotation but does occur with increased stimulation. This study describes a situation in which an individual cell senses a change in its physical environment and alters its cell biological behavior.

Gemcitabine sensitizes lung cancer cells to Fas/FasL system-mediated killing

PubMed Central

Siena, Liboria; Pace, Elisabetta; Ferraro, Maria; Di Sano, Caterina; Melis, Mario; Profita, Mirella; Spatafora, Mario; Gjomarkaj, Mark

2014-01-01

Gemcitabine is a chemotherapy agent commonly used in the treatment of non-small cell lung cancer (NSCLC) that has been demonstrated to induce apoptosis in NSCLC cells by increasing functionally active Fas expression. The aim of this study was to evaluate the Fas/Fas ligand (FasL) system involvement in gemcitabine-induced lung cancer cell killing. NSCLC H292 cells were cultured in the presence or absence of gemcitabine. FasL mRNA and protein were evaluated by real-time PCR, and by Western blot and flow cytometry, respectively. Apoptosis of FasL-expressing cells was evaluated by flow cytometry, and caspase-8 and caspase-3 activation by Western blot and a colorimetric assay. Cytotoxicity of lymphokine-activated killer (LAK) cells and malignant pleural fluid lymphocytes against H292 cells was analysed in the presence or absence of the neutralizing anti-Fas ZB4 antibody, by flow cytometry. Gemcitabine increased FasL mRNA and total protein expression, the percentage of H292 cells bearing membrane-bound FasL (mFasL) and of mFasL-positive apoptotic H292 cells, as well as caspase-8 and caspase-3 cleavage. Moreover, gemcitabine increased CH11-induced caspase-8 and caspase-3 cleavage and proteolytic activity. Cytotoxicity of LAK cells and pleural fluid lymphocytes was increased against gemcitabine-treated H292 cells and was partially inhibited by ZB4 antibody. These results demonstrate that gemcitabine: (i) induces up-regulation of FasL in lung cancer cells triggering cell apoptosis via an autocrine/paracrine loop; (ii) induces a Fas-dependent apoptosis mediated by caspase-8 and caspase-3 activation; (iii) enhances the sensitivity of lung cancer cells to cytotoxic activity of LAK cells and malignant pleural fluid lymphocytes, partially via Fas/FasL pathway. Our data strongly suggest an active involvement of the Fas/FasL system in gemcitabine-induced lung cancer cell killing. PMID:24128051

Interleukin-34 promotes tumor progression and metastatic process in osteosarcoma through induction of angiogenesis and macrophage recruitment.

PubMed

Ségaliny, Aude I; Mohamadi, Amel; Dizier, Blandine; Lokajczyk, Anna; Brion, Régis; Lanel, Rachel; Amiaud, Jérôme; Charrier, Céline; Boisson-Vidal, Catherine; Heymann, Dominique

2015-07-01

Interleukin-34 (IL-34) was recently characterized as the M-CSF "twin" cytokine, regulating the proliferation/differentiation/survival of myeloid cells. The implication of M-CSF in oncology was initially suspected by the reduced metastatic dissemination in knock-out mice, due to angiogenesis impairment. Based on this observation, our work studied the involvement of IL-34 in the pathogenesis of osteosarcoma. The in vivo effects of IL-34 were assessed on tissue vasculature and macrophage infiltration in a murine preclinical model based on a paratibial inoculation of human osteosarcoma cells overexpressing or not IL-34 or M-CSF. In vitro investigations using endothelial cell precursors and mature HUVEC cells were performed to analyse the involvement of IL-34 in angiogenesis and myeloid cell adhesion. The data revealed that IL-34 overexpression was associated with the progression of osteosarcoma (tumor growth, lung metastases) and an increase of neo-angiogenesis. In vitro analyses demonstrated that IL-34 stimulated endothelial cell proliferation and vascular cord formation. Pre-treatment of endothelial cells by chondroitinases/heparinases reduced the formation of vascular tubes and abolished the associated cell signalling. In addition, IL-34 increased the in vivo recruitment of M2 tumor-associated macrophages into the tumor tissue. IL-34 increased in vitro monocyte/CD34(+) cell adhesion to activated HUVEC monolayers under physiological shear stress conditions. This work also demonstrates that IL-34 is expressed by osteosarcoma cells, is regulated by TNF-α, IL-1β, and contributes to osteosarcoma growth by increasing the neo-angiogenesis and the recruitment of M2 macrophages. By promoting new vessel formation and extravasation of immune cells, IL-34 may play a key role in tumor development and inflammatory diseases. © 2014 UICC.

Dimethyl Sulfoxide (DMSO) Increases Percentage of CXCR4(+) Hematopoietic Stem/Progenitor Cells, Their Responsiveness to an SDF-1 Gradient, Homing Capacities, and Survival.

PubMed

Jarocha, Danuta; Zuba-Surma, Ewa; Majka, Marcin

2016-01-01

Cryopreservation of bone marrow (BM), mobilized peripheral blood (mPB), and cord blood (CB) hematopoietic stem/progenitor cells (HSPCs) is a routine procedure before transplantation. The most commonly used cryoprotectant for HSPCs is dimethyl sulfoxide (DMSO). The objective of this study was to evaluate the influence of DMSO on surface receptor expression and chemotactic activities of HSPCs. We found that 10 min of incubation of human mononuclear cells (MNCs) with 10% DMSO significantly increases the percentage of CXCR4(+), CD38(+), and CD34(+) cells, resulting in an increase of CD34(+), CD34(+)CXCR4(+), and CD34(+)CXCR4(+)CD38(-) subpopulations. Furthermore, DMSO significantly increased chemotactic responsiveness of MNCs and CXCR4(+) human hematopoietic Jurkat cell line to a stromal cell-derived factor-1 (SDF-1) gradient. Furthermore, we demonstrated enhanced chemotaxis of human clonogenic progenitor cells to an SDF-1 gradient, which suggests that DMSO directly enhances the chemotactic responsiveness of early human progenitors. DMSO preincubation also caused lower internalization of the CXCR4 receptor. In parallel experiments, we found that approximately 30% more of DMSO-preincubated human CD45(+) and CD45(+)CD34(+) cells homed to the mouse BM 24 h after transplantation in comparison to control cells. Finally, we demonstrated considerably higher (25 days) survival of mice transplanted with DMSO-exposed MNCs than those transplanted with the control cells. We show in this study an unexpected beneficial influence of DMSO on HSPC homing and suggest that a short priming with DMSO before transplantation could be considered a new strategy to enhance cell homing and engraftment.

Oxygen Nanobubble Tracking by Light Scattering in Single Cells and Tissues.

PubMed

Bhandari, Pushpak; Wang, Xiaolei; Irudayaraj, Joseph

2017-03-28

Oxygen nanobubbles (ONBs) have significant potential in targeted imaging and treatment in cancer diagnosis and therapy. Precise localization and tracking of single ONBs is demonstrated based on hyperspectral dark-field microscope (HSDFM) to image and track single oxygen nanobubbles in single cells. ONBs were proposed as promising contrast-generating imaging agents due to their strong light scattering generated from nonuniformity of refractive index at the interface. With this powerful platform, we have revealed the trajectories and quantities of ONBs in cells, and demonstrated the relation between the size and diffusion coefficient. We have also evaluated the presence of ONBs in the nucleus with respect to an increase in incubation time and have quantified the uptake in single cells in ex vivo tumor tissues. Our results demonstrate that HSDFM can be a versatile platform to detect and measure cellulosic nanoparticles at the single-cell level and to assess the dynamics and trajectories of this delivery system.

Sodium Lauryl Sulfate Stimulates the Generation of Reactive Oxygen Species through Interactions with Cell Membranes.

PubMed

Mizutani, Taeko; Mori, Ryota; Hirayama, Misaki; Sagawa, Yuki; Shimizu, Kenji; Okano, Yuri; Masaki, Hitoshi

2016-12-01

Sodium lauryl sulfate (SLS), a representative anionic surfactant, is well-known to induce rough skin following single or multiple topical applications. The mechanism by which SLS induces rough skin is thought to result from the disruption of skin moisture function consisting of NMF and epidermal lipids. However, a recent study demonstrated that topically applied SLS easily penetrates into the living cell layers of the epidermis, which suggests that physiological alterations of keratinocytes might cause the SLS-induced rough skin. This study was conducted to clarify the effects of SLS on keratinocytes to demonstrate the contribution of SLS to the induction of rough skin. In addition, the potentials of other widely used anionic surfactants to induce rough skin were evaluated. HaCaT keratinocytes treated with SLS had increased levels of intracellular ROS and IL-1α secretion. Application of SLS on the surface of a reconstructed epidermal equivalent also showed the increased generation of ROS. Further, SLS-treated cells showed an increase of intracellular calpain activity associated with the increase of intracellular Ca 2+ concentration. The increase of intracellular ROS was abolished by the addition of BAPTA-AM, a specific chelator of Ca 2+ . In addition, IL-1α also stimulated ROS generation by HaCaT keratinocytes. An ESR spin-labeling study demonstrated that SLS increased the fluidity of membranes of liposomes and cells. Together, those results indicate that SLS initially interacts with cell membranes, which results in the elevation of intracellular Ca 2+ influx. Ca 2+ stimulates the secretion of IL-1α due to the activation of calpain, and also increases ROS generation. IL-1α also stimulates ROS generation by HaCaT keratinocytes. We conclude from these results that the elevation of intracellular ROS levels is one of the causes of SLS-induced rough skin. Finally, among the other anionic surfactants tested, sodium lauryl phosphate has less potential to induce rough skin because of its lower generation of ROS.

Histone Deacetylase Inhibitors Stimulate Dedifferentiation of Human Breast Cancer Cells through WNT/β-catenin Signaling

PubMed Central

Debeb, Bisrat G; Lacerda, Lara; Xu, Wei; Larson, Richard; Solley, Travis; Atkinson, Rachel; Sulman, Erik P.; Ueno, Naoto T; Krishnamurthy, Savitri; Reuben, James M; Buchholz, Thomas A; Woodward, Wendy A

2015-01-01

Recent studies have shown that differentiated cancer cells can de-differentiate into cancer stem cells (CSCs) although to date no studies have reported whether this transition is influenced by systemic anti-cancer agents. Valproic acid (VA) is a histone deacetylase (HDAC) inhibitor that promotes self renewal and expansion of hematopietic stem cells and facilitates the generation of induced pluripotent stem cells from somatic cells and is currently being investigated in breast cancer clinical trials. We hypothesized that HDAC inhibitors reprogram differentiated cancer cells towards the more resistant stem cell-like state. Two highly aggressive breast cancer cell lines, SUM159 and MDA-231, were FACS-sorted based on ALDH activity and subsequently ALDH-negative and ALDH-positive cells were treated with one of two known HDAC inhibitors, VA or SAHA (suberoylanilide hydroxamic acid). In addition, primary tumor cells from patients with metastatic breast cancer were evaluated for ALDH activity following treatment with HDAC inhibitors. We demonstrate that single cell sorted ALDH- negative cells spontaneously generated ALDH-positive cells in vitro. Treatment of ALDH-negative cells with HDAC inhibitors promoted the expansion of ALDH-positive cells and increased mammosphere forming efficiency. Most importantly, it significantly increased the tumor-initiating capacity of ALDH- negative cells in limiting dilution outgrowth assays. Moreover, while HDAC inhibitors upregulated β-catenin expression and significantly increased WNT reporter activity, a TCF4 dominant negative construct abolished HDAC-inhibitor induced expansion of CSCs. These results demonstrate that HDAC inhibitors promote the expansion of breast CSCs through dedifferentiation and have important clinical implications for the use of HDAC inhibitors in the treatment of cancer. PMID:22961641

Transplantation of autologous bone marrow-derived mesenchymal stem cells for traumatic brain injury☆

PubMed Central

Jiang, Jindou; Bu, Xingyao; Liu, Meng; Cheng, Peixun

2012-01-01

Results from the present study demonstrated that transplantation of autologous bone marrow-derived mesenchymal stem cells into the lesion site in rat brain significantly ameliorated brain tissue pathological changes and brain edema, attenuated glial cell proliferation, and increased brain-derived neurotrophic factor expression. In addition, the number of cells double-labeled for 5-bromodeoxyuridine/glial fibrillary acidic protein and cells expressing nestin increased. Finally, blood vessels were newly generated, and the rats exhibited improved motor and cognitive functions. These results suggested that transplantation of autologous bone marrow-derived mesenchymal stem cells promoted brain remodeling and improved neurological functions following traumatic brain injury. PMID:25806058

Cell death sensitization of leukemia cells by opioid receptor activation

PubMed Central

Friesen, Claudia; Roscher, Mareike; Hormann, Inis; Fichtner, Iduna; Alt, Andreas; Hilger, Ralf A.; Debatin, Klaus-Michael; Miltner, Erich

2013-01-01

Cyclic AMP (cAMP) regulates a number of cellular processes and modulates cell death induction. cAMP levels are altered upon stimulation of specific G-protein-coupled receptors inhibiting or activating adenylyl cyclases. Opioid receptor stimulation can activate inhibitory Gi-proteins which in turn block adenylyl cyclase activity reducing cAMP. Opioids such as D,L-methadone induce cell death in leukemia cells. However, the mechanism how opioids trigger apoptosis and activate caspases in leukemia cells is not understood. In this study, we demonstrate that downregulation of cAMP induced by opioid receptor activation using the opioid D,L-methadone kills and sensitizes leukemia cells for doxorubicin treatment. Enhancing cAMP levels by blocking opioid-receptor signaling strongly reduced D,L-methadone-induced apoptosis, caspase activation and doxorubicin-sensitivity. Induction of cell death in leukemia cells by activation of opioid receptors using the opioid D,L-methadone depends on critical levels of opioid receptor expression on the cell surface. Doxorubicin increased opioid receptor expression in leukemia cells. In addition, the opioid D,L-methadone increased doxorubicin uptake and decreased doxorubicin efflux in leukemia cells, suggesting that the opioid D,L-methadone as well as doxorubicin mutually increase their cytotoxic potential. Furthermore, we found that opioid receptor activation using D,L-methadone alone or in addition to doxorubicin inhibits tumor growth significantly in vivo. These results demonstrate that opioid receptor activation via triggering the downregulation of cAMP induces apoptosis, activates caspases and sensitizes leukemia cells for doxorubicin treatment. Hence, opioid receptor activation seems to be a promising strategy to improve anticancer therapies. PMID:23633472

Mechanisms for Flow-Enhanced Cell Adhesion

PubMed Central

Zhu, Cheng; Yago, Tadayuki; Lou, Jizhong; Zarnitsyna, Veronika I.; McEver, Rodger P.

2009-01-01

Cell adhesion is mediated by specific receptor—ligand bonds. In several biological systems, increasing flow has been observed to enhance cell adhesion despite the increasing dislodging fluid shear forces. Flow-enhanced cell adhesion includes several aspects: flow augments the initial tethering of flowing cells to a stationary surface, slows the velocity and increases the regularity of rolling cells, and increases the number of rollingly adherent cells. Mechanisms for this intriguing phenomenon may include transport-dependent acceleration of bond formation and force-dependent deceleration of bond dissociation. The former includes three distinct transport modes: sliding of cell bottom on the surface, Brownian motion of the cell, and rotational diffusion of the interacting molecules. The latter involves a recently demonstrated counterintuitive behavior called catch bonds where force prolongs rather than shortens the lifetimes of receptor—ligand bonds. In this article, we summarize our recently published data that used dimensional analysis and mutational analysis to elucidate the above mechanisms for flow-enhanced leukocyte adhesion mediated by L-selectinligand interactions. PMID:18299992

Whole-body irradiation increases the magnitude and persistence of adoptively transferred T cells associated with tumor regression in a mouse model of prostate cancer

PubMed Central

Ward-Kavanagh, Lindsay K.; Zhu, Junjia; Cooper, Timothy K.; Schell, Todd D.

2014-01-01

Adoptive immunotherapy has demonstrated efficacy in a subset of clinical and preclinical studies, but the T cells used for therapy often are rendered rapidly non-functional in tumor-bearing hosts. Recent evidence indicates that prostate cancer can be susceptible to immunotherapy, but most studies using autochthonous tumor models demonstrate only short-lived T-cell responses in the tolerogenic prostate microenvironment. Here, we assessed the efficacy of sublethal whole-body irradiation (WBI) to enhance the magnitude and duration of adoptively transferred CD8+ T cells in the transgenic adenocarcinoma of the mouse prostate (TRAMP) model. We demonstrate that WBI promoted high-level accumulation of granzyme B (GzB)-expressing donor T cells both in lymphoid organs and in the prostate of TRAMP mice. Donor T cells remained responsive to vaccination in irradiated recipients, but a single round of WBI-enhanced adoptive immunotherapy failed to impact significantly the existing disease. Addition of a second round of immunotherapy promoted regression of established disease in half of the treated mice, with no progressions observed. Regression was associated with long-term persistence of effector/memory phenotype CD8+ donor cells. Administration of the second round of adoptive immunotherapy led to reacquisition of GzB expression by persistent T cells from the first transfer. These results indicate that WBI conditioning amplifies tumor-specific T cells in the TRAMP prostate and lymphoid tissue, and suggest that the initial treatment alters the tolerogenic microenvironment to increase antitumor activity by a second wave of donor cells. PMID:24801834

Antitumor Effect of KX-01 through Inhibiting Src Family Kinases and Mitosis.

PubMed

Kim, Seongyeong; Min, Ahrum; Lee, Kyung-Hun; Yang, Yaewon; Kim, Tae-Yong; Lim, Jee Min; Park, So Jung; Nam, Hyun-Jin; Kim, Jung Eun; Song, Sang-Hyun; Han, Sae-Won; Oh, Do-Youn; Kim, Jee Hyun; Kim, Tae-You; Hangauer, David; Lau, Johnson Yiu-Nam; Im, Kyongok; Lee, Dong Soon; Bang, Yung-Jue; Im, Seock-Ah

2017-07-01

KX-01 is a novel dual inhibitor of Src and tubulin. Unlike previous Src inhibitors that failed to show clinical benefit during treatment of breast cancer, KX-01 can potentially overcome the therapeutic limitations of current Src inhibitors through inhibition of both Src and tubulin. The present study further evaluates the activity and mechanism of KX-01 in vitro and in vivo . The antitumor effect of KX-01 in triple negative breast cancer (TNBC) cell lines was determined by MTT assay. Wound healing and immunofluorescence assays were performed to evaluate the action mechanisms of KX-01. Changes in the cell cycle and molecular changes induced by KX-01 were also evaluated. A MDA-MB-231 mouse xenograft model was used to demonstrate the in vivo effects. KX-01 effectively inhibited the growth of breast cancer cell lines. The expression of phospho-Src and proliferative-signaling molecules were down-regulated in KX-01-sensitive TNBC cell lines. In addition, migration inhibition was observed by wound healing assay. KX-01-induced G2/M cell cycle arrest and increased the aneuploid cell population in KX-01-sensitive cell lines. Multi-nucleated cells were significantly increased after KX-01 treatment. Furthermore, KX-01 effectively delayed tumor growth in a MDA-MB-231 mouse xenograft model. KX-01 effectively inhibited cell growth and migration of TNBC cells. Moreover, this study demonstrated that KX-01 showed antitumor effects through the inhibition of Src signaling and the induction of mitotic catastrophe. The antitumor effects of KX-01 were also demonstrated in vivo using a mouse xenograft model.

Enhanced performance of a structured cyclo olefin copolymer-based amorphous silicon solar cell

NASA Astrophysics Data System (ADS)

Zhan, Xinghua; Chen, Fei; Gao, Mengyu; Tie, Shengnian; Gao, Wei

2017-07-01

The submicron array was fabricated onto a cyclo olefin copolymer (COC) film by a hot embossing method. An amorphous silicon p-i-n junction and transparent conductive layers were then deposited onto it through a plasma enhanced chemical vapor deposition (PECVD) and magnetron sputtering. The efficiency of the fabricated COC-based solar cell was measured and the result demonstrated 18.6% increase of the solar cell efficiency when compared to the sample without array structure. The imprinted polymer solar cells with submicron array indeed increase their efficiency.

Low-level light therapy potentiates NPe6-mediated photodynamic therapy in a human osteosarcoma cell line via increased ATP.

PubMed

Tsai, Shang-Ru; Yin, Rui; Huang, Ying-Ying; Sheu, Bor-Ching; Lee, Si-Chen; Hamblin, Michael R

2015-03-01

Low-level light therapy (LLLT) is used to stimulate healing, reduce pain and inflammation, and preserve tissue from dying. LLLT has been shown to protect cells in culture from dying after various cytotoxic insults, and LLLT is known to increase the cellular ATP content. Previous studies have demonstrated that maintaining a sufficiently high ATP level is necessary for the efficient induction and execution of apoptosis steps after photodynamic therapy (PDT). We asked whether LLLT would protect cells from cytotoxicity due to PDT, or conversely whether LLLT would enhance the efficacy of PDT mediated by mono-l-aspartyl chlorin(e6) (NPe6). Increased ATP could lead to enhanced cell uptake of NPe6 by the energy dependent process of endocytosis, and also to more efficient apoptosis. In this study, human osteosarcoma cell line MG-63 was subjected to 1.5J/cm(2) of 810nm near infrared radiation (NIR) followed by addition of 10μM NPe6 and after 2h incubation by 1.5J/cm(2) of 652nm red light for PDT. PDT combined with LLLT led to higher cell death and increased intracellular reactive oxygen species compared to PDT alone. The uptake of NPe6 was moderately increased by LLLT, and cellular ATP was increased. The mitochondrial respiratory chain inhibitor antimycin A abrogated the LLLT-induced increase in cytotoxicity. Taken together, these results demonstrate that LLLT potentiates NPe6-mediated PDT via increased ATP synthesis and is a potentially promising strategy that could be applied in clinical PDT. Copyright © 2014 Elsevier B.V. All rights reserved.

Metformin Induces Apoptosis and Cell Cycle Arrest Mediated by Oxidative Stress, AMPK and FOXO3a in MCF-7 Breast Cancer Cells

PubMed Central

Queiroz, Eveline A. I. F.; Puukila, Stephanie; Eichler, Rosangela; Sampaio, Sandra C.; Forsyth, Heidi L.; Lees, Simon J.; Barbosa, Aneli M.; Dekker, Robert F. H.; Fortes, Zuleica B.; Khaper, Neelam

2014-01-01

Recent studies have demonstrated that the anti-diabetic drug, metformin, can exhibit direct antitumoral effects, or can indirectly decrease tumor proliferation by improving insulin sensitivity. Despite these recent advances, the underlying molecular mechanisms involved in decreasing tumor formation are not well understood. In this study, we examined the antiproliferative role and mechanism of action of metformin in MCF-7 cancer cells treated with 10 mM of metformin for 24, 48, and 72 hours. Using BrdU and the MTT assay, it was found that metformin demonstrated an antiproliferative effect in MCF-7 cells that occurred in a time- and concentration- dependent manner. Flow cytometry was used to analyze markers of cell cycle, apoptosis, necrosis and oxidative stress. Exposure to metformin induced cell cycle arrest in G0-G1 phase and increased cell apoptosis and necrosis, which were associated with increased oxidative stress. Gene and protein expression were determined in MCF-7 cells by real time RT-PCR and western blotting, respectively. In MCF-7 cells metformin decreased the activation of IRβ, Akt and ERK1/2, increased p-AMPK, FOXO3a, p27, Bax and cleaved caspase-3, and decreased phosphorylation of p70S6K and Bcl-2 protein expression. Co-treatment with metformin and H2O2 increased oxidative stress which was associated with reduced cell number. In the presence of metformin, treating with SOD and catalase improved cell viability. Treatment with metformin resulted in an increase in p-p38 MAPK, catalase, MnSOD and Cu/Zn SOD protein expression. These results show that metformin has an antiproliferative effect associated with cell cycle arrest and apoptosis, which is mediated by oxidative stress, as well as AMPK and FOXO3a activation. Our study further reinforces the potential benefit of metformin in cancer treatment and provides novel mechanistic insight into its antiproliferative role. PMID:24858012

Circulating CXCR5+PD-1+ICOS+ Follicular T Helper Cells Are Increased Close to the Diagnosis of Type 1 Diabetes in Children With Multiple Autoantibodies.

PubMed

Viisanen, Tyyne; Ihantola, Emmi-Leena; Näntö-Salonen, Kirsti; Hyöty, Heikki; Nurminen, Noora; Selvenius, Jenni; Juutilainen, Auni; Moilanen, Leena; Pihlajamäki, Jussi; Veijola, Riitta; Toppari, Jorma; Knip, Mikael; Ilonen, Jorma; Kinnunen, Tuure

2017-02-01

Although type 1 diabetes (T1D) is primarily perceived as a T cell-driven autoimmune disease, islet autoantibodies are the best currently available biomarker for autoimmunity and disease risk. These antibodies are produced by autoreactive B cells, the activation of which is largely dependent on the function of CD4 + CXCR5 + follicular T helper cells (Tfh). In this study, we have comprehensively characterized the Tfh- as well as B-cell compartments in a large cohort of children with newly diagnosed T1D or at different stages of preclinical T1D. We demonstrate that the frequency of CXCR5 + PD-1 + ICOS + -activated circulating Tfh cells is increased both in children with newly diagnosed T1D and in autoantibody-positive at-risk children with impaired glucose tolerance. Interestingly, this increase was only evident in children positive for two or more biochemical autoantibodies. No alterations in the circulating B-cell compartment were observed in children with either prediabetes or diabetes. Our results demonstrate that Tfh activation is detectable in the peripheral blood close to the presentation of clinical T1D but only in a subgroup of children identifiable by positivity for multiple autoantibodies. These findings suggest a role for Tfh cells in the pathogenesis of human T1D and carry important implications for targeting Tfh cells and/or B cells therapeutically. © 2017 by the American Diabetes Association.

[Studies on minimum antibiotic concentration of cephapirin against clinically isolated strain SMK-101 of Klebsiella pneumoniae].

PubMed

Takahashi, M; Usui, Y; Ichiman, Y; Yoshida, K; Yonaha, T

1985-01-01

Using strain SMK-101 of K. pneumoniae its nephelometric absorbencies, viable cell numbers and morphological changes were studied during the time course cultured in a broth medium containing cephapirin (CEPR), and following results were obtained. After 1 to 3 hours culture in the presence of varying concentration of the antibiotic, the absorbency increased in spite of without change in the viable cell number. Morphologically, elongation and swelling of central portion of the cells were observed though differences of the degree of these findings varied depending upon the concentration of the antibiotic. At the concentration higher than 1/4 MIC, indistinct structure was shown in cytoplasm. After 6 hours culture, 3 directions of absorbence curves, ascending, descending and no change, and 2 directions of viable cell numbers, decreasing and increasing were shown. As the morphological changes of the cells, filamentation, leaking of intracellular components were shown in rather upper concentration of the antibiotic. Fission was demonstrated around the end of cells cultured in rather lower concentration of the antibiotic. After 9 hours culture, absorbency and viable cell number were parallel. In this period, structural findings of cytoplasm became clear and fission was also demonstrated by light microscope except for the cells cultured in more than 1 MIC of the antibiotic. After 24 hours culture, both absorbency and viable cell number increased again and fission was observed in the cell which showed filamentation in 1 MIC of the antibiotic.

Comparison of human optimized bacterial luciferase, firefly luciferase, and green fluorescent protein for continuous imaging of cell culture and animal models

NASA Astrophysics Data System (ADS)

Close, Dan M.; Hahn, Ruth E.; Patterson, Stacey S.; Baek, Seung J.; Ripp, Steven A.; Sayler, Gary S.

2011-04-01

Bioluminescent and fluorescent reporter systems have enabled the rapid and continued growth of the optical imaging field over the last two decades. Of particular interest has been noninvasive signal detection from mammalian tissues under both cell culture and whole animal settings. Here we report on the advantages and limitations of imaging using a recently introduced bacterial luciferase (lux) reporter system engineered for increased bioluminescent expression in the mammalian cellular environment. Comparison with the bioluminescent firefly luciferase (Luc) system and green fluorescent protein system under cell culture conditions demonstrated a reduced average radiance, but maintained a more constant level of bioluminescent output without the need for substrate addition or exogenous excitation to elicit the production of signal. Comparison with the Luc system following subcutaneous and intraperitoneal injection into nude mice hosts demonstrated the ability to obtain similar detection patterns with in vitro experiments at cell population sizes above 2.5 × 104 cells but at the cost of increasing overall image integration time.

Muscle stem cell intramuscular delivery within hyaluronan methylcellulose improves engraftment efficiency and dispersion.

PubMed

Davoudi, Sadegh; Chin, Chih-Ying; Cooke, Michael J; Tam, Roger Y; Shoichet, Molly S; Gilbert, Penney M

2018-04-26

Adult skeletal muscle tissue harbors the capacity for self-repair due to the presence of tissue resident muscle stem cells (MuSCs). Advances in the area of prospective MuSC isolation demonstrated the potential of cell transplantation therapy as a regenerative medicine strategy to restore strength and long-term regenerative capacity to aged, injured, or diseased skeletal muscle tissue. However, cell loss during ejection, limits to post-injection proliferation, and poor donor cell dispersion distal to the injection site are amongst hurdles to overcome to maximize MuSC transplant impact. Here, we assess a physical blend of hyaluronan and methylcellulose (HAMC) as a bioactive, shear thinning hydrogel cell delivery system to improve MuSC transplantation efficiency. Using in vivo transplantation studies, we found that the HAMC delivery system results in a >45% increase in the number of donor-derived fibers as compared to saline delivery. We demonstrate that increases in donor-derived fibers when using HAMC are attributed to increased MuSC proliferation via a CD44-independent mechanism, preventing injected cell active clearance, and supporting in vivo expansion by delaying differentiation. Furthermore, we observed a significant improvement in donor fiber dispersion when MuSCs were delivered in HAMC. Our study results suggest that HAMC is a promising muscle stem cell delivery vehicle. Copyright © 2018 Elsevier Ltd. All rights reserved.

Colon Cancer-Upregulated Long Non-Coding RNA lincDUSP Regulates Cell Cycle Genes and Potentiates Resistance to Apoptosis.

PubMed

Forrest, Megan E; Saiakhova, Alina; Beard, Lydia; Buchner, David A; Scacheri, Peter C; LaFramboise, Thomas; Markowitz, Sanford; Khalil, Ahmad M

2018-05-09

Long non-coding RNAs (lncRNAs) are frequently dysregulated in many human cancers. We sought to identify candidate oncogenic lncRNAs in human colon tumors by utilizing RNA sequencing data from 22 colon tumors and 22 adjacent normal colon samples from The Cancer Genome Atlas (TCGA). The analysis led to the identification of ~200 differentially expressed lncRNAs. Validation in an independent cohort of normal colon and patient-derived colon cancer cell lines identified a novel lncRNA, lincDUSP, as a potential candidate oncogene. Knockdown of lincDUSP in patient-derived colon tumor cell lines resulted in significantly decreased cell proliferation and clonogenic potential, and increased susceptibility to apoptosis. The knockdown of lincDUSP affects the expression of ~800 genes, and NCI pathway analysis showed enrichment of DNA damage response and cell cycle control pathways. Further, identification of lincDUSP chromatin occupancy sites by ChIRP-Seq demonstrated association with genes involved in the replication-associated DNA damage response and cell cycle control. Consistent with these findings, lincDUSP knockdown in colon tumor cell lines increased both the accumulation of cells in early S-phase and γH2AX foci formation, indicating increased DNA damage response induction. Taken together, these results demonstrate a key role of lincDUSP in the regulation of important pathways in colon cancer.

Improved FCG-1 cell technology

NASA Astrophysics Data System (ADS)

Breault, R. D.; Congdon, J. V.; Coykendall, R. D.; Luoma, W. L.

1980-10-01

Fuel cell performance in the ribbed substrate cell configuration consistent with that projected for a commercial power plant is demonstrated. Tests were conducted on subscale cells and on two 20 cell stacks of 4.8 MW demonstrator size cell components. These tests evaluated cell stack materials, processes, components, and assembly configurations. The first task was to conduct a component development effort to introduce improvements in 3.7 square foot, ribbed substrate acid cell repeating parts which represented advances in performance, function, life, and lower cost for application in higher pressure and temperature power plants. Specific areas of change were the electrode substrate, catalyst, matrix, seals, separator plates, and coolers. Full sized ribbed substrate stack components incorporating more stable materials were evaluated at increased pressure (93 psia) and temperature (405 F) conditions. Two 20 cell stacks with a 3.7 square feet, ribbed substrate cell configuration were tested.

Tanshinone IIA Increases the Bystander Effect of Herpes Simplex Virus Thymidine Kinase/Ganciclovir Gene Therapy via Enhanced Gap Junctional Intercellular Communication

PubMed Central

Liu, Xijuan; Wu, Yingya; Du, Biaoyan; Li, Jiefen; Zhou, Jing; Li, Jingjing; Tan, Yuhui

2013-01-01

The bystander effect is an intriguing phenomenon by which adjacent cells become sensitized to drug treatment during gene therapy with herpes simplex virus thymidine kinase/ganciclovir (HSV-tk/GCV). This effect is reported to be mediated by gap junctional intercellular communication (GJIC), and therefore, we postulated that upregulation of genes that facilitate GJIC may enhance the HSV-tk/GCV bystander effect. Previous findings have shown Tanshinone IIA (Tan IIA), a chemical substance derived from a Chinese medicine herb, promotes the upregulation of the connexins Cx26 and Cx43 in B16 cells. Because gap junctions are formed by connexins, we hypothesized that Tan IIA might increase GJIC. Our results show that Tan IIA increased GJIC in B16 melanoma cells, leading to more efficient GCV-induced bystander killing in cells stably expressing HSV-tk. Additionally, in vivo experiments demonstrated that tumors in mice with 10% HSV-tk positive B16 cells and 90% wild-type B16 cells became smaller following treatment with the combination of GCV and Tan IIA as compared to GCV or Tan IIA alone. These data demonstrate that Tan IIA can augment the bystander effect of HSV-tk/GCV system through increased gap junction coupling, which adds strength to the promising strategy that develops connexins inducer to potentiate the effects of suicide gene therapy. PMID:23861780

Adipokine regulation of colon cancer: adiponectin attenuates interleukin-6-induced colon carcinoma cell proliferation via STAT-3

PubMed Central

Fenton, Jenifer I; Birmingham, Janette M

2010-01-01

Obesity results in increased circulating levels of specific adipokines which are associated with colon cancer risk. The disease state is associated with increased leptin, insulin, IGF-1, and IL-6. Conversely, adiponectin levels are decreased in obese individuals. Previously, we demonstrated adipokine-enhanced cell proliferation in preneoplastic, but not normal, colon epithelial cells, demonstrating a differential effect of adipokines on colon cancer progression in vitro. Using a model of late stage carcinoma cancer cell, namely murine MC-38 colon carcinoma cells, we compared the effect of obesity-associated adipokines (leptin, insulin and IGF-1 and IL-6) on MC-38 cell proliferation and determined whether adiponectin (full length or globular) could modulate adipokine-induced cell proliferation. We show that insulin and IL-6, but not leptin and IGF-1, induce proliferation in MC-38 cells. Adiponectin treatment of MC-38 cells did not inhibit insulin-induced cell proliferation but did inhibit IL-6-induced cell proliferation by decreasing STAT-3 phosphorylation and activation. Nitric oxide (NO) production was increased in MC-38 cells treated with IL-6; co-treatment with adiponectin blocked IL-6 induced iNOS and subsequent NO production. These data are compared to previously reported findings from our laboratory using the YAMC (model normal colon epithelial cells) and IMCE (model preneoplastic) cells. The cell lines are utilized to construct a model summarizing the hormonal consequences of obesity and the impact on the differential regulation of colon epithelial cells along the continuum to carcinoma. These data, taken together, highlight mechanisms involved in obesity-associated cancers and may lead to potential targeted therapies. PMID:20564347

Activation of the mitogen-activated protein kinase pathway by bone sialoprotein regulates osteoblast differentiation.

PubMed

Gordon, Jonathan A R; Hunter, Graeme K; Goldberg, Harvey A

2009-01-01

Bone sialoprotein (BSP) is an abundant protein in the extracellular matrix of bone that has been suggested to have several different physiological functions, including the nucleation of hydroxyapatite (HA), promotion of cell attachment and binding of collagen. Studies in our lab have demonstrated that increased expression of BSP in osteoblast cells can increase expression of the osteoblast-related genes Runx2 and Osx as well as alkaline phosphatase and osteocalcin and increase matrix mineralization. To determine the molecular mechanisms responsible for the BSP-mediated increase in osteoblastic differentiation, several functional domain mutants of BSP were expressed in primary rat bone osteoblastic cells, including the contiguous glutamic acid sequences (polyGlu) and the arginine-glycine-aspartic acid (RGD) motif. Markers of osteoblast differentiation, including matrix mineralization and alkaline phosphatase staining, were increased in cells expressing BSP mutants of the polyGlu sequences but not in cells expressing RGD-mutated BSP. We also determined the dependence on integrin-associated pathways in promoting BSP-mediated differentiation responses in osteoblasts by demonstrating the activation of focal adhesion kinase, MAP kinase-associated proteins ERK1/2, ribosomal s6 kinase 2 and the AP-1 protein cFos. Thus, the mechanism regulating osteoblast differentiation by BSP was determined to be dependent on integrin-mediated intracellular signaling pathways. Copyright 2008 S. Karger AG, Basel.

Cancer cells recovering from damage exhibit mitochondrial restructuring and increased aerobic glycolysis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Akakura, Shin; Ostrakhovitch, Elena; Sanokawa-Akakura, Reiko

2014-06-13

Highlights: • Some cancer cells recover from severe damage that causes cell death in majority of cells. • Damage-Recovered (DR) cancer cells show reduced mitochondria, mDNA and mitochondrial enzymes. • DR cells show increased aerobic glycolysis, ATP, cell proliferation, and resistance to damage. • DR cells recovered from in vivo damage also show increased glycolysis and proliferation rate. - Abstract: Instead of relying on mitochondrial oxidative phosphorylation, most cancer cells rely heavily on aerobic glycolysis, a phenomenon termed as “the Warburg effect”. We considered that this effect is a direct consequence of damage which persists in cancer cells that recovermore » from damage. To this end, we studied glycolysis and rate of cell proliferation in cancer cells that recovered from severe damage. We show that in vitro Damage-Recovered (DR) cells exhibit mitochondrial structural remodeling, display Warburg effect, and show increased in vitro and in vivo proliferation and tolerance to damage. To test whether cancer cells derived from tumor microenvironment can show similar properties, we isolated Damage-Recovered (T{sup DR}) cells from tumors. We demonstrate that T{sup DR} cells also show increased aerobic glycolysis and a high proliferation rate. These findings show that Warburg effect and its consequences are induced in cancer cells that survive severe damage.« less

Ultra-low dose naltrexone attenuates chronic morphine-induced gliosis in rats.

PubMed

Mattioli, Theresa-Alexandra M; Milne, Brian; Cahill, Catherine M

2010-04-16

The development of analgesic tolerance following chronic morphine administration can be a significant clinical problem. Preclinical studies demonstrate that chronic morphine administration induces spinal gliosis and that inhibition of gliosis prevents the development of analgesic tolerance to opioids. Many studies have also demonstrated that ultra-low doses of naltrexone inhibit the development of spinal morphine antinociceptive tolerance and clinical studies demonstrate that it has opioid sparing effects. In this study we demonstrate that ultra-low dose naltrexone attenuates glial activation, which may contribute to its effects on attenuating tolerance. Spinal cord sections from rats administered chronic morphine showed significantly increased immuno-labelling of astrocytes and microglia compared to saline controls, consistent with activation. 3-D images of astrocytes from animals administered chronic morphine had significantly larger volumes compared to saline controls. Co-injection of ultra-low dose naltrexone attenuated this increase in volume, but the mean volume differed from saline-treated and naltrexone-treated controls. Astrocyte and microglial immuno-labelling was attenuated in rats co-administered ultra-low dose naltrexone compared to morphine-treated rats and did not differ from controls. Glial activation, as characterized by immunohistochemical labelling and cell size, was positively correlated with the extent of tolerance developed. Morphine-induced glial activation was not due to cell proliferation as there was no difference observed in the total number of glial cells following chronic morphine treatment compared to controls. Furthermore, using 5-bromo-2-deoxyuridine, no increase in spinal cord cell proliferation was observed following chronic morphine administration. Taken together, we demonstrate a positive correlation between the prevention of analgesic tolerance and the inhibition of spinal gliosis by treatment with ultra-low dose naltrexone. This research provides further validation for using ultra-low dose opioid receptor antagonists in the treatment of various pain syndromes.

Latent KSHV Infected Endothelial Cells Are Glutamine Addicted and Require Glutaminolysis for Survival

PubMed Central

Sanchez, Erica L.; Carroll, Patrick A.; Thalhofer, Angel B.; Lagunoff, Michael

2015-01-01

Kaposi’s Sarcoma-associated Herpesvirus (KSHV) is the etiologic agent of Kaposi’s Sarcoma (KS). KSHV establishes a predominantly latent infection in the main KS tumor cell type, the spindle cell, which is of endothelial cell origin. KSHV requires the induction of multiple metabolic pathways, including glycolysis and fatty acid synthesis, for the survival of latently infected endothelial cells. Here we demonstrate that latent KSHV infection leads to increased levels of intracellular glutamine and enhanced glutamine uptake. Depletion of glutamine from the culture media leads to a significant increase in apoptotic cell death in latently infected endothelial cells, but not in their mock-infected counterparts. In cancer cells, glutamine is often required for glutaminolysis to provide intermediates for the tri-carboxylic acid (TCA) cycle and support for the production of biosynthetic and bioenergetic precursors. In the absence of glutamine, the TCA cycle intermediates alpha-ketoglutarate (αKG) and pyruvate prevent the death of latently infected cells. Targeted drug inhibition of glutaminolysis also induces increased cell death in latently infected cells. KSHV infection of endothelial cells induces protein expression of the glutamine transporter, SLC1A5. Chemical inhibition of SLC1A5, or knockdown by siRNA, leads to similar cell death rates as glutamine deprivation and, similarly, can be rescued by αKG. KSHV also induces expression of the heterodimeric transcription factors c-Myc-Max and related heterodimer MondoA-Mlx. Knockdown of MondoA inhibits expression of both Mlx and SLC1A5 and induces a significant increase in cell death of only cells latently infected with KSHV, again, fully rescued by the supplementation of αKG. Therefore, during latent infection of endothelial cells, KSHV activates and requires the Myc/MondoA-network to upregulate the glutamine transporter, SLC1A5, leading to increased glutamine uptake for glutaminolysis. These findings expand our understanding of the required metabolic pathways that are activated during latent KSHV infection of endothelial cells, and demonstrate a novel role for the extended Myc-regulatory network, specifically MondoA, during latent KSHV infection. PMID:26197457

Intact LKB1 activity is required for survival of dormant ovarian cancer spheroids.

PubMed

Peart, Teresa; Ramos Valdes, Yudith; Correa, Rohann J M; Fazio, Elena; Bertrand, Monique; McGee, Jacob; Préfontaine, Michel; Sugimoto, Akira; DiMattia, Gabriel E; Shepherd, Trevor G

2015-09-08

Metastatic epithelial ovarian cancer (EOC) cells can form multicellular spheroids while in suspension and disperse directly throughout the peritoneum to seed secondary lesions. There is growing evidence that EOC spheroids are key mediators of metastasis, and they use specific intracellular signalling pathways to control cancer cell growth and metabolism for increased survival. Our laboratory discovered that AKT signalling is reduced during spheroid formation leading to cellular quiescence and autophagy, and these may be defining features of tumour cell dormancy. To further define the phenotype of EOC spheroids, we have initiated studies of the Liver kinase B1 (LKB1)-5'-AMP-activated protein kinase (AMPK) pathway as a master controller of the metabolic stress response. We demonstrate that activity of AMPK and its upstream kinase LKB1 are increased in quiescent EOC spheroids as compared with proliferating adherent EOC cells. We also show elevated AMPK activity in spheroids isolated directly from patient ascites. Functional studies reveal that treatment with the AMP mimetic AICAR or allosteric AMPK activator A-769662 led to a cytostatic response in proliferative adherent ovarian cancer cells, but they fail to elicit an effect in spheroids. Targeted knockdown of STK11 by RNAi to reduce LKB1 expression led to reduced viability and increased sensitivity to carboplatin treatment in spheroids only, a phenomenon which was AMPK-independent. Thus, our results demonstrate a direct impact of altered LKB1-AMPK signalling function in EOC. In addition, this is the first evidence in cancer cells demonstrating a pro-survival function for LKB1, a kinase traditionally thought to act as a tumour suppressor.

NFAT5 Is Activated by Hypoxia: Role in Ischemia and Reperfusion in the Rat Kidney

PubMed Central

Villanueva, Sandra; Suazo, Cristian; Santapau, Daniela; Pérez, Francisco; Quiroz, Mariana; Carreño, Juan E.; Illanes, Sebastián; Lavandero, Sergio; Michea, Luis; Irarrazabal, Carlos E.

2012-01-01

The current hypothesis postulates that NFAT5 activation in the kidney's inner medulla is due to hypertonicity, resulting in cell protection. Additionally, the renal medulla is hypoxic (10–18 mmHg); however there is no information about the effect of hypoxia on NFAT5. Using in vivo and in vitro models, we evaluated the effect of reducing the partial pressure of oxygen (PO2) on NFAT5 activity. We found that 1) Anoxia increased NFAT5 expression and nuclear translocation in primary cultures of IMCD cells from rat kidney. 2) Anoxia increased transcriptional activity and nuclear translocation of NFAT5 in HEK293 cells. 3) The dose-response curve demonstrated that HIF-1α peaked at 2.5% and NFAT5 at 1% of O2. 4) At 2.5% of O2, the time-course curve of hypoxia demonstrated earlier induction of HIF-1α gene expression than NFAT5. 5) siRNA knockdown of NFAT5 increased the hypoxia-induced cell death. 6) siRNA knockdown of HIF-1α did not affect the NFAT5 induction by hypoxia. Additionally, HIF-1α was still induced by hypoxia even when NFAT5 was knocked down. 7) NFAT5 and HIF-1α expression were increased in kidney (cortex and medulla) from rats subjected to an experimental model of ischemia and reperfusion (I/R). 7) Experimental I/R increased the NFAT5-target gene aldose reductase (AR). 8) NFAT5 activators (ATM and PI3K) were induced in vitro (HEK293 cells) and in vivo (I/R kidneys) with the same timing of NFAT5. 8) Wortmannin, which inhibits ATM and PI3K, reduces hypoxia-induced NFAT5 transcriptional activation in HEK293 cells. These results demonstrate for the first time that NFAT5 is induced by hypoxia and could be a protective factor against ischemic damage. PMID:22768306

Voltage Preconditioning Allows Modulated Gene Expression in Neurons Using PEI-complexed siRNA

PubMed Central

Sridharan, Arati; Patel, Chetan; Muthuswamy, Jit

2013-01-01

We present here a high efficiency, high viability siRNA-delivery method using a voltage-controlled chemical transfection strategy to achieve modulated delivery of polyethylenimine (PEI) complexed with siRNA in an in vitro culture of neuro2A cells and neurons. Low voltage pulses were applied to adherent cells before the administration of PEI-siRNA complexes. Live assays of neuro2a cells transfected with fluorescently tagged siRNA showed an increase in transfection efficiency from 62 ± 14% to 98 ± 3.8% (after −1 V). In primary hippocampal neurons, transfection efficiencies were increased from 30 ± 18% to 76 ± 18% (after −1 V). Negligible or low-level transfection was observed after preconditioning at higher voltages, suggesting an inverse relationship with applied voltage. Experiments with propidium iodide ruled out the role of electroporation in the transfection of siRNAs suggesting an alternate electro-endocytotic mechanism. In addition, image analysis of preconditioned and transfected cells demonstrates siRNA uptake and loading that is tuned to preconditioning voltage levels. There is approximately a fourfold increase in siRNA loading after preconditioning at −1 V compared with the same at ±2–3 V. Modulated gene expression is demonstrated in a functional knockdown of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) in neuro2A cells using siRNA. Cell density and dendritic morphological changes are also demonstrated in modulated knockdown of brain derived neurotrophic factor (BDNF) in primary hippocampal neurons. The method reported here has potential applications in the development of high-throughput screening systems for large libraries of siRNA molecules involving difficult-to-transfect cells like neurons. PMID:23531602

Voltage Preconditioning Allows Modulated Gene Expression in Neurons Using PEI-complexed siRNA.

PubMed

Sridharan, Arati; Patel, Chetan; Muthuswamy, Jit

2013-03-26

We present here a high efficiency, high viability siRNA-delivery method using a voltage-controlled chemical transfection strategy to achieve modulated delivery of polyethylenimine (PEI) complexed with siRNA in an in vitro culture of neuro2A cells and neurons. Low voltage pulses were applied to adherent cells before the administration of PEI-siRNA complexes. Live assays of neuro2a cells transfected with fluorescently tagged siRNA showed an increase in transfection efficiency from 62 ± 14% to 98 ± 3.8% (after -1 V). In primary hippocampal neurons, transfection efficiencies were increased from 30 ± 18% to 76 ± 18% (after -1 V). Negligible or low-level transfection was observed after preconditioning at higher voltages, suggesting an inverse relationship with applied voltage. Experiments with propidium iodide ruled out the role of electroporation in the transfection of siRNAs suggesting an alternate electro-endocytotic mechanism. In addition, image analysis of preconditioned and transfected cells demonstrates siRNA uptake and loading that is tuned to preconditioning voltage levels. There is approximately a fourfold increase in siRNA loading after preconditioning at -1 V compared with the same at ±2-3 V. Modulated gene expression is demonstrated in a functional knockdown of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) in neuro2A cells using siRNA. Cell density and dendritic morphological changes are also demonstrated in modulated knockdown of brain derived neurotrophic factor (BDNF) in primary hippocampal neurons. The method reported here has potential applications in the development of high-throughput screening systems for large libraries of siRNA molecules involving difficult-to-transfect cells like neurons.Molecular Therapy-Nucleic Acids (2013) 2, e82; doi:10.1038/mtna.2013.10; published online 26 March 2013.

Compound C induces protective autophagy in human cholangiocarcinoma cells via Akt/mTOR-independent pathway.

PubMed

Zhao, Xiaofang; Luo, Guosong; Cheng, Ying; Yu, Wenjing; Chen, Run; Xiao, Bin; Xiang, Yuancai; Feng, Chunhong; Fu, Wenguang; Duan, Chunyan; Yao, Fuli; Xia, Xianming; Tao, Qinghua; Wei, Mei; Dai, Rongyang

2018-07-01

Compound C, a well-known inhibitor of AMP-activated protein kinase (AMPK), has been reported to exert antitumor activities in some types of cells. Whether compound C can exert antitumor effects in human cholangiocarcinoma (CCA) remains unknown. Here, we demonstrated that compound C is a potent inducer of cell death and autophagy in human CCA cells. Autophagy inhibitors increased the cytotoxicity of compound C towards human CCA cells, as confirmed by increased LDH release, and PARP cleavage. It is notable that compound C treatment increased phosphorylated Akt, sustained high levels of phosphorylated p70S6K, and decreased mTOR regulated p-ULK1 (ser757). Based on the data that blocking PI3K/Akt or mTOR had no apparent influence on autophagic response, we suggest that compound C induces autophagy independent of Akt/mTOR signaling in human CCA cells. Further study demonstrated that compound C inhibited the phosphorylation of JNK and its target c-Jun. Blocking JNK by SP600125 or siRNA suppressed autophagy induction upon compound C treatment. Moreover, compound C induced p38 MAPK activation, and its inhibition promoted autophagy induction via JNK activation. In addition, compound C induced p53 expression, and its inhibition attenuated compound C-induced autophagic response. Thus, compound C triggers autophagy, at least in part, via the JNK and p53 pathways in human CCA cells. In conclusion, suppresses autophagy could increase compound C sensitivity in human CCA. © 2018 Wiley Periodicals, Inc.

Allosteric Regulation of E-Cadherin Adhesion.

PubMed

Shashikanth, Nitesh; Petrova, Yuliya I; Park, Seongjin; Chekan, Jillian; Maiden, Stephanie; Spano, Martha; Ha, Taekjip; Gumbiner, Barry M; Leckband, Deborah E

2015-08-28

Cadherins are transmembrane adhesion proteins that maintain intercellular cohesion in all tissues, and their rapid regulation is essential for organized tissue remodeling. Despite some evidence that cadherin adhesion might be allosterically regulated, testing of this has been hindered by the difficulty of quantifying altered E-cadherin binding affinity caused by perturbations outside the ectodomain binding site. Here, measured kinetics of cadherin-mediated intercellular adhesion demonstrated quantitatively that treatment with activating, anti-E-cadherin antibodies or the dephosphorylation of a cytoplasmic binding partner, p120(ctn), increased the homophilic binding affinity of E-cadherin. Results obtained with Colo 205 cells, which express inactive E-cadherin and do not aggregate, demonstrated that four treatments, which induced Colo 205 aggregation and p120(ctn) dephosphorylation, triggered quantitatively similar increases in E-cadherin affinity. Several processes can alter cell aggregation, but these results directly demonstrated the allosteric regulation of cell surface E-cadherin by p120(ctn) dephosphorylation. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Chamomile confers protection against hydrogen peroxide-induced toxicity through activation of Nrf2-mediated defense response.

PubMed

Bhaskaran, Natarajan; Srivastava, Janmejai K; Shukla, Sanjeev; Gupta, Sanjay

2013-01-01

Oxidative stress plays an important role in the development of various human diseases. Aqueous chamomile extract is used as herbal medicine, in the form of tea, demonstrated to possess antiinflammatory and antioxidant properties. We demonstrate the cytoprotective effects of chamomile on hydrogen peroxide (H₂O₂)-induced cellular damage in macrophage RAW 264.7 cells. Pretreatment of cells with chamomile markedly attenuated H₂O₂-induced cell viability loss in a dose-dependent manner. The mechanisms by which chamomile-protected macrophages from oxidative stress was through the induction of several antioxidant enzymes including NAD(P)H:quinone oxidoreductase, superoxide dismutase, and catalase and increase nuclear accumulation of the transcription factor Nrf2 and its binding to antioxidant response elements. Furthermore, chamomile dose-dependently reduced H₂O₂-mediated increase in the intracellular levels of reactive oxygen species. Our results, for the first time, demonstrate that chamomile has protective effects against oxidative stress and might be beneficial to provide defense against cellular damage. Copyright © 2012 John Wiley & Sons, Ltd.

Chamomile Confers Protection against Hydrogen Peroxide-Induced Toxicity through Activation of Nrf2-Mediated Defense Response

PubMed Central

Bhaskaran, Natarajan; Srivastava, Janmejai K.; Shukla, Sanjeev; Gupta, Sanjay

2014-01-01

Oxidative stress plays an important role in the development of various human diseases. Aqueous chamomile extract is used as herbal medicine, in the form of tea, demonstrated to possess antiinflammatory and antioxidant properties. We demonstrate the cytoprotective effects of chamomile on hydrogen peroxide (H2O2)-induced cellular damage in macrophage RAW 264.7 cells. Pretreatment of cells with chamomile markedly attenuated H2O2-induced cell viability loss in a dose-dependent manner. The mechanisms by which chamomile-protected macrophages from oxidative stress was through the induction of several antioxidant enzymes including NAD (P)H:quinone oxidoreductase, superoxide dismutase, and catalase and increase nuclear accumulation of the transcription factor Nrf2 and its binding to antioxidant response elements. Furthermore, chamomile dose-dependently reduced H2O2-mediated increase in the intracellular levels of reactive oxygen species. Our results, for the first time, demonstrate that chamomile has protective effects against oxidative stress and might be beneficial to provide defense against cellular damage. PMID:22511316

Radioprotection by polysaccharides alone and in combination with aminothiols

NASA Astrophysics Data System (ADS)

Patchen, Myra L.; Macvittie, Thomas J.; Solberg, Brian D.; D'Alesandro, Michele M.; Brook, Itzhak

We demonstrated that glucan, a beta-1,3 polysaccharide immunomodulator, enhances survival of mice when administered before radiation exposure. Glucan's prophylactic survival-enhancing effects are mediated by several mechanisms including (1) increasing macrophage-mediated resistance to potentially lethal postirradiation opportunistic infections, (2) increasing the Do of hematopoietic progenitor cells, and (3) accelerating hematopoietic reconstitution. In addition, even when administered shortly after some otherwise lethal doses of radiation, glucan increases survival. Glucan's therapeutic survival-enhancing effects are also mediated through its ability to enhance macrophage function and to accelerate hematopoietic reconstitution; glucan's therapeutic potential, however, is ultimately dependent on the survival of a critical number of hematopoietic stem cells capable of responding to glucan's stimulatory effects. Preirradiation administration of the traditional aminothiol radioprotectants WR-2721 and WR-3689 has been previously demonstrated to be an extremely effective means to increase hematopoietic stem cell survival. Therapeutic glucan treatment administered in combination with preirradiation WR-2721 or WR-3689 treatment synergistically increases both hematopoietic reconstitution and survival. Such combined modality treatments offer new promise in treating acute radiation injury.

Mena invasive (MenaINV) promotes multicellular streaming motility and transendothelial migration in a mouse model of breast cancer

PubMed Central

Roussos, Evanthia T.; Balsamo, Michele; Alford, Shannon K.; Wyckoff, Jeffrey B.; Gligorijevic, Bojana; Wang, Yarong; Pozzuto, Maria; Stobezki, Robert; Goswami, Sumanta; Segall, Jeffrey E.; Lauffenburger, Douglas A.; Bresnick, Anne R.; Gertler, Frank B.; Condeelis, John S.

2011-01-01

We have shown previously that distinct Mena isoforms are expressed in invasive and migratory tumor cells in vivo and that the invasion isoform (MenaINV) potentiates carcinoma cell metastasis in murine models of breast cancer. However, the specific step of metastatic progression affected by this isoform and the effects on metastasis of the Mena11a isoform, expressed in primary tumor cells, are largely unknown. Here, we provide evidence that elevated MenaINV increases coordinated streaming motility, and enhances transendothelial migration and intravasation of tumor cells. We demonstrate that promotion of these early stages of metastasis by MenaINV is dependent on a macrophage–tumor cell paracrine loop. Our studies also show that increased Mena11a expression correlates with decreased expression of colony-stimulating factor 1 and a dramatically decreased ability to participate in paracrine-mediated invasion and intravasation. Our results illustrate the importance of paracrine-mediated cell streaming and intravasation on tumor cell dissemination, and demonstrate that the relative abundance of MenaINV and Mena11a helps to regulate these key stages of metastatic progression in breast cancer cells. PMID:21670198

Maintenance of CCL5 mRNA stores by post-effector and memory CD8 T cells is dependent on transcription and is coupled to increased mRNA stability.

PubMed

Marçais, Antoine; Tomkowiak, Martine; Walzer, Thierry; Coupet, Charles-Antoine; Ravel-Chapuis, Aymeric; Marvel, Jacqueline

2006-10-01

Immunological memory is associated with the display of improved effector functions by cells of the adaptive immune system. The storage of untranslated mRNA coding for the CCL5 chemokine by CD8 memory cells is a new process supporting the immediate display of an effector function. Here, we show that, after induction during the primary response, high CCL5 mRNA levels are specifically preserved in CD8 T cells. We have investigated the mechanisms involved in the long-term maintenance of CCL5 mRNA levels by memory CD8 T cells. We demonstrate that the CCL5 mRNA half-life is increased in memory CD8 T cells and that these cells constitutively transcribe ccl5 gene. By inhibiting ccl5 transcription using IL-4, we demonstrate the essential role of transcription in the maintenance of CCL5 mRNA stores. Finally, we show that these stores are spontaneously reconstituted when the inhibitory signal is removed, indicating that the transcription of ccl5 is a default feature of memory CD8 T cells imprinted in their genetic program.

Mena invasive (MenaINV) promotes multicellular streaming motility and transendothelial migration in a mouse model of breast cancer.

PubMed

Roussos, Evanthia T; Balsamo, Michele; Alford, Shannon K; Wyckoff, Jeffrey B; Gligorijevic, Bojana; Wang, Yarong; Pozzuto, Maria; Stobezki, Robert; Goswami, Sumanta; Segall, Jeffrey E; Lauffenburger, Douglas A; Bresnick, Anne R; Gertler, Frank B; Condeelis, John S

2011-07-01

We have shown previously that distinct Mena isoforms are expressed in invasive and migratory tumor cells in vivo and that the invasion isoform (Mena(INV)) potentiates carcinoma cell metastasis in murine models of breast cancer. However, the specific step of metastatic progression affected by this isoform and the effects on metastasis of the Mena11a isoform, expressed in primary tumor cells, are largely unknown. Here, we provide evidence that elevated Mena(INV) increases coordinated streaming motility, and enhances transendothelial migration and intravasation of tumor cells. We demonstrate that promotion of these early stages of metastasis by Mena(INV) is dependent on a macrophage-tumor cell paracrine loop. Our studies also show that increased Mena11a expression correlates with decreased expression of colony-stimulating factor 1 and a dramatically decreased ability to participate in paracrine-mediated invasion and intravasation. Our results illustrate the importance of paracrine-mediated cell streaming and intravasation on tumor cell dissemination, and demonstrate that the relative abundance of Mena(INV) and Mena11a helps to regulate these key stages of metastatic progression in breast cancer cells.

Bacterial Cell Enlargement Requires Control of Cell Wall Stiffness Mediated by Peptidoglycan Hydrolases

PubMed Central

Wheeler, Richard; Turner, Robert D.; Bailey, Richard G.; Salamaga, Bartłomiej; Mesnage, Stéphane; Mohamad, Sharifah A. S.; Hayhurst, Emma J.; Horsburgh, Malcolm; Hobbs, Jamie K.

2015-01-01

ABSTRACT Most bacterial cells are enclosed in a single macromolecule of the cell wall polymer, peptidoglycan, which is required for shape determination and maintenance of viability, while peptidoglycan biosynthesis is an important antibiotic target. It is hypothesized that cellular enlargement requires regional expansion of the cell wall through coordinated insertion and hydrolysis of peptidoglycan. Here, a group of (apparent glucosaminidase) peptidoglycan hydrolases are identified that are together required for cell enlargement and correct cellular morphology of Staphylococcus aureus, demonstrating the overall importance of this enzyme activity. These are Atl, SagA, ScaH, and SagB. The major advance here is the explanation of the observed morphological defects in terms of the mechanical and biochemical properties of peptidoglycan. It was shown that cells lacking groups of these hydrolases have increased surface stiffness and, in the absence of SagB, substantially increased glycan chain length. This indicates that, beyond their established roles (for example in cell separation), some hydrolases enable cellular enlargement by making peptidoglycan easier to stretch, providing the first direct evidence demonstrating that cellular enlargement occurs via modulation of the mechanical properties of peptidoglycan. PMID:26220963

Cell Motility and Invasiveness of Neurofibromin-Deficient Neural Crest Cells and Malignant Triton Tumor Lines

DTIC Science & Technology

2005-06-01

derived cells, we isolated first branchial arch mesenchymal populations, as well as trigeminal ganglion non-neuronal cells, from mouse embryos and measured...demonstrate that loss of neurofibromin affects the invasiveness of neural crest-derived (trigeminal ganglion) and cranial mesenchymal ( branchial arch) cell...trigeminal and branchial arch cells between El0 and El 2 indicates that the roles of neurofibromin in controlling motility may become increasingly

Expansion of myeloid immune suppressor Gr+CD11b+ cells in tumor-bearing host directly promotes tumor angiogenesis | Center for Cancer Research

Cancer.gov

We demonstrate a novel tumor-promoting role of myeloid immune suppressor Gr+CD11b+ cells, which are evident in cancer patients and tumor-bearing animals. These cells constitute approximately 5% of total cells in tumors. Tumors coinjected with Gr+CD11b+ cells exhibited increased vascular density, vascular maturation, and decreased necrosis. These immune cells produce high

Direct stimulation of immediate-early genes by intranuclear insulin in trypsin-treated H35 hepatoma cells.

PubMed Central

Lin, Y J; Harada, S; Loten, E G; Smith, R M; Jarett, L

1992-01-01

H35 hepatoma cells were treated with trypsin to abolish insulin binding and insulin-stimulated receptor kinase activity. Insulin was, however, internalized by fluid-phase endocytosis in trypsin-treated cells. Furthermore, nuclear accumulation of insulin was similar in control and trypsin-treated hepatoma cells. Northern blot analysis revealed insulin increased g33 and c-fos mRNA concentrations identically in control and trypsin-treated cells but had no effect on beta 2-microglobulin mRNA. Actinomycin D treatment prior to or after insulin addition demonstrated that insulin increased gene transcription and had no effect on mRNA degradation. These studies suggest that the accumulation of intact insulin in cell nuclei may be directly involved in the increased transcription of immediate-early genes. Images PMID:1409684

Effects and mechanisms of melatonin on the proliferation and neural differentiation of PC12 cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Liu, Yumei; Zhang, Ziqiang; Lv, Qiongxia

Melatonin, a lipophilic molecule that is mainly synthesized in the pineal gland, performs various neuroprotective functions. However, the detailed role and mechanisms of promoting neuronal differentiation remains limited. This study demonstrated that 10 μM melatonin led to significant increases in the proliferation and neurite outgrowth of PC12 cells. Increased expression of microtubule-associated protein 2 (MAP2, a neuron-specific protein) was also observed. However, luzindole (melatonin receptor antagonist) and PD98059 (MEK inhibitor) attenuated these increases. LY294002 (AKT inhibitor) inhibited melatonin-mediated proliferation in PC12 cells and did not affect melatonin-induced neural differentiation. The expression of p-ERK1/2/ERK1/2 was increased by melatonin treatment for 14 days in PC12 cells,more » whereas luzindole or PD98059 reduced the melatonin-induced increase. These results suggest that the activation of both the MEK/ERK and PI3K/AKT signaling pathways could potentially contribute to melatonin-mediated proliferation, but that only the MEK/ERK pathway participates in the melatonin-induced neural differentiation of PC12 cells. Altogether, our study demonstrates for the first time that melatonin may exert a positive effect on neural differentiation via melatonin receptor signalling and that the MEK/ERK1/2 signalling may act down stream from the melatonin pathway. - Highlights: • Melatonin improves the proliferation of PC12 cells. • Melatonin induces neural differentiation of PC12 cells. • Melatonin-mediated proliferation in PC12 cells relies on the ERK and AKT pathways. • Activation of ERK is essential for melatonin-induced neural differentiation of PC12.« less

Estradiol increases the expression of TNF-α and TNF receptor 1 in lactotropes.

PubMed

Zaldivar, Verónica; Magri, María Laura; Zárate, Sandra; Jaita, Gabriela; Eijo, Guadalupe; Radl, Daniela; Ferraris, Jimena; Pisera, Daniel; Seilicovich, Adriana

2011-01-01

Estrogens are recognized modulators of pituitary cell renewal, sensitizing cells to mitogenic and apoptotic signals. Tumor necrosis factor-α (TNF-α) is a proinflammatory cytokine that plays an important role in tissue homeostasis modulating cell proliferation, differentiation and death. We previously demonstrated that TNF-α-induced apoptosis of anterior pituitary cells from female rats is estrogen-dependent and predominant in cells from rats at proestrus when estradiol levels are the highest. Considering that one of the mechanisms involved in the apoptotic action of estrogens can result from increased expression of cytokines and/or their receptors, the aim of the present study was to evaluate the effect of estrogens on the expression of TNF-α and its receptor, TNF receptor 1 (TNFR1), in anterior pituitary cells. TNFR1 expression, determined by Western blot, was higher in anterior pituitary glands from rats at proestrus than at diestrus. Incubation of anterior pituitary cells from ovariectomized rats with 17β-estradiol enhanced TNFR1 protein expression. As determined by double immunocytochemistry, the expression of TNF-α and TNFR1 was detected in prolactin-, GH-, LH- and ACTH-bearing cells. 17β-estradiol increased the percentage of TNF-α and TNFR1-immunoreactive lactotropes but did not modify the number of GH-bearing cells expressing TNF-α or TNFR1. Our results demonstrate that estradiol increases the expression of TNF-α and TNFR1 in anterior pituitary cells, especially in lactotropes. The sensitizing action of estrogens to proapoptotic stimuli at proestrus in the anterior pituitary gland may involve changes in the expression of the TNF-α/TNFR1 system. Copyright © 2011 S. Karger AG, Basel.

Effects of Aged Stored Autologous Red Blood Cells on Human Endothelial Function

PubMed Central

Kanias, Tamir; Triulzi, Darrel; Donadee, Chenell; Barge, Suchitra; Badlam, Jessica; Jain, Shilpa; Belanger, Andrea M.; Kim-Shapiro, Daniel B.

2015-01-01

Rationale: A major abnormality that characterizes the red cell “storage lesion” is increased hemolysis and reduced red cell lifespan after infusion. Low levels of intravascular hemolysis after transfusion of aged stored red cells disrupt nitric oxide (NO) bioavailabity, via accelerated NO scavenging reaction with cell-free plasma hemoglobin. The degree of intravascular hemolysis post-transfusion and effects on endothelial-dependent vasodilation responses to acetylcholine have not been fully characterized in humans. Objectives: To evaluate the effects of blood aged to the limits of Food and Drug Administration–approved storage time on the human microcirculation and endothelial function. Methods: Eighteen healthy individuals donated 1 U of leukopheresed red cells, divided and autologously transfused into the forearm brachial artery 5 and 42 days after blood donation. Blood samples were obtained from stored blood bag supernatants and the antecubital vein of the infusion arm. Forearm blood flow measurements were performed using strain-gauge plethysmography during transfusion, followed by testing of endothelium-dependent blood flow with increasing doses of intraarterial acetylcholine. Measurements and Main Results: We demonstrate that aged stored blood has higher levels of arginase-1 and cell-free plasma hemoglobin. Compared with 5-day blood, the transfusion of 42-day packed red cells decreases acetylcholine-dependent forearm blood flows. Intravascular venous levels of arginase-1 and cell-free plasma hemoglobin increase immediately after red cell transfusion, with more significant increases observed after infusion of 42-day-old blood. Conclusions: We demonstrate that the transfusion of blood at the limits of Food and Drug Administration–approved storage has a significant effect on the forearm circulation and impairs endothelial function. Clinical trial registered with www.clinicaltrials.gov (NCT 01137656) PMID:26222884

Radiation-induced cyclooxygenase 2 up-regulation is dependent on redox status in prostate cancer cells.

PubMed

Li, Lingyun; Steinauer, Kirsten K; Dirks, Amie J; Husbeck, Bryan; Gibbs, Iris; Knox, Susan J

2003-12-01

Cyclooxygenase 2 (COX2) is the inducible isozyme of COX, a key enzyme in arachidonate metabolism and the conversion of arachidonic acid (AA) to prostaglandins (PGs) and other eicosanoids. Previous studies have demonstrated that the COX2 protein is up-regulated in prostate cancer cells after irradiation and that this results in elevated levels of PGE(2). In the present study, we further investigated whether radiation-induced COX2 up-regulation is dependent on the redox status of cells from the prostate cancer cell line PC-3. l-Buthionine sulfoximine (BSO), which inhibits gamma glutamyl cysteine synthetase (gammaGCS), and the antioxidants alpha-lipoic acid and N-acetyl-l-cysteine (NAC) were used to modulate the cellular redox status. BSO decreased the cellular GSH level and increased cellular reactive oxygen species (ROS) in PC-3 cells, whereas alpha-lipoic acid and NAC increased the GSH level and decreased cellular ROS. Both radiation and the oxidant H(2)O(2) had similar effects on COX2 up-regulation and PGE(2) production in PC-3 cells, suggesting that radiation-induced COX2 up-regulation is secondary to the production of ROS. The relative increases in COX2 expression and PGE(2) production induced by radiation and H(2)O(2) were even greater when PC-3 cells were pretreated with BSO. When the cells were pretreated with alpha-lipoic acid or NAC for 24 h, both radiation- and H(2)O(2)-induced COX2 up-regulation and PGE(2) production were markedly inhibited. These results demonstrate that radiation-induced COX2 up-regulation in prostate cancer cells is modulated by the cellular redox status. Radiation-induced increases in ROS levels contribute to the adaptive response of PC-3 cells, resulting in elevated levels of COX2.

Vitamin K2 improves proliferation and migration of bovine skeletal muscle cells in vitro.

PubMed

Rønning, Sissel Beate; Pedersen, Mona Elisabeth; Berg, Ragnhild Stenberg; Kirkhus, Bente; Rødbotten, Rune

2018-01-01

Skeletal muscle function is highly dependent on the ability to regenerate, however, during ageing or disease, the proliferative capacity is reduced, leading to loss of muscle function. We have previously demonstrated the presence of vitamin K2 in bovine skeletal muscles, but whether vitamin K has a role in muscle regulation and function is unknown. In this study, we used primary bovine skeletal muscle cells, cultured in monolayers in vitro, to assess a potential effect of vitamin K2 (MK-4) during myogenesis of muscle cells. Cell viability experiments demonstrate that the amount of ATP produced by the cells was unchanged when MK-4 was added, indicating viable cells. Cytotoxicity analysis show that MK-4 reduced the lactate dehydrogenase (LDH) released into the media, suggesting that MK-4 was beneficial to the muscle cells. Cell migration, proliferation and differentiation was characterised after MK-4 incubation using wound scratch analysis, immunocytochemistry and real-time PCR analysis. Adding MK-4 to the cells led to an increased muscle proliferation, increased gene expression of the myogenic transcription factor myod as well as increased cell migration. In addition, we observed a reduction in the fusion index and relative gene expression of muscle differentiation markers, with fewer complex myotubes formed in MK-4 stimulated cells compared to control cells, indicating that the MK-4 plays a significant role during the early phases of muscle proliferation. Likewise, we see the same pattern for the relative gene expression of collagen 1A, showing increased gene expression in proliferating cells, and reduced expression in differentiating cells. Our results also suggest that MK-4 incubation affect low density lipoprotein receptor-related protein 1 (LRP1) and the low-density lipoprotein receptor (LDLR) with a peak in gene expression after 45 min of MK-4 incubation. Altogether, our experiments show that MK-4 has a positive effect on muscle cell migration and proliferation, which are two important steps during early myogenesis.

Regulation of bone sialoprotein mRNA by steroid hormones

PubMed Central

1989-01-01

In this report we demonstrate an increase in the steady-state level of bone sialoprotein (BSP) mRNA in rat calvaria and a rat osteosarcoma cell line (ROS 17/2.8) after treatment with the synthetic glucocorticoid, dexamethasone. In contrast, 1.25-dihydroxyvitamin D3 reduced the amount of BSP mRNA in calvaria and inhibited the dexamethasone induction in ROS 17/2.8 cells. The increase in BSP mRNA is most likely due to an increase in the transcriptional rate. The stability of mRNA was unchanged after dexamethasone treatment with a half-life of approximately 5 h. Nuclear transcription experiments with nuclei isolated from ROS 17/2.8 cells showed an increased BSP mRNA synthesis in cells treated with dexamethasone. PMID:2592421

Multimodal imaging of lung cancer and its microenvironment (Conference Presentation)

NASA Astrophysics Data System (ADS)

Hariri, Lida P.; Niederst, Matthew J.; Mulvey, Hillary; Adams, David C.; Hu, Haichuan; Chico Calero, Isabel; Szabari, Margit V.; Vakoc, Benjamin J.; Hasan, Tayyaba; Bouma, Brett E.; Engelman, Jeffrey A.; Suter, Melissa J.

2016-03-01

Despite significant advances in targeted therapies for lung cancer, nearly all patients develop drug resistance within 6-12 months and prognosis remains poor. Developing drug resistance is a progressive process that involves tumor cells and their microenvironment. We hypothesize that microenvironment factors alter tumor growth and response to targeted therapy. We conducted in vitro studies in human EGFR-mutant lung carcinoma cells, and demonstrated that factors secreted from lung fibroblasts results in increased tumor cell survival during targeted therapy with EGFR inhibitor, gefitinib. We also demonstrated that increased environment stiffness results in increased tumor survival during gefitinib therapy. In order to test our hypothesis in vivo, we developed a multimodal optical imaging protocol for preclinical intravital imaging in mouse models to assess tumor and its microenvironment over time. We have successfully conducted multimodal imaging of dorsal skinfold chamber (DSC) window mice implanted with GFP-labeled human EGFR mutant lung carcinoma cells and visualized changes in tumor development and microenvironment facets over time. Multimodal imaging included structural OCT to assess tumor viability and necrosis, polarization-sensitive OCT to measure tissue birefringence for collagen/fibroblast detection, and Doppler OCT to assess tumor vasculature. Confocal imaging was also performed for high-resolution visualization of EGFR-mutant lung cancer cells labeled with GFP, and was coregistered with OCT. Our results demonstrated that stromal support and vascular growth are essential to tumor progression. Multimodal imaging is a useful tool to assess tumor and its microenvironment over time.

Lipopolysaccharide-induced endothelial barrier breakdown is cyclic adenosine monophosphate dependent in vivo and in vitro.

PubMed

Schlegel, Nicolas; Baumer, Yvonne; Drenckhahn, Detlev; Waschke, Jens

2009-05-01

To determine whether cyclic adenosine monophosphate (cAMP) is critically involved in lipopolysaccharide (LPS)-induced breakdown of endothelial barrier functions in vivo and in vitro. Experimental laboratory research. Research laboratory. Wistar rats and cultured human microvascular endothelial cells. Permeability measurements in single postcapillary venules in vivo and permeability measurements and cell biology techniques in vitro. We demonstrate that within 120 minutes LPS increased endothelial permeability in rat mesenteric postcapillary venules in vivo and caused a barrier breakdown in human dermal microvascular endothelial cells in vitro. This was associated with the formation of large intercellular gaps and fragmentation of vascular endothelial cadherin immunostaining. Furthermore, claudin 5 immunostaining at cell borders was drastically reduced after LPS treatment. Interestingly, activity of the small GTPase Rho A, which has previously been suggested to mediate the LPS-induced endothelial barrier breakdown, was not increased after 2 hours. However, activity of Rac 1, which is known to be important for maintenance of endothelial barrier functions, was significantly reduced to 64 +/- 8% after 2 hours. All LPS-induced changes of endothelial cells were blocked by a forskolin-mediated or rolipram-mediated increase of cAMP. Consistently, enzyme-linked immunosorbent assay-based measurements demonstrated that LPS significantly decreased intracellular cAMP. In summary, our data demonstrate that LPS disrupts endothelial barrier properties by decreasing intracellular cAMP. This mechanism may involve inactivation of Rac 1 rather than activation of Rho A.

Novel daidzein analogs enhance osteogenic activity of bone marrow-derived mesenchymal stem cells and adipose-derived stromal/stem cells through estrogen receptor dependent and independent mechanisms

USDA-ARS?s Scientific Manuscript database

Osteoporosis is a disease characterized by low bone mineral density (BMD) and increased risk of fractures. Studies have demonstrated the use of phytoestrogens, or plant-derived estrogens, such as genistein anddaidzein, to effectively increase osteogenic activity of bone marrow-derived mesenchymal s...

Interleukin 6 trigged ataxia-telangiectasia mutated activation facilitates lung cancer metastasis via MMP-3/MMP-13 up-regulation.

PubMed

Jiang, Yi Na; Yan, Hong Qiong; Huang, Xiao Bo; Wang, Yi Nan; Li, Qing; Gao, Feng Guang

2015-12-01

Our previous studies show that the phosphorylation of ataxia-telangiectasia mutated (ATM) induced by interleukin 6 (IL-6) treatment contributes to multidrug resistance formation in lung cancer cells, but the exact role of ATM activation in IL-6 increased metastasis is still elusive. In the present study, matrix metalloproteinase-3 (MMP-3) and MMP-13 were firstly demonstrated to be involved in IL-6 correlated cell migration. Secondly, IL-6 treatment not only increased MMP-3/MMP-13 expression but also augmented its activities. Thirdly, the inhibition of ATM phosphorylation efficiently abolished IL-6 up-regulating MMP-3/MMP-13 expression and increasing abilities of cell migration. Most importantly, the in vivo test showed that the inhibition of ATM abrogate the effect of IL-6 on lung cancer metastasis via MMP-3/MMP-13 down-regulation. Taken together, these findings demonstrate that IL-6 inducing ATM phosphorylation increases the expression of MMP-3/MMP-13, augments the abilities of cell migration, and promotes lung cancer metastasis, indicating that ATM is a potential target molecule to overcome IL-6 correlated lung cancer metastasis.

Targeting the chromatin remodeling enzyme BRG1 increases the efficacy of chemotherapy drugs in breast cancer cells

PubMed Central

Wu, Qiong; Sharma, Soni; Cui, Hang; LeBlanc, Scott E.; Zhang, Hong; Muthuswami, Rohini; Nickerson, Jeffrey A.; Imbalzano, Anthony N.

2016-01-01

Brahma related gene product 1 (BRG1) is an ATPase that drives the catalytic activity of a subset of the mammalian SWI/SNF chromatin remodeling enzymes. BRG1 is overexpressed in most human breast cancer tumors without evidence of mutation and is required for breast cancer cell proliferation. We demonstrate that knockdown of BRG1 sensitized triple negative breast cancer cells to chemotherapeutic drugs used to treat breast cancer. An inhibitor of the BRG1 bromodomain had no effect on breast cancer cell viability, but an inhibitory molecule that targets the BRG1 ATPase activity recapitulated the increased drug efficacy observed in the presence of BRG1 knockdown. We further demonstrate that inhibition of BRG1 ATPase activity blocks the induction of ABC transporter genes by these chemotherapeutic drugs and that BRG1 binds to ABC transporter gene promoters. This inhibition increased intracellular concentrations of the drugs, providing a likely mechanism for the increased chemosensitivity. Since ABC transporters and their induction by chemotherapy drugs are a major cause of chemoresistance and treatment failure, these results support the idea that targeting the enzymatic activity of BRG1 would be an effective adjuvant therapy for breast cancer. PMID:27029062

Borrelia burgdorferi adhere to blood vessels in the dura mater and are associated with increased meningeal T cells during murine disseminated borreliosis

PubMed Central

Divan, Ali; Casselli, Timothy; Narayanan, S. Anand; Mukherjee, Sanjib; Zawieja, David C.; Watt, John A.

2018-01-01

Borrelia burgdorferi, the causative agent of Lyme disease, is a vector-borne bacterial infection that is transmitted through the bite of an infected tick. If not treated with antibiotics during the early stages of infection, disseminated infection can spread to the central nervous system (CNS). In non-human primates (NHPs) it has been demonstrated that the leptomeninges are among the tissues colonized by B. burgdorferi spirochetes. Although the NHP model parallels aspects of human borreliosis, a small rodent model would be ideal to study the trafficking of spirochetes and immune cells into the CNS. Here we show that during early and late disseminated infection, B. burgdorferi infects the meninges of intradermally infected mice, and is associated with concurrent increases in meningeal T cells. We found that the dura mater was consistently culture positive for spirochetes in transcardially perfused mice, independent of the strain of B. burgdorferi used. Within the dura mater, spirochetes were preferentially located in vascular regions, but were also present in perivascular, and extravascular regions, as late as 75 days post-infection. At the same end-point, we observed significant increases in the number of CD3+ T cells within the pia and dura mater, as compared to controls. Flow cytometric analysis of leukocytes isolated from the dura mater revealed that CD3+ cell populations were comprised of both CD4 and CD8 T cells. Overall, our data demonstrate that similarly to infection in peripheral tissues, spirochetes adhere to the dura mater during disseminated infection, and are associated with increases in the number of meningeal T cells. Collectively, our results demonstrate that there are aspects of B. burgdorferi meningeal infection that can be modelled in laboratory mice, suggesting that mice may be useful for elucidating mechanisms of meningeal pathogenesis by B. burgdorferi. PMID:29723263

Modulating EGFR Signaling by Targeting the Deacetylase HDAC6-Hsp90 Complex in Breast Tumors

DTIC Science & Technology

2007-06-01

concomitant increase in 4 directed cell migration (15). Analysis of fibroblasts derived from WAVE2 knockout mice 5 demonstrates deficiency in ruffle...Takenawa. 2003. Differential 1 roles of WAVE1 and WAVE2 in dorsal and peripheral ruffle formation for 2 fibroblast cell migration. Dev Cell 5:595

A distinct first replication cycle of DNA introduced in mammalian cells

PubMed Central

Chandok, Gurangad S.; Kapoor, Kalvin K.; Brick, Rachel M.; Sidorova, Julia M.; Krasilnikova, Maria M.

2011-01-01

Many mutation events in microsatellite DNA sequences were traced to the first embryonic divisions. It was not known what makes the first replication cycles of embryonic DNA different from subsequent replication cycles. Here we demonstrate that an unusual replication mode is involved in the first cycle of replication of DNA introduced in mammalian cells. This alternative replication starts at random positions, and occurs before the chromatin is fully assembled. It is detected in various cell lines and primary cells. The presence of single-stranded regions increases the efficiency of this alternative replication mode. The alternative replication cannot progress through the A/T-rich FRA16B fragile site, while the regular replication mode is not affected by it. A/T-rich microsatellites are associated with the majority of chromosomal breakpoints in cancer. We suggest that the alternative replication mode may be initiated at the regions with immature chromatin structure in embryonic and cancer cells resulting in increased genomic instability. This work demonstrates, for the first time, differences in the replication progression during the first and subsequent replication cycles in mammalian cells. PMID:21062817

Synaptic connectivity and spatial memory: a topological approach

NASA Astrophysics Data System (ADS)

Milton, Russell; Babichev, Andrey; Dabaghian, Yuri

2015-03-01

In the hippocampus, a network of place cells generates a cognitive map of space, in which each cell is responsive to a particular area of the environment - its place field. The peak response of each cell and the size of each place field have considerable variability. Experimental evidence suggests that place cells encode a topological map of space that serves as a basis of spatial memory and spatial awareness. Using a computational model based on Persistent Homology Theory we demonstrate that if the parameters of the place cells spiking activity fall inside of the physiological range, the network correctly encodes the topological features of the environment. We next introduce parameters of synaptic connectivity into the model and demonstrate that failures in synapses that detect coincident neuronal activity lead to spatial learning deficiencies similar to the ones that are observed in rodent models of neurodegenerative diseases. Moreover, we show that these learning deficiencies may be mitigated by increasing the number of active cells and/or by increasing their firing rate, suggesting the existence of a compensatory mechanism inherent to the cognitive map.

Cancer Stem-Like Cells Enriched in Panc-1 Spheres Possess Increased Migration Ability and Resistance to Gemcitabine

PubMed Central

Yin, Tao; Wei, Hongji; Gou, Shanmiao; Shi, Pengfei; Yang, Zhiyong; Zhao, Gang; Wang, Chunyou

2011-01-01

Pancreatic cancer is one of the most lethal malignancies with poor prognosis. Previously, we found that a subpopulation of cancer stem cells (CSCs) in the Panc-1 pancreatic cancer cell line could propagate to form spheres. Here we characterized the malignant phenotypes of the pancreatic cancer stem CD44+/CD24+ cells, which were enriched under sphere forming conditions as analyzed by flow cytometry. These cells demonstrated increased resistance to gemcitabine and increased migration ability. Moreover, these cells exhibited epithelial to mesenchymal transition characterized by a decreased level of the epithelial marker E-cadherin and an increased level of the mesenchymal marker vimentin. Notably, abnormal expression of Bmi-1, ABCG2, Cyclin D1 and p16 were found in Panc-1 CSCs. Our results suggest that targeted inhibition of CSCs represents a novel therapeutic approach to overcome chemoresistance and metastasis of pancreatic cancer. PMID:21673909

The same drug but a different mechanism of action: comparison of free doxorubicin with two different N-(2-hydroxypropyl)methacrylamide copolymer-bound doxorubicin conjugates in EL-4 cancer cell line.

PubMed

Kovár, Lubomír; Strohalm, Jirí; Chytil, Petr; Mrkvan, Tomás; Kovár, Marek; Hovorka, Ondrej; Ulbrich, Karel; Ríhová, Blanka

2007-01-01

Doxorubicin is one of the most potent anti-tumor drugs with a broad spectrum of use. To reduce its toxic effect and improve its pharmacokinetics, we conjugated it to an HPMA copolymer carrier that enhances its passive accumulation within solid tumors via the EPR effect and decreases its cytotoxicity to normal, noncancer cells. In this study, we compared the antiproliferative, pro-survival, and death signals triggered in EL-4 cancer cells exposed to free doxorubicin and doxorubicin conjugated to a HPMA copolymer carrier via either enzymatically (PK1) or hydrolytically (HYD) degradable bonds. We have previously shown that the intracellular distribution of free doxorubicin, HYD, and PK1 is markedly different. Here, we demonstrated that these three agents greatly differ also in the antiproliferative effect and cell death signals they trigger. JNK phosphorylation sharply increased in cells treated with HYD, while treatment with free doxorubicin moderately decreased and treatment with PK1 even strongly decreased it. On the other hand, treatment with free doxorubicin greatly increased p38 phosphorylation, while PK1 and HYD increased it slightly. PK1 also significantly increased ERK phosphorylation, while both the free doxorubicin and HYD conjugate slightly decreased it. Long-term inhibition of JNK significantly increased both proliferation and viability of EL-4 cells treated with free doxorubicin, showing that the JNK signaling pathway could be critical for mediating cell death in EL-4 cells exposed to free doxorubicin. Both activation of caspase 3 and decreased binding activity of the p50 subunit of NFkappaB were observed in cells treated with free doxorubicin and HYD, while no such effects were seen in cells incubated with PK1. Analysis of the expression of genes involved in apoptosis and regulation of the cell cycle demonstrated that free doxorubicin and HYD have very similar mechanisms of action, while PK1 has very different characteristics.

Increased levels of B1 and B2 SINE transcripts in mouse fibroblast cells due to minute virus of mice infection.

PubMed

Williams, Warren P; Tamburic, Lillian; Astell, Caroline R

2004-10-01

Minute virus of mice (MVM), an autonomous parvovirus, has served as a model for understanding parvovirus infection including host cell response to infection. In this paper, we report the effect of MVM infection on host cell gene expression in mouse fibroblast cells (LA9 cells), analyzed by differential display. Somewhat surprisingly, our data reveal that few cellular protein-coding genes appear to be up- or downregulated and identify the murine B1 and B2 short interspersed element (SINE) transcripts as being increased upon MVM infection. Primer extension assays confirm the effect of MVM infection on SINE expression and demonstrate that both SINEs are upregulated in a roughly linear fashion throughout MVM infection. They also demonstrate that the SINE response was due to RNA polymerase III transcription and not contaminating DNA or RNA polymerase II transcription. Furthermore, expression of MVM NS1, the major nonstructural protein, by transient transfection also leads to an increase in both murine SINEs. We believe this is the first time that the B1 and B2 SINEs have been shown to be altered by viral infection and the first time parvovirus infection has been shown to increase SINE expression. The increase in SINE transcripts caused by MVM infection does not appear to be due to an increase in either of the basal transcription factors TFIIIC110 or 220, in contrast to that which has been shown for other viruses.

Ketogenic diets as an adjuvant cancer therapy: History and potential mechanism

PubMed Central

Allen, Bryan G.; Bhatia, Sudershan K.; Anderson, Carryn M.; Eichenberger-Gilmore, Julie M.; Sibenaller, Zita A.; Mapuskar, Kranti A.; Schoenfeld, Joshua D.; Buatti, John M.; Spitz, Douglas R.; Fath, Melissa A.

2014-01-01

Cancer cells, relative to normal cells, demonstrate significant alterations in metabolism that are proposed to result in increased steady-state levels of mitochondrial-derived reactive oxygen species (ROS) such as O2•−and H2O2. It has also been proposed that cancer cells increase glucose and hydroperoxide metabolism to compensate for increased levels of ROS. Given this theoretical construct, it is reasonable to propose that forcing cancer cells to use mitochondrial oxidative metabolism by feeding ketogenic diets that are high in fats and low in glucose and other carbohydrates, would selectively cause metabolic oxidative stress in cancer versus normal cells. Increased metabolic oxidative stress in cancer cells would in turn be predicted to selectively sensitize cancer cells to conventional radiation and chemotherapies. This review summarizes the evidence supporting the hypothesis that ketogenic diets may be safely used as an adjuvant therapy to conventional radiation and chemotherapies and discusses the proposed mechanisms by which ketogenic diets may enhance cancer cell therapeutic responses. PMID:25460731

Stochastic loss and gain of symmetric divisions in the C. elegans epidermis perturbs robustness of stem cell number

PubMed Central

Katsanos, Dimitris; Koneru, Sneha L.; Mestek Boukhibar, Lamia; Gritti, Nicola; Ghose, Ritobrata; Appleford, Peter J.; Doitsidou, Maria; Woollard, Alison; van Zon, Jeroen S.; Poole, Richard J.

2017-01-01

Biological systems are subject to inherent stochasticity. Nevertheless, development is remarkably robust, ensuring the consistency of key phenotypic traits such as correct cell numbers in a certain tissue. It is currently unclear which genes modulate phenotypic variability, what their relationship is to core components of developmental gene networks, and what is the developmental basis of variable phenotypes. Here, we start addressing these questions using the robust number of Caenorhabditis elegans epidermal stem cells, known as seam cells, as a readout. We employ genetics, cell lineage tracing, and single molecule imaging to show that mutations in lin-22, a Hes-related basic helix-loop-helix (bHLH) transcription factor, increase seam cell number variability. We show that the increase in phenotypic variability is due to stochastic conversion of normally symmetric cell divisions to asymmetric and vice versa during development, which affect the terminal seam cell number in opposing directions. We demonstrate that LIN-22 acts within the epidermal gene network to antagonise the Wnt signalling pathway. However, lin-22 mutants exhibit cell-to-cell variability in Wnt pathway activation, which correlates with and may drive phenotypic variability. Our study demonstrates the feasibility to study phenotypic trait variance in tractable model organisms using unbiased mutagenesis screens. PMID:29108019

Comparison of Vibrio parahaemolyticus grown in estuarine water and rich medium.

PubMed Central

Pace, J; Chai, T J

1989-01-01

Cell envelope composition and selected physiological traits of Vibrio parahaemolyticus were studied in regard to the Kanagawa phenomenon and growth conditions. Cell envelopes were prepared from cells cultured in Proteose Peptone-beef extract (Difco Laboratories, Detroit, Mich.) medium or filtered estuarine water. Protein, phospholipid, and lipopolysaccharide contents varied with culture conditions. The phospholipids present in the cell envelopes were identified as phosphatidylethanolamine, phosphatidylglycerol, and cardiolipin. Phosphatidylethanolamine decreased and phosphatidylglycerol increased in cells grown in estuarine water. Profiles of proteins separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis demonstrated numerous protein species, with four to six predominant proteins ranging from 26,000 to 120,000 in molecular weight. The profile of V. parahaemolyticus cell envelope proteins was unique and might be useful in the identification of the organism. Alkaline phosphatase activity was slightly higher in Kanagawa-negative strains and was higher in cells grown in estuarine water than in cells grown in rich laboratory medium. The DNA levels in estuarine water-grown cells increased, while RNA levels and cell volume decreased. Bacteriophage sensitivity typing demonstrated a close intraspecies relationship. Results indicated that Kanagawa-positive and -negative strains were closely related, but they could be grouped separately and may have undergone starvation-related physiological changes when cultured in estuarine water. Images PMID:2782869

A Nitric Oxide Storage and Transport System That Protects Activated Macrophages from Endogenous Nitric Oxide Cytotoxicity*

PubMed Central

Lok, Hiu Chuen; Sahni, Sumit; Jansson, Patric J.; Kovacevic, Zaklina; Hawkins, Clare L.; Richardson, Des R.

2016-01-01

Nitric oxide (NO) is integral to macrophage cytotoxicity against tumors due to its ability to induce iron release from cancer cells. However, the mechanism for how activated macrophages protect themselves from endogenous NO remains unknown. We previously demonstrated by using tumor cells that glutathione S-transferase P1 (GSTP1) sequesters NO as dinitrosyl-dithiol iron complexes (DNICs) and inhibits NO-mediated iron release from cells via the transporter multidrug resistance protein 1 (MRP1/ABCC1). These prior studies also showed that MRP1 and GSTP1 protect tumor cells against NO cytotoxicity, which parallels their roles in defending cancer cells from cytotoxic drugs. Considering this, and because GSTP1 and MRP1 are up-regulated during macrophage activation, this investigation examined whether this NO storage/transport system protects macrophages against endogenous NO cytotoxicity in two well characterized macrophage cell types (J774 and RAW 264.7). MRP1 expression markedly increased upon macrophage activation, and the role of MRP1 in NO-induced 59Fe release was demonstrated by Mrp1 siRNA and the MRP1 inhibitor, MK571, which inhibited NO-mediated iron efflux. Furthermore, Mrp1 silencing increased DNIC accumulation in macrophages, indicating a role for MRP1 in transporting DNICs out of cells. In addition, macrophage 59Fe release was enhanced by silencing Gstp1, suggesting GSTP1 was responsible for DNIC binding/storage. Viability studies demonstrated that GSTP1 and MRP1 protect activated macrophages from NO cytotoxicity. This was confirmed by silencing nuclear factor-erythroid 2-related factor 2 (Nrf2), which decreased MRP1 and GSTP1 expression, concomitant with reduced 59Fe release and macrophage survival. Together, these results demonstrate a mechanism by which macrophages protect themselves against NO cytotoxicity. PMID:27866158

Molecular Mechanism of Flocculation Self-Recognition in Yeast and Its Role in Mating and Survival

PubMed Central

Goossens, Katty V. Y.; Ielasi, Francesco S.; Nookaew, Intawat; Stals, Ingeborg; Alonso-Sarduy, Livan; Daenen, Luk; Van Mulders, Sebastiaan E.; Stassen, Catherine; van Eijsden, Rudy G. E.; Siewers, Verena; Delvaux, Freddy R.; Kasas, Sandor; Nielsen, Jens; Devreese, Bart

2015-01-01

ABSTRACT We studied the flocculation mechanism at the molecular level by determining the atomic structures of N-Flo1p and N-Lg-Flo1p in complex with their ligands. We show that they have similar ligand binding mechanisms but distinct carbohydrate specificities and affinities, which are determined by the compactness of the binding site. We characterized the glycans of Flo1p and their role in this binding process and demonstrate that glycan-glycan interactions significantly contribute to the cell-cell adhesion mechanism. Therefore, the extended flocculation mechanism is based on the self-interaction of Flo proteins and this interaction is established in two stages, involving both glycan-glycan and protein-glycan interactions. The crucial role of calcium in both types of interaction was demonstrated: Ca2+ takes part in the binding of the carbohydrate to the protein, and the glycans aggregate only in the presence of Ca2+. These results unify the generally accepted lectin hypothesis with the historically first-proposed “Ca2+-bridge” hypothesis. Additionally, a new role of cell flocculation is demonstrated; i.e., flocculation is linked to cell conjugation and mating, and survival chances consequently increase significantly by spore formation and by introduction of genetic variability. The role of Flo1p in mating was demonstrated by showing that mating efficiency is increased when cells flocculate and by differential transcriptome analysis of flocculating versus nonflocculating cells in a low-shear environment (microgravity). The results show that a multicellular clump (floc) provides a uniquely organized multicellular ultrastructure that provides a suitable microenvironment to induce and perform cell conjugation and mating. PMID:25873380

Runx2- and histone deacetylase 3-mediated repression is relieved in differentiating human osteoblast cells to allow high bone sialoprotein expression.

PubMed

Lamour, Virginie; Detry, Cédric; Sanchez, Christelle; Henrotin, Yves; Castronovo, Vincent; Bellahcène, Akeila

2007-12-14

Bone sialoprotein (BSP) is a bone matrix glycoprotein whose expression coincides with terminal osteoblastic differentiation and the onset of mineralization. In this study we show that BSP expression is considerably increased in confluent Saos-2 human osteosarcoma cells and in differentiating normal human osteoblasts, concomitantly with the decrease of Runx2, a key transcription factor controlling bone formation. Therefore, we investigated the role of Runx2 in the regulation of BSP expression in Saos-2 cells. Using a mobility shift assay, we demonstrated that Runx2 binds to the BSP promoter only in preconfluent cells. Histone deacetylase 3 (HDAC3) has been recently shown to act as a Runx2 co-repressor. Chromatin immunoprecipitation assays demonstrated that both Runx2 and HDAC3 are detectable at the BSP promoter in preconfluent Saos-2 cells but not when they are confluent and overexpress BSP. Consistently, nuclear Runx2 protein level is down-regulated, whereas Saos-2 cells became increasingly confluent. Finally, the suppression of HDAC3, Runx2, or both by RNA interference induced the expression of BSP at both mRNA and protein levels in Saos-2 cells. Our data demonstrate that Runx2 and HDAC3 repress BSP gene expression and that this repression is suspended upon osteoblastic cell differentiation. Both the nuclear disappearance of Runx2 and the non-recruitment of HDAC3 represent new means to relieve Runx2-mediated suppression of BSP expression, thus allowing the acquisition of a fully differentiated and mineralization-competent phenotype by osteoblast cells.

3D heterogeneous islet organoid generation from human embryonic stem cells using a novel engineered hydrogel platform.

PubMed

Candiello, Joseph; Grandhi, Taraka Sai Pavan; Goh, Saik Kia; Vaidya, Vimal; Lemmon-Kishi, Maya; Eliato, Kiarash Rahmani; Ros, Robert; Kumta, Prashant N; Rege, Kaushal; Banerjee, Ipsita

2018-05-25

Organoids, which exhibit spontaneous organ specific organization, function, and multi-cellular complexity, are in essence the in vitro reproduction of specific in vivo organ systems. Recent work has demonstrated human pluripotent stem cells (hPSCs) as a viable regenerative cell source for tissue-specific organoid engineering. This is especially relevant for engineering islet organoids, due to the recent advances in generating functional beta-like cells from human pluripotent stem cells. In this study, we report specific engineering of regenerative islet organoids of precise size and cellular heterogeneity, using a novel hydrogel system, Amikagel. Amikagel facilitated controlled and spontaneous aggregation of human embryonic stem cell derived pancreatic progenitor cells (hESC-PP) into robust homogeneous spheroids. This platform further allowed fine control over the integration of multiple cell populations to produce heterogeneous spheroids, which is a necessity for complex organoid engineering. Amikagel induced hESC-PP spheroid formation enhanced pancreatic islet-specific Pdx-1 and NKX6.1 gene and protein expression, while also increasing the percentage of committed population. hESC-PP spheroids were further induced towards mature beta-like cells which demonstrated increased Beta-cell specific INS1 gene and C-peptide protein expression along with functional insulin production in response to in vitro glucose challenge. Further integration of hESC-PP with biologically relevant supporting endothelial cells resulted in multicellular organoids which demonstrated spontaneous maturation towards islet-specific INS1 gene and C-peptide protein expression along with a significantly developed extracellular matrix support system. These findings establish Amikagel -facilitated platform ideal for islet organoid engineering. Copyright © 2018. Published by Elsevier Ltd.

Metformin directly acts on mitochondria to alter cellular bioenergetics

PubMed Central

2014-01-01

Background Metformin is widely used in the treatment of diabetes, and there is interest in ‘repurposing’ the drug for cancer prevention or treatment. However, the mechanism underlying the metabolic effects of metformin remains poorly understood. Methods We performed respirometry and stable isotope tracer analyses on cells and isolated mitochondria to investigate the impact of metformin on mitochondrial functions. Results We show that metformin decreases mitochondrial respiration, causing an increase in the fraction of mitochondrial respiration devoted to uncoupling reactions. Thus, cells treated with metformin become energetically inefficient, and display increased aerobic glycolysis and reduced glucose metabolism through the citric acid cycle. Conflicting prior studies proposed mitochondrial complex I or various cytosolic targets for metformin action, but we show that the compound limits respiration and citric acid cycle activity in isolated mitochondria, indicating that at least for these effects, the mitochondrion is the primary target. Finally, we demonstrate that cancer cells exposed to metformin display a greater compensatory increase in aerobic glycolysis than nontransformed cells, highlighting their metabolic vulnerability. Prevention of this compensatory metabolic event in cancer cells significantly impairs survival. Conclusions Together, these results demonstrate that metformin directly acts on mitochondria to limit respiration and that the sensitivity of cells to metformin is dependent on their ability to cope with energetic stress. PMID:25184038

Involvement of caspase-dependent and -independent apoptotic pathways in cisplatin-induced apoptosis

NASA Astrophysics Data System (ADS)

Liu, Lei; Zhang, Yingjie; Wang, Xianwang

2009-02-01

Cisplatin, an efficient anticancer agent, can trigger multiple apoptotic pathways in cancer cells. However, the signal transduction pathways in response to cisplatin-based chemotherapy are complicated, and the mechanism is not fully understood. In current study, we showed that, during cisplatin-induced apoptosis of human lung adenocarcinoma cells, both the caspase-dependent and -independent pathways were activated. Herein, we reported that after cisplatin treatment, the activities of caspase-9/-3 were sharply increased; pre-treatment with Z-LEHD-fmk (inhibitor of caspase-9), Z-DEVD-fmk (inhibitor of caspase-3), and Z-VAD-fmk (a pan-caspase inhibitor) increased cell viability and decreased apoptosis, suggesting that caspase-mediated apoptotic pathway was activated following cisplatin treatment. Confocal imaging of the cells transfected with AIF-GFP demonstrated that AIF release occurred about 9 h after cisplatin treatment. The event proceeded progressively over time, coinciding with a nuclear translocation and lasting for more than 2 hours. Down-regulation of AIF by siRNA also significantly increased cell viability and decreased apoptosis, these results suggested that AIF-mediated caspase-independent apoptotic pathway was involved in cispatin-induced apoptosis. In conclusion, the current study demonstrated that both caspase-dependent and -independent apoptotic pathways were involved in cisplatin-induced apoptosis in human lung adenocarcinoma cells.

N-acetyl-cysteine increases cellular dysfunction in progressive chronic kidney damage after acute kidney injury by dampening endogenous antioxidant responses.

PubMed

Small, David M; Sanchez, Washington Y; Roy, Sandrine F; Morais, Christudas; Brooks, Heddwen L; Coombes, Jeff S; Johnson, David W; Gobe, Glenda C

2018-05-01

Oxidative stress and mitochondrial dysfunction exacerbate acute kidney injury (AKI), but their role in any associated progress to chronic kidney disease (CKD) remains unclear. Antioxidant therapies often benefit AKI, but their benefits in CKD are controversial since clinical and preclinical investigations often conflict. Here we examined the influence of the antioxidant N-acetyl-cysteine (NAC) on oxidative stress and mitochondrial function during AKI (20-min bilateral renal ischemia plus reperfusion/IR) and progression to chronic kidney pathologies in mice. NAC (5% in diet) was given to mice 7 days prior and up to 21 days post-IR (21d-IR). NAC treatment resulted in the following: prevented proximal tubular epithelial cell apoptosis at early IR (40-min postischemia), yet enhanced interstitial cell proliferation at 21d-IR; increased transforming growth factor-β1 expression independent of IR time; and significantly dampened nuclear factor-like 2-initiated cytoprotective signaling at early IR. In the long term, NAC enhanced cellular metabolic impairment demonstrated by increased peroxisome proliferator activator-γ serine-112 phosphorylation at 21d-IR. Intravital multiphoton microscopy revealed increased endogenous fluorescence of nicotinamide adenine dinucleotide (NADH) in cortical tubular epithelial cells during ischemia, and at 21d-IR that was not attenuated with NAC. Fluorescence lifetime imaging microscopy demonstrated persistent metabolic impairment by increased free/bound NADH in the cortex at 21d-IR that was enhanced by NAC. Increased mitochondrial dysfunction in remnant tubular cells was demonstrated at 21d-IR by tetramethylrhodamine methyl ester fluorimetry. In summary, NAC enhanced progression to CKD following AKI not only by dampening endogenous cellular antioxidant responses at time of injury but also by enhancing persistent kidney mitochondrial and metabolic dysfunction.

Cell-mediated fibre recruitment drives extracellular matrix mechanosensing in engineered fibrillar microenvironments

NASA Astrophysics Data System (ADS)

Baker, Brendon M.; Trappmann, Britta; Wang, William Y.; Sakar, Mahmut S.; Kim, Iris L.; Shenoy, Vivek B.; Burdick, Jason A.; Chen, Christopher S.

2015-12-01

To investigate how cells sense stiffness in settings structurally similar to native extracellular matrices, we designed a synthetic fibrous material with tunable mechanics and user-defined architecture. In contrast to flat hydrogel surfaces, these fibrous materials recapitulated cell-matrix interactions observed with collagen matrices including stellate cell morphologies, cell-mediated realignment of fibres, and bulk contraction of the material. Increasing the stiffness of flat hydrogel surfaces induced mesenchymal stem cell spreading and proliferation; however, increasing fibre stiffness instead suppressed spreading and proliferation for certain network architectures. Lower fibre stiffness permitted active cellular forces to recruit nearby fibres, dynamically increasing ligand density at the cell surface and promoting the formation of focal adhesions and related signalling. These studies demonstrate a departure from the well-described relationship between material stiffness and spreading established with hydrogel surfaces, and introduce fibre recruitment as a previously undescribed mechanism by which cells probe and respond to mechanics in fibrillar matrices.

Androgen receptor (AR) promotes clear cell renal cell carcinoma (ccRCC) migration and invasion via altering the circHIAT1/miR-195-5p/29a-3p/29c-3p/CDC42 signals.

PubMed

Wang, Kefeng; Sun, Yin; Tao, Wei; Fei, Xiang; Chang, Chawnshang

2017-05-28

Increasing evidence has demonstrated that the androgen receptor (AR) plays important roles to promote the metastasis of clear cell renal cell carcinoma (ccRCC). The detailed mechanisms, especially how AR functions via altering the circular RNAs (circRNAs) remain unclear. Here we identified a new circRNA (named as circHIAT1) whose expression was lower in ccRCCs than adjacent normal tissues. Targeting AR could suppress ccRCC cell progression via increasing circHIAT1 expression. ChIP assay and luciferase assay demonstrated that AR suppressed circHIAT1 expression via regulating its host gene, Hippocampus Abundant Transcript 1 (HIAT1) expression at the transcriptional level. The consequences of AR-suppressed circHIAT1 resulted in deregulating miR-195-5p/29a-3p/29c-3p expressions, which increased CDC42 expression to enhance ccRCC cell migration and invasion. Increasing this newly identified signal via circHIAT1 suppressed AR-enhanced ccRCC cell migration and invasion. Together, these results suggested that circHIAT1 functioned as a metastatic inhibitor to suppress AR-enhanced ccRCC cell migration and invasion. Targeting this newly identified AR-circHIAT1-mediated miR-195-5p/29a-3p/29c-3p/CDC42 signals may help us develop potential new therapies to better suppress ccRCC metastasis. Copyright © 2017 Elsevier B.V. All rights reserved.

SASH1 mediates sensitivity of breast cancer cells to chloropyramine and is associated with prognosis in breast cancer

PubMed Central

Burgess, Joshua T.; Bolderson, Emma; Saunus, Jodi M.; Zhang, Shu-Dong; Reid, Lynne E.; McNicol, Anne Marie; Lakhani, Sunil R.; Cuff, Katharine; Richard, Kerry; Richard, Derek J.; O'Byrne, Kenneth J.

2016-01-01

Expression of the SASH1 protein is reduced in a range of human cancers and has been implicated in apoptotic cancer cell death. This study investigated whether increasing SASH1 expression could be a useful therapeutic strategy in breast cancer. Ectopic SASH1 expression increased apoptosis in 7/8 breast cancer cell lines. Subsequent in silico connectivity screening demonstrated that the clinically approved antihistamine drug, chloropyramine, increased SASH1 mRNA levels. Chloropyramine has previously been shown to have anti-tumour activity in breast cancer in part through modulation of FAK signalling, a pathway also regulated by SASH1. This study demonstrated that chloropyramine increased SASH1 protein levels in breast cancer cells. Consistent with this the agent reduced cell confluency in 7/8 cell lines treated irrespective of their ER status but not apoptosis incompetent MCF7 cells. In contrast SASH1 siRNA-transfected breast cancer cells exhibited reduced chloropyramine sensitivity. The prognostic significance of SASH1 expression was also investigated in two breast cancer cohorts. Expression was associated with favourable outcome in ER-positive cases, but only those of low histological grade/proliferative status. Conversely, we found a very strong inverse association in HER2+ disease irrespective of ER status, and in triple-negative, basal-like cases. Overall, the data suggest that SASH1 is prognostic in breast cancer and could have subtype-dependent effects on breast cancer progression. Pharmacologic induction of SASH1 by chloropyramine treatment of breast cancer warrants further preclinical and clinical investigation. PMID:27637080

SASH1 mediates sensitivity of breast cancer cells to chloropyramine and is associated with prognosis in breast cancer.

PubMed

Burgess, Joshua T; Bolderson, Emma; Saunus, Jodi M; Zhang, Shu-Dong; Reid, Lynne E; McNicol, Anne Marie; Lakhani, Sunil R; Cuff, Katharine; Richard, Kerry; Richard, Derek J; O'Byrne, Kenneth J

2016-11-08

Expression of the SASH1 protein is reduced in a range of human cancers and has been implicated in apoptotic cancer cell death. This study investigated whether increasing SASH1 expression could be a useful therapeutic strategy in breast cancer. Ectopic SASH1 expression increased apoptosis in 7/8 breast cancer cell lines. Subsequent in silico connectivity screening demonstrated that the clinically approved antihistamine drug, chloropyramine, increased SASH1 mRNA levels. Chloropyramine has previously been shown to have anti-tumour activity in breast cancer in part through modulation of FAK signalling, a pathway also regulated by SASH1. This study demonstrated that chloropyramine increased SASH1 protein levels in breast cancer cells. Consistent with this the agent reduced cell confluency in 7/8 cell lines treated irrespective of their ER status but not apoptosis incompetent MCF7 cells. In contrast SASH1 siRNA-transfected breast cancer cells exhibited reduced chloropyramine sensitivity. The prognostic significance of SASH1 expression was also investigated in two breast cancer cohorts. Expression was associated with favourable outcome in ER-positive cases, but only those of low histological grade/proliferative status. Conversely, we found a very strong inverse association in HER2+ disease irrespective of ER status, and in triple-negative, basal-like cases. Overall, the data suggest that SASH1 is prognostic in breast cancer and could have subtype-dependent effects on breast cancer progression. Pharmacologic induction of SASH1 by chloropyramine treatment of breast cancer warrants further preclinical and clinical investigation.

Phenformin has anti-tumorigenic effects in human ovarian cancer cells and in an orthotopic mouse model of serous ovarian cancer.

PubMed

Jackson, Amanda L; Sun, Wenchuan; Kilgore, Joshua; Guo, Hui; Fang, Ziwei; Yin, Yajie; Jones, Hannah M; Gilliam, Timothy P; Zhou, Chunxiao; Bae-Jump, Victoria L

2017-11-21

Obesity and diabetes have been associated with increased risk and worse outcomes in ovarian cancer (OC). The biguanide metformin is used in the treatment of type 2 diabetes and is also believed to have anti-tumorigenic benefits. Metformin is highly hydrophilic and requires organic cation transporters (OCTs) for entry into human cells. Phenformin, another biguanide, was taken off the market due to an increased risk of lactic acidosis over metformin. However, phenformin is not reliant on transporters for cell entry; and thus, may have increased potency as both an anti-diabetic and anti-tumorigenic agent than metformin. Thus, our goal was to evaluate the effect of phenformin on established OC cell lines, primary cultures of human OC cells and in an orthotopic mouse model of high grade serous OC. In three OC cell lines, phenformin significantly inhibited cellular proliferation, induced cell cycle G1 arrest and apoptosis, caused cellular stress, inhibited adhesion and invasion, and activation of AMPK and inhibition of the mTOR pathway. Phenformin also exerted anti-proliferative effects in seven primary cell cultures of human OC. Lastly, phenformin inhibited tumor growth in an orthotopic mouse model of serous OC, coincident with decreased Ki-67 staining and phosphorylated-S6 expression and increased expression of caspase 3 and phosphorylated-AMPK. Our findings demonstrate that phenformin has anti-tumorigenic effects in OC as previously demonstrated by metformin but it is yet to be determined if it is superior to metformin for the potential treatment of this disease.

Phenformin has anti-tumorigenic effects in human ovarian cancer cells and in an orthotopic mouse model of serous ovarian cancer

PubMed Central

Jackson, Amanda L.; Sun, Wenchuan; Kilgore, Joshua; Guo, Hui; Fang, Ziwei; Yin, Yajie; Jones, Hannah M.; Gilliam, Timothy P.; Zhou, Chunxiao; Bae-Jump, Victoria L.

2017-01-01

Obesity and diabetes have been associated with increased risk and worse outcomes in ovarian cancer (OC). The biguanide metformin is used in the treatment of type 2 diabetes and is also believed to have anti-tumorigenic benefits. Metformin is highly hydrophilic and requires organic cation transporters (OCTs) for entry into human cells. Phenformin, another biguanide, was taken off the market due to an increased risk of lactic acidosis over metformin. However, phenformin is not reliant on transporters for cell entry; and thus, may have increased potency as both an anti-diabetic and anti-tumorigenic agent than metformin. Thus, our goal was to evaluate the effect of phenformin on established OC cell lines, primary cultures of human OC cells and in an orthotopic mouse model of high grade serous OC. In three OC cell lines, phenformin significantly inhibited cellular proliferation, induced cell cycle G1 arrest and apoptosis, caused cellular stress, inhibited adhesion and invasion, and activation of AMPK and inhibition of the mTOR pathway. Phenformin also exerted anti-proliferative effects in seven primary cell cultures of human OC. Lastly, phenformin inhibited tumor growth in an orthotopic mouse model of serous OC, coincident with decreased Ki-67 staining and phosphorylated-S6 expression and increased expression of caspase 3 and phosphorylated-AMPK. Our findings demonstrate that phenformin has anti-tumorigenic effects in OC as previously demonstrated by metformin but it is yet to be determined if it is superior to metformin for the potential treatment of this disease. PMID:29245964

Proliferation of protease-enriched mast cells in sarcoptic skin lesions of raccoon dogs.

PubMed

Noviana, D; W Harjanti, D; Otsuka, Y; Horii, Y

2004-07-01

Skin sites, tongue, lung, liver, jejunum and rectum from two raccoon dogs with Sarcoptes scabiei infestation and five normal (control) raccoon dogs were examined in terms of the distribution, proteoglycan properties and protease activity of mast cells. Infestation with S. scabiei caused a significant increase in the number of dermal mast cells. While the number of mast cells (average +/- standard deviation) in specimens of skin from the dorsum, dorsal neck, dorsal hind foot and dorsal fore foot was 40.0 +/- 19.8/mm2 in control animals, it was 236.1 +/- 58.9/mm2 in the skin of mange-infested animals. Histochemical analysis revealed the glycosaminoglycan, heparin, within the mast cells of all organs examined in both control and affected animals. Enzyme-histochemical detection of serine proteases demonstrated an increase in mast-cell-specific protease activity (i.e., chymase and tryptase) in the skin of infested animals. The percentage of mast cells demonstrating chymase activity was 53.0 +/- 27.4% in control animals and 73.8 +/- 19.4% in mite-infested animals. The corresponding results for tryptase activity were 53.5 +/- 25.2% and 89.4 +/- 9.8%. Increases in mast cell chymase or tryptase activity, or both, were also observed within other organs of the infected animals, but the total number of mast cells found at such sites (with the exception of liver and ventrolateral pinna) did not differ from those of control animals. Copyright 2004 Elsevier Ltd.

Cell wall properties play an important role in the emergence of lateral root primordia from the parent root.

PubMed

Roycewicz, Peter S; Malamy, Jocelyn E

2014-05-01

Plants adapt to their unique soil environments by altering the number and placement of lateral roots post-embryonic. Mutants were identified in Arabidopsis thaliana that exhibit increased lateral root formation. Eight mutants were characterized in detail and were found to have increased lateral root formation due to at least three distinct mechanisms. The causal mutation in one of these mutants was found in the XEG113 gene, recently shown to be involved in plant cell wall biosynthesis. Lateral root primordia initiation is unaltered in this mutant. In contrast, synchronization of lateral root initiation demonstrated that mutation of XEG113 increases the rate at which lateral root primordia develop and emerge to form lateral roots. The effect of the XEG113 mutation was specific to the root system and had no apparent effect on shoot growth. Screening of 17 additional cell wall mutants, altering a myriad of cell wall components, revealed that many (but not all) types of cell wall defects promote lateral root formation. These results suggest that proper cell wall biosynthesis is necessary to constrain lateral root primordia emergence. While previous reports have shown that lateral root emergence is accompanied by active remodelling of cell walls overlying the primordia, this study is the first to demonstrate that alteration of the cell wall is sufficient to promote lateral root formation. Therefore, inherent cell wall properties may play a previously unappreciated role in regulation of root system architecture.

Increased expression of fatty acid synthase provides a survival advantage to colorectal cancer cells via upregulation of cellular respiration

PubMed Central

Zaytseva, Yekaterina Y.; Harris, Jennifer W.; Mitov, Mihail I.; Kim, Ji Tae; Butterfield, D. Allan; Lee, Eun Y.; Weiss, Heidi L.; Gao, Tianyan; Evers, B. Mark

2015-01-01

Fatty acid synthase (FASN), a lipogenic enzyme, is upregulated in colorectal cancer (CRC). Increased de novo lipid synthesis is thought to be a metabolic adaptation of cancer cells that promotes survival and metastasis; however, the mechanisms for this phenomenon are not fully understood. We show that FASN plays a role in regulation of energy homeostasis by enhancing cellular respiration in CRC. We demonstrate that endogenously synthesized lipids fuel fatty acid oxidation, particularly during metabolic stress, and maintain energy homeostasis. Increased FASN expression is associated with a decrease in activation of energy-sensing pathways and accumulation of lipid droplets in CRC cells and orthotopic CRCs. Immunohistochemical evaluation demonstrated increased expression of FASN and p62, a marker of autophagy inhibition, in primary CRCs and liver metastases compared to matched normal colonic mucosa. Our findings indicate that overexpression of FASN plays a crucial role in maintaining energy homeostasis in CRC via increased oxidation of endogenously synthesized lipids. Importantly, activation of fatty acid oxidation and consequent downregulation of stress-response signaling pathways may be key adaptation mechanisms that mediate the effects of FASN on cancer cell survival and metastasis, providing a strong rationale for targeting this pathway in advanced CRC. PMID:25970773

Increased expression of fatty acid synthase provides a survival advantage to colorectal cancer cells via upregulation of cellular respiration.

PubMed

Zaytseva, Yekaterina Y; Harris, Jennifer W; Mitov, Mihail I; Kim, Ji Tae; Butterfield, D Allan; Lee, Eun Y; Weiss, Heidi L; Gao, Tianyan; Evers, B Mark

2015-08-07

Fatty acid synthase (FASN), a lipogenic enzyme, is upregulated in colorectal cancer (CRC). Increased de novo lipid synthesis is thought to be a metabolic adaptation of cancer cells that promotes survival and metastasis; however, the mechanisms for this phenomenon are not fully understood. We show that FASN plays a role in regulation of energy homeostasis by enhancing cellular respiration in CRC. We demonstrate that endogenously synthesized lipids fuel fatty acid oxidation, particularly during metabolic stress, and maintain energy homeostasis. Increased FASN expression is associated with a decrease in activation of energy-sensing pathways and accumulation of lipid droplets in CRC cells and orthotopic CRCs. Immunohistochemical evaluation demonstrated increased expression of FASN and p62, a marker of autophagy inhibition, in primary CRCs and liver metastases compared to matched normal colonic mucosa. Our findings indicate that overexpression of FASN plays a crucial role in maintaining energy homeostasis in CRC via increased oxidation of endogenously synthesized lipids. Importantly, activation of fatty acid oxidation and consequent downregulation of stress-response signaling pathways may be key adaptation mechanisms that mediate the effects of FASN on cancer cell survival and metastasis, providing a strong rationale for targeting this pathway in advanced CRC.

Lactoferrin affects the adherence and invasion of Streptococcus dysgalactiae ssp. dysgalactiae in mammary epithelial cells.

PubMed

O'Halloran, Fiona; Beecher, Christine; Chaurin, Valerie; Sweeney, Torres; Giblin, Linda

2016-06-01

Streptococcus dysgalactiae ssp. dysgalactiae is an important causative agent of bovine mastitis worldwide. Lactoferrin is an innate immune protein that is associated with many functions including immunomodulatory, antiproliferative, and antimicrobial properties. This study aimed to investigate the interactions between lactoferrin and a clinical bovine mastitis isolate, Strep. dysgalactiae ssp. dysgalactiae DPC5345. Initially a deliberate in vivo bovine intramammary challenge was performed with Strep. dysgalactiae DPC5345. Results demonstrated a significant difference in lactoferrin mRNA levels in milk cells between the control and infused quarters 7h postinfusion. Milk lactoferrin levels in the Strep. dysgalactiae DPC5345 infused quarters were significantly increased compared with control quarters at 48h postinfusion. In vitro studies demonstrated that lactoferrin had a bacteriostatic effect on the growth of Strep. dysgalactiae DPC5345 and significantly decreased the ability of the bacteria to internalize into HC-11 mammary epithelial cells. Confocal microscopy images of HC-11 cells exposed to Strep. dysgalactiae and lactoferrin further supported this effect by demonstrating reduced invasion of bacteria to HC-11 cells. The combined data suggest that a bovine immune response to Strep. dysgalactiae infection includes a significant increase in lactoferrin expression in vivo, and based on in vitro data, lactoferrin limits mammary cell invasion of this pathogen by binding to the bacteria and preventing its adherence. Copyright © 2016 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

Arctigenin, a natural lignan compound, induces G0/G1 cell cycle arrest and apoptosis in human glioma cells.

PubMed

Maimaitili, Aisha; Shu, Zunhua; Cheng, Xiaojiang; Kaheerman, Kadeer; Sikandeer, Alifu; Li, Weimin

2017-02-01

The aim of the current study was to investigate the anticancer potential of arctigenin, a natural lignan compound, in malignant gliomas. The U87MG and T98G human glioma cell lines were treated with various concentrations of arctigenin for 48 h and the effects of arctigenin on the aggressive phenotypes of glioma cells were assessed. The results demonstrated that arctigenin dose-dependently inhibited the growth of U87MG and T98G cells, as determined using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and bromodeoxyuridine incorporation assays. Arctigenin exposure also induced a 60-75% reduction in colony formation compared with vehicle-treated control cells. However, arctigenin was not observed to affect the invasiveness of glioma cells. Arctigenin significantly increased the proportion of cells in the G 0 /G 1 phase and reduced the number of cells in the S phase, as compared with the control group (P<0.05). Western blot analysis demonstrated that arctigenin increased the expression levels of p21, retinoblastoma and p53 proteins, and significantly decreased the expression levels of cyclin D1 and cyclin-dependent kinase 4 proteins. Additionally, arctigenin was able to induce apoptosis in glioma cells, coupled with increased expression levels of cleaved caspase-3 and the pro-apoptotic BCL2-associated X protein. Furthermore, arctigenin-induced apoptosis was significantly suppressed by the pretreatment of cells with Z-DEVD-FMK, a caspase-3 inhibitor. In conclusion, the results suggest that arctigenin is able to inhibit cell proliferation and may induce apoptosis and cell cycle arrest at the G 0 /G 1 phase in glioma cells. These results warrant further investigation of the anticancer effects of arctigenin in animal models of gliomas.

Arctigenin, a natural lignan compound, induces G0/G1 cell cycle arrest and apoptosis in human glioma cells

PubMed Central

Maimaitili, Aisha; Shu, Zunhua; Cheng, Xiaojiang; Kaheerman, Kadeer; Sikandeer, Alifu; Li, Weimin

2017-01-01

The aim of the current study was to investigate the anticancer potential of arctigenin, a natural lignan compound, in malignant gliomas. The U87MG and T98G human glioma cell lines were treated with various concentrations of arctigenin for 48 h and the effects of arctigenin on the aggressive phenotypes of glioma cells were assessed. The results demonstrated that arctigenin dose-dependently inhibited the growth of U87MG and T98G cells, as determined using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and bromodeoxyuridine incorporation assays. Arctigenin exposure also induced a 60–75% reduction in colony formation compared with vehicle-treated control cells. However, arctigenin was not observed to affect the invasiveness of glioma cells. Arctigenin significantly increased the proportion of cells in the G0/G1 phase and reduced the number of cells in the S phase, as compared with the control group (P<0.05). Western blot analysis demonstrated that arctigenin increased the expression levels of p21, retinoblastoma and p53 proteins, and significantly decreased the expression levels of cyclin D1 and cyclin-dependent kinase 4 proteins. Additionally, arctigenin was able to induce apoptosis in glioma cells, coupled with increased expression levels of cleaved caspase-3 and the pro-apoptotic BCL2-associated X protein. Furthermore, arctigenin-induced apoptosis was significantly suppressed by the pretreatment of cells with Z-DEVD-FMK, a caspase-3 inhibitor. In conclusion, the results suggest that arctigenin is able to inhibit cell proliferation and may induce apoptosis and cell cycle arrest at the G0/G1 phase in glioma cells. These results warrant further investigation of the anticancer effects of arctigenin in animal models of gliomas. PMID:28356992

Human Cancer and Platelet Interaction, a Potential Therapeutic Target.

PubMed

Wang, Shike; Li, Zhenyu; Xu, Ren

2018-04-20

Cancer patients experience a four-fold increase in thrombosis risk, indicating that cancer development and progression are associated with platelet activation. Xenograft experiments and transgenic mouse models further demonstrate that platelet activation and platelet-cancer cell interaction are crucial for cancer metastasis. Direct or indirect interaction of platelets induces cancer cell plasticity and enhances survival and extravasation of circulating cancer cells during dissemination. In vivo and in vitro experiments also demonstrate that cancer cells induce platelet aggregation, suggesting that platelet-cancer interaction is bidirectional. Therefore, understanding how platelets crosstalk with cancer cells may identify potential strategies to inhibit cancer metastasis and to reduce cancer-related thrombosis. Here, we discuss the potential function of platelets in regulating cancer progression and summarize the factors and signaling pathways that mediate the cancer cell-platelet interaction.

Depletion of regulatory T cells by anti-ICOS antibody enhances anti-tumor immunity of tumor cell vaccine in prostate cancer.

PubMed

Mo, Lijun; Chen, Qianmei; Zhang, Xinji; Shi, Xiaojun; Wei, Lili; Zheng, Dianpeng; Li, Hongwei; Gao, Jimin; Li, Jinlong; Hu, Zhiming

2017-10-13

ICOS + Treg cells exert important immunosuppressive effects in tumor immunity. We adopt a combination approach of ICOS + Treg cells depletion with tumor cell vaccine to evaluate anti-tumor immunity in mouse prostate cancer model. Streptavidin (SA)-mGM-CSF surface-modified RM-1 cells were prepared as the vaccine and the mouse subcutaneous prostate tumor model was used to evaluate the immunity. Tumor growth, flow cytometry, immunohistochemistry, immunofluorescence and enzyme linked immunosorbent assay (ELISA) were performed to evaluate the therapeutic effects. Our results demonstrated that SA-mGM-CSF vaccine was prepared successfully and tumor growth was inhibited. The tumor size in the combination group was much smaller than that in the vaccine with IgG mAb group. The portions of dendritic cells, CD8 + and CD4 + T cells in the mice blood and tumor tissues were increased after treatment with vaccine. There were more immune-suppressing Tregs infiltrated into tumor after treatment with tumor cell vaccine, and ICOS blocking could deplete the infiltrated Tregs, and T lymphocytes increased more dramatically in the combination therapy group. The concentrations of interferon-γ were increased in all vaccine group, the concentrations of Interleukin-10 and Interleukin-4 were much lower in the combination group. Our study demonstrated that ICOS blocking could deplete the tumor-infiltrated ICOS + Treg cells. Combining GM-CSF surface-modified RM-1 cell vaccine with Anti-ICOS antibody could induce better antitumor immunity than a vaccine alone. Copyright © 2017 Elsevier Ltd. All rights reserved.

Fatty acid regulates gene expression and growth of human prostate cancer PC-3 cells

NASA Technical Reports Server (NTRS)

Hughes-Fulford, M.; Chen, Y.; Tjandrawinata, R. R.

2001-01-01

It has been proposed that the omega-6 fatty acids increase the rate of tumor growth. Here we test that hypothesis in the PC-3 human prostate tumor. We found that the essential fatty acids, linoleic acid (LA) and arachidonic acid (AA), and the AA metabolite PGE(2) stimulate tumor growth while oleic acid (OA) and the omega-3 fatty acid, eicosapentaenoic acid (EPA) inhibited growth. In examining the role of AA in growth response, we extended our studies to analyze changes in early gene expression induced by AA. We demonstrate that c-fos expression is increased within minutes of addition in a dose-dependent manner. Moreover, the immediate early gene cox-2 is also increased in the presence of AA in a dose-dependent manner, while the constitutive cox-1 message was not increased. Three hours after exposure to AA, the synthesis of PGE(2) via COX-2 was also increased. Previous studies have demonstrated that AA was primarily delivered by low density lipoprotein (LDL) via its receptor (LDLr). Since it is known that hepatomas, acute myelogenous leukemia and colorectal tumors lack normal cholesterol feedback, we examined the role of the LDLr in growth regulation of the PC-3 prostate cancer cells. Analysis of ldlr mRNA expression and LDLr function demonstrated that human PC-3 prostate cancer cells lack normal feedback regulation. While exogenous LDL caused a significant stimulation of cell growth and PGE(2) synthesis, no change was seen in regulation of the LDLr by LDL. Taken together, these data show that normal cholesterol feedback of ldlr message and protein is lost in prostate cancer. These data suggest that unregulated over-expression of LDLr in tumor cells would permit increased availability of AA, which induces immediate early genes c-fos and cox-2 within minutes of uptake.

Fatty acid regulates gene expression and growth of human prostate cancer PC-3 cells.

PubMed

Hughes-Fulford, M; Chen, Y; Tjandrawinata, R R

2001-05-01

It has been proposed that the omega-6 fatty acids increase the rate of tumor growth. Here we test that hypothesis in the PC-3 human prostate tumor. We found that the essential fatty acids, linoleic acid (LA) and arachidonic acid (AA), and the AA metabolite PGE(2) stimulate tumor growth while oleic acid (OA) and the omega-3 fatty acid, eicosapentaenoic acid (EPA) inhibited growth. In examining the role of AA in growth response, we extended our studies to analyze changes in early gene expression induced by AA. We demonstrate that c-fos expression is increased within minutes of addition in a dose-dependent manner. Moreover, the immediate early gene cox-2 is also increased in the presence of AA in a dose-dependent manner, while the constitutive cox-1 message was not increased. Three hours after exposure to AA, the synthesis of PGE(2) via COX-2 was also increased. Previous studies have demonstrated that AA was primarily delivered by low density lipoprotein (LDL) via its receptor (LDLr). Since it is known that hepatomas, acute myelogenous leukemia and colorectal tumors lack normal cholesterol feedback, we examined the role of the LDLr in growth regulation of the PC-3 prostate cancer cells. Analysis of ldlr mRNA expression and LDLr function demonstrated that human PC-3 prostate cancer cells lack normal feedback regulation. While exogenous LDL caused a significant stimulation of cell growth and PGE(2) synthesis, no change was seen in regulation of the LDLr by LDL. Taken together, these data show that normal cholesterol feedback of ldlr message and protein is lost in prostate cancer. These data suggest that unregulated over-expression of LDLr in tumor cells would permit increased availability of AA, which induces immediate early genes c-fos and cox-2 within minutes of uptake.

Narcolepsy Type 1 Is Associated with a Systemic Increase and Activation of Regulatory T Cells and with a Systemic Activation of Global T Cells

PubMed Central

Pitoiset, Fabien; Regnault, Armelle; Tran, Tu Anh; Liblau, Roland; Klatzmann, David; Rosenzwajg, Michelle

2017-01-01

Narcolepsy is a rare neurologic disorder characterized by excessive daytime sleepiness, cataplexy and disturbed nocturnal sleep patterns. Narcolepsy type 1 (NT1) has been shown to result from a selective loss of hypothalamic hypocretin-secreting neurons with patients typically showing low CSF-hypocretin levels (<110 pg/ml). This specific loss of hypocretin and the strong association with the HLA-DQB1*06:02 allele led to the hypothesis that NT1 could be an immune-mediated pathology. Moreover, susceptibility to NT1 has recently been associated with several pathogens, particularly with influenza A H1N1 virus either through infection or vaccination. The goal of this study was to compare peripheral blood immune cell populations in recent onset pediatric NT1 subjects (post or non-post 2009-influenza A H1N1 vaccination) to healthy donors. We demonstrated an increased number of central memory CD4+ T cells (CD62L+ CD45RA-) associated to an activated phenotype (increase in CD69 and CD25 expression) in NT1 patients. Percentage and absolute count of regulatory T cells (Tregs) in NT1 patients were increased associated with an activated phenotype (increase in GITR and LAP expression), and of activated memory phenotype. Cytokine production by CD4+ and CD8+ T cells after activation was not modified in NT1 patients. In H1N1 vaccinated NT1 patients, absolute counts of CD3+, CD8+ T cells, and B cells were increased compared to non-vaccinated NT1 patients. These results support a global T cell activation in NT1 patients and thus support a T cell-mediated autoimmune origin of NT1, but do not demonstrate the pathological role of H1N1 prophylactic vaccination. They should prompt further studies of T cells, particularly of Tregs (such as suppression and proliferation antigen specific assays, and also T-cell receptor sequencing), in NT1. PMID:28107375

Nutrient supplemented serum-free medium increases cardiomyogenesis efficiency of human pluripotent stem cells.

PubMed

Ting, Sherwin; Lecina, Marti; Chan, Yau-Chi; Tse, Hung Fat; Reuveny, Shaul; Oh, Steve Kw

2013-07-26

To development of an improved p38 MAPK inhibitor-based serum-free medium for embryoid body cardiomyocyte differentiation of human pluripotent stem cells. Human embryonic stem cells (hESC) differentiated to cardiomyocytes (CM) using a p38 MAPK inhibitor (SB203580) based serum-free medium (SB media). Nutrient supplements known to increase cell viability were added to SB medium. The ability of these supplements to improve cardiomyogenesis was evaluated by measurements of cell viability, total cell count, and the expression of cardiac markers via flow cytometry. An improved medium containing Soy hydrolysate (HySoy) and bovine serum albumin (BSA) (SupSB media) was developed and tested on 2 additional cell lines (H1 and Siu-hiPSC). Characterization of the cardiomyocytes was done by immunohistochemistry, electrophysiology and quantitative real-time reverse transcription-polymerase chain reaction. hESC cell line, HES-3, differentiating in SB medium for 16 d resulted in a cardiomyocyte yield of 0.07 ± 0.03 CM/hESC. A new medium (SupSB media) was developed with the addition of HySoy and BSA to SB medium. This medium resulted in 2.6 fold increase in cardiomyocyte yield (0.21 ± 0.08 CM/hESC). The robustness of SupSB medium was further demonstrated using two additional pluripotent cell lines (H1, hESC and Siu1, hiPSC), showing a 15 and 9 fold increase in cardiomyocyte yield respectively. The age (passage number) of the pluripotent cells did not affect the cardiomyocyte yields. Embryoid body (EB) cardiomyocytes formed in SupSB medium expressed canonical cardiac markers (sarcomeric α-actinin, myosin heavy chain and troponin-T) and demonstrated all three major phenotypes: nodal-, atrial- and ventricular-like. Electrophysiological characteristics (maximum diastolic potentials and action potential durations) of cardiomyocytes derived from SB and SupSB media were similar. The nutrient supplementation (HySoy and BSA) leads to increase in cell viability, cell yield and cardiac marker expression during cardiomyocyte differentiation, translating to an overall increase in cardiomyocyte yield.

Macrophage-mediated trogocytosis leads to death of antibody-opsonized tumor cells

PubMed Central

Velmurugan, Ramraj; Challa, Dilip K.; Ram, Sripad; Ober, Raimund J.; Ward, E. Sally

2016-01-01

Understanding the complex behavior of effector cells such as monocytes or macrophages in regulating cancerous growth is of central importance for cancer immunotherapy. Earlier studies using CD20-specific antibodies have demonstrated that the Fcγ receptor (FcγR)-mediated transfer of the targeted receptors from tumor cells to these effector cells through trogocytosis can enable escape from antibody therapy, leading to the viewpoint that this process is pro-tumorigenic. In the current study we demonstrate that persistent trogocytic attack results in the killing of HER2-overexpressing breast cancer cells. Further, antibody engineering to increase FcγR interactions enhances this tumoricidal activity. These studies extend the complex repertoire of activities of macrophages to trogocytic-mediated cell death of HER2-overexpressing target cells and have implications for the development of effective antibody-based therapies. PMID:27226489

Real-time and non-invasive measurements of cell mechanical behaviour with optical coherence phase microscopy

NASA Astrophysics Data System (ADS)

Gillies, D.; Gamal, W.; Downes, A.; Reinwald, Y.; Yang, Y.; El Haj, A.; Bagnaninchi, P. O.

2017-02-01

There is an unmet need in tissue engineering for non-invasive, label-free monitoring of cell mechanical behaviour in their physiological environment. Here, we describe a novel optical coherence phase microscopy (OCPM) set-up which can map relative cell mechanical behaviour in monolayers and 3D systems non-invasively, and in real-time. 3T3 and MCF-7 cells were investigated, with MCF-7 demonstrating an increased response to hydrostatic stimulus indicating MCF-7 being softer than 3T3. Thus, OCPM shows the ability to provide qualitative data on cell mechanical behaviour. Quantitative measurements of 6% agarose beads have been taken with commercial Cell Scale Microsquisher system demonstrating that their mechanical properties are in the same order of magnitude of cells, indicating that this is an appropriate test sample for the novel method described.

The Absence of NOD1 Enhances Killing of Aspergillus fumigatus Through Modulation of Dectin-1 Expression.

PubMed

Gresnigt, Mark S; Jaeger, Martin; Subbarao Malireddi, R K; Rasid, Orhan; Jouvion, Grégory; Fitting, Catherine; Melchers, Willem J G; Kanneganti, Thirumala-Devi; Carvalho, Agostinho; Ibrahim-Granet, Oumaima; van de Veerdonk, Frank L

2017-01-01

One of the major life-threatening infections for which severely immunocompromised patients are at risk is invasive aspergillosis (IA). Despite the current treatment options, the increasing antifungal resistance and poor outcome highlight the need for novel therapeutic strategies to improve outcome of patients with IA. In the current study, we investigated whether and how the intracellular pattern recognition receptor NOD1 is involved in host defense against Aspergillus fumigatus . When exploring the role of NOD1 in an experimental mouse model, we found that Nod1 -/- mice were protected against IA and demonstrated reduced fungal outgrowth in the lungs. We found that macrophages derived from bone marrow of Nod1 -/- mice were more efficiently inducing reactive oxygen species and cytokines in response to Aspergillus . Most strikingly, these cells were highly potent in killing A. fumigatus compared with wild-type cells. In line, human macrophages in which NOD1 was silenced demonstrated augmented Aspergillus killing and NOD1 stimulation decreased fungal killing. The differentially altered killing capacity of NOD1 silencing versus NOD1 activation was associated with alterations in dectin-1 expression, with activation of NOD1 reducing dectin-1 expression. Furthermore, we were able to demonstrate that Nod1 -/- mice have elevated dectin-1 expression in the lung and bone marrow, and silencing of NOD1 gene expression in human macrophages increases dectin-1 expression. The enhanced dectin-1 expression may be the mechanism of enhanced fungal killing of Nod1 -/- cells and human cells in which NOD1 was silenced, since blockade of dectin-1 reversed the augmented killing in these cells. Collectively, our data demonstrate that NOD1 receptor plays an inhibitory role in the host defense against Aspergillus . This provides a rationale to develop novel immunotherapeutic strategies for treatment of aspergillosis that target the NOD1 receptor, to enhance the efficiency of host immune cells to clear the infection by increasing fungal killing and cytokine responses.

Post-Spaceflight (STS-135) Mouse Splenocytes Demonstrate Altered Activation Properties and Surface Molecule Expression

PubMed Central

Crucian, Brian; Sams, Clarence

2015-01-01

Alterations in immune function have been documented during or post-spaceflight and in ground based models of microgravity. Identification of immune parameters that are dysregulated during spaceflight is an important step in mitigating crew health risks during deep space missions. The in vitro analysis of leukocyte activity post-spaceflight in both human and animal species is primarily focused on lymphocytic function. This report completes a broader spectrum analysis of mouse lymphocyte and monocyte changes post 13 days orbital flight (mission STS-135). Analysis includes an examination in surface markers for cell activation, and antigen presentation and co-stimulatory molecules. Cytokine production was measured after stimulation with T-cell mitogen or TLR-2, TLR-4, or TLR-5 agonists. Splenocyte surface marker analysis immediate post-spaceflight and after in vitro culture demonstrated unique changes in phenotypic populations between the flight mice and matched treatment ground controls. Post-spaceflight splenocytes (flight splenocytes) had lower expression intensity of CD4+CD25+ and CD8+CD25+ cells, lower percentage of CD11c+MHC II+ cells, and higher percentage of CD11c+MHC I+ populations compared to ground controls. The flight splenocytes demonstrated an increase in phagocytic activity. Stimulation with ConA led to decrease in CD4+ population but increased CD4+CD25+ cells compared to ground controls. Culturing with TLR agonists led to a decrease in CD11c+ population in splenocytes isolated from flight mice compared to ground controls. Consequently, flight splenocytes with or without TLR-agonist stimulation showed a decrease in CD11c+MHC I+, CD11c+MHC II+, and CD11c+CD86+ cells compared to ground controls. Production of IFN-γ was decreased and IL-2 was increased from ConA stimulated flight splenocytes. This study demonstrated that expression of surface molecules can be affected by conditions of spaceflight and impaired responsiveness persists under culture conditions in vitro. PMID:25970640

Preclinical Evaluation of Efficacy and Safety of an Improved Lentiviral Vector for the Treatment of β-Thalassemia and Sickle Cell Disease

PubMed Central

Negre, Olivier; Bartholomae, Cynthia; Beuzard, Yves; Cavazzana, Marina; Christiansen, Lauryn; Courne, Céline; Deichmann, Annette; Denaro, Maria; de Dreuzy, Edouard; Finer, Mitchell; Fronza, Raffaele; Gillet-Legrand, Béatrix; Joubert, Christophe; Kutner, Robert; Leboulch, Philippe; Maouche, Leïla; Paulard, Anaïs; Pierciey, Francis J.; Rothe, Michael; Ryu, Byoung; Schmidt, Manfred; von Kalle, Christof; Payen, Emmanuel; Veres, Gabor

2015-01-01

A previously published clinical trial demonstrated the benefit of autologous CD34+ cells transduced with a self-inactivating lentiviral vector (HPV569) containing an engineered β-globin gene (βA-T87Q-globin) in a subject with β-thalassemia major. This vector has been modified to increase transduction efficacy without compromising safety. In vitro analyses indicated that the changes resulted in both increased vector titers (3 to 4 fold) and increased transduction efficacy (2 to 3 fold). An in vivo study in which 58 β-thalassemic mice were transplanted with vector- or mock-transduced syngenic bone marrow cells indicated sustained therapeutic efficacy. Secondary transplantations involving 108 recipients were performed to evaluate long-term safety. The six month study showed no hematological or biochemical toxicity. Integration site (IS) profile revealed an oligo/polyclonal hematopoietic reconstitution in the primary transplants and reduced clonality in secondary transplants. Tumor cells were detected in the secondary transplant mice in all treatment groups (including the control group), without statistical differences in the tumor incidence. Immunohistochemistry and quantitative PCR demonstrated that tumor cells were not derived from transduced donor cells. This comprehensive efficacy and safety data provided the basis for initiating two clinical trials with this second generation vector (BB305) in Europe and in the USA in patients with β-thalassemia major and sickle cell disease. PMID:25429463

Multiple scale model for cell migration in monolayers: Elastic mismatch between cells enhances motility

NASA Astrophysics Data System (ADS)

Palmieri, Benoit; Bresler, Yony; Wirtz, Denis; Grant, Martin

2015-07-01

We propose a multiscale model for monolayer of motile cells that comprise normal and cancer cells. In the model, the two types of cells have identical properties except for their elasticity; cancer cells are softer and normal cells are stiffer. The goal is to isolate the role of elasticity mismatch on the migration potential of cancer cells in the absence of other contributions that are present in real cells. The methodology is based on a phase-field description where each cell is modeled as a highly-deformable self-propelled droplet. We simulated two types of nearly confluent monolayers. One contains a single cancer cell in a layer of normal cells and the other contains normal cells only. The simulation results demonstrate that elasticity mismatch alone is sufficient to increase the motility of the cancer cell significantly. Further, the trajectory of the cancer cell is decorated by several speed “bursts” where the cancer cell quickly relaxes from a largely deformed shape and consequently increases its translational motion. The increased motility and the amplitude and frequency of the bursts are in qualitative agreement with recent experiments.

CH3NH3I post-treatment improves the performance of perovskite solar cells via eliminating the impure phases

NASA Astrophysics Data System (ADS)

Liang, Jingjing; Liang, Chunjun; Zhang, Huimin; Sun, Mengjie; Liu, Hong; Ji, Chao; Zhang, Xuewen; Li, Dan; He, Zhiqun

Organic-inorganic halide perovskites are currently generating extensive interest for applications in solar cells. The perovskite morphology and composition have significant roles in solar cells. Impure phases, which will influence the performance of solar cells, are inevitably present in the film of perovskite. We found that another MAI deposition on the previous perovskite could ameliorate the film. The post-deposited MAI participates in the reconstruction of the perovskite, leading to reduced amount of impure phase, increased grain size, increased absorption and significantly improved power conversion efficiency. The results demonstrate a treatment approach to fabricate efficient planar heterojunction perovskite solar cells.

Salt Inactivates Endothelial Nitric Oxide Synthase in Endothelial Cells12

PubMed Central

Li, Juan; White, James; Guo, Ling; Zhao, Xiaomin; Wang, Jiafu; Smart, Eric J.; Li, Xiang-An

2009-01-01

There is a 1–4 mmol/L rise in plasma sodium concentrations in individuals with high salt intake and in patients with essential hypertension. In this study, we used 3 independent assays to determine whether such a small increase in sodium concentrations per se alters endothelial nitric oxide synthase (eNOS) function and contributes to hypertension. By directly measuring NOS activity in living bovine aortic endothelial cells, we demonstrated that a 5-mmol/L increase in salt concentration (from 137 to 142 mmol/L) caused a 25% decrease in NOS activity. Importantly, the decrease in NOS activity was in a salt concentration-dependent manner. The NOS activity was decreased by 25, 45, and 70%, with the increase of 5, 10, and 20 mmol/L of NaCl, respectively. Using Chinese hamster ovary cells stably expressing eNOS, we confirmed the inhibitory effects of salt on eNOS activity. The eNOS activity was unaffected in the presence of equal milliosmol of mannitol, which excludes an osmotic effect. Using an ex vivo aortic angiogenesis assay, we demonstrated that salt attenuated the nitric oxide (NO)-dependent proliferation of endothelial cells. By directly monitoring blood pressure changes in response to salt infusion, we found that in vivo infusion of salt induced an acute increase in blood pressure in a salt concentration-dependent manner. In conclusion, our findings demonstrated that eNOS is sensitive to changes in salt concentration. A 5-mmol/L rise in salt concentration, within the range observed in essential hypertension patients or in individuals with high salt intake, could significantly suppress eNOS activity. This salt-induced reduction in NO generation in endothelial cells may contribute to the development of hypertension. PMID:19176751

Salt inactivates endothelial nitric oxide synthase in endothelial cells.

PubMed

Li, Juan; White, James; Guo, Ling; Zhao, Xiaomin; Wang, Jiafu; Smart, Eric J; Li, Xiang-An

2009-03-01

There is a 1-4 mmol/L rise in plasma sodium concentrations in individuals with high salt intake and in patients with essential hypertension. In this study, we used 3 independent assays to determine whether such a small increase in sodium concentrations per se alters endothelial nitric oxide synthase (eNOS) function and contributes to hypertension. By directly measuring NOS activity in living bovine aortic endothelial cells, we demonstrated that a 5-mmol/L increase in salt concentration (from 137 to 142 mmol/L) caused a 25% decrease in NOS activity. Importantly, the decrease in NOS activity was in a salt concentration-dependent manner. The NOS activity was decreased by 25, 45, and 70%, with the increase of 5, 10, and 20 mmol/L of NaCl, respectively. Using Chinese hamster ovary cells stably expressing eNOS, we confirmed the inhibitory effects of salt on eNOS activity. The eNOS activity was unaffected in the presence of equal milliosmol of mannitol, which excludes an osmotic effect. Using an ex vivo aortic angiogenesis assay, we demonstrated that salt attenuated the nitric oxide (NO)-dependent proliferation of endothelial cells. By directly monitoring blood pressure changes in response to salt infusion, we found that in vivo infusion of salt induced an acute increase in blood pressure in a salt concentration-dependent manner. In conclusion, our findings demonstrated that eNOS is sensitive to changes in salt concentration. A 5-mmol/L rise in salt concentration, within the range observed in essential hypertension patients or in individuals with high salt intake, could significantly suppress eNOS activity. This salt-induced reduction in NO generation in endothelial cells may contribute to the development of hypertension.

Autocrine stimulation of VEGFR-2 activates human leukemic cell growth and migration

PubMed Central

Dias, Sergio; Hattori, Koichi; Zhu, Zhenping; Heissig, Beate; Choy, Margaret; Lane, William; Wu, Yan; Chadburn, Amy; Hyjek, Elizabeth; Gill, Muhammad; Hicklin, Daniel J.; Witte, Larry; Moore, M.A.S.; Rafii, Shahin

2000-01-01

Emerging data suggest that VEGF receptors are expressed by endothelial cells as well as hematopoietic stem cells. Therefore, we hypothesized that functional VEGF receptors may also be expressed in malignant counterparts of hematopoietic stem cells such as leukemias. We demonstrate that certain leukemias not only produce VEGF but also express functional VEGFR-2 in vivo and in vitro, resulting in the generation of an autocrine loop that may support leukemic cell survival and proliferation. Approximately 50% of freshly isolated leukemias expressed mRNA and protein for VEGFR-2. VEGF165 induced phosphorylation of VEGFR-2 and increased proliferation of leukemic cells, demonstrating these receptors were functional. VEGF165 also induced the expression of MMP-9 by leukemic cells and promoted their migration through reconstituted basement membrane. The neutralizing mAb IMC-1C11, specific to human VEGFR-2, inhibited leukemic cell survival in vitro and blocked VEGF165-mediated proliferation of leukemic cells and VEGF-induced leukemic cell migration. Xenotransplantation of primary leukemias and leukemic cell lines into immunocompromised nonobese diabetic mice resulted in significant elevation of human, but not murine, VEGF in plasma and death of inoculated mice within 3 weeks. Injection of IMC-1C11 inhibited proliferation of xenotransplanted human leukemias and significantly increased the survival of inoculated mice. Interruption of signaling by VEGFRs, particularly VEGFR-2, may provide a novel strategy for inhibiting leukemic cell proliferation. PMID:10953026

PAR-2 receptor-induced effects on human eccrine sweat gland cells.

PubMed

L Bovell, Douglas; Kofler, Barbara; Lang, Roland

2009-01-01

Serine proteases can induce cell signaling by stimulating G-protein-coupled receptors, called proteinase-activated receptors (PAR's) on a variety of epithelial cells. While PAR-2, one such receptor, activates cell signaling in a secretory cell line derived from human sweat glands, there was no information on their presence and effects on intact sweat glands. PAR-2 presence and activation of eccrine sweat glands isolated from human skin samples was investigated using Western blot analysis, immunohistochemistry, electron microscopy (EM) and Ca(2+) imaging. Anti-human PAR-2 antibody demonstrated the presence of these receptors in eccrine sweat glands. EM showed that PAR-2 activation resulted in degranulation of secretory cells. Ca(2+) imaging using PAR-2 activators demonstrated a two phase increase in [Ca(2+)](i) which was dependent on extracellular Ca(2+) for the second phase, and that the response could be blocked by prior incubation with xestospongin, the IP(3) receptor blocker. The results demonstrated that PAR-2 receptors are present in human sweat gland secretory cells and that these receptors are functionally active and can induce changes associated with secretory events in eccrine glands.

Biological effects of two successive shock waves focused on liver tissues and melanoma cells.

PubMed

Benes, J; Sunka, P; Králová, J; Kaspar, J; Poucková, P

2007-01-01

A new generator of two successive shock waves focused to a common focal point has been developed. Cylindrical pressure waves created by multichannel electrical discharges on two cylindrical composite anodes are focused by a metallic parabolic reflector - cathode, and near the focus they are transformed to strong shock waves. Schlieren photos of the focal region have demonstrated that mutual interaction of the two waves results in generation of a large number of secondary short-wavelength shocks. Interaction of the focused shockwaves with liver tissues and cancer cell suspensions was investigated. Localized injury of rabbit liver induced by the shock waves was demonstrated by magnetic resonance imaging. Histological analysis of liver samples taken from the injured region revealed that the transition between the injured and the healthy tissues is sharp. Suspension of melanoma B16 cells was exposed and the number of the surviving cells rapidly decreased with increasing number of shocks and only 8 % of cells survived 350 shocks. Photographs of cells demonstrate that even small number of shocks results in perforation of cell membranes.

Leukemia cells demonstrate a different metabolic perturbation provoked by 2-deoxyglucose.

PubMed

Miwa, Hiroshi; Shikami, Masato; Goto, Mineaki; Mizuno, Shohei; Takahashi, Miyuki; Tsunekawa-Imai, Norikazu; Ishikawa, Takamasa; Mizutani, Motonori; Horio, Tomohiro; Gotou, Mayuko; Yamamoto, Hidesuke; Wakabayashi, Motohiro; Watarai, Masaya; Hanamura, Ichiro; Imamura, Akira; Mihara, Hidetsugu; Nitta, Masakazu

2013-05-01

The shift in energy metabolism from oxidative phosphorylation to glycolysis can serve as a target for the inhibition of cancer growth. Here, we examined the metabolic changes induced by 2-deoxyglucose (2-DG), a glycolysis inhibitor, in leukemia cells by metabolome analysis. NB4 cells mainly utilized glucose as an energy source by glycolysis and oxidative phosphorylation in mitochondria, since metabolites in the glycolytic pathway and in the tricarboxylic acid (TCA) cycle were significantly decreased by 2-DG. In THP-1 cells, metabolites in the TCA cycle were not decreased to the same extent by 2-DG as in NB4 cells, which indicates that THP-1 utilizes energy sources other than glucose. TCA cycle metabolites in THP-1 cells may be derived from acetyl-CoA by fatty acid β-oxidation, which was supported by abundant detection of carnitine and acetylcarnitine in THP-1 cells. 2-DG treatment increased the levels of pentose phosphate pathway (PPP) metabolites and augmented the generation of NADPH by glucose-6-phosphate dehydrogenase. An increase in NADPH and upregulation of glutathione synthetase expression resulted in the increase in the reduced form of glutathione by 2-DG in NB4 cells. We demonstrated that a combination of 2-DG and inhibition of PPP by dehydroepiandrosterone (DHEA) effectively suppressed the growth of NB4 cells. The replenishment of the TCA cycle by fatty acid oxidation by carnitine palmitoyltransferase in THP-1 cells, treated by 2-DG, might be regulated by AMPK, as the combination of 2-DG and inhibition of AMPK by compound C potently suppressed the growth of THP-1 cells. Although 2-DG has been effective in preclinical and clinical studies, this treatment has not been fully explored due to concerns related to potential toxicities such as brain toxicity at high doses. We demonstrated that a combination of 2-DG and DHEA or compound C at a relatively low concentration effectively inhibits the growth of NB4 and THP-1 cells, respectively. These observations may aid in the identification of appropriate combinations of metabolic inhibitors at low concentrations which do not cause toxicities.

Sirtuin1 and autophagy protect cells from fluoride-induced cell stress.

PubMed

Suzuki, Maiko; Bartlett, John D

2014-02-01

Sirtuin1 (SIRT1) is a nicotinamide adenine dinucleotide (NAD(+))-dependent deacetylase functioning in the regulation of metabolism, cell survival and organismal lifespan. Active SIRT1 regulates autophagy during cell stress, including calorie restriction, endoplasmic reticulum (ER) stress and oxidative stress. Previously, we reported that fluoride induces ER-stress in ameloblasts responsible for enamel formation, suggesting that ER-stress plays a role in dental fluorosis. However, the molecular mechanism of how cells respond to fluoride-induced cell stress is unclear. Here, we demonstrate that fluoride activates SIRT1 and initiates autophagy to protect cells from fluoride exposure. Fluoride treatment of ameloblast-derived cells (LS8) significantly increased Sirt1 expression and induced SIRT1 phosphorylation resulting in the augmentation of SIRT1 deacetylase activity. To demonstrate that fluoride exposure initiates autophagy, we characterized the expression of autophagy related genes (Atg); Atg5, Atg7 and Atg8/LC3 and showed that both their transcript and protein levels were significantly increased following fluoride treatment. To confirm that SIRT1 plays a protective role in fluoride toxicity, we used resveratrol (RES) to augment SIRT1 activity in fluoride treated LS8 cells. RES increased autophagy, inhibited apoptosis, and decreased fluoride cytotoxicity. Rats treated with fluoride (0, 50, 100 and 125ppm) in drinking water for 6weeks had significantly elevated expression levels of Sirt1, Atg5, Atg7 and Atg8/LC3 in their maturation stage enamel organs. Increased protein levels of p-SIRT1, ATG5 and ATG8/LC3 were present in fluoride-treated rat maturation stage ameloblasts. Therefore, the SIRT1/autophagy pathway may play a critical role as a protective response to help prevent dental fluorosis. Copyright © 2013 Elsevier B.V. All rights reserved.

Sirtuin1 and autophagy protect cells from fluoride-induced cell stress

PubMed Central

Suzuki, Maiko; Bartlett, John D.

2014-01-01

Sirtuin1 (SIRT1) is an (NAD+)-dependent deacetylase functioning in the regulation of metabolism, cell survival and organismal lifespan. Active SIRT1 regulates autophagy during cell stress, including calorie restriction, endoplasmic reticulum stress and oxidative stress. Previously, we reported that fluoride induces endoplasmic reticulum (ER) stress in ameloblasts responsible for enamel formation, suggesting that ER-stress plays a role in dental fluorosis. However, the molecular mechanism of how cells respond to fluoride-induced cell stress is unclear. Here, we demonstrate that fluoride activates SIRT1 and initiates autophagy to protect cells from fluoride exposure. Fluoride treatment of ameloblast-derived cells (LS8) significantly increased Sirt1 expression and induced SIRT1 phosphorylation resulting in the augmentation of SIRT1 deacetylase activity. To demonstrate that fluoride exposure initiates autophagy, we characterized the expression of autophagy related genes (Atg); Atg5, Atg7 and Atg8/LC3 and showed that both their transcript and protein levels were significantly increased following fluoride treatment. To confirm that SIRT1 plays a protective role in fluoride toxicity, we used resveratrol (RES) to augmented SIRT1 activity in fluoride treated LS8 cells. RES increased autophagy, inhibited apoptosis, and decreased fluoride cytotoxicity. Rats treated with fluoride (0, 50 and 100 ppm) in drinking water for 6 weeks had significantly elevated expression levels of Sirt1, Atg5, Atg7 and Atg8/LC3 in their maturation stage enamel organs. Increased protein levels of p-SIRT1, ATG5 and ATG8/LC3 were present in fluoride-treated rat maturation stage ameloblasts. Therefore, the SIRT1/autophagy pathway may play a critical role as a protective response to help prevent dental fluorosis. PMID:24296261

Pigment epithelium-derived factor stimulates skeletal muscle glycolytic activity through NADPH oxidase-dependent reactive oxygen species production.

PubMed

Carnagarin, Revathy; Carlessi, Rodrigo; Newsholme, Philip; Dharmarajan, Arun M; Dass, Crispin R

2016-09-01

Pigment epithelium-derived factor is a multifunctional serpin implicated in insulin resistance in metabolic disorders. Recent evidence suggests that exposure of peripheral tissues such as skeletal muscle to PEDF has profound metabolic consequences with predisposition towards chronic conditions such as obesity, type 2 diabetes, metabolic syndrome and polycystic ovarian syndrome. Chronic inflammation shifts muscle metabolism towards increased glycolysis and decreased oxidative metabolism. In the present study, we demonstrate a novel effect of PEDF on cellular metabolism in mouse cell line (C2C12) and human primary skeletal muscle cells. PEDF addition to skeletal muscle cells induced enhanced phospholipase A2 activity. This was accompanied with increased production of reactive oxygen species in a nicotinamide adenine dinucleotide phosphate (NADPH) oxidase-dependent manner that triggered a shift towards a more glycolytic phenotype. Extracellular flux analysis and glucose consumption assays demonstrated that PEDF treatment resulted in enhanced glycolysis but did not change mitochondrial respiration. Our results demonstrate that skeletal muscle cells express a PEDF-inducible oxidant generating system that enhances glycolysis but is sensitive to antioxidants and NADPH oxidase inhibition. Copyright © 2016 Elsevier Ltd. All rights reserved.

Target molecules in 3T3-L1 adipocytes differentiation are regulated by maslinic acid, a natural triterpene from Olea europaea.

PubMed

Pérez-Jiménez, Amalia; Rufino-Palomares, Eva E; Fernández-Gallego, Nieves; Ortuño-Costela, M Carmen; Reyes-Zurita, Fernando J; Peragón, Juan; García-Salguero, Leticia; Mokhtari, Khalida; Medina, Pedro P; Lupiáñez, José A

2016-11-15

Metabolic syndrome is a set of pathologies among which stand out the obesity, which is related to the lipid droplet accumulation and changes to cellular morphology regulated by several molecules and transcription factors. Maslinic acid (MA) is a natural product with demonstrated pharmacological functions including anti-inflammation, anti-tumor and anti-oxidation, among others. Here we report the effects of MA on the adipogenesis process in 3T3-L1 cells. Cell viability, glucose uptake, cytoplasmic triglyceride droplets, triglycerides quantification, gene transcription factors such as peroxisome proliferator-activated receptor γ (PPARγ) and adipocyte fatty acid-binding protein (aP2) and intracellular Ca 2+ levels were determined in pre-adipocytes and adipocytes of 3T3-L1 cells. MA increased glucose uptake. MA also decreased lipid droplets and triglyceride levels, which is in concordance with the down-regulation of PPARγ and aP2. Finally, MA increased the intracellular Ca 2+ concentration, which could also be involved in the demonstrated antiadipogenic effect of this triterpene. MA has been demonstrated as potential antiadipogenic compound in 3T3-L1 cells. Copyright © 2016 Elsevier GmbH. All rights reserved.

Dose-dependent and cell type-specific cell death and proliferation following in vitro exposure to radial extracorporeal shock waves

PubMed Central

Hochstrasser, Tanja; Frank, Hans-Georg; Schmitz, Christoph

2016-01-01

Radial extracorporeal shock wave (rESW) therapy is widely used in musculoskeletal disorders and wound repair. However, the mechanisms of action are still largely unknown. The current study compared the effects of rESWs on two cell types. Human fetal foreskin fibroblasts (HFFF2) and human placental choriocarcinoma cell line JEG-3 were exposed to 0, 100, 200, 500 or 5000 rESWs generated with a Swiss DolorClast device (2.5 bar, 1 Hz). FACS analysis immediately after rESW exposure showed that initially, rESWs rather induced mechanical cell destruction than regulated or programmed cell death. Cell damage was nearly negated by reducing cavitation. Furthermore, cell viability decreased progressively with higher numbers of rESWs. Exposure to rESWs had no impact on growth potential of JEG-3 cells, but dose-dependently increased growth potential of HFFF2 cells. Cultivation of cells that were initially exposed to sham-rESWs in conditioned media increased the growth potential of HFFF2 cells, nevertheless, an even stronger effect was achieved by direct exposure to rESWs. Additionally, cell cycle distribution analysis demonstrated a shift in proportion from G0/G1 to G2/M phase in HFFF2 cells, but not in JEG-3 cells. These data demonstrate that rESWs leads to initial and subsequent dose-dependent and cell type-specific effects in vitro. PMID:27477873

Enhanced metastatic capacity of breast cancer cells after interaction and hybrid formation with mesenchymal stroma/stem cells (MSC).

PubMed

Melzer, Catharina; von der Ohe, Juliane; Hass, Ralf

2018-01-05

Fusion of breast cancer cells with tumor-associated populations of the microenvironment including mesenchymal stroma/stem-like cells (MSC) represents a rare event in cell communication whereby the metastatic capacity of those hybrid cells remains unclear. Functional changes were investigated in vitro and in vivo following spontaneous fusion and hybrid cell formation between primary human MSC and human MDA-MB-231 breast cancer cells. Thus, lentiviral eGFP-labeled MSC and breast cancer cells labeled with mcherry resulted in dual-fluorescing hybrid cells after co-culture. Double FACS sorting and single cell cloning revealed two different aneuploid male hybrid populations (MDA-hyb1 and MDA-hyb2) with different STR profiles, pronounced telomerase activities, and enhanced proliferative capacities as compared to the parental cells. Microarray-based mRNA profiling demonstrated marked regulation of genes involved in epithelial-mesenchymal transition and increased expression of metastasis-associated genes including S100A4. In vivo studies following subcutaneous injection of the breast cancer and the two hybrid populations substantiated the in vitro findings by a significantly elevated tumor growth of the hybrid cells. Moreover, both hybrid populations developed various distant organ metastases in a much shorter period of time than the parental breast cancer cells. Together, these data demonstrate spontaneous development of new tumor cell populations exhibiting different parental properties after close interaction and subsequent fusion of MSC with breast cancer cells. This formation of tumor hybrids contributes to continuously increasing tumor heterogeneity and elevated metastatic capacities.

[The implementation of computer model in research of dynamics of proliferation of cells of thyroid gland follicle].

PubMed

Abduvaliev, A A; Gil'dieva, M S; Khidirov, B N; Saĭdalieva, M; Khasanov, A A; Musaeva, Sh N; Saatov, T S

2012-04-01

The article deals with the results of computational experiments in research of dynamics of proliferation of cells of thyroid gland follicle in normal condition and in the case of malignant neoplasm. The model studies demonstrated that the chronic increase of parameter of proliferation of cells of thyroid gland follicle results in abnormal behavior of numbers of cell cenosis of thyroid gland follicle. The stationary state interrupts, the auto-oscillations occur with transition to irregular oscillations with unpredictable cell proliferation and further to the "black hole" effect. It is demonstrated that the present medical biologic experimental data and theory propositions concerning the structural functional organization of thyroid gland on cell level permit to develop mathematical models for quantitative analysis of numbers of cell cenosis of thyroid gland follicle in normal conditions. The technique of modeling of regulative mechanisms of living systems and equations of cell cenosis regulations was used

Experimental demonstration of programmable multi-functional spin logic cell based on spin Hall effect

NASA Astrophysics Data System (ADS)

Zhang, X.; Wan, C. H.; Yuan, Z. H.; Fang, C.; Kong, W. J.; Wu, H.; Zhang, Q. T.; Tao, B. S.; Han, X. F.

2017-04-01

Confronting with the gigantic volume of data produced every day, raising integration density by reducing the size of devices becomes harder and harder to meet the ever-increasing demand for high-performance computers. One feasible path is to actualize more logic functions in one cell. In this respect, we experimentally demonstrate a prototype spin-orbit torque based spin logic cell integrated with five frequently used logic functions (AND, OR, NOT, NAND and NOR). The cell can be easily programmed and reprogrammed to perform desired function. Furthermore, the information stored in cells is symmetry-protected, making it possible to expand into logic gate array where the cell can be manipulated one by one without changing the information of other undesired cells. This work provides a prospective example of multi-functional spin logic cell with reprogrammability and nonvolatility, which will advance the application of spin logic devices.

Defects in intracellular trafficking of fungal cell wall synthases lead to aberrant host immune recognition.

PubMed

Esher, Shannon K; Ost, Kyla S; Kohlbrenner, Maria A; Pianalto, Kaila M; Telzrow, Calla L; Campuzano, Althea; Nichols, Connie B; Munro, Carol; Wormley, Floyd L; Alspaugh, J Andrew

2018-06-01

The human fungal pathogen, Cryptococcus neoformans, dramatically alters its cell wall, both in size and composition, upon entering the host. This cell wall remodeling is essential for host immune avoidance by this pathogen. In a genetic screen for mutants with changes in their cell wall, we identified a novel protein, Mar1, that controls cell wall organization and immune evasion. Through phenotypic studies of a loss-of-function strain, we have demonstrated that the mar1Δ mutant has an aberrant cell surface and a defect in polysaccharide capsule attachment, resulting in attenuated virulence. Furthermore, the mar1Δ mutant displays increased staining for exposed cell wall chitin and chitosan when the cells are grown in host-like tissue culture conditions. However, HPLC analysis of whole cell walls and RT-PCR analysis of cell wall synthase genes demonstrated that this increased chitin exposure is likely due to decreased levels of glucans and mannans in the outer cell wall layers. We observed that the Mar1 protein differentially localizes to cellular membranes in a condition dependent manner, and we have further shown that the mar1Δ mutant displays defects in intracellular trafficking, resulting in a mislocalization of the β-glucan synthase catalytic subunit, Fks1. These cell surface changes influence the host-pathogen interaction, resulting in increased macrophage activation to microbial challenge in vitro. We established that several host innate immune signaling proteins are required for the observed macrophage activation, including the Card9 and MyD88 adaptor proteins, as well as the Dectin-1 and TLR2 pattern recognition receptors. These studies explore novel mechanisms by which a microbial pathogen regulates its cell surface in response to the host, as well as how dysregulation of this adaptive response leads to defective immune avoidance.

Analysis of re-replication from deregulated origin licensing by DNA fiber spreading

PubMed Central

Dorn, Elizabeth S.; Chastain, Paul D.; Hall, Jonathan R.; Cook, Jeanette Gowen

2009-01-01

A major challenge each human cell-division cycle is to ensure that DNA replication origins do not initiate more than once, a phenomenon known as re-replication. Acute deregulation of replication control ultimately causes extensive DNA damage, cell-cycle checkpoint activation and cell death whereas moderate deregulation promotes genome instability and tumorigenesis. In the absence of detectable increases in cellular DNA content however, it has been difficult to directly demonstrate re-replication or to determine if the ability to re-replicate is restricted to a particular cell-cycle phase. Using an adaptation of DNA fiber spreading we report the direct detection of re-replication on single DNA molecules from human chromosomes. Using this method we demonstrate substantial re-replication within 1 h of S phase entry in cells overproducing the replication factor, Cdt1. Moreover, a comparison of the HeLa cancer cell line to untransformed fibroblasts suggests that HeLa cells produce replication signals consistent with low-level re-replication in otherwise unperturbed cell cycles. Re-replication after depletion of the Cdt1 inhibitor, geminin, in an untransformed fibroblast cell line is undetectable by standard assays but readily quantifiable by DNA fiber spreading analysis. Direct evaluation of re-replicated DNA molecules will promote increased understanding of events that promote or perturb genome stability. PMID:19010964

Homologous recombination mediates S-phase-dependent radioresistance in cells deficient in DNA polymerase eta.

PubMed

Nicolay, Nils H; Carter, Rebecca; Hatch, Stephanie B; Schultz, Niklas; Prevo, Remko; McKenna, W Gillies; Helleday, Thomas; Sharma, Ricky A

2012-11-01

DNA polymerase eta (pol η) is the only DNA polymerase causally linked to carcinogenesis in humans. Inherited deficiency of pol η in the variant form of xeroderma pigmentosum (XPV) predisposes to UV-light-induced skin cancer. Pol η-deficient cells demonstrate increased sensitivity to cisplatin and oxaliplatin chemotherapy. We have found that XP30R0 fibroblasts derived from a patient with XPV are more resistant to cell kill by ionising radiation (IR) than the same cells complemented with wild-type pol η. This phenomenon has been confirmed in Burkitt's lymphoma cells, which either expressed wild-type pol η or harboured a pol η deletion. Pol η deficiency was associated with accumulation of cells in S-phase, which persisted after IR. Cells deficient in pol η demonstrated increased homologous recombination (HR)-directed repair of double strand breaks created by IR. Depletion of the HR protein, X-ray repair cross-complementing protein 3 (XRCC3), abrogated the radioresistance observed in pol η-deficient cells as compared with pol η-complemented cells. These findings suggest that HR mediates S-phase-dependent radioresistance associated with pol η deficiency. We propose that pol η protein levels in tumours may potentially be used to identify patients who require treatment with chemo-radiotherapy rather than radiotherapy alone for adequate tumour control.

Investigation of dynamic driving cycle effect on the degradation of proton exchange membrane fuel cell by segmented cell technology

NASA Astrophysics Data System (ADS)

Lin, R.; Xiong, F.; Tang, W. C.; Técher, L.; Zhang, J. M.; Ma, J. X.

2014-08-01

Durability is one of the most important limiting factors for the commercialization of proton exchange membrane fuel cell (PEMFC). Fuel cells are more vulnerable to degradation under operating conditions as dynamic load cycle or start up/shut down. The purpose of this study is to evaluate influences of driving cycles on the durability of fuel cells through analyzing the degradation mechanism of a segmented cell in real time. This study demonstrates that the performance of the fuel cell significantly decreases after 200 cycles. The segmented cell technology is used to measure the local current density distribution, which shows that the current density at the exit region and the inlet region declines much faster than the other parts. Meanwhile, electro-chemical impedance spectroscopy (EIS) reveals that after 200 cycles the ohmic resistance of fuel cell increases, especially at the cathode, and electro-chemical surface area (ESA) decreases from 392 to 307 cm2 mg-1. Furthermore, scanning electron microscopy (SEM) images of the membrane-electrode assembly (MEA) in cross-section demonstrate crackle flaw on the surface of the catalyst layer and the delamination of the electrodes from the membrane. Transmission electron microscope (TEM) results also show that the Pt particle size increases distinctly after driving cycles.

Combined antitumor activity of the nitroreductase/CB1954 suicide gene system and γ-rays in HeLa cells in vitro

PubMed Central

Teng, Geling; Ju, Yuanrong; Yang, Yepeng; Hua, Hu; Chi, Jingyu; Mu, Xiuan

2016-01-01

Escherichia coli nitroreductase (NTR) may convert the prodrug CB1954 (5-(aziridin-1-yl)-2,4-dinitrobenzamide) into a bifunctional alkylating agent, which may lead to DNA crosslinks and the apoptosis of cancer cells. NTR/CB1954 has been demonstrated to be an effective gene therapy in cancer cells. The present study examined whether the NTR/CB1954 suicide gene system had cytotoxic effects on HeLa cells and may improve the radiosensitivity of HeLa cells to γ-rays. It was observed that the NTR/CB1954 suicide gene system exerted marked cytotoxic effects on HeLa cells. The combined therapeutic effects of NTR/CB1954 and γ-rays on HeLa cells demonstrated a synergistic effect. CB1954 at concentrations of 12.5 and 25 µmol/l increased the sensitization enhancement ratio of HeLa cells to 1.54 and 1.66, respectively. Therefore, when compared with monotherapy, the combined therapy of NTR/CB1954 and γ-rays may increase the apoptotic rate and enhance the radiosensitivity of HeLa cells. The combined therapy of γ-ray radiation and the NTR/CB1954 suicide gene system may be a novel and potent therapeutic method for the treatment of cervical carcinoma. PMID:27840931

Combined antitumor activity of the nitroreductase/CB1954 suicide gene system and γ-rays in HeLa cells in vitro.

PubMed

Teng, Geling; Ju, Yuanrong; Yang, Yepeng; Hua, Hu; Chi, Jingyu; Mu, Xiuan

2016-12-01

Escherichia coli nitroreductase (NTR) may convert the prodrug CB1954 (5-(aziridin-1-yl)-2,4-dinitrobenzamide) into a bifunctional alkylating agent, which may lead to DNA crosslinks and the apoptosis of cancer cells. NTR/CB1954 has been demonstrated to be an effective gene therapy in cancer cells. The present study examined whether the NTR/CB1954 suicide gene system had cytotoxic effects on HeLa cells and may improve the radiosensitivity of HeLa cells to γ‑rays. It was observed that the NTR/CB1954 suicide gene system exerted marked cytotoxic effects on HeLa cells. The combined therapeutic effects of NTR/CB1954 and γ‑rays on HeLa cells demonstrated a synergistic effect. CB1954 at concentrations of 12.5 and 25 µmol/l increased the sensitization enhancement ratio of HeLa cells to 1.54 and 1.66, respectively. Therefore, when compared with monotherapy, the combined therapy of NTR/CB1954 and γ‑rays may increase the apoptotic rate and enhance the radiosensitivity of HeLa cells. The combined therapy of γ‑ray radiation and the NTR/CB1954 suicide gene system may be a novel and potent therapeutic method for the treatment of cervical carcinoma.

T Cell Activation Thresholds are Affected by Gravitational

NASA Technical Reports Server (NTRS)

Adams, Charley; Gonzalez, M.; Nelman-Gonzalez, M.

1999-01-01

T cells stimulated in space flight by various mitogenic signals show a dramatic reduction in proliferation and expression of early activation markers. Similar results are also obtained in a ground based model of microgravity, clinorotation, which provides a vector-averaged reduction of the apparent gravity on cells without significant shear force. Here we demonstrate that T cell inhibition is due to an increase in the required threshold for activation. Dose response curves indicate that cells activated during clinorotation require higher stimulation to achieve the same level of activation, as measured by CD69 expression. Interleukin 2 receptor expression, and DNA synthesis. The amount of stimulation necessary for 50% activation is 5 fold in the clinostat relative to static. Correlation of TCR internalization with activation also exhibit a dramatic right shift in clinorotation, demonstrating unequivocally that signal transduction mechanism independent of TCR triggering account for the increased activation threshold. Previous results from space flight experiments are consistent with the dose response curves obtained for clinorotation. Activation thresholds are important aspects of T cell memory, autoimmunity and tolerance Clinorotation is a useful, noninvasive tool for the study of cellular and biochemical event regulating T cell activation threshold and the effects of gravitation forces on these systems.

Increased levels of the long noncoding RNA, HOXA-AS3, promote proliferation of A549 cells.

PubMed

Zhang, Hongyue; Liu, Ying; Yan, Lixin; Zhang, Min; Yu, Xiufeng; Du, Wei; Wang, Siqi; Li, Qiaozhi; Chen, He; Zhang, Yafeng; Sun, Hanliang; Tang, Zhidong; Zhu, Daling

2018-06-13

Many long noncoding RNAs (lncRNAs) have been identified as powerful regulators of lung adenocarcinoma (LAD). However, the role of HOXA-AS3, a novel lncRNA, in LAD is largely unknown. In this study, we showed that HOXA-AS3 was significantly upregulated in LAD tissues and A549 cells. After knockdown of HOXA-AS3, cell proliferation, migration, and invasion were inhibited. Xenografts derived from A549 cells transfected with shRNA/HOXA-AS3 had significantly lower tumor weights and smaller tumor volumes. We also demonstrated that HOXA-AS3 increased HOXA6 mRNA stability by forming an RNA duplex. In addition, HOXA6 promoted cell proliferation, migration, and invasion in vitro. Using a RNA pull-down assay, we found that HOXA-AS3 bonded with NF110, which regulated the cell localization of HOXA-AS3. Moreover, histone acetylation was involved in upregulation of HOXA-AS3. These results demonstrate that HOXA-AS3 was activated in LAD and supported cancer cell progression. Therefore, inhibition of HOXA-AS3 could be an effective targeted therapy for patients with LAD.

Ebola virus glycoprotein directly triggers T lymphocyte death despite of the lack of infection.

PubMed

Iampietro, Mathieu; Younan, Patrick; Nishida, Andrew; Dutta, Mukta; Lubaki, Ndongala Michel; Santos, Rodrigo I; Koup, Richard A; Katze, Michael G; Bukreyev, Alexander

2017-05-01

Fatal outcomes of Ebola virus (EBOV) infections are typically preceded by a 'sepsis-like' syndrome and lymphopenia despite T cells being resistant to Ebola infection. The mechanisms that lead to T lymphocytes death remain largely unknown; however, the degree of lymphopenia is highly correlative with fatalities. Here we investigated whether the addition of EBOV or its envelope glycoprotein (GP) to isolated primary human CD4+ T cells induced cell death. We observed a significant decrease in cell viability in a GP-dependent manner, which is suggestive of a direct role of GP in T cell death. Using immunoprecipitation assays and flow cytometry, we demonstrate that EBOV directly binds to CD4+ T cells through interaction of GP with TLR4. Transcriptome analysis revealed that the addition of EBOV to CD4+ T cells results in the significant upregulation of pathways associated with interferon signaling, pattern recognition receptors and intracellular activation of NFκB signaling pathway. Both transcriptome analysis and specific inhibitors allowed identification of apoptosis and necrosis as mechanisms associated with the observed T cell death following exposure to EBOV. The addition of the TLR4 inhibitor CLI-095 significantly reduced CD4+ T cell death induced by GP. EBOV stimulation of primary CD4+ T cells resulted in a significant increase in secreted TNFα; inhibition of TNFα-mediated signaling events significantly reduced T cell death while inhibitors of both necrosis and apoptosis similarly reduced EBOV-induced T cell death. Lastly, we show that stimulation with EBOV or GP augments monocyte maturation as determined by an overall increase in expression levels of markers of differentiation. Subsequently, the increased rates of cellular differentiation resulted in higher rates of infection further contributing to T cell death. These results demonstrate that GP directly subverts the host's immune response by increasing the susceptibility of monocytes to EBOV infection and triggering lymphopenia through direct and indirect mechanisms.

High-performance and environmentally stable planar heterojunction perovskite solar cells based on a solution-processed copper-doped nickel oxide hole-transporting layer.

PubMed

Kim, Jong H; Liang, Po-Wei; Williams, Spencer T; Cho, Namchul; Chueh, Chu-Chen; Glaz, Micah S; Ginger, David S; Jen, Alex K-Y

2015-01-27

An effective approach to significantly increase the electrical conductivity of a NiOx hole-transporting layer (HTL) to achieve high-efficiency planar heterojunction perovskite solar cells is demonstrated. Perovskite solar cells based on using Cu-doped NiOx HTL show a remarkably improved power conversion efficiency up to 15.40% due to the improved electrical conductivity and enhanced perovskite film quality. General applicability of Cu-doped NiOx to larger bandgap perovskites is also demonstrated in this study. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Intravenous administration of bone marrow-derived multipotent mesenchymal stromal cells enhances the recruitment of CD11b(+) myeloid cells to the lungs and facilitates B16-F10 melanoma colonization.

PubMed

Souza, Lucas E B; Almeida, Danilo C; Yaochite, Juliana N U; Covas, Dimas T; Fontes, Aparecida M

2016-07-15

The discovery that the regenerative properties of bone marrow multipotent mesenchymal stromal cells (BM-MSCs) could collaterally favor neoplastic progression has led to a great interest in the function of these cells in tumors. However, the effect of BM-MSCs on colonization, a rate-limiting step of the metastatic cascade, is unknown. In this study, we investigated the effect of BM-MSCs on metastatic outgrowth of B16-F10 melanoma cells. In in vitro experiments, direct co-culture assays demonstrated that BM-MSCs stimulated the proliferation of B16-F10 cells in a dose-dependent manner. For in vivo experiments, luciferase-expressing B16-F10 cells were injected through tail vein and mice were subsequently treated with four systemic injections of BM-MSCs. In vivo bioluminescent imaging during 16 days demonstrated that BM-MSCs enhanced the colonization of lungs by B16-F10 cells, which correlated with a 2-fold increase in the number of metastatic foci. Flow cytometry analysis of lungs demonstrated that although mice harboring B16-F10 metastases displayed more endothelial cells, CD4 T and CD8 T lymphocytes in the lungs in comparison to metastases-free mice, BM-MSCs did not alter the number of these cells. Interestingly, BM-MSCs inoculation resulted in a 2-fold increase in the number of CD11b(+) myeloid cells in the lungs of melanoma-bearing animals, a cell population previously described to organize "premetastatic niches" in experimental models. These findings indicate that BM-MSCs provide support to B16-F10 cells to overcome the constraints that limit metastatic outgrowth and that these effects might involve the interplay between BM-MSCs, CD11b(+) myeloid cells and tumor cells. Copyright © 2015 Elsevier Inc. All rights reserved.

Nuclear Countermeasure Activity of TP508 Linked to Restoration of Endothelial Function and Acceleration of DNA Repair

PubMed Central

Olszewska-Pazdrak, Barbara; McVicar, Scott D.; Rayavara, Kempaiah; Moya, Stephanie M.; Kantara, Carla; Gammarano, Chris; Olszewska, Paulina; Fuller, Gerald M.; Sower, Laurie E.; Carney, Darrell H.

2016-01-01

There is increasing evidence that radiation-induced damage to endothelial cells and loss of endothelial function may contribute to both acute radiation syndromes and long-term effects of whole-body nuclear irradiation. Therefore, several drugs are being developed to mitigate the effects of nuclear radiation, most of these drugs will target and protect or regenerate leukocytes and platelets. Our laboratory has demonstrated that TP508, a 23-amino acid thrombin peptide, activates endothelial cells and stem cells to revascularize and regenerate tissues. We now show that TP508 can mitigate radiation-induced damage to endothelial cells in vitro and in vivo. Our in vitro results demonstrate that human endothelial cells irradiation attenuates nitric oxide (NO) signaling, disrupts tube formation and induces DNA double-strand breaks (DSB). TP508 treatment reverses radiation effects on NO signaling, restores tube formation and accelerates the repair of radiation-induced DSB. The radiation-mitigating effects of TP508 on endothelial cells were also seen in CD-1 mice where systemic injection of TP508 stimulated endothelial cell sprouting from aortic explants after 8 Gy irradiation. Systemic doses of TP508 that mitigated radiation-induced endothelial cell damage, also significantly increased survival of CD-1 mice when injected 24 h after 8.5 Gy exposure. These data suggest that increased survival observed with TP508 treatment may be due to its effects on vascular and microvascular endothelial cells. Our study supports the usage of a regenerative drug such as TP508 to activate endothelial cells as a countermeasure for mitigating the effects of nuclear radiation. PMID:27388041

Hexon modification to improve the activity of oncolytic adenovirus vectors against neoplastic and stromal cells in pancreatic cancer.

PubMed

Lucas, Tanja; Benihoud, Karim; Vigant, Frédéric; Schmidt, Christoph Q; Schmidt, Christoph Q Andreas; Wortmann, Andreas; Bachem, Max G; Simmet, Thomas; Kochanek, Stefan

2015-01-01

Primary pancreatic carcinoma has an unfavourable prognosis and standard treatment strategies mostly fail in advanced cases. Virotherapy might overcome this resistance to current treatment modalities. However, data from clinical studies with oncolytic viruses, including replicating adenoviral (Ad) vectors, have shown only limited activity against pancreatic cancer and other carcinomas. Since pancreatic carcinomas have a complex tumor architecture and frequently a strong stromal compartment consisting of non-neoplastic cell types (mainly pancreatic stellate cells = hPSCs) and extracellular matrix, it is not surprising that Ad vectors replicating in neoplastic cells will likely fail to eradicate this aggressive tumor type. Because the TGFβ receptor (TGFBR) is expressed on both neoplastic cells and hPSCs we inserted the TGFBR targeting peptide CKS17 into the hypervariable region 5 (HVR5) of the capsid protein hexon with the aim to generate a replicating Ad vector with improved activity in complex tumors. We demonstrated increased transduction of both pancreatic cancer cell lines and of hPSCs and enhanced cytotoxicity in co-cultures of both cell types. Surface plasmon resonance analysis demonstrated decreased binding of coagulation factor X to CKS17-modified Ad particles and in vivo biodistribution studies performed in mice indicated decreased transduction of hepatocytes. Thus, to increase activity of replicating Ad vectors we propose to relax tumor cell selectivity by genetic hexon-mediated targeting to the TGFBR (or other receptors present on both neoplastic and non-neoplastic cells within the tumor) to enable replication also in the stromal cell compartment of tumors, while abolishing hepatocyte transduction, and thereby increasing safety.

IAP Antagonists Enhance Apoptotic Response to Enzalutamide in Castration-Resistant Prostate Cancer Cells via Autocrine TNF-α Signaling.

PubMed

Pilling, Amanda B; Hwang, Ok; Boudreault, Alain; Laurent, Alain; Hwang, Clara

2017-06-01

Castration-resistant prostate cancer (CRPC) remains incurable and identifying effective treatments continues to present a clinical challenge. Although treatment with enzalutamide, a second generation androgen receptor (AR) antagonist, prolongs survival in prostate cancer patients, responses can be limited by intrinsic resistance or acquired resistance. A potential mechanism of resistance to androgen axis inhibition is evasion of apoptosis. Inhibitor of apoptosis proteins (IAPs) are found to be overexpressed in prostate cancer and function to block apoptosis and promote survival signaling. Novel, small-molecule IAP antagonists, such as AEG40995, are emerging as a strategy to induce apoptosis and increase therapeutic response in cancer. Human prostate cancer cell lines LNCaP and C4-2 were treated with enzalutamide with or without addition of IAP antagonist AEG40995 and proliferation and survival were determined by MTS and clonogenic assay. Western blot was used to evaluate IAP protein expression changes and PARP-1 cleavage was assessed as indication of apoptosis. Flow cytometry was performed to analyze apoptosis in treated cells. Caspase activity was determined by luminescence assay. Quantitative real-time PCR and immunometric ELISA was used to assess TNF-α (transcript and protein levels, respectively) in response to treatment. In this study, we demonstrate that IAP antagonist AEG40995 exhibits minimal effects on prostate cancer cell proliferation or survival, but rapidly degrades cIAP1 protein. Combination treatment with enzalutamide demonstrates that AEG40995 increases apoptosis and reduces proliferation and clonogenic survival in cell line models of prostate cancer. Mechanistically, we demonstrate that apoptosis in response to enzalutamide and IAP antagonist requires activation of caspase-8, suggesting extrinsic/death receptor apoptosis signaling. Assessment of TNF-α in response to combination treatment with enzalutamide and AEG40995 reveals increased mRNA expression and autocrine protein secretion. Blocking TNF-α signaling abrogates the apoptotic response demonstrating that TNF-α plays a critical role in executing cell death in response to this drug combination. These findings suggest that IAP antagonists can increase sensitivity and amplify the caspase-mediated apoptotic response to enzalutamide through TNF-α signaling mechanisms. Combination with an IAP antagonist increases enzalutamide sensitivity, lowers the apoptotic threshold and may combat drug resistance in patients with prostate cancer. Prostate 77:866-877, 2017. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

Interneuron- and GABAA receptor-specific inhibitory synaptic plasticity in cerebellar Purkinje cells

NASA Astrophysics Data System (ADS)

He, Qionger; Duguid, Ian; Clark, Beverley; Panzanelli, Patrizia; Patel, Bijal; Thomas, Philip; Fritschy, Jean-Marc; Smart, Trevor G.

2015-07-01

Inhibitory synaptic plasticity is important for shaping both neuronal excitability and network activity. Here we investigate the input and GABAA receptor subunit specificity of inhibitory synaptic plasticity by studying cerebellar interneuron-Purkinje cell (PC) synapses. Depolarizing PCs initiated a long-lasting increase in GABA-mediated synaptic currents. By stimulating individual interneurons, this plasticity was observed at somatodendritic basket cell synapses, but not at distal dendritic stellate cell synapses. Basket cell synapses predominantly express β2-subunit-containing GABAA receptors; deletion of the β2-subunit ablates this plasticity, demonstrating its reliance on GABAA receptor subunit composition. The increase in synaptic currents is dependent upon an increase in newly synthesized cell surface synaptic GABAA receptors and is abolished by preventing CaMKII phosphorylation of GABAA receptors. Our results reveal a novel GABAA receptor subunit- and input-specific form of inhibitory synaptic plasticity that regulates the temporal firing pattern of the principal output cells of the cerebellum.

Improving the outcome of adoptive cell transfer by targeting tumor escape

PubMed Central

Kaluza, Karen M.; Vile, Richard

2013-01-01

Adoptive T-cell transfer is among the most promising immunotherapies against cancer. To continue increasing the potential of this therapy, our studies focus on the inhibition of tumor recurrence. Recently, we have demonstrated several ways in which combination therapies involving multiple T-cell populations and immunostimulatory chemotherapy can enhance long-term survival. PMID:23483796

Glutamic acid ameliorates estrogen deficiency-induced menopausal-like symptoms in ovariectomized mice.

PubMed

Han, Na-Ra; Kim, Hee-Yun; Yang, Woong Mo; Jeong, Hyun-Ja; Kim, Hyung-Min

2015-09-01

Some amino acids are considered alternative therapies for improving menopausal symptoms. Glutamic acid (GA), which is abundant in meats, fish, and protein-rich plant foods, is known to be a neurotransmitter or precursor of γ-aminobutyric acid. Although it is unclear if GA functions in menopausal symptoms, we hypothesized that GA would attenuate estrogen deficiency-induced menopausal symptoms. The objective to test our hypothesis was to examine an estrogenic effect of GA in ovariectomized (OVX) mice, estrogen receptor (ER)-positive human osteoblast-like MG-63 cells, and ER-positive human breast cancer MCF-7 cells. The results demonstrated that administration with GA to mice suppressed body weight gain and vaginal atrophy when compared with the OVX mice. A microcomputed tomographic analysis of the trabecular bone showed increases in bone mineral density, trabecular number, and connectivity density as well as a significant decrease in total porosity of the OVX mice treated with GA. In addition, GA increased serum levels of alkaline phosphatase and estrogen compared with the OVX mice. Furthermore, GA induced proliferation and increased ER-β messenger RNA (mRNA) expression, estrogen response element (ERE) activity, extracellular signal-regulated kinase phosphorylation, and alkaline phosphatase activity in MG-63 cells. In MCF-7 cells, GA also increased proliferation, Ki-67 mRNA expression, ER-β mRNA expression, and ERE activity. Estrogen response element activity increased by GA was inhibited by an estrogen antagonist. Taken together, our data demonstrated that GA has estrogenic and osteogenic activities in OVX mice, MG-63 cells, and MCF-7 cells. Copyright © 2015 Elsevier Inc. All rights reserved.

Biocompatibility of Synthetic Poly(ester urethane)/Polyhedral Oligomeric Silsesquioxane Matrices with Embryonic Stem Cell Proliferation and Differentiation

PubMed Central

Guo, Yan-Lin; Wang, Wenshou; Otaigbe, Joshua U.

2010-01-01

Incorporation of polyhedral oligomeric silsesquioxanes (POSS) into poly (ester urethane)s (PEU) as a building block results in a PEU/POSS hybrid polymer with increased mechanical strength and thermostability. An attractive feature of the new polymer is that it forms a porous matrix when cast in the form of a thin film, making it potentially useful in tissue engineering. In this study, we present detailed microscopic analysis of the PEU/POSS matrix and demonstrate its biocompatibility with cell culture. The PEU/POSS polymer forms a continuous porous matrix with open pores and interconnected grooves. From SEM image analysis, it is calculated that there are about 950 pores per mm2 of the matrix area with pore size ranging from 1 to 15 μm in diameter. The area occupied by the pores represents approximately 7.6 % of matrix area. Using mouse embryonic stem cells (ESCs), we demonstrate that the PEU/POSS matrix provides excellent support for cell proliferation and differentiation. Under the cell culture condition optimized to maintain self-renewal, ESCs grown on a PEU/POSS matrix exhibit undifferentiated morphology, express pluripotency markers, and have similar growth rate to cells grown on gelatin. When induced for differentiation, ESCs underwent dramatic morphological change, characterized by the loss of clonogenecity and increased cell size with well-expanded cytoskeleton networks. Differentiated cells are able to form a continuous monolayer that is closely embedded on the matrix. The excellent compatibility between the PEU/POSS matrix and ESC proliferation/differentiation demonstrates the potential of using PEU/POSS polymers in future ESC-based tissue engineering. PMID:20213627

Coriolus versicolor (Yunzhi) extract attenuates growth of human leukemia xenografts and induces apoptosis through the mitochondrial pathway.

PubMed

Ho, Cheong-Yip; Kim, Chi-Fai; Leung, Kwok-Nam; Fung, Kwok-Pui; Tse, Tak-Fu; Chan, Helen; Lau, Clara Bik-San

2006-09-01

Coriolus versicolor (CV), also called Yunzhi, has been demonstrated to exert anti-tumor effects on various types of cancer cells. Our previous studies have demonstrated that a standardized aqueous ethanol extract prepared from CV inhibited the proliferation of human leukemia cells via induction of apoptosis. The present study aimed to evaluate the underlying mechanisms of apoptosis through modulation of Bax, Bcl-2 and cytochrome c protein expressions in a human pro-myelocytic leukemia (HL-60) cell line, as well as the potential of the CV extract as anti-leukemia agent using the athymic mouse xenograft model. Our results demonstrated that the CV extract dose-dependently suppressed the proliferation of HL-60 cells (IC50 = 150.6 microg/ml), with increased nucleosome production from apoptotic cells. Expression of pro-apoptotic protein Bax was significantly up-regulated in HL-60 cells treated with the CV extract, especially after 16 and 24 h. Meanwhile, expression of anti-apoptotic protein Bcl-2 was concomitantly down-regulated, as reflected by the increased Bax/Bcl-2 ratio. The CV extract markedly, but transiently, promoted the release of cytochrome c from mitochondria to cytosol after 24-h incubation. In vivo studies in the athymic nude mouse xenograft model also confirmed the growth-inhibitory activity of the CV extract on human leukemia cells. In conclusion, the CV extract attenuated the human leukemia cell proliferation in vivo, and in vitro possibly by inducing apoptosis through the mitochondrial pathway. The CV extract is likely to be valuable for the treatment of some forms of human leukemia.

Changes in Nutritional Status Impact Immune Cell Metabolism and Function.

PubMed

Alwarawrah, Yazan; Kiernan, Kaitlin; MacIver, Nancie J

2018-01-01

Immune cell function and metabolism are closely linked. Many studies have now clearly demonstrated that alterations in cellular metabolism influence immune cell function and that, conversely, immune cell function determines the cellular metabolic state. Less well understood, however, are the effects of systemic metabolism or whole organism nutritional status on immune cell function and metabolism. Several studies have demonstrated that undernutrition is associated with immunosuppression, which leads to both increased susceptibility to infection and protection against several types of autoimmune disease, whereas overnutrition is associated with low-grade, chronic inflammation that increases the risk of metabolic and cardiovascular disease, promotes autoreactivity, and disrupts protective immunity. Here, we review the effects of nutritional status on immunity and highlight the effects of nutrition on circulating cytokines and immune cell populations in both human studies and mouse models. As T cells are critical members of the immune system, which direct overall immune response, we will focus this review on the influence of systemic nutritional status on T cell metabolism and function. Several cytokines and hormones have been identified which mediate the effects of nutrition on T cell metabolism and function through the expression and action of key regulatory signaling proteins. Understanding how T cells are sensitive to both inadequate and overabundant nutrients may enhance our ability to target immune cell metabolism and alter immunity in both malnutrition and obesity.

Modeling Hematopoiesis and Responses to Radiation Countermeasures in a Bone Marrow-on-a-Chip.

PubMed

Torisawa, Yu-Suke; Mammoto, Tadanori; Jiang, Elisabeth; Jiang, Amanda; Mammoto, Akiko; Watters, Alexander L; Bahinski, Anthony; Ingber, Donald E

2016-05-01

Studies on hematopoiesis currently rely on animal models because in vitro culture methods do not accurately recapitulate complex bone marrow physiology. We recently described a bone marrow-on-a-chip microfluidic device that enables the culture of living hematopoietic bone marrow and mimics radiation toxicity in vitro. In the present study, we used this microdevice to demonstrate continuous blood cell production in vitro and model bone marrow responses to potential radiation countermeasure drugs. The device maintained mouse hematopoietic stem and progenitor cells in normal proportions for at least 2 weeks in culture. Increases in the number of leukocytes and red blood cells into the microfluidic circulation also could be detected over time, and addition of erythropoietin induced a significant increase in erythrocyte production. Exposure of the bone marrow chip to gamma radiation resulted in reduction of leukocyte production, and treatment of the chips with two potential therapeutics, granulocyte-colony stimulating factor or bactericidal/permeability-increasing protein (BPI), induced significant increases in the number of hematopoietic stem cells and myeloid cells in the fluidic outflow. In contrast, BPI was not found to have any effect when analyzed using static marrow cultures, even though it has been previously shown to accelerate recovery from radiation-induced toxicity in vivo. These findings demonstrate the potential value of the bone marrow-on-a-chip for modeling blood cell production, monitoring responses to hematopoiesis-modulating drugs, and testing radiation countermeasures in vitro.

Signaling via the CD2 receptor enhances HTLV-1 replication in T lymphocytes.

PubMed

Guyot, D J; Newbound, G C; Lairmore, M D

1997-07-21

Human T lymphotropic virus type 1 (HTLV-1) is considered the etiologic agent of adult T cell leukemia/lymphoma and several chronic progressive immune-mediated diseases. Approximately 1-4% of infected individuals develop disease, generally decades following infection. Increased proviral transcription, mediated by the viral 40-kDa trans-activating protein, Tax, has been implicated in the pathogenesis of HTLV-1-associated diseases. Since the HTLV-1 promoter contains sequences responsive to cyclic AMP and protein kinase C, we hypothesized that lymphocyte activation signals initiated through the TCR/CD3 complex or CD2 receptor promote viral replication in HTLV-1-infected lymphocytes. We demonstrate that mAbs directed against the CD2, but not the CD3 receptor increase viral p24 capsid protein 1.5- to 5.7-fold in CD2/CD3+ HTLV-1-infected cell culture supernatants. Northern blot analysis demonstrated a 2.5- to 4-fold increase in all species of viral mRNA following CD2 cross-linking of OSP2/4 cells, an immortalized HTLV-1 cell line. Consistent with transcriptional regulation, reporter gene activity increased approximately 11-fold in CD2-stimulated Jurkat T cells cotransfected with a Tax-expressing plasmid and a CAT reporter gene construct under control of the HTLV-1 promoter. These data suggest a possible physiologic mechanism, whereby CD2-mediated cell adhesion and lymphocyte activation may promote viral transcription in infected lymphocytes.

Development of a 3D cell printed construct considering angiogenesis for liver tissue engineering.

PubMed

Lee, Jin Woo; Choi, Yeong-Jin; Yong, Woon-Jae; Pati, Falguni; Shim, Jin-Hyung; Kang, Kyung Shin; Kang, In-Hye; Park, Jaesung; Cho, Dong-Woo

2016-01-12

Several studies have focused on the regeneration of liver tissue in a two-dimensional (2D) planar environment, whereas actual liver tissue is three-dimensional (3D). Cell printing technology has been successfully utilized for building 3D structures; however, the poor mechanical properties of cell-laden hydrogels are a major concern. Here, we demonstrate the printing of a 3D cell-laden construct and its application to liver tissue engineering using 3D cell printing technology through a multi-head tissue/organ building system. Polycaprolactone (PCL) was used as a framework material because of its excellent mechanical properties. Collagen bioink containing three different types of cells-hepatocytes (HCs), human umbilical vein endothelial cells , and human lung fibroblasts--was infused into the canals of a PCL framework to induce the formation of capillary--like networks and liver cell growth. A co-cultured 3D microenvironment of the three types of cells was successfully established and maintained. The vascular formation and functional abilities of HCs (i.e., albumin secretion and urea synthesis) demonstrated that the heterotypic interaction among HCs and nonparenchymal cells increased the survivability and functionality of HCs within the collagen gel. Therefore, our results demonstrate the prospect of using cell printing technology for the creation of heterotypic cellular interaction within a structure for liver tissue engineering.

The VP40 protein of Marburg virus exhibits impaired budding and increased sensitivity to human tetherin following mouse adaptation.

PubMed

Feagins, Alicia R; Basler, Christopher F

2014-12-01

The Marburg virus VP40 protein is a viral matrix protein that spontaneously buds from cells. It also functions as an interferon (IFN) signaling antagonist by targeting Janus kinase 1 (JAK1). A previous study demonstrated that the VP40 protein of the Ravn strain of Marburg virus (Ravn virus [RAVV]) failed to block IFN signaling in mouse cells, whereas the mouse-adapted RAVV (maRAVV) VP40 acquired the ability to inhibit IFN responses in mouse cells. The increased IFN antagonist function of maRAVV VP40 mapped to residues 57 and 165, which were mutated during the mouse adaptation process. In the present study, we demonstrate that maRAVV VP40 lost the capacity to efficiently bud from human cell lines, despite the fact that both parental and maRAVV VP40s bud efficiently from mouse cell lines. The impaired budding in human cells corresponds with the appearance of protrusions on the surface of maRAVV VP40-expressing Huh7 cells and with an increased sensitivity of maRAVV VP40 to restriction by human tetherin but not mouse tetherin. However, transfer of the human tetherin cytoplasmic tail to mouse tetherin restored restriction of maRAVV VP40. Residues 57 and 165 were demonstrated to contribute to the failure of maRAVV VP40 to bud from human cells, and residue 57 was demonstrated to alter VP40 oligomerization, as assessed by coprecipitation assay, and to determine sensitivity to human tetherin. This suggests that RAVV VP40 acquired, during adaptation to mice, changes in its oligomerization potential that enhanced IFN antagonist function. However, this new capacity impaired RAVV VP40 budding from human cells. Filoviruses, which include Marburg viruses and Ebola viruses, are zoonotic pathogens that cause severe disease in humans and nonhuman primates but do not cause similar disease in wild-type laboratory strains of mice unless first adapted to these animals. Although mouse adaptation has been used as a method to develop small animal models of pathogenesis, the molecular determinants associated with filovirus mouse adaptation are poorly understood. Our study demonstrates how genetic changes that accrued during mouse adaptation of the Ravn strain of Marburg virus have impacted the budding function of the viral VP40 matrix protein. Strikingly, we find impairment of mouse-adapted VP40 budding function in human but not mouse cell lines, and we correlate the impairment with an increased sensitivity of VP40 to restriction by human but not mouse tetherin and with changes in VP40 oligomerization. These data suggest that there are functional costs associated with filovirus adaptation to new hosts and implicate tetherin as a filovirus host restriction factor. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

In vitro and in vivo study of the application of volvox spheres to co-culture vehicles in liver tissue engineering.

PubMed

Chang, Siou Han; Huang, Han Hsiang; Kang, Pei Leun; Wu, Yu Chian; Chang, Ming-Huang; Kuo, Shyh Ming

2017-11-01

Volvox sphere is a biomimetic concept of a natural Volvox, wherein a large outer sphere contains smaller inner spheres, which can encapsulate cells and provide a double-layer three-dimensional environment for culturing cells. This study simultaneously encapsulated rat mesenchymal stem cells (MSCs) and AML12 hepatocytes in volvox spheres and extensively evaluated the effects of various culturing modes on cell functions and fates. The results showed that compared with a static flask culture, MSCs encapsulated in volvox spheres differentiated into hepatocyte-like cells with a 2-fold increase in albumin (ALB) expression and a 2.5-fold increase in cytokeratin 18 expression in a dynamic bioreactor. Moreover, the restorative effects of volvox spheres encapsulating cells on retrorsine-exposed CCl 4 -induced liver injuries in rats were evaluated. The data presented significant reductions in AST and ALT levels after the implantation of volvox spheres encapsulating both MSCs and AML12 hepatocytes in vivo. In contrast to the negative control group, histopathological analysis demonstrated liver repair and formation of the new liver tissue in groups implanted with volvox spheres containing cells. These results demonstrate that liver cells implanted with volvox spheres encapsulating both MSCs and AML12 hepatocytes promote liver repair and liver tissue regeneration in liver failure caused by necrotizing agents such as retrorsine and CCl 4 . Hence, volvox spheres encapsulating MSCs and liver cells can be a promising and clinically effective therapy for liver injury. In this study, we used a volvox sphere, which is a unique design that mimics the natural Volvox, that consists of a large outer sphere that contains smaller inner spheres, which provide a three-dimensional environment to culture cells. The purpose of this study is to co-culture mesenchymal stem cells (MSCs) and AML12 liver cells in volvox spheres and evaluate two different culture methods, dynamic bioreactor and static culture flask,on the cultured cells. In addition, we aimed to evaluate the restorative effects of volvox spheres encapsulating MSCs and/or AML12 liver cells on rats with retrorsine-exposed CCl 4 -induced liver injuries. The results showed that MSCs encapsulated in volvox spheres differentiated into hepatocyte-like cells with a 2-fold increase in albumin expression and a 2.5-fold increase in cytokeratin 18 expression ina dynamic bioreactor. Moreover, the data presented significant reductions in AST and ALT levels after the implantation of volvox spheres encapsulating both MSCs and AML12 hepatocytes in vivo. In contrast to the negative control group, histopathological analysis demonstrated liver repair and formation of new liver tissue in groups implanted with volvox spheres containing cells. These results demonstrate that liver cells implanted with volvox spheres encapsulating both MSCs and AML12 hepatocytes promote liver repair and liver tissue regeneration in liver failure caused by necrotizing agents such as retrorsine and CCl 4 . Hence, volvox spheres encapsulating MSCs and liver cells can be a promising and clinically effective therapy for liver injury. Copyright © 2017 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Hepatocellular toxicity of oxalicumone A via oxidative stress injury and mitochondrial dysfunction in healthy human liver cells.

PubMed

Shi, Si; Yao, Limei; Guo, Kunbin; Wang, Xiangyu; Wang, Qi; Li, Weirong

2018-01-01

The marine‑derived oxalicumone A (POA) has been demonstrated as a potent anti‑tumor bioactive agent for a variety of human carcinoma, but to the best of our knowledge, remains to be evaluated in healthy liver cells. As many drugs distribute preferentially in the liver, the present study aimed to investigate the effects of POA on apoptosis, oxidative stress and mitochondrial function in L‑02 healthy liver cells. A Cell‑Counting kit‑8 assay demonstrated that POA inhibits the proliferation of L‑02 cells in a dose‑ and time‑dependent manner. Furthermore, POA induced apoptosis by increasing the percentage of cells in early apoptosis and the sub‑G1 cell cycle, along with causing S‑phase arrest in L‑02 cells. Additionally, POA activated caspase 3, increased the protein expression levels of Fas ligand and B‑cell lymphoma X‑associated protein, and decreased the expression of the anti‑apoptotic protein B‑cell lymphoma 2. POA additionally reduced the content of GSH and the activity of superoxide dismutase, elevated malondialdehyde and nitric oxide levels, increased reactive oxygen species production and the levels of alanine aminotransferase and aspartate aminotransferase, which suggested that POA induced lipid peroxidation injury in L‑02 cells and that oxidative stress serves an important role. Furthermore, POA caused alternations of mitochondrial function, including an abrupt depletion of adenosine triphosphate synthesis, mitochondrial permeability transition pore opening and depletion of mitochondrial membrane potential in L‑02 cells. These data suggested that POA exerts cytotoxicity, at least in part, by inducing oxidative stress, mitochondrial dysfunction, and eventually apoptosis. Changes in mitochondrial function and oxidative stress by POA may therefore be critical in POA‑induced toxicity in L‑02 cells.

Intact LKB1 activity is required for survival of dormant ovarian cancer spheroids

PubMed Central

Peart, Teresa; Valdes, Yudith Ramos; Correa, Rohann J. M.; Fazio, Elena; Bertrand, Monique; McGee, Jacob; Préfontaine, Michel; Sugimoto, Akira; DiMattia, Gabriel E.; Shepherd, Trevor G.

2015-01-01

Metastatic epithelial ovarian cancer (EOC) cells can form multicellular spheroids while in suspension and disperse directly throughout the peritoneum to seed secondary lesions. There is growing evidence that EOC spheroids are key mediators of metastasis, and they use specific intracellular signalling pathways to control cancer cell growth and metabolism for increased survival. Our laboratory discovered that AKT signalling is reduced during spheroid formation leading to cellular quiescence and autophagy, and these may be defining features of tumour cell dormancy. To further define the phenotype of EOC spheroids, we have initiated studies of the Liver kinase B1 (LKB1)-5′-AMP-activated protein kinase (AMPK) pathway as a master controller of the metabolic stress response. We demonstrate that activity of AMPK and its upstream kinase LKB1 are increased in quiescent EOC spheroids as compared with proliferating adherent EOC cells. We also show elevated AMPK activity in spheroids isolated directly from patient ascites. Functional studies reveal that treatment with the AMP mimetic AICAR or allosteric AMPK activator A-769662 led to a cytostatic response in proliferative adherent ovarian cancer cells, but they fail to elicit an effect in spheroids. Targeted knockdown of STK11 by RNAi to reduce LKB1 expression led to reduced viability and increased sensitivity to carboplatin treatment in spheroids only, a phenomenon which was AMPK-independent. Thus, our results demonstrate a direct impact of altered LKB1-AMPK signalling function in EOC. In addition, this is the first evidence in cancer cells demonstrating a pro-survival function for LKB1, a kinase traditionally thought to act as a tumour suppressor. PMID:26068970

Reactive Oxygen Species Alter Autocrine and Paracrine Signaling

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zangar, Richard C.; Bollinger, Nikki; Weber, Thomas J.

2011-12-01

Cytochrome P450 (P450) 3A4 (CYP3A4) is the most abundant P450 protein in human liver and intestine and is highly inducible by a variety of drugs and other compounds. The P450 catalytic cycle is known to uncouple and release reactive oxygen species (ROS), but the effects of ROS from P450 and other enzymes in the endo-plasmic reticulum have been poorly studied from the perspective of effects on cell biology. In this study, we expressed low levels of CYP3A4 in HepG2 cells, a human hepatocarcinoma cell line, and examined effects on intracellular levels of ROS and on the secretion of a varietymore » of growth factors that are important in extracellular communication. Using the redox-sensitive dye RedoxSensor red, we demonstrate that CYP3A4 expression increases levels of ROS in viable cells. A customELISA microarray platform was employed to demonstrate that expression of CYP3A4 increased secretion of amphiregulin, intracellular adhesion molecule 1, matrix metalloprotease 2, platelet-derived growth factor (PDGF), and vascular endothelial growth factor, but suppressed secretion of CD14. The antioxidant N-acetylcysteine suppressed all P450-dependent changes in protein secretion except for CD14. Quantitative RT-PCR demonstrated that changes in protein secretion were consistently associated with corresponding changes in gene expression. Inhibition of the NF-{kappa}B pathway blocked P450 effects on PDGF secretion. CYP3A4 expression also altered protein secretion in human mammary epithelial cells and C10 mouse lung cells. Overall, these results suggest that increased ROS production in the endoplasmic reticulum alters the secretion of proteins that have key roles in paracrine and autocrine signaling.« less

Silybin counteracts lipid excess and oxidative stress in cultured steatotic hepatic cells

PubMed Central

Vecchione, Giulia; Grasselli, Elena; Voci, Adriana; Baldini, Francesca; Grattagliano, Ignazio; Wang, David QH; Portincasa, Piero; Vergani, Laura

2016-01-01

AIM: To investigate in vitro the therapeutic effect and mechanisms of silybin in a cellular model of hepatic steatosis. METHODS: Rat hepatoma FaO cells were loaded with lipids by exposure to 0.75 mmol/L oleate/palmitate for 3 h to mimic liver steatosis. Then, the steatotic cells were incubated for 24 h with different concentrations (25 to 100 μmol/L) of silybin as phytosome complex with vitamin E. The effects of silybin on lipid accumulation and metabolism, and on indices of oxidative stress were evaluated by absorption and fluorescence microscopy, quantitative real-time PCR, Western blot, spectrophotometric and fluorimetric assays. RESULTS: Lipid-loading resulted in intracellular triglyceride (TG) accumulation inside lipid droplets, whose number and size increased. TG accumulation was mediated by increased levels of peroxisome proliferator-activated receptors (PPARs) and sterol regulatory element-binding protein-1c (SREBP-1c). The lipid imbalance was associated with higher production of reactive oxygen species (ROS) resulting in increased lipid peroxidation, stimulation of catalase activity and activation of nuclear factor kappa-B (NF-κB). Incubation of steatotic cells with silybin 50 μmol/L significantly reduced TG accumulation likely by promoting lipid catabolism and by inhibiting lipogenic pathways, as suggested by the changes in carnitine palmitoyltransferase 1 (CPT-1), PPAR and SREBP-1c levels. The reduction in fat accumulation exerted by silybin in the steatotic cells was associated with the improvement of the oxidative imbalance caused by lipid excess as demonstrated by the reduction in ROS content, lipid peroxidation, catalase activity and NF-κB activation. CONCLUSION: We demonstrated the direct anti-steatotic and anti-oxidant effects of silybin in steatotic cells, thus elucidating at a cellular level the encouraging results demonstrated in clinical and animal studies. PMID:27468193

TAR-independent transactivation by Tat in cells derived from the CNS: a novel mechanism of HIV-1 gene regulation.

PubMed Central

Taylor, J P; Pomerantz, R; Bagasra, O; Chowdhury, M; Rappaport, J; Khalili, K; Amini, S

1992-01-01

The Tat protein of human immunodeficiency virus type 1 (HIV-1) is essential for productive infection and is a potential target for antiviral therapy. Tat, a potent activator of HIV-1 gene expression, serves to greatly increase the rate of transcription directed by the viral promoter. This induction, which seems to be an important component in the progression of acquired immune deficiency syndrome (AIDS), may be due to increased transcriptional initiation, increased transcriptional elongation, or a combination of these processes. Much attention has been focused on the interaction of Tat with a specific RNA target termed TAR (transactivation responsive) which is present in the leader sequence of all HIV-1 mRNAs. This interaction is believed to be an important component of the mechanism of transactivation. In this report we demonstrate that in certain CNS-derived cells Tat is capable of activating HIV-1 through a TAR-independent pathway. A Tat-responsive element is found upstream within the viral promoter that in glial-derived cell lines allows transactivation in the absence of TAR. Deletion mapping and hybrid promoter constructs demonstrate that the newly identified Tat-responsive element corresponds to a sequence within the viral long terminal repeat (LTR) previously identified as the HIV-1 enhancer, or NF-kappa B domain. DNA band-shift analysis reveals NF-kappa B binding activity in glial cells that differs from that present in T lymphoid cells. Further, we observe that TAR-deleted mutants of HIV-1 demonstrate normal late gene expression in glial cells as evidenced by syncytia formation and production of viral p24 antigen.(ABSTRACT TRUNCATED AT 250 WORDS) Images PMID:1505523

CD21+ (B2 antigen+) cell decrement and CD4+CD29+ (helper-inducer) cell increment suggest an activation of cell immune reactivity in multiple sclerosis.

PubMed

Gambi, D; Porrini, A M; Giampietro, A; Macor, S

1991-08-01

Two-color flow cytometric analysis on peripheral blood lymphocytes of 35 untreated multiple sclerosis (MS) patients, 17 other medical disease (OMD) patients and 14 healthy control (HC) subjects was performed to evaluate the levels of different T and B cell subpopulations. In MS patients we observed an increase in CD4+CD29+ helper-inducer cells but this increase was not related to the different phases of the disease. We hypothesize that this change is related to the reduction of CD21+ cells expressing B2 antigen, a 140 kDa molecule disappearing after B cell activation. An increased level of CD4+CD45RA- (helper-inducer-like cells) and a reduction of CD4+CD29- (suppressor-inducer-like cells) were also present in our patients. These findings demonstrate an immune 'disequilibrium' in MS, which is linked with an increased level of CD25+ cells expressing the interleukin-2 (IL-2) receptor. IL-2, besides being a T cell growth factor, is also a B cell growth factor. These data let us hypothesize that an activation of the immune response is present in MS.

The effect of Platelet Lysate on osteoblast proliferation associated with a transient increase of the inflammatory response in bone regeneration.

PubMed

Ruggiu, Alessandra; Ulivi, Valentina; Sanguineti, Francesca; Cancedda, Ranieri; Descalzi, Fiorella

2013-12-01

Platelet Lysate (PL) contains a cocktail of growth factors and cytokines, which actively participates in tissue repair and its clinical application has been broadly described. The aim of this study was to assess the regenerative potential of PL for bone repair. We demonstrated that PL stimulation induces a transient increase of the inflammatory response in quiescent human osteoblasts, via NF-kB activation, COX-2 induction, PGE2 production and secretion of pro-inflammatory cytokines. Furthermore, we showed that long-term PL stimulation enhances proliferation of actively replicating osteoblasts, without affecting their differentiation potential, along with changes of cell morphology, resulting in increased cell density at confluence. In confluent resting osteoblasts, PL treatment induced resumption of proliferation, change in cell morphology and increase of cell density at confluence. A burst of PL treatment (24-h) was sufficient to trigger such processes in both conditions. These results correlated with up-regulation of the proliferative and survival pathways ERKs and Akt and with cell cycle re-activation via induction of CyclinD1 and phosphorylation of Rb, following PL stimulation. Our findings demonstrate that PL treatment results in activation and expansion of resting osteoblasts, without affecting their differentiation potential. Therefore PL represents a good therapeutic candidate in regenerative medicine for bone repair. Copyright © 2013 Elsevier Ltd. All rights reserved.

The Possible Mechanism of Advanced Glycation End Products (AGEs) for Alzheimer’s Disease

PubMed Central

Ko, Shun-Yao; Ko, Hshin-An; Chu, Kuo-Hsiung; Shieh, Tzong-Ming; Chi, Tzong-Cherng; Chen, Hong-I; Chang, Weng-Cheng; Chang, Shu-Shing

2015-01-01

Amyloid precursor protein (APP) has been modified by β and γ-secretase that cause amyloid deposits (plaques) in neuronal cells. Glyceraldhyde-derived AGEs has been identified as a major source of neurotoxicity in Alzheimer’s disease (AD). In a previous study, we demonstrated that glyceraldehyde-derived AGEs increase APP and Aβ via ROS. Furthermore, the combination of AGEs and Aβ has been shown to enhance neurotoxicity. In mice, APP expression is increased by tail vein injection of AGEs. This evidence suggests a correlation between AGEs and the development of AD. However, the role played by AGEs in the pathogenesis of AD remains unclear. In this report, we demonstrate that AGEs up-regulate APP processing protein (BACE and PS1) and Sirt1 expression via ROS, but do not affect the expression of downstream antioxidant genes HO-1 and NQO-1. Moreover, we found that AGEs increase GRP78 expression and enhance the cell death-related pathway p53, bcl-2/bax ratio, caspase 3. These results indicate that AGEs impair the neuroprotective effects of Sirt1 and lead to neuronal cell death via ER stress. Our findings suggest that AGEs increase ROS production, which stimulates downstream pathways related to APP processing, Aβ production, Sirt1, and GRP78, resulting in the up-regulation of cell death related pathway. This in-turn enhances neuronal cell death, which leads to the development of AD. PMID:26587989

The Possible Mechanism of Advanced Glycation End Products (AGEs) for Alzheimer's Disease.

PubMed

Ko, Shun-Yao; Ko, Hshin-An; Chu, Kuo-Hsiung; Shieh, Tzong-Ming; Chi, Tzong-Cherng; Chen, Hong-I; Chang, Weng-Cheng; Chang, Shu-Shing

2015-01-01

Amyloid precursor protein (APP) has been modified by β and γ-secretase that cause amyloid deposits (plaques) in neuronal cells. Glyceraldhyde-derived AGEs has been identified as a major source of neurotoxicity in Alzheimer's disease (AD). In a previous study, we demonstrated that glyceraldehyde-derived AGEs increase APP and Aβ via ROS. Furthermore, the combination of AGEs and Aβ has been shown to enhance neurotoxicity. In mice, APP expression is increased by tail vein injection of AGEs. This evidence suggests a correlation between AGEs and the development of AD. However, the role played by AGEs in the pathogenesis of AD remains unclear. In this report, we demonstrate that AGEs up-regulate APP processing protein (BACE and PS1) and Sirt1 expression via ROS, but do not affect the expression of downstream antioxidant genes HO-1 and NQO-1. Moreover, we found that AGEs increase GRP78 expression and enhance the cell death-related pathway p53, bcl-2/bax ratio, caspase 3. These results indicate that AGEs impair the neuroprotective effects of Sirt1 and lead to neuronal cell death via ER stress. Our findings suggest that AGEs increase ROS production, which stimulates downstream pathways related to APP processing, Aβ production, Sirt1, and GRP78, resulting in the up-regulation of cell death related pathway. This in-turn enhances neuronal cell death, which leads to the development of AD.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Gao, Zhan

Cleft palate is caused by the failure of palatal midline epithelial cells to disintegrate, which is necessary for palatal mesenchymal confluence. Although 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) exposure is linked to cleft palate at a high rate, the mechanism remains to be elucidated. The present study was designed to determine the effects of TCDD on the fate of epithelial cell isolated from human fetal palatal shelves (hFPECs). We demonstrate that TCDD increased cell proliferation and promoted the progression of cells from G1 to S phase as well as increased the number of cells entering the G2/M phase. We found that TCDD has nomore » measurable effect on apoptosis of hFPECs. The protein level assays revealed that TCDD increased cyclin-dependent kinases 4 (cdk4), cyclin D1, cyclin E and p21 (Waf1/Cip1) but not cdk2, bcl-2, cyclin B1 and cyclin A. Furthermore, TCDD activated PI3K/AKT signaling, and the PI3K inhibitor, LY294002, partially abrogated TCDD-induced cell proliferation and gene modulations. TCDD treatment increased CYP1A1 mRNA and protein levels, which indicated the activation of AhR signaling. Knockdown of the AhR with siRNA suppressed TCDD-induced cell proliferation and PI3K/AKT signaling activation. Taken together, these data demonstrated that TCDD is able to promote growth of hFPECs through AhR-dependent activation of the PI3K/AKT pathway, which may account for the underlying mechanism by which TCDD causes a failure of palatal fusion. - Highlights: • TCDD promoted the cell growth with a character of significant accumulation of cells in G2/M. • TCDD treatment induced a various profile of cell cycle regulatory proteins. • PI3K/AKT pathway was involved in TCDD-induced cell proliferation and gene modifications. • AhR knockdown blocked TCDD-induced cell proliferation and PI3K/Akt signaling activation.« less

4-Methoxyestradiol-induced oxidative injuries in human lung epithelial cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Cheng Yahsin; Chang, Louis W.; Cheng Lichuan

2007-05-01

Epidemiological studies indicated that people exposed to dioxins were prone to the development of lung diseases including lung cancer. Animal studies demonstrated that 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) increased liver tumors and promoted lung metaplasia in females. Metabolic changes in 17{beta}-estradiol (E{sub 2}) resulted from an interaction between TCDD and E{sub 2} could be associated with gender difference. Previously, we reported that methoxylestradiols (MeOE{sub 2}), especially 4-MeOE{sub 2}, accumulated in human lung cells (BEAS-2B) co-treated with TCDD and E{sub 2}. In the present study, we demonstrate unique accumulation of 4-MeOE{sub 2}, as a result of TCDD/E{sub 2} interaction and revealed its bioactivity inmore » human lung epithelial cell line (H1355). 4-Methoxyestradiol treatment significantly decreased cell growth and increased mitotic index. Elevation of ROS and SOD activity, with a concomitant decrease in the intracellular GSH/GSSG ratio, was also detected in 4-MeOE{sub 2}-treated cells. Quantitative comet assay showed increased oxidative DNA damage in the 4-MeOE{sub 2}-treated H1355 cells, which could be significantly reduced by the anti-oxidant N-acetylcysteine (NAC). However, inhibition of cell growth and increase in mitotic arrest induced by 4-MeOE{sub 2} were unaffected by NAC. We concluded that 4-MeOE{sub 2} accumulation resulting from TCDD and E{sub 2} interaction would contribute to the higher vulnerability on lung pathogenesis in females when exposed to TCDD.« less

Induction of apoptosis by pinostrobin in human cervical cancer cells: Possible mechanism of action.

PubMed

Jaudan, Alka; Sharma, Sapna; Malek, Sri Nurestri Abd; Dixit, Aparna

2018-01-01

Pinostrobin (PN) is a naturally occurring dietary bioflavonoid, found in various medicinal herbs/plants. Though anti-cancer potential of many such similar constituents has been demonstrated, critical biochemical targets and exact mechanism for their apoptosis-inducing actions have not been fully elucidated. The present study was aimed to investigate if PN induced apoptosis in cervical cancer cells (HeLa) of human origin. It is demonstrated that PN at increasing dose effectivity reduced the cell viability as well as GSH and NO2- levels. Condensed nuclei with fragmented chromatin and changes in mitochondrial matrix morphology clearly indicated the role of mitochondria in PN induced apoptosis. A marked reduction in mitochondrial membrane potential and increased ROS production after PN treatment showed involvement of free radicals, which in turn further augment ROS levels. PN treatment resulted in DNA damage, which could have been triggered by an increase in ROS levels. Decrease in apoptotic cells in the presence of caspase 3 inhibitor in PN-treated cells suggested that PN induced apoptosis via caspase dependent pathways. Additionally, a significant increase in the expression of proteins of extrinsic (TRAIL R1/DR4, TRAIL R2/DR5, TNF RI/TNFRSF1A, FADD, Fas/TNFRSF6) and intrinsic pathway (Bad, Bax, HTRA2/Omi, SMAC/Diablo, cytochrome C, Pro-Caspase-3, Cleaved Caspase-3) was observed in the cells exposed to PN. Taken together, these observations suggest that PN efficiently induces apoptosis through ROS mediated extrinsic and intrinsic dependent signaling pathways, as well as ROS mediated mitochondrial damage in HeLa cells.

Combination of miRNA499 and miRNA133 Exerts a Synergic Effect on Cardiac Differentiation

PubMed Central

Pisano, Federica; Altomare, Claudia; Cervio, Elisabetta; Barile, Lucio; Rocchetti, Marcella; Ciuffreda, Maria Chiara; Malpasso, Giuseppe; Copes, Francesco; Mura, Manuela; Danieli, Patrizia; Viarengo, Gianluca; Zaza, Antonio; Gnecchi, Massimiliano

2015-01-01

Several studies have demonstrated that miRNA are involved in cardiac development, stem cell maintenance, and differentiation. In particular, it has been shown that miRNA133, miRNA1, and miRNA499 are involved in progenitor cell differentiation into cardiomyocytes. However, it is unknown whether different miRNA may act synergistically to improve cardiac differentiation. We used mouse P19 cells as a cardiogenic differentiation model. miRNA499, miRNA1, or miRNA133 were transiently over-expressed in P19 cells individually or in different combinations. The over-expression of miRNA499 alone increased the number of beating cells and the association of miRNA499 with miRNA133 exerted a synergistic effect, further increasing the number of beating cells. Real-time polymerase chain reaction showed that the combination of miRNA499 + 133 enhanced the expression of cardiac genes compared with controls. Western blot and immunocytochemistry for connexin43 and cardiac troponin T confirmed these findings. Importantly, caffeine responsiveness, a clear functional parameter of cardiac differentiation, was increased by miRNA499 in association with miRNA133 and was directly correlated with the activation of the cardiac troponin I isoform promoter. Cyclic contractions were reversibly abolished by extracellular calcium depletion, nifedipine, ryanodine, and IP3R blockade. Finally, we demonstrated that the use of miRNA499 + 133 induced cardiac differentiation even in the absence of dimethyl sulfoxide. Our results show that the areas spontaneously contracting possess electrophysiological and pharmacological characteristics compatible with true cardiac excitation-contraction coupling. The translational relevance of our findings was reinforced by the demonstration that the over-expression of miRNA499 and miRNA133 was also able to induce the differentiation of human mesenchymal stromal cells toward the cardiac lineage. Stem Cells 2015;33:1187–1199 PMID:25534971

Reduced hematopoietic stem cell frequency predicts outcome in acute myeloid leukemia.

PubMed

Wang, Wenwen; Stiehl, Thomas; Raffel, Simon; Hoang, Van T; Hoffmann, Isabel; Poisa-Beiro, Laura; Saeed, Borhan R; Blume, Rachel; Manta, Linda; Eckstein, Volker; Bochtler, Tilmann; Wuchter, Patrick; Essers, Marieke; Jauch, Anna; Trumpp, Andreas; Marciniak-Czochra, Anna; Ho, Anthony D; Lutz, Christoph

2017-09-01

In patients with acute myeloid leukemia and low percentages of aldehyde-dehydrogenase-positive cells, non-leukemic hematopoietic stem cells can be separated from leukemic cells. By relating hematopoietic stem cell frequencies to outcome we detected poor overall- and disease-free survival of patients with low hematopoietic stem cell frequencies. Serial analysis of matched diagnostic and follow-up samples further demonstrated that hematopoietic stem cells increased after chemotherapy in patients who achieved durable remissions. However, in patients who eventually relapsed, hematopoietic stem cell numbers decreased dramatically at the time of molecular relapse demonstrating that hematopoietic stem cell levels represent an indirect marker of minimal residual disease, which heralds leukemic relapse. Upon transplantation in immune-deficient mice cases with low percentages of hematopoietic stem cells of our cohort gave rise to leukemic or no engraftment, whereas cases with normal hematopoietic stem cell levels mostly resulted in multi-lineage engraftment. Based on our experimental data, we propose that leukemic stem cells have increased niche affinity in cases with low percentages of hematopoietic stem cells. To validate this hypothesis, we developed new mathematical models describing the dynamics of healthy and leukemic cells under different regulatory scenarios. These models suggest that the mechanism leading to decreases in hematopoietic stem cell frequencies before leukemic relapse must be based on expansion of leukemic stem cells with high niche affinity and the ability to dislodge hematopoietic stem cells. Thus, our data suggest that decreasing numbers of hematopoietic stem cells indicate leukemic stem cell persistence and the emergence of leukemic relapse. Copyright© 2017 Ferrata Storti Foundation.

Increased plasma membrane cholesterol in cystic fibrosis cells correlates with CFTR genotype and depends on de novo cholesterol synthesis

PubMed Central

2010-01-01

Background Previous observations demonstrate that Cftr-null cells and tissues exhibit alterations in cholesterol processing including perinuclear cholesterol accumulation, increased de novo synthesis, and an increase in plasma membrane cholesterol accessibility compared to wild type controls. The hypothesis of this study is that membrane cholesterol accessibility correlates with CFTR genotype and is in part influenced by de novo cholesterol synthesis. Methods Electrochemical detection of cholesterol at the plasma membrane is achieved with capillary microelectrodes with a modified platinum coil that accepts covalent attachment of cholesterol oxidase. Modified electrodes absent cholesterol oxidase serves as a baseline control. Cholesterol synthesis is determined by deuterium incorporation into lipids over time. Incorporation into cholesterol specifically is determined by mass spectrometry analysis. All mice used in the study are on a C57Bl/6 background and are between 6 and 8 weeks of age. Results Membrane cholesterol measurements are elevated in both R117H and ΔF508 mouse nasal epithelium compared to age-matched sibling wt controls demonstrating a genotype correlation to membrane cholesterol detection. Expression of wt CFTR in CF epithelial cells reverts membrane cholesterol to WT levels further demonstrating the impact of CFTR on these processes. In wt epithelial cell, the addition of the CFTR inhibitors, Gly H101 or CFTRinh-172, for 24 h surprisingly results in an initial drop in membrane cholesterol measurement followed by a rebound at 72 h suggesting a feedback mechanism may be driving the increase in membrane cholesterol. De novo cholesterol synthesis contributes to membrane cholesterol accessibility. Conclusions The data in this study suggest that CFTR influences cholesterol trafficking to the plasma membrane, which when depleted, leads to an increase in de novo cholesterol synthesis to restore membrane content. PMID:20487541

Advanced glycation end products increase carbohydrate responsive element binding protein expression and promote cancer cell proliferation.

PubMed

Chen, Hanbei; Wu, Lifang; Li, Yakui; Meng, Jian; Lin, Ning; Yang, Dianqiang; Zhu, Yemin; Li, Xiaoyong; Li, Minle; Xu, Ye; Wu, Yuchen; Tong, Xuemei; Su, Qing

2014-09-01

Diabetic patients have increased levels of advanced glycation end products (AGEs) and the role of AGEs in regulating cancer cell proliferation is unclear. Here, we found that treating colorectal and liver cancer cells with AGEs promoted cell proliferation. AGEs stimulated both the expression and activation of a key transcription factor called carbohydrate responsive element binding protein (ChREBP) which had been shown to promote glycolytic and anabolic activity as well as proliferation of colorectal and liver cancer cells. Using siRNAs or the antagonistic antibody for the receptor for advanced glycation end-products (RAGE) blocked AGEs-induced ChREBP expression or cell proliferation in cancer cells. Suppressing ChREBP expression severely impaired AGEs-induced cancer cell proliferation. Taken together, these results demonstrate that AGEs-RAGE signaling enhances cancer cell proliferation in which AGEs-mediated ChREBP induction plays an important role. These findings may provide new explanation for increased cancer progression in diabetic patients. Copyright © 2014. Published by Elsevier Ireland Ltd.

Barium-cross-linked alginate-gelatine microcapsule as a potential platform for stem cell production and modular tissue formation.

PubMed

Alizadeh Sardroud, Hamed; Nemati, Sorour; Baradar Khoshfetrat, Ali; Nabavinia, Mahbobeh; Beygi Khosrowshahi, Younes

2017-08-01

Influence of gelatine concentration and cross-linker ions of Ca 2+ and Ba 2+ was evaluated on characteristics of alginate hydrogels and proliferation behaviours of model adherent and suspendable stem cells of fibroblast and U937 embedded in alginate microcapsules. Increasing gelatine concentration to 2.5% increased extent of swelling to 15% and 25% for barium- and calcium-cross-linked hydrogels, respectively. Mechanical properties also decreased with increasing swelling of hydrogels. Both by increasing gelatine concentration and using barium ions increased considerably the proliferation of encapsulated model stem cells. Barium-cross-linked alginate-gelatine microcapsule tested for bone building block showed a 13.5 ± 1.5-fold expansion for osteoblast cells after 21 days with deposition of bone matrix. The haematopoietic stem cells cultured in the microcapsule after 7 days also showed up to 2-fold increase without adding any growth factor. The study demonstrates that barium-cross-linked alginate-gelatine microcapsule has potential for use as a simple and efficient 3D platform for stem cell production and modular tissue formation.

Metabolite toxicity determines the pace of molecular evolution within microbial populations.

PubMed

Lilja, Elin E; Johnson, David R

2017-02-14

The production of toxic metabolites has shaped the spatial and temporal arrangement of metabolic processes within microbial cells. While diverse solutions to mitigate metabolite toxicity have evolved, less is known about how evolution itself is affected by metabolite toxicity. We hypothesized that the pace of molecular evolution should increase as metabolite toxicity increases. At least two mechanisms could cause this. First, metabolite toxicity could increase the mutation rate. Second, metabolite toxicity could increase the number of available mutations with large beneficial effects that selection could act upon (e.g., mutations that provide tolerance to toxicity), which consequently would increase the rate at which those mutations increase in frequency. We tested this hypothesis by experimentally evolving the bacterium Pseudomonas stutzeri under denitrifying conditions. The metabolite nitrite accumulates during denitrification and has pH-dependent toxic effects, which allowed us to evolve P. stutzeri at different magnitudes of nitrite toxicity. We demonstrate that increased nitrite toxicity results in an increased pace of molecular evolution. We further demonstrate that this increase is generally due to an increased number of available mutations with large beneficial effects and not to an increased mutation rate. Our results demonstrate that the production of toxic metabolites can have important impacts on the evolutionary processes of microbial cells. Given the ubiquity of toxic metabolites, they could also have implications for understanding the evolutionary histories of biological organisms.

Single cell systems biology by super-resolution imaging and combinatorial labeling

PubMed Central

Lubeck, Eric; Cai, Long

2012-01-01

Fluorescence microscopy is a powerful quantitative tool for exploring regulatory networks in single cells. However, the number of molecular species that can be measured simultaneously is limited by the spectral separability of fluorophores. Here we demonstrate a simple but general strategy to drastically increase the capacity for multiplex detection of molecules in single cells by using optical super-resolution microscopy (SRM) and combinatorial labeling. As a proof of principle, we labeled mRNAs with unique combinations of fluorophores using Fluorescence in situ Hybridization (FISH), and resolved the sequences and combinations of fluorophores with SRM. We measured the mRNA levels of 32 genes simultaneously in single S. cerevisiae cells. These experiments demonstrate that combinatorial labeling and super-resolution imaging of single cells provides a natural approach to bring systems biology into single cells. PMID:22660740

Preclinical Antileukemia Activity of Tramesan: A Newly Identified Bioactive Fungal Metabolite.

PubMed

Ricciardi, M R; Licchetta, R; Mirabilii, S; Scarpari, M; Parroni, A; Fabbri, A A; Cescutti, P; Reverberi, M; Fanelli, C; Tafuri, A

2017-01-01

Despite improvements that occurred in the last decades in the acute myeloid leukemia (AML) treatment, clinical results are still unsatisfactory. More effective therapies are required, and innovative approaches are ongoing, including the discovery of novel antileukemia natural compounds. Several studies have described the activity of extracts from mushrooms which produce compounds that exhibited immunological and antitumor activities. The latter has been demonstrated to be promoted in vitro by mushroom polysaccharides via induction of apoptosis. However, the antileukemia activity of these compounds on primary cells is still not reported. In the present study, we examined the in vitro effects of Tramesan (TR), a bioactive compound extracted from Trametes versicolor , on leukemic cell lines and primary cells. Our results demonstrated that TR induced a marked growth inhibition of leukemic cell lines and primary cells from AML patients. The antiproliferative effects of TR were associated in primary AML cells with a significant increase of apoptosis. No significant cytotoxic effects were observed in normal peripheral blood mononuclear cells (MNC) from healthy donors. Our data demonstrated a cytotoxic activity of TR on leukemia cells prompting further translational applications. Ongoing studies are elucidating the molecular mechanisms underlying its antileukemic activity.

Preclinical Antileukemia Activity of Tramesan: A Newly Identified Bioactive Fungal Metabolite

PubMed Central

Scarpari, M.; Parroni, A.; Fabbri, A. A.; Cescutti, P.; Reverberi, M.; Fanelli, C.

2017-01-01

Despite improvements that occurred in the last decades in the acute myeloid leukemia (AML) treatment, clinical results are still unsatisfactory. More effective therapies are required, and innovative approaches are ongoing, including the discovery of novel antileukemia natural compounds. Several studies have described the activity of extracts from mushrooms which produce compounds that exhibited immunological and antitumor activities. The latter has been demonstrated to be promoted in vitro by mushroom polysaccharides via induction of apoptosis. However, the antileukemia activity of these compounds on primary cells is still not reported. In the present study, we examined the in vitro effects of Tramesan (TR), a bioactive compound extracted from Trametes versicolor, on leukemic cell lines and primary cells. Our results demonstrated that TR induced a marked growth inhibition of leukemic cell lines and primary cells from AML patients. The antiproliferative effects of TR were associated in primary AML cells with a significant increase of apoptosis. No significant cytotoxic effects were observed in normal peripheral blood mononuclear cells (MNC) from healthy donors. Our data demonstrated a cytotoxic activity of TR on leukemia cells prompting further translational applications. Ongoing studies are elucidating the molecular mechanisms underlying its antileukemic activity. PMID:29270245

The Gardos channel is responsible for CDNB-induced dense sickle cell formation.

PubMed

Shartava, A; McIntyre, J; Shah, A K; Goodman, S R

2000-07-01

The red blood cells (RBCs) derived from blood taken from homozygous sickle cell (SS) patients demonstrate densities that are inversely proportional to the intracellular reduced glutathione (GSH) content. Addition of 1 mM 1-chloro-2,4-dinitrobenzene (CDNB) to low-density sickle cells (LDSS), at 4 degrees C, results in a shift of LDSS erythrocytes to high-density sickle cells (HDSS), with corresponding decreases in GSH. We have previously demonstrated that this CDNB effect was due to increased K(+) leakage and that dense cell formation could be inhibited by clotrimazole (specific for the Gardos channel) but not DIOA (specific for the K(+)-Cl(-) co-transport system) at pH 7.4 (Shartava et al. Am. J. Hematol. 1999;62:19-24). Here we demonstrate that clotrimazole (10 microM) inhibits dense cell formation at pH 7.1 and 6.8, while DIOA (1 mM) has no effect. As pH 6.8 is the optimal pH for the K(+)-Cl(-) co-transport system, we can now reasonably conclude that damage to the Gardos channel is responsible for CDNB-induced dense cell formation. Copyright 2000 Wiley-Liss, Inc.

Increased numbers of total nucleated and CD34+ cells in blood group O cord blood: an analysis of neonatal innate factors in the Korean population.

PubMed

Lee, Hye Ryun; Park, Jeong Su; Shin, Sue; Roh, Eun Youn; Yoon, Jong Hyun; Han, Kyou Sup; Kim, Byung Jae; Storms, Robert W; Chao, Nelson J

2012-01-01

We analyzed neonatal factors that could affect hematopoietic variables of cord blood (CB) donated from Korean neonates. The numbers of total nucleated cells (TNCs), CD34+ cells, and CD34+ cells/TNCs of CB in neonates were compared according to sex, gestational age, birth weight, birth weight centile for gestational age, and ABO blood group. With 11,098 CB units analyzed, blood group O CB showed an increased number of TNCs, CD34+ cells, and CD34+ cells/TNCs compared with other blood groups. Although TNC counts were lower in males, no difference in the number of CD34+ cells was demonstrated because the number of CD34+ cells/TNCs was higher in males. An increase in the gestational age resulted in an increase in the number of TNCs and decreases in the number of CD34+ cells and CD34+ cells/TNCs. The numbers of TNCs, CD34+ cells, and CD34+ cells/TNCs increased according to increased birth weight centile as well as birth weight. CB with blood group O has unique hematologic variables in this large-scale analysis of Korean neonates, although the impact on the storage policies of CB banks or the clinical outcome of transplantation remains to be determined. © 2011 American Association of Blood Banks.

Chimeric antigen receptor T cells: a novel therapy for solid tumors.

PubMed

Yu, Shengnan; Li, Anping; Liu, Qian; Li, Tengfei; Yuan, Xun; Han, Xinwei; Wu, Kongming

2017-03-29

The chimeric antigen receptor T (CAR-T) cell therapy is a newly developed adoptive antitumor treatment. Theoretically, CAR-T cells can specifically localize and eliminate tumor cells by interacting with the tumor-associated antigens (TAAs) expressing on tumor cell surface. Current studies demonstrated that various TAAs could act as target antigens for CAR-T cells, for instance, the type III variant epidermal growth factor receptor (EGFRvIII) was considered as an ideal target for its aberrant expression on the cell surface of several tumor types. CAR-T cell therapy has achieved gratifying breakthrough in hematological malignancies and promising outcome in solid tumor as showed in various clinical trials. The third generation of CAR-T demonstrates increased antitumor cytotoxicity and persistence through modification of CAR structure. In this review, we summarized the preclinical and clinical progress of CAR-T cells targeting EGFR, human epidermal growth factor receptor 2 (HER2), and mesothelin (MSLN), as well as the challenges for CAR-T cell therapy.

Optimal matrix rigidity for stress fiber polarization in stem cells

PubMed Central

Rehfeldt, F.; Brown, A. E. X.; Discher, D. E.; Safran, S. A.

2010-01-01

The shape and differentiation of human mesenchymal stem cells is especially sensitive to the rigidity of their environment; the physical mechanisms involved are unknown. A theoretical model and experiments demonstrate here that the polarization/alignment of stress-fibers within stem cells is a non-monotonic function of matrix rigidity. We treat the cell as an active elastic inclusion in a surrounding matrix whose polarizability, unlike dead matter, depends on the feedback of cellular forces that develop in response to matrix stresses. The theory correctly predicts the monotonic increase of the cellular forces with the matrix rigidity and the alignment of stress-fibers parallel to the long axis of cells. We show that the anisotropy of this alignment depends non-monotonically on matrix rigidity and demonstrate it experimentally by quantifying the orientational distribution of stress-fibers in stem cells. These findings offer a first physical insight for the dependence of stem cell differentiation on tissue elasticity. PMID:20563235

Pilot line report: Development of a high efficiency thin silicon solar cell

NASA Technical Reports Server (NTRS)

1978-01-01

Experimental technology advances were implemented to increase the conversion efficiency of ultrathin 2cm x 2cm cells, to demonstrate a capability for fabricating such cells at a rate of 10,000 per month, and to fabricate 200 large-area ultrathin cells to determine their feasibility of manufacture. A production rate of 10,000 50 micron m cells per month with lot average AM0 efficiencies of 11.5% was demonstrated, with peak efficiencies of 13.5% obtained. Losses in most stages of the processing were minimized, the remaining exceptions being in the photolithography and metallization steps for front contact generation and breakage handling. The 5cm x 5cm cells were fabricated with a peak yield in excess of 40% for over 10% AM0 efficiency. Greater fabrication volume is needed to fully evaluate the expected yield and efficiency levels for large cells.

Novel culture system of mesenchymal stromal cells from human subcutaneous adipose tissue.

PubMed

Iwashima, Shigejiro; Ozaki, Takenori; Maruyama, Shoichi; Saka, Yousuke; Kobori, Masato; Omae, Kaoru; Yamaguchi, Hirotake; Niimi, Tomoaki; Toriyama, Kazuhiro; Kamei, Yuzuru; Torii, Shuhei; Murohara, Toyoaki; Yuzawa, Yukio; Kitagawa, Yasuo; Matsuo, Seiichi

2009-05-01

Accumulating evidence suggests that the delivery of human adipose tissue-derived stromal cells (hASCs) has great potential as regenerative therapy. This was performed to develop a method for expanding hASCs by reducing the amount of serum required. We demonstrate that hASCs were able to expand efficiently in media containing 2% serum and fibroblast growth factor-2. These cells, or low serum cultured hASCs (hLASCs), expressed cell surface markers similar to those on bone marrow-derived mesenchymal stem cells, and could be differentiated into cells of mesenchymal lineage. Of interest, hLASCs secreted higher levels of vascular endothelial growth factor (VEGF) and hepatocyte growth factor (HGF) than hASCs cultured in 20% serum (hHASCs). Moreover, hLASC-conditioned media significantly increased endothelial cell (EC) proliferation and decreased EC apoptosis compared to that obtained from hHASCs or control media only. Antibodies against VEGF and HGF virtually negated these effects. When hASCs were administered into the ischemic hindlimbs of nude rats, hLASCs improved blood flow, increased capillary density, and raised the levels of VEGF and HGF in the muscles as compared with hHASCs. In conclusion, we demonstrate a novel low serum culture system for hASCs, which may have great potential in regenerative cell therapy for damaged organs in the clinical setting.

NF-κB mediates the antiproliferative and proapoptotic effects of bergamot juice in HepG2 cells.

PubMed

Ferlazzo, Nadia; Cirmi, Santa; Russo, Marina; Trapasso, Elena; Ursino, Maria Rita; Lombardo, Giovanni Enrico; Gangemi, Sebastiano; Calapai, Gioacchino; Navarra, Michele

2016-02-01

Among cancers, hepatocellular carcinoma is one of the commonest worldwide, and its incidence is increasing around the world. A lot of evidence underlines that natural substances usually consumed in the diet can have an important role in the prevention of cancer. In this study we investigated the molecular mechanisms underlying the antiproliferative activity of Citrus bergamia (bergamot) juice (BJ) in human hepatocellular carcinoma HepG2 cells. HepG2 cells were exposed to BJ and then cell proliferation, cell cycle progression, apoptosis and NF-κB nuclear translocation were evaluated. Here we present results demonstrating that BJ reduced the growth rate of human hepatocellular carcinoma HepG2 cells in a time- and concentration-dependent manner, by a mechanism involving the activation of apoptotic machinery via both intrinsic and extrinsic pathways. Moreover, BJ increased expression of P53 and P21 proteins that may be responsible for the HepG2 cell cycle arrest in G2 phase. In addition, BJ reduced NF-κB nuclear translocation. Our data demonstrate the ability of BJ in reducing the growth of HepG2 cells, revealing its mechanism of action and suggesting a promising role as anticancer drugs. Copyright © 2016 Elsevier Inc. All rights reserved.

Mitochondrial matrix pH controls oxidative phosphorylation and metabolism-secretion coupling in INS-1E clonal beta cells.

PubMed

Akhmedov, Dmitry; Braun, Matthias; Mataki, Chikage; Park, Kyu-Sang; Pozzan, Tullio; Schoonjans, Kristina; Rorsman, Patrik; Wollheim, Claes B; Wiederkehr, Andreas

2010-11-01

Glucose-evoked mitochondrial signals augment ATP synthesis in the pancreatic β cell. This activation of energy metabolism increases the cytosolic ATP/ADP ratio, which stimulates plasma membrane electrical activity and insulin granule exocytosis. We have recently demonstrated that matrix pH increases during nutrient stimulation of the pancreatic β cell. Here, we have tested whether mitochondrial matrix pH controls oxidative phosphorylation and metabolism-secretion coupling in the rat β-cell line INS-1E. Acidification of the mitochondrial matrix pH by nigericin blunted nutrient-dependent respiratory and ATP responses (continuously monitored in intact cells). Using electrophysiology and single cell imaging, we find that the associated defects in energy metabolism suppress glucose-stimulated plasma membrane electrical activity and cytosolic calcium transients. The same parameters were unaffected after direct stimulation of electrical activity with tolbutamide, which bypasses mitochondrial function. Furthermore, lowered matrix pH strongly inhibited sustained, but not first-phase, insulin secretion. Our results demonstrate that the matrix pH exerts a control function on oxidative phosphorylation in intact cells and that this mode of regulation is of physiological relevance for the generation of downstream signals leading to insulin granule exocytosis. We propose that matrix pH serves a novel signaling role in sustained cell activation.

Levels of Ycg1 Limit Condensin Function during the Cell Cycle

PubMed Central

Arsenault, Heather E.; Benanti, Jennifer A.

2016-01-01

During mitosis chromosomes are condensed to facilitate their segregation, through a process mediated by the condensin complex. Although several factors that promote maximal condensin activity during mitosis have been identified, the mechanisms that downregulate condensin activity during interphase are largely unknown. Here, we demonstrate that Ycg1, the Cap-G subunit of budding yeast condensin, is cell cycle-regulated with levels peaking in mitosis and decreasing as cells enter G1 phase. This cyclical expression pattern is established by a combination of cell cycle-regulated transcription and constitutive degradation. Interestingly, overexpression of YCG1 and mutations that stabilize Ycg1 each result in delayed cell-cycle entry and an overall proliferation defect. Overexpression of no other condensin subunit impacts the cell cycle, suggesting that Ycg1 is limiting for condensin complex formation. Consistent with this possibility, we find that levels of intact condensin complex are reduced in G1 phase compared to mitosis, and that increased Ycg1 expression leads to increases in both levels of condensin complex and binding to chromatin in G1. Together, these results demonstrate that Ycg1 levels limit condensin function in interphase cells, and suggest that the association of condensin with chromosomes must be reduced following mitosis to enable efficient progression through the cell cycle. PMID:27463097

Enrichment of prostate cancer stem-like cells from human prostate cancer cell lines by culture in serum-free medium and chemoradiotherapy.

PubMed

Wang, Lei; Huang, Xing; Zheng, Xinmin; Wang, Xinghuan; Li, Shiwen; Zhang, Lin; Yang, Zhonghua; Xia, Zhiping

2013-01-01

The discovery of rare subpopulations of cancer stem cells (CSCs) has created a new focus in cancer research. As CSCs demonstrate resistance to chemoradiation therapy relative to other cancer cells, this allows the enrichment of CSC populations by killing apoptosis-susceptible cancer cells. In this study, three commonly used human prostate cancer (PCa) cell lines (DU145, PC-3 and LNCaP) were examined for their expression of the putative stem cell markers CD133 and CD44 via flow cytometric analysis. Under normal culture conditions, CD133(+)/CD44(+) cells were only present in the DU145 cell line, and comprised only a minor percentage (0.1% ± 0.01%) of the total population. However, the proportion of these CD133(+)/CD44(+) prostate CSCs could be increased in these cell lines via culture in serum-free medium (SFM), or through chemotherapy or radiotherapy. Indeed, after culture in SFM, the proportion of CD133(+)/CD44(+) cells in DU145 and PC-3 had increased to 10.3% and 3.0%, respectively. Moreover, the proportion had increased to 9.8% enriched by chemotherapy and 3.5% by radiotherapy in DU145. Colony-formation tests, cell invasion assays, and tumor xenografts in BALB/c nude mice were used to evaluate the stem cell properties of CD133(+)/CD44(+) PCa cells that were isolated via fluorescence-activated cell sorting (FACS). CD133(+)/CD44(+) cells had an enhanced colony-formation capability and invasive ability in vitro, and displayed greater tumorigenic properties in vivo. These results demonstrate the presence of CD133(+)/CD44(+) prostate CSCs in established PCa cell lines and that populations of these cells can be enriched by culture in SFM or chemoradiotherapy. Finding novel therapies to override chemoradiation resistance in the prostate CSCs is the key to improve long-term results in PCa management.

The insect repellent N,N-diethyl-m-toluamide (DEET) induces angiogenesis via allosteric modulation of the M3 muscarinic receptor in endothelial cells.

PubMed

Legeay, Samuel; Clere, Nicolas; Hilairet, Grégory; Do, Quoc-Tuan; Bernard, Philippe; Quignard, Jean-François; Apaire-Marchais, Véronique; Lapied, Bruno; Faure, Sébastien

2016-06-27

The insect repellent N,N-diethyl-m-toluamide (DEET) has been reported to inhibit AChE (acetylcholinesterase) and to possess potential carcinogenic properties with excessive vascularization. In the present paper, we demonstrate that DEET specifically stimulates endothelial cells that promote angiogenesis which increases tumor growth. DEET activates cellular processes that lead to angiogenesis including proliferation, migration and adhesion. This is associated with an enhancement of NO production and VEGF expression in endothelial cells. M3 silencing or the use of a pharmacological M3 inhibitor abrogates all of these effects which reveals that DEET-induced angiogenesis is M3 sensitive. The experiments involving calcium signals in both endothelial and HEK cells overexpressing M3 receptors, as well as binding and docking studies demonstrate that DEET acts as an allosteric modulator of the M3 receptor. In addition, DEET inhibited AChE which increased acetylcholine bioavailability and binding to M3 receptors and also strengthened proangiogenic effects by an allosteric modulation.

Activation of β-adrenergic receptors is required for elevated α1A-adrenoreceptors expression and signaling in mesenchymal stromal cells

PubMed Central

Tyurin-Kuzmin, Pyotr A.; Fadeeva, Julia I.; Kanareikina, Margarita A.; Kalinina, Natalia I.; Sysoeva, Veronika Yu.; Dyikanov, Daniyar T.; Stambolsky, Dmitriy V.; Tkachuk, Vsevolod A.

2016-01-01

Sympathetic neurons are important components of mesenchymal stem cells (MSCs) niche and noradrenaline regulates biological activities of these cells. Here we examined the mechanisms of regulation of MSCs responsiveness to noradrenaline. Using flow cytometry, we demonstrated that α1A adrenergic receptors isoform was the most abundant in adipose tissue-derived MSCs. Using calcium imaging in single cells, we demonstrated that only 6.9 ± 0.8% of MSCs responded to noradrenaline by intracellular calcium release. Noradrenaline increases MSCs sensitivity to catecholamines in a transitory mode. Within 6 hrs after incubation with noradrenaline the proportion of cells responding by Ca2+ release to the fresh noradrenaline addition has doubled but declined to the baseline after 24 hrs. Increased sensitivity was due to the elevated quantities of α1A-adrenergic receptors on MSCs. Such elevation depended on the stimulation of β-adrenergic receptors and adenylate cyclase activation. The data for the first time clarify mechanisms of regulation of MSCs sensitivity to noradrenaline. PMID:27596381

Apoptotic cell death in the central nervous system of Bufo arenarum tadpoles induced by cypermethrin.

PubMed

Casco, V H; Izaguirre, M F; Marín, L; Vergara, M N; Lajmanovich, R C; Peltzer, P; Soler, A Peralta

2006-05-01

Tadpoles of the toad Bufo arenarum treated with cypermethrin (CY) at concentrations above 39 mug CY/L showed dose-dependent apoptotic cell death in immature cells of the central nervous system as demonstrated by morphometric analysis, the TUNEL method, and DNA fragmentation assay. Light-and electron-microscopic studies showed structural alterations in the intermediate and marginal layers of the brain. Immature cerebral tissue showed cellular shrinkage, nuclear fragmentation and increase of intercellular spaces. In this study we demonstrated high toxicity of CY to larval stages of Bufo arenarum. Our results show that doses lower than those used in routine insecticide applications can cause massive apoptosis in the immature cells of the central nervous system. These results coincide with our previous studies in Physalaemus biligonigerus, confirming the severe toxic effects of CY to the central nervous system of anuran species from Argentina. This may increase the mortality index in wild animals and contribute to the loss of biodiversity in our agroecosystems. We postulate that CY induces apoptosis in central nervous system cells of Bufo arenarum tadpoles by specific neurotoxic mechanisms.

Effects of direct current electric fields on lung cancer cell electrotaxis in a PMMA-based microfluidic device.

PubMed

Li, Yaping; Xu, Tao; Chen, Xiaomei; Lin, Shin; Cho, Michael; Sun, Dong; Yang, Mengsu

2017-03-01

Tumor metastasis is the primary cause of cancer death. Numerous studies have demonstrated the electrotactic responses of various cancer cell types, and suggested its potential implications in metastasis. In this study, we used a microfluidic device to emulate endogenous direct current electric field (dcEF) environment, and studied the electrotactic migration of non-small cell lung cancer cell lines (H460, HCC827, H1299, and H1975) and the underlying mechanisms. These cell lines exhibited greatly different response in applied dcEFs (2-6 V/cm). While H460 cells (large cell carcinoma) showed slight migration toward cathode, H1299 cells (large cell carcinoma) showed increased motility and dcEF-dependent anodal migration with cell reorientation. H1975 cells (adenocarcinoma) showed dcEF-dependent cathodal migration with increased motility, and HCC827 cells (adenocarcinoma) responded positively in migration speed and reorientation but minimally in migrating directions to dcEF. Activation of MAPK and PI3K signaling pathways was found to be associated with the realignment and directed migration of lung cancer cells. In addition, both Ca 2+ influx through activated stretch-activated calcium channels (SACCs) (but not voltage-gated calcium channels, VGCCs) and Ca 2+ release from intracellular storage were involved in lung cancer cell electrotactic responses. The results demonstrated that the microfluidic device provided a stable and controllable microenvironment for cell electrotaxis study, and revealed that the electrotactic responses of lung cancer cells were heterogeneous and cell-type dependent, and multiple signals contributed to lung cancer cells electrotaxis.

Effect of UVB radiation exposure in the expression of genes and proteins related to apoptosis in freshwater prawn embryos.

PubMed

Schramm, Heloísa; Jaramillo, Michael L; Quadros, Thaline de; Zeni, Eliane C; Müller, Yara M R; Ammar, Dib; Nazari, Evelise M

2017-10-01

Our previous studies showed that embryos of the freshwater prawn Macrobrachium olfersii exposed to ultraviolet B (UVB) radiation exhibited DNA damage, excessive ROS production, mitochondrial dysfunction and increased hsp70 expression, which are able, independently or together, to induce apoptosis. Thus, we attempted to elucidate some key apoptosis-related genes (ARG) and apoptosis-related proteins (ARP) and their expression during different stages of embryonic development, as well as to characterize the chronology of ARG expression and ARP contents after UVB radiation insult. We demonstrate that p53, Bax and Caspase3 genes are active in the embryonic cells at early embryonic developmental stages, and that the Bcl2 gene is active from the mid-embryonic stage. After UVB radiation exposure, we found an increase in ARP such as p53 and Bak after 3h of exposure. Moreover, an increase in ARG transcript levels for p53, Bax, Bcl2 and Caspase3 was observed at 6h after UVB exposure. Then, after 12h of UVB radiation exposure, an increase in Caspase3 gene expression and protein was observed, concomitantly with an increased number of apoptotic cells. Our data reveal that ARG and ARP are developmentally regulated in embryonic cells of M. olfersii and that UVB radiation causes apoptosis after 12h of exposure. Overall, we demonstrate that embryonic cells of M. olfersii are able to active the cell machinery against environmental changes, such as increased incidence of UVB radiation in aquatic ecosystems. Copyright © 2017 Elsevier B.V. All rights reserved.

Enhanced autophagy in pulmonary endothelial cells on exposure to HIV-Tat and morphine: Role in HIV-related pulmonary arterial hypertension.

PubMed

Dalvi, Pranjali; Sharma, Himanshu; Chinnappan, Mahendran; Sanderson, Miles; Allen, Julie; Zeng, Ruoxi; Choi, Augustine; O'Brien-Ladner, Amy; Dhillon, Navneet K

2016-12-01

Intravenous drug use is one of the major risk factors for HIV-infection in HIV-related pulmonary arterial hypertension patients. We previously demonstrated exaggerated pulmonary vascular remodeling with enhanced apoptosis followed by increased proliferation of pulmonary endothelial cells on simultaneous exposure to both opioids and HIV protein(s). Here we hypothesize that the exacerbation of autophagy may be involved in the switching of endothelial cells from an early apoptotic state to later hyper-proliferative state. Treatment of human pulmonary microvascular endothelial cells (HPMECs) with both the HIV-protein Tat and morphine resulted in an oxidative stress-dependent increase in the expression of various markers of autophagy and formation of autophagosomes when compared to either Tat or morphine monotreatments as demonstrated by western blot, transmission electron microscopy and immunofluorescence. Autophagy flux experiments suggested increased formation rather than decreased clearance of autolysosomes. Inhibition of autophagy resulted in a significant increase in apoptosis and reduction in proliferation of HPMECs with combined morphine and Tat (M+T) treatment compared to monotreatments whereas stimulation of autophagy resulted in opposite effects. Significant increases in the expression of autophagy markers as well as the number of autophagosomes and autolysosomes was observed in the lungs of SIV-infected macaques and HIV-infected humans exposed to opioids. Overall our findings indicate that morphine in combination with viral protein(s) results in the induction of autophagy in pulmonary endothelial cells that may lead to an increase in severity of angio-proliferative remodeling of the pulmonary vasculature on simian and human immunodeficiency virus infection in the presence of opioids.

Zika Virus Induces Autophagy in Human Umbilical Vein Endothelial Cells.

PubMed

Peng, Haoran; Liu, Bin; Yves, Toure Doueu; He, Yanhua; Wang, Shijie; Tang, Hailin; Ren, Hao; Zhao, Ping; Qi, Zhongtian; Qin, Zhaoling

2018-05-15

Autophagy is a common strategy for cell protection; however, some viruses can in turn adopt cellular autophagy to promote viral replication. Zika virus (ZIKV) is the pathogen that causes Zika viral disease, and it is a mosquito-borne virus. However, its pathogenesis, especially the interaction between ZIKV and target cells during the early stages of infection, is still unclear. In this study, we demonstrate that infecting human umbilical vein endothelial cells (HUVEC) with ZIKV triggers cellular autophagy. We observed both an increase in the conversion of LC3-I to LC3-II and increased accumulation of fluorescent cells with LC3 dots, which are considered to be the two key indicators of autophagy. The ratio of LC3-II/GAPDH in each group was significantly increased at different times after ZIKV infection at different MOIs, indicating that the production of lipidated LC3-II increased. Moreover, both the ratio of LC3-II/GAPDH and the expression of viral NS3 protein increased with increasing time of viral infection. The expression level of p62 decreased gradually from 12 h post-infection. Expression profile of double fluorescent protein labelling LC3 indicated that the autophagy induced by ZIKV infection was a complete process. We further investigated the role of autophagy in ZIKV replication. We demonstrated that either the treatment with inhibitors of autophagosomes formation or short hairpin RNA targeting the Beclin-1 gene, which is critical for the formation of autophagosomes, significantly reduced viral production. Taken together, our results indicate that ZIKV infection induces autophagy of HUVEC, and inhibition of ZIKV-induced autophagy restrains viral replication.

Electrolytes for Use in High Energy Lithium-ion Batteries with Wide Operating Temperature Range

NASA Technical Reports Server (NTRS)

Smart, Marshall C.; Ratnakumar, B. V.; West, W. C.; Whitcanack, L. D.; Huang, C.; Soler, J.; Krause, F. C.

2012-01-01

Met programmatic milestones for program. Demonstrated improved performance with wide operating temperature electrolytes containing ester co-solvents (i.e., methyl butyrate) containing electrolyte additives in A123 prototype cells: Previously demonstrated excellent low temperature performance, including 11C rates at -30 C and the ability to perform well down to -60 C. Excellent cycle life at room temperature has been displayed, with over 5,000 cycles being demonstrated. Good high temperature cycle life performance has also been achieved. Demonstrated improved performance with methyl propionate-containing electrolytes in large capacity prototype cells: Demonstrated the wide operating temperature range capability in large cells (12 Ah), successfully scaling up technology from 0.25 Ah size cells. Demonstrated improved performance at low temperature and good cycle life at 40 C with methyl propionate-based electrolyte containing increasing FEC content and the use of LiBOB as an additive. Utilized three-electrode cells to investigate the electrochemical characteristics of high voltage systems coupled with wide operating temperature range electrolytes: From Tafel polarization measurements on each electrode, it is evident the NMC-based cathode displays poor lithium kinetics (being the limiting electrode). The MB-based formulations containing LiBOB delivered the best rate capability at low temperature, which is attributed to improved cathode kinetics. Whereas, the use of lithium oxalate as an additive lead to the highest reversible capacity and lower irreversible losses.

Myeloid cells are capable of synthesizing aldosterone to exacerbate damage in muscular dystrophy.

PubMed

Chadwick, Jessica A; Swager, Sarah A; Lowe, Jeovanna; Welc, Steven S; Tidball, James G; Gomez-Sanchez, Celso E; Gomez-Sanchez, Elise P; Rafael-Fortney, Jill A

2016-12-01

FDA-approved mineralocorticoid receptor (MR) antagonists are used to treat heart failure. We have recently demonstrated efficacy of MR antagonists for skeletal muscles in addition to heart in Duchenne muscular dystrophy mouse models and that mineralocorticoid receptors are present and functional in skeletal muscles. The goal of this study was to elucidate the underlying mechanisms of MR antagonist efficacy on dystrophic skeletal muscles. We demonstrate for the first time that infiltrating myeloid cells clustered in damaged areas of dystrophic skeletal muscles have the capacity to produce the natural ligand of MR, aldosterone, which in excess is known to exacerbate tissue damage. Aldosterone synthase protein levels are increased in leukocytes isolated from dystrophic muscles compared with controls and local aldosterone levels in dystrophic skeletal muscles are increased, despite normal circulating levels. All genes encoding enzymes in the pathway for aldosterone synthesis are expressed in muscle-derived leukocytes. 11β-HSD2, the enzyme that inactivates glucocorticoids to increase MR selectivity for aldosterone, is also increased in dystrophic muscle tissues. These results, together with the demonstrated preclinical efficacy of antagonists, suggest MR activation is in excess of physiological need and likely contributes to the pathology of muscular dystrophy. This study provides new mechanistic insight into the known contribution of myeloid cells to muscular dystrophy pathology. This first report of myeloid cells having the capacity to produce aldosterone may have implications for a wide variety of acute injuries and chronic diseases with inflammation where MR antagonists may be therapeutic. © The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

MiR-199a-3p enhances cisplatin sensitivity of cholangiocarcinoma cells by inhibiting mTOR signaling pathway and expression of MDR1.

PubMed

Li, Qiang; Xia, Xuefeng; Ji, Jie; Ma, Jianghui; Tao, Liang; Mo, Linjun; Chen, Wei

2017-05-16

Several studies have reported reduced miRNA-199a-3p (miR-199a-3p) in different human malignancies, however, little is known about miR-199a-3p in cholangiocarcinoma cells. In this study, we demonstrate the essential role and mechanism of miR-199a-3p in regulating cisplatin sensitivity in cholangiocarcinoma cell lines. Using a CCK-8 cell counting assay we found that expression of miR-199a-3p was positively correlated with cisplatin sensitivity in cholangiocarcinoma cell lines. MiR-199a-3p overexpression could decrease the proliferation rate and increase apoptosis of cholangiocarcinoma cells in the presence of cisplatin, while miR-199a-3p inhibition had the opposite effect. Further study demonstrated that mTOR was the target gene of miR-199a-3p, and that miR-199a-3p mimics could inhibit expression of mTOR, which consequently reduced the phosphorylation of its downstream proteins 4EBP1 and p70s6k. Rescue experiments proved that miR-199a-3p could increase the cisplatin sensitivity of cholangiocarcinoma cell lines by regulating mTOR expression. Moreover, we also found that miR-199a-3p overexpression could reduce cisplatin induced MDR1 expression by decreasing the synthesis and increasing the degradation of MDR1, thus enhancing the effectiveness of cisplatin in cholangiocarcinoma. In conclusion, miR-199a-3p could increase cisplatin sensitivity of cholangiocarcinoma cell lines by inhibiting the activity of the mTOR signaling pathway and decreasing the expression of MDR1.

Classical and alternative activation of rat hepatic sinusoidal endothelial cells by inflammatory stimuli.

PubMed

Liu, Yinglin; Gardner, Carol R; Laskin, Jeffrey D; Laskin, Debra L

2013-02-01

The ability of rat hepatic sinusoidal endothelial cells (HSEC) to become activated in response to diverse inflammatory stimuli was analyzed. Whereas the classical macrophage activators, IFNγ and/or LPS upregulated expression of iNOS in HSEC, the alternative macrophage activators, IL-10 or IL-4+IL-13 upregulated arginase-1 and mannose receptor. Similar upregulation of iNOS and arginase-1 was observed in classically and alternatively activated Kupffer cells, respectively. Removal of inducing stimuli from the cells had no effect on expression of these markers, demonstrating that activation is persistent. Washing and incubation of IFNγ treated cells with IL-4+IL-13 resulted in decreased iNOS and increased arginase-1 expression, while washing and incubation of IL-4+IL-13 treated cells with IFNγ resulted in decreased arginase-1 and increased iNOS, indicating that classical and alternative activation of the cells is reversible. HSEC were more sensitive to phenotypic switching than Kupffer cells, suggesting greater functional plasticity. Hepatocyte viability and expression of PCNA, β-catenin and MMP-9 increased in the presence of alternatively activated HSEC. In contrast, the viability of hepatocytes pretreated for 2 h with 5 mM acetaminophen decreased in the presence of classically activated HSEC. These data demonstrate that activated HSEC can modulate hepatocyte responses following injury. The ability of hepatocytes to activate HSEC was also investigated. Co-culture of HSEC with acetaminophen-injured hepatocytes, but not control hepatocytes, increased the sensitivity of HSEC to classical and alternative activating stimuli. The capacity of HSEC to respond to phenotypic activators may represent an important mechanism by which they participate in inflammatory responses associated with hepatotoxicity. Copyright © 2012 Elsevier Inc. All rights reserved.

Effect of sialic acid loss on dendritic cell maturation

PubMed Central

Crespo, Hélio J; Guadalupe Cabral, M; Teixeira, Alexandra V; Lau, Joseph T Y; Trindade, Hélder; Videira, Paula A

2009-01-01

Sialic acids are key structural determinants and contribute to the functionality of a number of immune cell receptors. Previously, we demonstrated that differentiation of human dendritic cells (DCs) is accompanied by an increased expression of sialylated cell surface structures, putatively through the activity of the ST3Gal.I and ST6Gal.I sialyltransferases. Furthermore, DC endocytosis was reduced upon removal of the cell surface sialic acid residues by neuraminidase. In the present work, we evaluate the contribution of the sialic acid modifications in DC maturation. We demonstrate that neuraminidase-treated human DCs have increased expression of major histocompatibility complex (MHC) and costimulatory molecules, increased gene expression of specific cytokines and induce a higher proliferative response of T lymphocytes. Together, the data suggest that clearance of cell surface sialic acids contributes to the development of a T helper type 1 proinflammatory response. This postulate is supported by mouse models, where elevated MHC class II and increased maturation of specific DC subsets were observed in DCs harvested from ST3Gal.I−/− and ST6Gal.I−/− mice. Moreover, important qualitative differences, particularly in the extent of reduced endocytosis and in the peripheral distribution of DC subsets, existed between the ST3Gal.I−/− and ST6Gal.I−/− strains. Together, the data strongly suggest not only a role of cell surface sialic acid modifications in maturation and functionality of DCs, but also that the sialic acid linkages created by different sialyltransferases are functionally distinct. Consequently, with particular relevance to DC-based therapies, cell surface sialylation, mediated by individual sialyltransferases, can influence the immunogenicity of DCs upon antigen loading. PMID:19740323

Progesterone facilitates chromosome instability (aneuploidy) in p53 null normal mammary epithelial cells

NASA Technical Reports Server (NTRS)

Goepfert, T. M.; McCarthy, M.; Kittrell, F. S.; Stephens, C.; Ullrich, R. L.; Brinkley, B. R.; Medina, D.

2000-01-01

Mammary epithelial cells from p53 null mice have been shown recently to exhibit an increased risk for tumor development. Hormonal stimulation markedly increased tumor development in p53 null mammary cells. Here we demonstrate that mammary tumors arising in p53 null mammary cells are highly aneuploid, with greater than 70% of the tumor cells containing altered chromosome number and a mean chromosome number of 56. Normal mammary cells of p53 null genotype and aged less than 14 wk do not exhibit aneuploidy in primary cell culture. Significantly, the hormone progesterone, but not estrogen, increases the incidence of aneuploidy in morphologically normal p53 null mammary epithelial cells. Such cells exhibited 40% aneuploidy and a mean chromosome number of 54. The increase in aneuploidy measured in p53 null tumor cells or hormonally stimulated normal p53 null cells was not accompanied by centrosome amplification. These results suggest that normal levels of progesterone can facilitate chromosomal instability in the absence of the tumor suppressor gene, p53. The results support the emerging hypothesis based both on human epidemiological and animal model studies that progesterone markedly enhances mammary tumorigenesis.

Vaccination with OK-432 followed by TC-1 tumor lysate leads to significant antitumor effects.

PubMed

Chen, I-Ju; Yen, Chih-Feng; Lin, Kun-Ju; Lee, Chyi-Long; Soong, Yung-Kuei; Lai, Chyong-Huey; Lin, Cheng-Tao

2011-07-01

Human papillomavirus (HPV) infects large numbers of women worldwide and is present in more than 99% of all cervical cancer. TC-1 cell is a cell line with high expression of E7 antigen of HPV type 16 and its cell lysate has been demonstrated as an ideal inducer of E7-specific, antitumor immunity. OK-432 (Picibanil), a penicillin-killed Streptococcus pyogenes, has been reported with potent immunomodulation properties in cancer treatment by stimulating the maturation of dendritic cells (DCs) and secretion of Th-1 type cytokines. The current study demonstrated that a protocol to immunize the C57BL/6 mice with OK-432 followed by treatment with TC-1 lysate can generate markedly increased immune responses of E7-specific CD4(+) T cells and a moderate increase of natural killer (NK) cell, as well as a satisfactorily protective and therapeutic antitumor effect by triggering the DCs to prime T cells. Depletion of lymphocyte subset in vivo suggested that the antitumor effects could be dominantly executed by CD8+ T cells and followed by NK cells, and both of these reactions were induced by the generation of robust E7-specific CD4(+) T helper cell response. These findings warrant OK-432 combination with tumor-lysate as an effective and safe vaccine in future clinical application of cervical cancer.

Direct glass bonded high specific power silicon solar cells for space applications

NASA Technical Reports Server (NTRS)

Dinetta, L. C.; Rand, J. A.; Cummings, J. R.; Lampo, S. M.; Shreve, K. P.; Barnett, Allen M.

1991-01-01

A lightweight, radiation hard, high performance, ultra-thin silicon solar cell is described that incorporates light trapping and a cover glass as an integral part of the device. The manufacturing feasibility of high specific power, radiation insensitive, thin silicon solar cells was demonstrated experimentally and with a model. Ultra-thin, light trapping structures were fabricated and the light trapping demonstrated experimentally. The design uses a micro-machined, grooved back surface to increase the optical path length by a factor of 20. This silicon solar cell will be highly tolerant to radiation because the base width is less than 25 microns making it insensitive to reduction in minority carrier lifetime. Since the silicon is bonded without silicone adhesives, this solar cell will also be insensitive to UV degradation. These solar cells are designed as a form, fit, and function replacement for existing state of the art silicon solar cells with the effect of simultaneously increasing specific power, power/area, and power supply life. Using a 3-mil thick cover glass and a 0.3 g/sq cm supporting Al honeycomb, a specific power for the solar cell plus cover glass and honeycomb of 80.2 W/Kg is projected. The development of this technology can result in a revolutionary improvement in high survivability silicon solar cell products for space with the potential to displace all existing solar cell technologies for single junction space applications.

Non-Thermal Atmospheric Pressure Plasma Preferentially Induces Apoptosis in p53-Mutated Cancer Cells by Activating ROS Stress-Response Pathways

PubMed Central

Ma, Yonghao; Ha, Chang Seung; Hwang, Seok Won; Lee, Hae June; Kim, Gyoo Cheon; Lee, Kyo-Won; Song, Kiwon

2014-01-01

Non-thermal atmospheric pressure plasma (NTAPP) is an ionized gas at room temperature and has potential as a new apoptosis-promoting cancer therapy that acts by generating reactive oxygen species (ROS). However, it is imperative to determine its selectivity and standardize the components and composition of NTAPP. Here, we designed an NTAPP-generating apparatus combined with a He gas feeding system and demonstrated its high selectivity toward p53-mutated cancer cells. We first determined the proper conditions for NTAPP exposure to selectively induce apoptosis in cancer cells. The apoptotic effect of NTAPP was greater for p53-mutated cancer cells; artificial p53 expression in p53-negative HT29 cells decreased the pro-apoptotic effect of NTAPP. We also examined extra- and intracellular ROS levels in NTAPP-treated cells to deduce the mechanism of NTAPP action. While NTAPP-mediated increases in extracellular nitric oxide (NO) did not affect cell viability, intracellular ROS increased under NTAPP exposure and induced apoptotic cell death. This effect was dose-dependently reduced following treatment with ROS scavengers. NTAPP induced apoptosis even in doxorubicin-resistant cancer cell lines, demonstrating the feasibility of NTAPP as a potent cancer therapy. Collectively, these results strongly support the potential of NTAPP as a selective anticancer treatment, especially for p53-mutated cancer cells. PMID:24759730

Autocrine HBEGF expression promotes breast cancer intravasation, metastasis and macrophage-independent invasion in vivo

PubMed Central

Zhou, Zhen Ni; Sharma, Ved P.; Beaty, Brian T.; Roh-Johnson, Minna; Peterson, Esther A.; Van Rooijen, Nico; Kenny, Paraic A.; Wiley, H. Steven; Condeelis, John S.; Segall, Jeffrey E.

2013-01-01

Increased expression of HBEGF in ER negative breast tumors is correlated with enhanced metastasis to distant organ sites and more rapid disease recurrence upon removal of the primary tumor. Our previous work has demonstrated a paracrine loop between breast cancer cells and macrophages in which the tumor cells are capable of stimulating macrophages through the secretion of CSF-1 while the tumor associated macrophages (TAMs) in turn aid in tumor cell invasion by secreting EGF. To determine how the autocrine expression of EGFR ligands by carcinoma cells would affect this paracrine loop mechanism, and in particular whether tumor cell invasion depends on spatial ligand gradients generated by TAMs, we generated cell lines with increased HBEGF expression. We find that autocrine HBEGF expression enhanced in vivo intravasation and metastasis, and resulted in a novel phenomenon in which macrophages were no longer required for in vivo invasion of breast cancer cells. In vitro studies revealed that expression of HBEGF enhanced invadopodium formation, thus providing a mechanism for cell autonomous invasion. The increased invadopodium formation was directly dependent on EGFR signaling, as demonstrated by a rapid decrease in invadopodia upon inhibition of autocrine HBEGF/EGFR signaling as well as inhibition of signaling downstream of EGFR activation. HBEGF expression also resulted in enhanced invadopodium function via upregulation of MMP2 and MMP9 expression. We conclude that high levels of HBEGF expression can short-circuit the tumor cell/macrophage paracrine invasion loop, resulting in enhanced tumor invasion that is independent of macrophage signaling. PMID:24013225

Autocrine HBEGF expression promotes breast cancer intravasation, metastasis and macrophage-independent invasion in vivo.

PubMed

Zhou, Z N; Sharma, V P; Beaty, B T; Roh-Johnson, M; Peterson, E A; Van Rooijen, N; Kenny, P A; Wiley, H S; Condeelis, J S; Segall, J E

2014-07-17

Increased expression of HBEGF in estrogen receptor-negative breast tumors is correlated with enhanced metastasis to distant organ sites and more rapid disease recurrence upon removal of the primary tumor. Our previous work has demonstrated a paracrine loop between breast cancer cells and macrophages in which the tumor cells are capable of stimulating macrophages through the secretion of colony-stimulating factor-1 while the tumor-associated macrophages (TAMs), in turn, aid in tumor cell invasion by secreting epidermal growth factor. To determine how the autocrine expression of epidermal growth factor receptor (EGFR) ligands by carcinoma cells would affect this paracrine loop mechanism, and in particular whether tumor cell invasion depends on spatial ligand gradients generated by TAMs, we generated cell lines with increased HBEGF expression. We found that autocrine HBEGF expression enhanced in vivo intravasation and metastasis and resulted in a novel phenomenon in which macrophages were no longer required for in vivo invasion of breast cancer cells. In vitro studies revealed that expression of HBEGF enhanced invadopodium formation, thus providing a mechanism for cell autonomous invasion. The increased invadopodium formation was directly dependent on EGFR signaling, as demonstrated by a rapid decrease in invadopodia upon inhibition of autocrine HBEGF/EGFR signaling as well as inhibition of signaling downstream of EGFR activation. HBEGF expression also resulted in enhanced invadopodium function via upregulation of matrix metalloprotease 2 (MMP2) and MMP9 expression levels. We conclude that high levels of HBEGF expression can short-circuit the tumor cell/macrophage paracrine invasion loop, resulting in enhanced tumor invasion that is independent of macrophage signaling.

Inhibition of cyclin D1 enhances sensitivity to radiotherapy and reverses epithelial to mesenchymal transition for esophageal cancer cells.

PubMed

Su, Huafang; Jin, Xiance; Shen, Lanxiao; Fang, Ya; Fei, Zhenghua; Zhang, Xuebang; Xie, Congying; Chen, Xiaolei

2016-04-01

Acquired radioresistance during radiotherapy has significantly affected the treatment efficacy in esophageal cancer. Many of radioresistant cancer cells demonstrated epithelial-mesenchymal transition (EMT).We found in previous study that a radioresistant cell line (KYSE-150R) possessed EMT characteristic with cyclin D1 overexpression. Cyclin D1 has been demonstrated to affect the radiation sensitivity in cancer cells. To elucidate the molecular functions of cyclin D1 on EMT phenotypes and esophageal cancer radiosensitivity, we treated the radioresistant esophageal cancer cells (KYSE-150R) and parental cells (KYSE-150) with cyclin D1 small interfering RNA (siRNA). The cell proliferation rate of KYSE-150R and the radiation survival fraction were significantly decreased in cyclin D1 siRNA treatment group. Knocking down cyclin D1 resulted in G0/G1 arrest in KYSE-150R cells. The average number of irradiation-induced γ-H2AX foci increased in the cells treated with cyclin D1 siRNA, indicating impaired DNA double-strand break (DSB) repair in KYSE-150R cells. Cyclin D1 also reversed EMT phenotypes with significantly increased expression of E-cadherin in KYSE-150R cells. However, cyclin D1 siRNA have no radiosensitizing effects on KYSE-150 cells, with no obvious change in EMT marker expression .Our work showed that EMT phenotypes can be reduced and the radiosensitivity of esophageal cancer cells can be enhanced by inhibiting cyclin D1.

B cells promote tumor progression in a mouse model of HPV-mediated cervical cancer.

PubMed

Tang, Alexandre; Dadaglio, Gilles; Oberkampf, Marine; Di Carlo, Selene; Peduto, Lucie; Laubreton, Daphné; Desrues, Belinda; Sun, Cheng-Ming; Montagutelli, Xavier; Leclerc, Claude

2016-09-15

Enhancing anti-tumor immunity and preventing tumor escape are efficient strategies to increase the efficacy of therapeutic cancer vaccines. However, the treatment of advanced tumors remains difficult, mainly due to the immunosuppressive tumor microenvironment. Regulatory T cells and myeloid-derived suppressor cells have been extensively studied, and their role in suppressing tumor immunity is now well established. In contrast, the role of B lymphocytes in tumor immunity remains unclear because B cells can promote tumor immunity or display regulatory functions to control excessive inflammation, mainly through IL-10 secretion. Here, in a mouse model of HPV-related cancer, we demonstrate that B cells accumulated in the draining lymph node of tumor-bearing mice, due to a prolonged survival, and showed a decreased expression of MHC class II and CD86 molecules and an increased expression of Ly6A/E, PD-L1 and CD39, suggesting potential immunoregulatory properties. However, B cells from tumor-bearing mice did not show an increased ability to secrete IL-10 and a deficiency in IL-10 production did not impair tumor growth. In contrast, in B cell-deficient μMT mice, tumor rejection occurred due to a strong T cell-dependent anti-tumor response. Genetic analysis based on single nucleotide polymorphisms identified genetic variants associated with tumor rejection in μMT mice, which could potentially affect reactive oxygen species production and NK cell activity. Our results demonstrate that B cells play a detrimental role in anti-tumor immunity and suggest that targeting B cells could enhance the anti-tumor response and improve the efficacy of therapeutic cancer vaccines. © 2016 UICC.

Drug-Induced Trafficking of P-Glycoprotein in Human Brain Capillary Endothelial Cells as Demonstrated by Exposure to Mitomycin C

PubMed Central

Noack, Andreas; Noack, Sandra; Hoffmann, Andrea; Maalouf, Katia; Buettner, Manuela; Couraud, Pierre-Olivier; Romero, Ignacio A.; Weksler, Babette; Alms, Dana; Römermann, Kerstin; Naim, Hassan Y.; Löscher, Wolfgang

2014-01-01

P-glycoprotein (Pgp; ABCB1/MDR1) is a major efflux transporter at the blood-brain barrier (BBB), restricting the penetration of various compounds. In other tissues, trafficking of Pgp from subcellular stores to the cell surface has been demonstrated and may constitute a rapid way of the cell to respond to toxic compounds by functional membrane insertion of the transporter. It is not known whether drug-induced Pgp trafficking also occurs in brain capillary endothelial cells that form the BBB. In this study, trafficking of Pgp was investigated in human brain capillary endothelial cells (hCMEC/D3) that were stably transfected with a doxycycline-inducible MDR1-EGFP fusion plasmid. In the presence of doxycycline, these cells exhibited a 15-fold increase in Pgp-EGFP fusion protein expression, which was associated with an increased efflux of the Pgp substrate rhodamine 123 (Rho123). The chemotherapeutic agent mitomycin C (MMC) was used to study drug-induced trafficking of Pgp. Confocal fluorescence microscopy of single hCMEC/D3-MDR1-EGFP cells revealed that Pgp redistribution from intracellular pools to the cell surface occurred within 2 h of MMC exposure. Pgp-EGFP exhibited a punctuate pattern at the cell surface compatible with concentrated regions of the fusion protein in membrane microdomains, i.e., lipid rafts, which was confirmed by Western blot analysis of biotinylated cell surface proteins in Lubrol-resistant membranes. MMC exposure also increased the functionality of Pgp as assessed in three functional assays with Pgp substrates (Rho123, eFluxx-ID Gold, calcein-AM). However, this increase occurred with some delay after the increased Pgp expression and coincided with the release of Pgp from the Lubrol-resistant membrane complexes. Disrupting rafts by depleting the membrane of cholesterol increased the functionality of Pgp. Our data present the first direct evidence of drug-induced Pgp trafficking at the human BBB and indicate that Pgp has to be released from lipid rafts to gain its full functionality. PMID:24505408

Drug-induced trafficking of p-glycoprotein in human brain capillary endothelial cells as demonstrated by exposure to mitomycin C.

PubMed

Noack, Andreas; Noack, Sandra; Hoffmann, Andrea; Maalouf, Katia; Buettner, Manuela; Couraud, Pierre-Olivier; Romero, Ignacio A; Weksler, Babette; Alms, Dana; Römermann, Kerstin; Naim, Hassan Y; Löscher, Wolfgang

2014-01-01

P-glycoprotein (Pgp; ABCB1/MDR1) is a major efflux transporter at the blood-brain barrier (BBB), restricting the penetration of various compounds. In other tissues, trafficking of Pgp from subcellular stores to the cell surface has been demonstrated and may constitute a rapid way of the cell to respond to toxic compounds by functional membrane insertion of the transporter. It is not known whether drug-induced Pgp trafficking also occurs in brain capillary endothelial cells that form the BBB. In this study, trafficking of Pgp was investigated in human brain capillary endothelial cells (hCMEC/D3) that were stably transfected with a doxycycline-inducible MDR1-EGFP fusion plasmid. In the presence of doxycycline, these cells exhibited a 15-fold increase in Pgp-EGFP fusion protein expression, which was associated with an increased efflux of the Pgp substrate rhodamine 123 (Rho123). The chemotherapeutic agent mitomycin C (MMC) was used to study drug-induced trafficking of Pgp. Confocal fluorescence microscopy of single hCMEC/D3-MDR1-EGFP cells revealed that Pgp redistribution from intracellular pools to the cell surface occurred within 2 h of MMC exposure. Pgp-EGFP exhibited a punctuate pattern at the cell surface compatible with concentrated regions of the fusion protein in membrane microdomains, i.e., lipid rafts, which was confirmed by Western blot analysis of biotinylated cell surface proteins in Lubrol-resistant membranes. MMC exposure also increased the functionality of Pgp as assessed in three functional assays with Pgp substrates (Rho123, eFluxx-ID Gold, calcein-AM). However, this increase occurred with some delay after the increased Pgp expression and coincided with the release of Pgp from the Lubrol-resistant membrane complexes. Disrupting rafts by depleting the membrane of cholesterol increased the functionality of Pgp. Our data present the first direct evidence of drug-induced Pgp trafficking at the human BBB and indicate that Pgp has to be released from lipid rafts to gain its full functionality.

Pulsed-dosing with oral sodium phenylbutyrate increases hemoglobin F in a patient with sickle cell anemia.

PubMed

Hines, Patrick; Dover, George J; Resar, Linda M S

2008-02-01

Increasing hemoglobin F (HbF) appears to be beneficial for patients with sickle cell anemia. We previously demonstrated that daily, oral sodium phenylbutyrate (OSPB) induces HbF synthesis in pediatric and adult patients with hemoglobin SS (HbSS). The high doses and need for daily therapy, however, have limited its use. Here, we report a patient treated with pulsed-dosing of OSPB for over 3 years. This patient developed a modest, but sustained elevation in HbF over the course of therapy without side effects. Although larger studies are needed, this case demonstrates that pulsed-dosing with OSPB enhances HbF synthesis. (c) 2007 Wiley-Liss, Inc.

Study of stem cell homing & self-renewal marker gene profile of ex vivo expanded human CD34+ cells manipulated with a mixture of cytokines & stromal cell-derived factor 1

PubMed Central

Kode, Jyoti; Khattry, Navin; Bakshi, Ashish; Amrutkar, Vasanti; Bagal, Bhausaheb; Karandikar, Rohini; Rane, Pallavi; Fujii, Nobutaka; Chiplunkar, Shubhada

2017-01-01

Background & objectives: Next generation transplantation medicine aims to develop stimulating cocktail for increased ex vivo expansion of primitive hematopoietic stem and progenitor cells (HSPC). The present study was done to evaluate the cocktail GF (Thrombopoietin + Stem Cell factor + Flt3-ligand) and homing-defining molecule Stromal cell-derived factor 1 (SDF1) for HSPC ex vivo expansion. Methods: Peripheral blood stem cell (n=74) harvests were analysed for CD34hi CD45lo HSPC. Immunomagnetically enriched HSPC were cultured for eight days and assessed for increase in HSPC, colony forming potential in vitro and in vivo engrafting potential by analyzing human CD45+ cells. Expression profile of genes for homing and stemness were studied using microarray analysis. Expression of adhesion/homing markers were validated by flow cytometry/ confocal microscopy. Results: CD34hi CD45lo HSPC expansion cultures with GF+SDF1 demonstrated increased nucleated cells (n=28, P< 0.001), absolute CD34+ cells (n=8, P=0.021) and increased colony forming units (cfu) compared to unstimulated and GF-stimulated HSPC. NOD-SCID mice transplanted with GF+SDF1-HSPC exhibited successful homing/engraftment (n=24, P< 0.001). Microarray analysis of expanded HSPC demonstrated increased telomerase activity and many homing-associated genes (35/49) and transcription factors for stemness/self-renewal (49/56) were significantly upregulated in GF+SDF1 stimulated HSPC when compared to GF-stimulated HSPC. Expression of CD44, CXCR4, CD26, CD14, CD45 and soluble IL-6 in expanded cultures were validated by flow cytometry and confocal microscopy. Interpretation & conclusions: Cocktail of cytokines and SDF1 showed good potential to successfully expand HSPC which exhibited enhanced ability to generate multilineage cells in short-term and long-term repopulation assay. This cocktail-mediated stem cell expansion has potential to obviate the need for longer and large volume apheresis procedure making it convenient for donors. PMID:29168461

Overexpression of the human DEK oncogene reprograms cellular metabolism and promotes glycolysis

PubMed Central

Watanabe, Miki; Muraleedharan, Ranjithmenon; Lambert, Paul F.; Lane, Andrew N.; Romick-Rosendale, Lindsey E.; Wells, Susanne I.

2017-01-01

The DEK oncogene is overexpressed in many human malignancies including at early tumor stages. Our reported in vitro and in vivo models of squamous cell carcinoma have demonstrated that DEK contributes functionally to cellular and tumor survival and to proliferation. However, the underlying molecular mechanisms remain poorly understood. Based on recent RNA sequencing experiments, DEK expression was necessary for the transcription of several metabolic enzymes involved in anabolic pathways. This identified a possible mechanism whereby DEK may drive cellular metabolism to enable cell proliferation. Functional metabolic Seahorse analysis demonstrated increased baseline and maximum extracellular acidification rates, a readout of glycolysis, in DEK-overexpressing keratinocytes and squamous cell carcinoma cells. DEK overexpression also increased the maximum rate of oxygen consumption and therefore increased the potential for oxidative phosphorylation (OxPhos). To detect small metabolites that participate in glycolysis and the tricarboxylic acid cycle (TCA) that supplies substrate for OxPhos, we carried out NMR-based metabolomics studies. We found that high levels of DEK significantly reprogrammed cellular metabolism and altered the abundances of amino acids, TCA cycle intermediates and the glycolytic end products lactate, alanine and NAD+. Taken together, these data support a scenario whereby overexpression of the human DEK oncogene reprograms keratinocyte metabolism to fulfill energy and macromolecule demands required to enable and sustain cancer cell growth. PMID:28558019

Regulation of collagenase-3 and osteocalcin gene expression by collagen and osteopontin in differentiating MC3T3-E1 cells

NASA Technical Reports Server (NTRS)

D'Alonzo, Richard C.; Kowalski, Aaron J.; Denhardt, David T.; Nickols, G. Allen; Partridge, Nicola C.

2002-01-01

Both collagenase-3 and osteocalcin mRNAs are expressed maximally during the later stages of osteoblast differentiation. Here, we demonstrate that collagenase-3 mRNA expression in differentiating MC3T3-E1 cells is dependent upon the presence of ascorbic acid, is inhibited in the presence of the collagen synthesis inhibitor, 3,4-dehydroproline, and is stimulated by growth on collagen in the absence of ascorbic acid. Transient transfection studies show that collagenase-3 promoter activity increases during cell differentiation and requires the presence of ascorbic acid. Additionally, we show that, in differentiating MC3T3-E1 cells, collagenase-3 gene expression increases in the presence of an anti-osteopontin monoclonal antibody that binds near the RGD motif of this protein, whereas osteocalcin expression is inhibited. Furthermore, an RGD peptidomimetic compound, designed to block interaction of ligands to the alpha(v) integrin subunit, increases osteocalcin expression and inhibits collagenase-3 expression, suggesting that the RGD peptidomimetic initiates certain alpha(v) integrin signaling in osteoblastic cells. Overall, these studies demonstrate that stimulation of collagenase-3 expression during osteoblast differentiation requires synthesis of a collagenous matrix and that osteopontin and alpha(v) integrins exert divergent regulation of collagenase-3 and osteocalcin expression during osteoblast differentiation.

Overexpression of the human DEK oncogene reprograms cellular metabolism and promotes glycolysis.

PubMed

Matrka, Marie C; Watanabe, Miki; Muraleedharan, Ranjithmenon; Lambert, Paul F; Lane, Andrew N; Romick-Rosendale, Lindsey E; Wells, Susanne I

2017-01-01

The DEK oncogene is overexpressed in many human malignancies including at early tumor stages. Our reported in vitro and in vivo models of squamous cell carcinoma have demonstrated that DEK contributes functionally to cellular and tumor survival and to proliferation. However, the underlying molecular mechanisms remain poorly understood. Based on recent RNA sequencing experiments, DEK expression was necessary for the transcription of several metabolic enzymes involved in anabolic pathways. This identified a possible mechanism whereby DEK may drive cellular metabolism to enable cell proliferation. Functional metabolic Seahorse analysis demonstrated increased baseline and maximum extracellular acidification rates, a readout of glycolysis, in DEK-overexpressing keratinocytes and squamous cell carcinoma cells. DEK overexpression also increased the maximum rate of oxygen consumption and therefore increased the potential for oxidative phosphorylation (OxPhos). To detect small metabolites that participate in glycolysis and the tricarboxylic acid cycle (TCA) that supplies substrate for OxPhos, we carried out NMR-based metabolomics studies. We found that high levels of DEK significantly reprogrammed cellular metabolism and altered the abundances of amino acids, TCA cycle intermediates and the glycolytic end products lactate, alanine and NAD+. Taken together, these data support a scenario whereby overexpression of the human DEK oncogene reprograms keratinocyte metabolism to fulfill energy and macromolecule demands required to enable and sustain cancer cell growth.

Serglycin as a potential biomarker for glioma: association of serglycin expression, extent of mast cell recruitment and glioblastoma progression

PubMed Central

Roy, Ananya; Attarha, Sanaz; Weishaupt, Holger; Edqvist, Per-Henrik; Swartling, Fredrik J.; Bergqvist, Michael; Siebzehnrubl, Florian A.; Smits, Anja; Pontén, Fredrik; Tchougounova, Elena

2017-01-01

Serglycin is an intracellular proteoglycan with a unique ability to adopt highly divergent structures by glycosylation with variable types of glycosaminoglycans (GAGs) when expressed by different cell types. Serglycin is overexpressed in aggressive cancers suggesting its protumorigenic role. In this study, we explored the expression of serglycin in human glioma and its correlation with survival and immune cell infiltration. We demonstrate that serglycin is expressed in glioma and that increased expression predicts poor survival of patients. Analysis of serglycin expression in a large cohort of low- and high-grade human glioma samples reveals that its expression is grade dependent and is positively correlated with mast cell (MC) infiltration. Moreover, serglycin expression in patient-derived glioma cells is significantly increased upon MC co-culture. This is also accompanied by increased expression of CXCL12, CXCL10, as well as markers of cancer progression, including CD44, ZEB1 and vimentin. In conclusion, these findings indicate the importance of infiltrating MCs in glioma by modulating signaling cascades involving serglycin, CD44 and ZEB1. The present investigation reveals serglycin as a potential prognostic marker for glioma and demonstrates an association with the extent of MC recruitment and glioma progression, uncovering potential future therapeutic opportunities for patients. PMID:28445977

Endogenous bax translocation in SH-SY5Y human neuroblastoma cells and cerebellar granule neurons undergoing apoptosis.

PubMed

McGinnis, K M; Gnegy, M E; Wang, K K

1999-05-01

Changes at the mitochondria are an early, required step in apoptosis in various cell types. We used western blot analysis to demonstrate that the proapoptotic protein Bax translocated from the cytosolic to the mitochondrial fraction in SH-SY5Y human neuroblastoma cells undergoing staurosporine- or EGTA-mediated apoptosis. Levels of mitochondrial Bax increased 15 min after staurosporine treatment. In EGTA-treated cells, increased levels of mitochondrial Bax were seen at 4 h, consistent with a slower onset of apoptosis in EGTA versus staurosporine treatments. We also demonstrate the concomitant translocation of cytochrome c from the mitochondrial to the cytosolic fractions. We correlated these translocations with changes in caspase-3-like activity. An increase in caspase-3-like activity was evident 2 h after staurosporine treatment. Inhibition of the mitochondrial permeability transition had no effect on Bax translocation or caspase-3-like activity in staurosporine-treated SH-SY5Y cells. In primary cultures of cerebellar granule neurons undergoing low K(+)-mediated apoptosis, Bax translocation to the mitochondrial fraction was evident at 3 h. Cytochrome c release into the cytosol was not significant until 8 h after treatment. These data support a model of apoptosis in which Bax acts directly at the mitochondria to allow the release of cytochrome c.

In vitro culture of stress erythroid progenitors identifies distinct progenitor populations and analogous human progenitors.

PubMed

Xiang, Jie; Wu, Dai-Chen; Chen, Yuanting; Paulson, Robert F

2015-03-12

Tissue hypoxia induces a systemic response designed to increase oxygen delivery to tissues. One component of this response is increased erythropoiesis. Steady-state erythropoiesis is primarily homeostatic, producing new erythrocytes to replace old erythrocytes removed from circulation by the spleen. In response to anemia, the situation is different. New erythrocytes must be rapidly made to increase hemoglobin levels. At these times, stress erythropoiesis predominates. Stress erythropoiesis is best characterized in the mouse, where it is extramedullary and utilizes progenitors and signals that are distinct from steady-state erythropoiesis. In this report, we use an in vitro culture system that recapitulates the in vivo development of stress erythroid progenitors. We identify cell-surface markers that delineate a series of stress erythroid progenitors with increasing maturity. In addition, we use this in vitro culture system to expand human stress erythroid progenitor cells that express analogous cell-surface markers. Consistent with previous suggestions that human stress erythropoiesis is similar to fetal erythropoiesis, we demonstrate that human stress erythroid progenitors express fetal hemoglobin upon differentiation. These data demonstrate that similar to murine bone marrow, human bone marrow contains cells that can generate BMP4-dependent stress erythroid burst-forming units when cultured under stress erythropoiesis conditions. © 2015 by The American Society of Hematology.

Histone deacetylase inhibitors epigenetically promote reparative events in primary dental pulp cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Duncan, Henry F., E-mail: Hal.Duncan@dental.tcd.ie; Smith, Anthony J.; Fleming, Garry J.P.

Application of histone deacetylase inhibitors (HDACi) to cells epigenetically alters their chromatin structure and induces transcriptional and cellular reparative events. This study investigated the application of two HDACi, valproic acid (VPA) and trichostatin A (TSA) on the induction of repair-associated responses in primary dental pulp cell (DPC) cultures. Flow cytometry demonstrated that TSA (100 nM, 400 nM) significantly increased cell viability. Neither HDACi was cytotoxic, although cell growth analysis revealed significant anti-proliferative effects at higher concentrations for VPA (>0.5 mM) and TSA (>50 nM). While high-content-analysis demonstrated that HDACi did not significantly induce caspase-3 or p21 activity, p53-expression was increasedmore » by VPA (3 mM, 5 mM) at 48 h. HDACi-exposure induced mineralization per cell dose-dependently to a plateau level (VPA-0.125 mM and TSA-25 nM) with accompanying increases in mineralization/dentinogenic-associated gene expression at 5 days (DMP-1, BMP-2/-4, Nestin) and 10 days (DSPP, BMP-2/-4). Both HDACis, at a range of concentrations, significantly stimulated osteopontin and BMP-2 protein expression at 10 and 14 days further supporting the ability of HDACi to promote differentiation. HDACi exert different effects on primary compared with transformed DPCs and promote mineralization and differentiation events without cytotoxic effects. These novel data now highlight the potential in restorative dentistry for applying low concentrations of HDACi in vital pulp treatment. -- Highlights: • Valproic acid and trichostatin A promoted mineralization in primary pulp cells. • Cell viability, apoptosis, caspase-3, p21 unaltered; p53 increased by valproic acid. • Trichostatin A increased cell viability at 24 h at selected concentrations. • Altered cell toxicity and differentiation between primary and transformed cells. • HDACi-induced the differentiation marker proteins osteopontin and BMP-2.« less

Blockade of the Programmed Death-1 Pathway Restores Sarcoidosis CD4+ T-Cell Proliferative Capacity

PubMed Central

Braun, Nicole A.; Celada, Lindsay J.; Herazo-Maya, Jose D.; Abraham, Susamma; Shaginurova, Guzel; Sevin, Carla M.; Grutters, Jan; Culver, Daniel A.; Dworski, Ryszard; Sheller, James; Massion, Pierre P.; Polosukhin, Vasiliy V.; Johnson, Joyce E.; Kaminski, Naftali; Wilkes, David S.; Oswald-Richter, Kyra A.

2014-01-01

Rationale: Effective therapeutic interventions for chronic, idiopathic lung diseases remain elusive. Normalized T-cell function is an important contributor to spontaneous resolution of pulmonary sarcoidosis. Up-regulation of inhibitor receptors, such as programmed death-1 (PD-1) and its ligand, PD-L1, are important inhibitors of T-cell function. Objectives: To determine the effects of PD-1 pathway blockade on sarcoidosis CD4+ T-cell proliferative capacity. Methods: Gene expression profiles of sarcoidosis and healthy control peripheral blood mononuclear cells were analyzed at baseline and follow-up. Flow cytometry was used to measure ex vivo expression of PD-1 and PD-L1 on systemic and bronchoalveolar lavage–derived cells of subjects with sarcoidosis and control subjects, as well as the effects of PD-1 pathway blockade on cellular proliferation after T-cell receptor stimulation. Immunohistochemistry analysis for PD-1/PD-L1 expression was conducted on sarcoidosis, malignant, and healthy control lung specimens. Measurements and Main Results: Microarray analysis demonstrates longitudinal increase in PDCD1 gene expression in sarcoidosis peripheral blood mononuclear cells. Immunohistochemistry analysis revealed increased PD-L1 expression within sarcoidosis granulomas and lung malignancy, but this was absent in healthy lungs. Increased numbers of sarcoidosis PD-1+ CD4+ T cells are present systemically, compared with healthy control subjects (P < 0.0001). Lymphocytes with reduced proliferative capacity exhibited increased proliferation with PD-1 pathway blockade. Longitudinal analysis of subjects with sarcoidosis revealed reduced PD-1+ CD4+ T cells with spontaneous clinical resolution but not with disease progression. Conclusions: Analogous to the effects in other chronic lung diseases, these findings demonstrate that the PD-1 pathway is an important contributor to sarcoidosis CD4+ T-cell proliferative capacity and clinical outcome. Blockade of the PD-1 pathway may be a viable therapeutic target to optimize clinical outcomes. PMID:25073001

A Demonstration of Simultaneous Electrochemiluminescence

ERIC Educational Resources Information Center

Ibanez, Jorge G.; Zavala-Araiza, Daniel; Sotomayor-Martinez Barranco, Biaani; Torres-Perez, Jonatan; Camacho-Zuniga, Claudia; Bohrmann-Linde, Claudia; Tausch, Michael W.

2013-01-01

Paired (simultaneous) electrochemical processes can increase energy savings in selected cases by using the reactions at both electrodes of an electrochemical cell to perform a desired process, as is the case in the commercially successful chlor-alkali process. In the demonstration described herein, simultaneous blue electrochemiluminescence (ECL)…

Triggering of the TRPV2 channel by cannabidiol sensitizes glioblastoma cells to cytotoxic chemotherapeutic agents.

PubMed

Nabissi, Massimo; Morelli, Maria Beatrice; Santoni, Matteo; Santoni, Giorgio

2013-01-01

The aggressive behavior of Glioblastoma multiforme (GBM) is mainly due to high invasiveness and proliferation rate as well as to high resistance to standard chemotherapy. Several chemotherapeutic agents like temozolomide (TMZ), carmustine (BCNU) or doxorubicin (DOXO) have been employed for treatment of GBM, but they display limited efficacy. Therefore, it is important to identify new treatment modalities to improve therapeutic effects and enhance GBM chemosensitivity. Recently, activation of the transient receptor potential vanilloid type 2 (TRPV2) has been found to inhibit human GBM cell proliferation and overcome BCNU resistance of GBM cells. Herein, we evaluated the involvement of cannabidiol (CBD)-induced TRPV2 activation, in the modulation of glioma cell chemosensitivity to TMZ, BCNU and DOXO. We found that CBD increases TRPV2 expression and activity. CBD by triggering TRPV2-dependent Ca(2+) influx increases drug uptake and synergizes with cytotoxic agents to induce apoptosis of glioma cells, whereas no effects were observed in normal human astrocytes. Moreover, as the pore region of transient receptor potential (TRP) channels is critical for ion channel permeation, we demonstrated that deletion of TRPV2 poredomain inhibits CBD-induced Ca(2+) influx, drug uptake and cytotoxic effects. Overall, we demonstrated that co-administration of cytotoxic agents together with the TRPV2 agonist CBD increases drug uptake and parallelly potentiates cytotoxic activity in human glioma cells.

Promotion of mouse fibroblast collagen gene expression by mast cells stimulated via the Fc epsilon RI. Role for mast cell-derived transforming growth factor beta and tumor necrosis factor alpha

PubMed Central

1994-01-01

Chronic allergic diseases and other disorders associated with mast cell activation can also be associated with tissue fibrosis, but a direct link between mast cell mediator release and fibroblast collagen gene expression has not been established. Using in situ hybridization, we show that the elicitation of an IgE-dependent passive cutaneous anaphylaxis (PCA) reaction in mice results in a transient, but marked augmentation of steady state levels of type alpha-1 (I) collagen mRNA in the dermis. While peak levels of collagen mRNA expression in the skin are observed 16-24 h after mast cell activation, substantial numbers of dermal cells are strongly positive for collagen mRNA at 1 and 2 h after antigen challenge, before circulating inflammatory cells are recruited into the tissues. Furthermore, experiments in mast cell- reconstituted or genetically mast cell-deficient WBB6F1-W/Wv mice demonstrate that the increased expression of collagen mRNA at sites of PCA reactions is entirely mast cell dependent. In vitro studies show that the supernatants of mouse serosal mast cells activated via the Fc epsilon RI markedly increase type alpha-1 (I) collagen mRNA levels in mouse embryonic skin fibroblasts, and also upregulate collagen secretion by these cells. The ability of mast cell supernatants to induce increased steady state levels of collagen mRNA in mouse skin fibroblasts is markedly diminished by absorption with antibodies specific for either of two mast cell-derived cytokines, transforming growth factor beta (TGF-beta 1) or tumor necrosis factor alpha (TNF- alpha), and is eliminated entirely by absorption with antibodies against both cytokines. Taken together, these findings demonstrate that IgE-dependent mouse mast cell activation can induce a transient and marked increase in steady state levels of type alpha-1 (I) collagen mRNA in dermal fibroblasts and that mast cell-derived TGF-beta 1 and TNF-alpha importantly contribute to this effect. PMID:7964480

Enhancing effects of indirubin on the arsenic disulfide-induced apoptosis of human diffuse large B-cell lymphoma cells

PubMed Central

WANG, LING; LI, XIANGLU; LIU, XINYU; LU, KANG; CHEN, NA; LI, PEIPEI; LV, XIAO; WANG, XIN

2015-01-01

The aim of the present study was to investigate the indirubin-enhanced effects of arsenic disulfide (As2S2) on the proliferation and apoptosis of diffuse large B-cell lymphoma (DLBCL) cells in order to identify an optimum combination therapy. The human DLBCL cells, LY1 and LY8, were treated with different concentrations of indirubin for 24, 48 and 72 h. Next, the cells were treated with 10 μM As2S2 or a combination of 10 μM As2S2 and 20 μM indirubin for 48 h. Cell proliferation inhibition was detected using cell counting kit-8 and cell apoptosis was determined using flow cytometry. The expression levels of Bcl-2, Bcl-2-associated X protein (Bax) and caspase-3 were analyzed by quantitative polymerase chain reaction (qPCR) and western blotting. The DLBCL cell viability exhibited no significant changes at 24, 48 or 72 h with increasing indirubin concentration. In addition, the apoptotic rates of the LY1 and LY8 cells demonstrated no noticeable effects at 48 h with increasing indirubin concentration. Following treatment with the combination of indirubin and As2S2, the inhibitory and apoptotic rates of the cells were notably increased compared with those of the As2S2-treated group. The qPCR results revealed that indirubin alone had no enhancing effect upon the Bax/Bcl-2 mRNA expression ratio and caspase-3 mRNA expression. Western blot analysis revealed that indirubin alone had an enhancing effect upon the Bax/Bcl-2 protein ratio and procaspase-3 protein expression. In addition, the results demonstrated that the 21-KDa Bax protein was proteolytically cleaved into an 18-KDa Bax in the DLBCL cells treated with the combination of indirubin and As2S2. Indirubin alone did not inhibit proliferation or induce the apoptosis of the LY1 and LY8 cells. However, the combination of indirubin and As2S2 yielded enhancing effects. Therefore, the results of the present study demonstrated that with regard to antitumor activities, As2S2 served as the principal drug, whereas indirubin served as the adjuvant drug. The enhancing effect was due, in part, to the induction of the mitochondrial apoptotic pathway, which involves the cleavage of Bax. PMID:25789073

Use of a Novel Cell Adhesion Method and Digital Measurement to Show Stimulus-dependent Variation in Somatic and Oral Ciliary Beat Frequency in Paramecium

PubMed Central

Bell, Wade E.; Hallworth, Richard; Wyatt, Todd A.; Sisson, Joseph H.

2015-01-01

When Paramecium encounters positive stimuli, the membrane hyperpolarizes and ciliary beat frequency increases. We adapted an established immobilization protocol using a biological adhesive and a novel digital analysis system to quantify beat frequency in immobilized Paramecium. Cells showed low mortality and demonstrated beat frequencies consistent with previous studies. Chemoattractant molecules, reduction in external potassium, and posterior stimulation all increased somatic beat frequency. In all cases, the oral groove cilia maintained a higher beat frequency than mid-body cilia, but only oral cilia from cells stimulated with chemoattactants showed an increase from basal levels. PMID:25066640

Characterization of Cell Wall Components and Their Modifications during Postharvest Storage of Asparagus officinalis L.: Storage-Related Changes in Dietary Fiber Composition.

PubMed

Schäfer, Judith; Wagner, Steffen; Trierweiler, Bernhard; Bunzel, Mirko

2016-01-20

Changes in cell wall composition during storage of plant foods potentially alter the physiological effects of dietary fiber components. To investigate postharvest cell wall modifications of asparagus and their consequences in terms of insoluble dietary fiber structures, asparagus was stored at 20 and 1 °C for different periods of time. Structural analyses demonstrated postharvest changes in the polysaccharide profile, dominated by decreased portions of galactans. Increasing lignin contents correlated with compositional changes (monolignol ratios and linkage types) of the lignin polymer as demonstrated by chemical and two-dimensional nuclear magnetic resonance (2D-NMR) methods. Depending on the storage time and temperature, syringyl units were preferentially incorporated into the lignin polymer. Furthermore, a drastic increase in the level of ester-linked phenolic monomers (i.e., p-coumaric acid and ferulic acid) and polymer cross-links (di- and triferulic acids) was detected. The attachment of p-coumaric acid to lignin was demonstrated by 2D-NMR experiments. Potential consequences of postharvest modifications on physiological effects of asparagus dietary fiber are discussed.

Beta-cell function, incretin effect, and incretin hormones in obese youth along the span of glucose tolerance from normal to prediabetes to Type 2 diabetes

USDA-ARS?s Scientific Manuscript database

Using the hyperglycemic and euglycemic clamp, we demonstrated impaired Beta-cell function in obese youth with increasing dysglycemia. Herein we describe oral glucose tolerance test (OGTT)-modeled Beta-cell function and incretin effect in obese adolescents spanning the range of glucose tolerance. Bet...

Role of polyamines in DNA synthesis of Catharanthus roseus cells grown in suspension culture

Treesearch

Rakesh Minocha; Subhash C. Minocha; Atsushi Komamine; Walter C. Shortle

1990-01-01

The requirement for polyamines in the proliferation of cells was first demonstrated in bacteria (3). While significant progress has been made in this field using animal cell cultures, only preliminary studies have been reported with plant tissues. Serafini-Fracassini et al. (9) showed a marked increase in polyamine synthesis early during the G 1 phase, concomitant with...

Withaferin A induces heme oxygenase (HO-1) expression in endothelial cells via activation of the Keap1/Nrf2 pathway.

PubMed

Heyninck, Karen; Sabbe, Linde; Chirumamilla, Chandra Sekhar; Szarc Vel Szic, Katarzyna; Vander Veken, Pieter; Lemmens, Kristien J A; Lahtela-Kakkonen, Maija; Naulaerts, Stefan; Op de Beeck, Ken; Laukens, Kris; Van Camp, Guy; Weseler, Antje R; Bast, Aalt; Haenen, Guido R M M; Haegeman, Guy; Vanden Berghe, Wim

2016-06-01

Withaferin A (WA), a natural phytochemical derived from the plant Withania somnifera, is a well-studied bioactive compound exerting a broad spectrum of health promoting effects. To gain better insight in the potential therapeutic capacity of WA, we evaluated the transcriptional effects of WA on primary human umbilical vein endothelial cells (HUVECs) and an endothelial cell line (EA.hy926). RNA microarray analysis of WA treated HUVEC cells demonstrated increased expression of the antioxidant gene heme oxygenase (HO-1). Transcriptional regulation of this gene is strongly dependent on the transcription factor NF-E2-related factor 2 (Nrf2), which senses chemical changes in the cell and coordinates transcriptional responses to maintain chemical homeostasis via expression of antioxidant genes and cytoprotective Phase II detoxifying enzymes. Under normal conditions, Nrf2 is kept in the cytoplasm by Kelch-like ECH-associated protein 1 (Keap1), an adaptor protein controlling the half-life of Nrf2 via constant proteasomal degradation. In this study we demonstrate that WA time- and concentration-dependently induces HO-1 expression in endothelial cells via upregulation and increased nuclear translocation of Nrf2. According to the crucial negative regulatory role of Keap1 in Nrf2 expression levels, a direct interaction of WA with Keap1 could be demonstrated. In vitro and in silico evaluations suggest that specific cysteine residues in Keap1 might be involved in the interaction with WA. Copyright © 2016 Elsevier Inc. All rights reserved.

Proliferative effects of apical, but not basal, matrix metalloproteinase-7 activity in polarized MDCK cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Harrell, Permila C.; McCawley, Lisa J.; Fingleton, Barbara

2005-02-15

Matrix metalloproteinase-7 (MMP-7) is primarily expressed in glandular epithelium. Therefore, its mechanism of action may be influenced by its regulated vectorial release to either the apical and/or basolateral compartments, where it would act on its various substrates. To gain a better understanding of where MMP-7 is released in polarized epithelium, we have analyzed its pattern of secretion in polarized MDCK cells expressing stably transfected human MMP-7 (MDCK-MMP-7), and HCA-7 and Caco2 human colon cancer cell lines. In all cell lines, latent MMP-7 was secreted to both cellular compartments, but was 1.5- to 3-fold more abundant in the basolateral compartment asmore » compared to the apical. However, studies in the MDCK system demonstrated that MMP-7 activity was 2-fold greater in the apical compartment of MDCK-MMP-7{sup HIGH}-polarized monolayers, which suggests the apical co-release of an MMP-7 activator. In functional assays, MMP-7 over-expression increased cell saturation density as a result of increased cell proliferation with no effect on apoptosis. Apical MMP-7 activity was shown to be responsible for the proliferative effect, which occurred, as demonstrated by media transfer experiments, through cleavage of an apical substrate and not through the generation of a soluble factor. Taken together, our findings demonstrate the importance of MMP-7 secretion in relation to its mechanism of action when expressed in a polarized epithelium.« less

MRG15, a component of HAT and HDAC complexes, is essential for proliferation and differentiation of neural precursor cells.

PubMed

Chen, Meizhen; Takano-Maruyama, Masumi; Pereira-Smith, Olivia M; Gaufo, Gary O; Tominaga, Kaoru

2009-05-15

Neurogenesis during development depends on the coordinated regulation of self-renewal and differentiation of neural precursor cells (NPCs). Chromatin regulation is a key step in self-renewal activity and fate decision of NPCs. However, the molecular mechanism or mechanisms of this regulation is not fully understood. Here, we demonstrate for the first time that MRG15, a chromatin regulator, is important for proliferation and neural fate decision of NPCs. Neuroepithelia from Mrg15-deficient embryonic brain are much thinner than those from control, and apoptotic cells increase in this region. We isolated NPCs from Mrg15-deficient and wild-type embryonic whole brains and produced neurospheres to measure the self-renewal and differentiation abilities of these cells in vitro. Neurospheres culture from Mrg15-deficient embryo grew less efficiently than those from wild type. Measurement of proliferation by means of BrdU (bromodeoxyuridine) incorporation revealed that Mrg15-deficient NPCs have reduced proliferation ability and apoptotic cells do not increase during in vitro culture. The reduced proliferation of Mrg15-deficient NPCs most likely accounts for the thinner neuroepithelia in Mrg15-deficient embryonic brain. Moreover, we also demonstrate Mrg15-deficient NPCs are defective in differentiation into neurons in vitro. Our results demonstrate that MRG15 has more than one function in neurogenesis and defines a novel role for this chromatin regulator that integrates proliferation and cell-fate determination in neurogenesis during development. Copyright 2008 Wiley-Liss, Inc.

Rapamycin promotes differentiation increasing βIII-tubulin, NeuN, and NeuroD while suppressing nestin expression in glioblastoma cells

PubMed Central

Lenzi, Paola; Gambardella, Stefano; Ferese, Rosangela; Calierno, Maria Teresa; Falleni, Alessandra; Grimaldi, Alfonso; Frati, Alessandro; Esposito, Vincenzo; Limatola, Cristina; Fornai, Francesco

2017-01-01

Glioblastoma cells feature mammalian target of rapamycin (mTOR) up-regulation which relates to a variety of effects such as: lower survival, higher infiltration, high stemness and radio- and chemo-resistance. Recently, it was demonstrated that mTOR may produce a gene shift leading to altered protein expression. Therefore, in the present study we administered different doses of the mTOR inhibitor rapamycin to explore whether the transcription of specific genes are modified. By using a variety of methods we demonstrate that rapamycin stimulates gene transcription related to neuronal differentiation while inhibiting stemness related genes such as nestin. In these experimental conditions, cell phenotype shifts towards a pyramidal neuron-like shape owing long branches. Rapamycin suppressed cell migration when exposed to fetal bovine serum (FBS) while increasing the cell adhesion protein phospho-FAK (pFAK). The present study improves our awareness of basic mechanisms which relate mTOR activity to the biology of glioblastoma cells. These findings apply to a variety of effects which can be induced by mTOR regulation in the brain. In fact, the ability to promote neuronal differentiation might be viewed as a novel therapeutic pathway to approach neuronal regeneration. PMID:28418837

Rapamycin promotes differentiation increasing βIII-tubulin, NeuN, and NeuroD while suppressing nestin expression in glioblastoma cells.

PubMed

Ferrucci, Michela; Biagioni, Francesca; Lenzi, Paola; Gambardella, Stefano; Ferese, Rosangela; Calierno, Maria Teresa; Falleni, Alessandra; Grimaldi, Alfonso; Frati, Alessandro; Esposito, Vincenzo; Limatola, Cristina; Fornai, Francesco

2017-05-02

Glioblastoma cells feature mammalian target of rapamycin (mTOR) up-regulation which relates to a variety of effects such as: lower survival, higher infiltration, high stemness and radio- and chemo-resistance. Recently, it was demonstrated that mTOR may produce a gene shift leading to altered protein expression. Therefore, in the present study we administered different doses of the mTOR inhibitor rapamycin to explore whether the transcription of specific genes are modified. By using a variety of methods we demonstrate that rapamycin stimulates gene transcription related to neuronal differentiation while inhibiting stemness related genes such as nestin. In these experimental conditions, cell phenotype shifts towards a pyramidal neuron-like shape owing long branches. Rapamycin suppressed cell migration when exposed to fetal bovine serum (FBS) while increasing the cell adhesion protein phospho-FAK (pFAK). The present study improves our awareness of basic mechanisms which relate mTOR activity to the biology of glioblastoma cells. These findings apply to a variety of effects which can be induced by mTOR regulation in the brain. In fact, the ability to promote neuronal differentiation might be viewed as a novel therapeutic pathway to approach neuronal regeneration.

Histone deacetylase inhibitor valproic acid promotes the induction of pluripotency in mouse fibroblasts by suppressing reprogramming-induced senescence stress

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhai, Yingying; Chen, Xi; Yu, Dehai

2015-09-10

Histone deacetylase inhibitor valproic acid (VPA) has been used to increase the reprogramming efficiency of induced pluripotent stem cell (iPSC) from somatic cells, yet the specific molecular mechanisms underlying this effect is unknown. Here, we demonstrate that reprogramming with lentiviruses carrying the iPSC-inducing factors (Oct4-Sox2-Klf4-cMyc, OSKM) caused senescence in mouse fibroblasts, establishing a stress barrier for cell reprogramming. Administration of VPA protected cells from reprogramming-induced senescent stress. Using an in vitro pre-mature senescence model, we found that VPA treatment increased cell proliferation and inhibited apoptosis through the suppression of the p16/p21 pathway. In addition, VPA also inhibited the G2/M phasemore » blockage derived from the senescence stress. These findings highlight the role of VPA in breaking the cell senescence barrier required for the induction of pluripotency. - Highlights: • Histone deacetylase inhibitor valproic acid enhances iPSC induction. • Valproic acid suppresses reprogramming-induced senescence stress. • Valproic acid downregulates the p16/p21 pathway in reprogramming. • This study demonstrates a new mechanistic role of valproic acid in enhancing reprogramming.« less

Biological significance of PinX1 telomerase inhibitor in esophageal carcinoma treatment

PubMed Central

Fan, Xiang-Kui; Yan, Rui-Hua; Geng, Xiang-Qun; Li, Jing-Shan; Chen, Xiang-Ming; Li, Jian-Zhe

2016-01-01

In the present study, to investigate the expression of PinX1 gene and its functional effects in human esophageal carcinoma (Eca)-109 cell line, expression vectors of human PinX1 (pEGFP-C3-PinX1) and its small interfering RNA (PinX1-FAM-siRNA) were constructed and transfected into Eca-109 cells using Lipofectamine 2000. Firstly, the mRNA expression level of PinX1 was examined using reverse transcription-polymerase chain reaction (RT-PCR). Once successful transfection was achieved, the effects on the mRNA level of human telomerase reverse transcriptase (hTERT), telomerase activity, cell proliferation and apoptosis were examined by semi-quantitative RT-PCR, stretch PCR, MTT assay and flow cytometry, respectively. Analysis of restriction and sequencing demonstrated that the recombining plasmids were successfully constructed. The results also indicated that transfection with pEGFP-C3-PinX1 and PinX1-FAM-siRNA into Eca-109 cells significantly increased PinX1 mRNA, decreased hTERT mRNA by 29.9% (P<0.05), and significantly reduced telomerase activity (P<0.05), inhibited cell growth, and increased the cell apoptotic index from 19.27±0.76 to 49.73±2%. The transfected PinX1-FAM-SiRNA exhibited PinX1 mRNA expression levels that were significantly decreased by 70% (P<0.05), whereas the remaining characteristics of Eca-109 cells, including cell growth, mRNA level of hTERT, telomerase activity and cell apoptotic index were not altered. Exogenous PinX1 has been demonstrated to be highly expressed in human Eca. PinX1 can inhibit human telomerase activity and the expression of hTERT mRNA, reduce tumor cell growth and induce apoptosis. Notably, these inhibitory functions were inhibited by silencing PinX1 in Eca with PinX1-FAM-siRNA. PinX1 was successfully increased and decreased in the present study, demonstrating that it may be a potential telomerase activity inhibitor. As PinX1 is an endogenous telomerase inhibitor, it may be used as a novel tumor-targeted gene therapy. PMID:27698711

Analysis of epothilone B-induced cell death in normal ovarian cells.

PubMed

Rogalska, Aneta; Gajek, Arkadiusz; Marczak, Agnieszka

2013-12-01

We have investigated the mode of cell death induced by a new microtubule-stabilizing agent, epothilone B (EpoB, patupilone), and a clinically used medicine, paclitaxel (PTX), in normal ovarian cells. Using fluorescence microscopy, polyacrylamide gel electrophoresis preceding Western blot analysis, as well as spectrofluorimetric and colorimetric detection, we demonstrate that, compared to EpoB, PTX induced high time-dependent morphological and biochemical changes typical of apoptosis. Induction of apoptosis followed an early increase in p53 levels. Apoptosis reached its maximum at 24-48 h. At the same time, there was a significant increase in caspase-9 and -3 activity and PARP fragmentation, which suggests that an intrinsic path was involved. Apoptosis in MM14 cells was increased more by PTX than EpoB, and also induced more necrosis responsible for inflammation (1.4-fold) than EpoB. © 2013 International Federation for Cell Biology.

High-yield cell-free synthesis of human EGFR by IRES-mediated protein translation in a continuous exchange cell-free reaction format

PubMed Central

Quast, Robert B.; Sonnabend, Andrei; Stech, Marlitt; Wüstenhagen, Doreen A.; Kubick, Stefan

2016-01-01

Cell-free protein synthesis systems derived from eukaryotic sources often provide comparatively low amounts of several μg per ml of de novo synthesized membrane protein. In order to overcome this, we herein demonstrate the high-yield cell-free synthesis of the human EGFR in a microsome-containing system derived from cultured Sf21 cells. Yields were increased more than 100-fold to more than 285 μg/ml by combination of IRES-mediated protein translation with a continuous exchange cell-free reaction format that allowed for prolonged reaction lifetimes exceeding 24 hours. In addition, an orthogonal cell-free translation system is presented that enabled the site-directed incorporation of p-Azido-L-phenylalanine by amber suppression. Functionality of cell-free synthesized receptor molecules is demonstrated by investigation of autophosphorylation activity in the absence of ligand and interaction with the cell-free synthesized adapter molecule Grb2. PMID:27456041

Increased viability of fibroblasts when pretreated with ceria nanoparticles during serum deprivation.

PubMed

Genier, Francielli S; Bizanek, Maximilian; Webster, Thomas J; Roy, Amit K

2018-01-01

Conditions of cellular stress are often the cause of cell death or dysfunction. Sustained cell stress can lead to several health complications, such as extensive inflammatory responses, tumor growth, and necrosis. To prevent disease and protect human tissue during these conditions and to avoid medication side effects, nanomaterials with unique characteristics have been applied to biological systems. This paper introduces the pretreatment in human dermal fibroblasts with cerium oxide nanoparticles during nutritional stress. For this purpose, human dermal fibroblast cells received cell culture media with concentrations of 250 µg/mL and 500 µg/mL of nano-cerium oxide before being exposed to 24, 48, and 72 hours of serum starvation. Contrast images demonstrated higher cell confluence and cell integrity in cells pretreated with ceria nanoparticles compared to untreated cells. It was confirmed by MTS assay after 72 hours of serum starvation that higher cell viability was achieved with ceria nanoparticles. The results demonstrate the potential of cerium oxide nanoparticles as protective agents during cellular starvation.

SOCS3: an essential regulator of LIF receptor signaling in trophoblast giant cell differentiation

PubMed Central

Takahashi, Yutaka; Carpino, Nick; Cross, James C.; Torres, Miguel; Parganas, Evan; Ihle, James N.

2003-01-01

Suppressor of cytokine signaling 3 (SOCS3) binds cytokine receptors and thereby suppresses cytokine signaling. Deletion of SOCS3 causes an embryonic lethality that is rescued by a tetraploid rescue approach, demonstrating an essential role in placental development and a non-essential role in embryo development. Rescued SOCS3-deficient mice show a perinatal lethality with cardiac hypertrophy. SOCS3-deficient placentas have reduced spongiotrophoblasts and increased trophoblast secondary giant cells. Enforced expression of SOCS3 in a trophoblast stem cell line (Rcho-1) suppresses giant cell differentiation. Conversely, SOCS3-deficient trophoblast stem cells differentiate more readily to giant cells in culture, demonstrating that SOCS3 negatively regulates trophoblast giant cell differentiation. Leukemia inhibitory factor (LIF) promotes giant cell differentiation in vitro, and LIF receptor (LIFR) deficiency results in loss of giant cell differentiation in vivo. Finally, LIFR deficiency rescues the SOCS3-deficient placental defect and embryonic lethality. The results establish SOCS3 as an essential regulator of LIFR signaling in trophoblast differentiation. PMID:12554639

Epstein-Barr virus-associated peripheral T-Cell lymphoma involving spleen in a renal transplant patient.

PubMed Central

Lee, Hye Kyung; Kim, Hee Jung; Lee, Eun Hee; Kim, Suk Young; Park, Tae In; Kang, Chang Suk; Yang, Woo Ick

2003-01-01

The incidence of posttransplantation lymphoproliferative disorders (PTLDs) has increased in recent years. Although rare, various types of T-cell lymphoma have been reported and their association with Epstein-Barr virus (EBV) has been compared with B-cell PTLDs. We report a case of splenic peripheral T-cell lymphoma occurring in a 47-yr-old male patient 7 yr after renal allograft transplantation. The spleen showed sinusoidal proliferation of focal CD30 positive, large, atypical lymphoid cells. Positivity for CD3 and cytolytic granule-associated proteins was also demonstrated in the tumor cells, while anaplastic large cell lymphoma kinase (ALK) and CD8 were not expressed. Strong nuclear signals for EBV mRNA were noted by EBER1 in situ hybridization. A molecular genetic study demonstrated a rearrangement of the gamma T-cell receptor gene. To our knowledge, this case is unique in terms of a posttransplant T-cell lymphoma that shows focal CD30, cytolytic granule-associated proteins, and EBV positivity. PMID:12692428

Additive effect of red blood cell rigidity and adherence to endothelial cells in inducing vascular resistance.

PubMed

Kaul, D K; Koshkaryev, A; Artmann, G; Barshtein, G; Yedgar, S

2008-10-01

To explore the contribution of red blood cell (RBC) deformability and interaction with endothelial cells (ECs) to circulatory disorders, these RBC properties were modified by treatment with hydrogen peroxide (H(2)O(2)), and their effects on vascular resistance were monitored following their infusion into rat mesocecum vasculature. Treatment with 0.5 mM H(2)O(2) increased RBC/EC adherence without significant alteration of RBC deformability. At 5.0 mM H(2)O(2), RBC deformability was considerably reduced, inducing a threefold increase in the number of undeformable cells, whereas RBC/EC adherence was not further affected by the increased H(2)O(2) concentration. This enabled the selective manipulation of RBC adherence and deformability and the testing of their differential effect on vascular resistance. Perfusion of RBCs with enhanced adherence and unchanged deformability (treatment with 0.5 mM H(2)O(2)) increased vascular resistance by about 35% compared with untreated control RBCs. Perfusion of 5.0 mM H(2)O(2)-treated RBCs, with reduced deformability (without additional increase of adherence), further increased vascular resistance by about 60% compared with untreated control RBCs. These results demonstrate the specific effects of elevated adherence and reduced deformability of oxidized RBCs on vascular resistance. These effects can be additive, depending on the oxidation conditions. The oxidation-induced changes applied in this study are moderate compared with those observed in RBCs in pathological states. Yet, they caused a considerable increase in vascular resistance, thus demonstrating the potency of RBC/EC adherence and RBC deformability in determining resistance to blood flow in vivo.

Correlated parameter fit of arrhenius model for thermal denaturation of proteins and cells.

PubMed

Qin, Zhenpeng; Balasubramanian, Saravana Kumar; Wolkers, Willem F; Pearce, John A; Bischof, John C

2014-12-01

Thermal denaturation of proteins is critical to cell injury, food science and other biomaterial processing. For example protein denaturation correlates strongly with cell death by heating, and is increasingly of interest in focal thermal therapies of cancer and other diseases at temperatures which often exceed 50 °C. The Arrhenius model is a simple yet widely used model for both protein denaturation and cell injury. To establish the utility of the Arrhenius model for protein denaturation at 50 °C and above its sensitivities to the kinetic parameters (activation energy E a and frequency factor A) were carefully examined. We propose a simplified correlated parameter fit to the Arrhenius model by treating E a, as an independent fitting parameter and allowing A to follow dependently. The utility of the correlated parameter fit is demonstrated on thermal denaturation of proteins and cells from the literature as a validation, and new experimental measurements in our lab using FTIR spectroscopy to demonstrate broad applicability of this method. Finally, we demonstrate that the end-temperature within which the denaturation is measured is important and changes the kinetics. Specifically, higher E a and A parameters were found at low end-temperature (50 °C) and reduce as end-temperatures increase to 70 °C. This trend is consistent with Arrhenius parameters for cell injury in the literature that are significantly higher for clonogenics (45-50 °C) vs. membrane dye assays (60-70 °C). Future opportunities to monitor cell injury by spectroscopic measurement of protein denaturation are discussed.

Correlated Parameter Fit of Arrhenius Model for Thermal Denaturation of Proteins and Cells

PubMed Central

Qin, Zhenpeng; Balasubramanian, Saravana Kumar; Wolkers, Willem F.; Pearce, John A.; Bischof, John C.

2014-01-01

Thermal denaturation of proteins is critical to cell injury, food science and other biomaterial processing. For example protein denaturation correlates strongly with cell death by heating, and is increasingly of interest in focal thermal therapies of cancer and other diseases at temperatures which often exceed 50 °C. The Arrhenius model is a simple yet widely used model for both protein denaturation and cell injury. To establish the utility of the Arrhenius model for protein denaturation at 50 °C and above its sensitivities to the kinetic parameters (activation energy Ea and frequency factor A) were carefully examined. We propose a simplified correlated parameter fit to the Arrhenius model by treating Ea, as an independent fitting parameter and allowing A to follow dependently. The utility of the correlated parameter fit is demonstrated on thermal denaturation of proteins and cells from the literature as a validation, and new experimental measurements in our lab using FTIR spectroscopy to demonstrate broad applicability of this method. Finally, we demonstrate that the end-temperature within which the denaturation is measured is important and changes the kinetics. Specifically, higher Ea and A parameters were found at low end-temperature (50°C) and reduce as end-temperatures increase to 70 °C. This trend is consistent with Arrhenius parameters for cell injury in the literature that are significantly higher for clonogenics (45 – 50 °C) vs. membrane dye assays (60 –70 °C). Future opportunities to monitor cell injury by spectroscopic measurement of protein denaturation are discussed. PMID:25205396

Transcriptional and Cell Cycle Alterations Mark Aging of Primary Human Adipose-Derived Stem Cells.

PubMed

Shan, Xiaoyin; Roberts, Cleresa; Kim, Eun Ji; Brenner, Ariana; Grant, Gregory; Percec, Ivona

2017-05-01

Adult stem cells play a critical role in the maintenance of tissue homeostasis and prevention of aging. While the regenerative potential of stem cells with low cellular turnover, such as adipose-derived stem cells (ASCs), is increasingly recognized, the study of chronological aging in ASCs is technically difficult and remains poorly understood. Here, we use our model of chronological aging in primary human ASCs to examine genome-wide transcriptional networks. We demonstrate first that the transcriptome of aging ASCs is distinctly more stable than that of age-matched fibroblasts, and further, that age-dependent modifications in cell cycle progression and translation initiation specifically characterize aging ASCs in conjunction with increased nascent protein synthesis and a distinctly shortened G1 phase. Our results reveal novel chronological aging mechanisms in ASCs that are inherently different from differentiated cells and that may reflect an organismal attempt to meet the increased demands of tissue and organ homeostasis during aging. Stem Cells 2017;35:1392-1401. © 2017 AlphaMed Press.

The NLRP3 Inflammasome Promotes Age-related Thymic Demise and Immunosenescence

PubMed Central

Youm, Yun-Hee; Kanneganti, Thirumala-Devi; Vandanmagsar, Bolormaa; Zhu, Xuewei; Ravussin, Anthony; Adijiang, Ayinuer; Owen, John S.; Thomas, Michael J.; Francis, Joseph; Parks, John S.; Dixit, Vishwa Deep

2013-01-01

The collapse of thymic stromal cell microenvironment with age and resultant inability of the thymus to produce naïve T cells contributes to lower immune-surveillance in the elderly. Here we show that age-related increase in ‘lipotoxic danger signals’ such as free cholesterol (FC) and ceramides, leads to thymic caspase-1 activation via the Nlrp3 inflammasome. Elimination of Nlrp3 and Asc, a critical adaptor required for inflammasome assembly, reduces age-related thymic atrophy and results in an increase in cortical thymic epithelial cells, T cell progenitors and maintenance of T cell repertoire diversity. Using a mouse model of irradiation and hematopoietic stem cell transplantation (HSCT), we show that deletion of the Nlrp3 inflammasome accelerates T cell reconstitution and immune recovery in middle-aged animals. Collectively, these data demonstrate that lowering inflammasome-dependent caspase-1 activation increases thymic lymphopoiesis and suggest that Nlrp3 inflammasome inhibitors may aid the reestablishment of a diverse T cell repertoire in middle-aged or elderly patients undergoing HSCT. PMID:22832107

Mast cells boost myeloid-derived suppressor cell activity and contribute to the development of tumor-favoring microenvironment.

PubMed

Danelli, Luca; Frossi, Barbara; Gri, Giorgia; Mion, Francesca; Guarnotta, Carla; Bongiovanni, Lucia; Tripodo, Claudio; Mariuzzi, Laura; Marzinotto, Stefania; Rigoni, Alice; Blank, Ulrich; Colombo, Mario P; Pucillo, Carlo E

2015-01-01

Inflammation plays crucial roles at different stages of tumor development and may lead to the failure of immune surveillance and immunotherapy. Myeloid-derived suppressor cells (MDSC) are one of the major components of the immune-suppressive network that favors tumor growth, and their interaction with mast cells is emerging as critical for the outcome of the tumor-associated immune response. Herein, we showed the occurrence of cell-to-cell interactions between MDSCs and mast cells in the mucosa of patients with colon carcinoma and in the colon and spleen of tumor-bearing mice. Furthermore, we demonstrated that the CT-26 colon cancer cells induced the accumulation of CD11b(+)Gr1(+) immature MDSCs and the recruitment of protumoral mast cells at the tumor site. Using ex vivo analyses, we showed that mast cells have the ability to increase the suppressive properties of spleen-derived monocytic MDSCs, through a mechanism involving IFNγ and nitric oxide production. In addition, we demonstrated that the CD40:CD40L cross-talk between the two cell populations is responsible for the instauration of a proinflammatory microenvironment and for the increase in the production of mediators that can further support MDSC mobilization and tumor growth. In light of these results, interfering with the MDSC:mast cell axis could be a promising approach to abrogate MDSC-related immune suppression and to improve the antitumor immune response. ©2014 American Association for Cancer Research.

Erythrocyte Enrichment in Hematopoietic Progenitor Cell Cultures Based on Magnetic Susceptibility of the Hemoglobin

PubMed Central

Jin, Xiaoxia; Abbot, Stewart; Zhang, Xiaokui; Kang, Lin; Voskinarian-Berse, Vanessa; Zhao, Rui; Kameneva, Marina V.; Moore, Lee R.; Chalmers, Jeffrey J.; Zborowski, Maciej

2012-01-01

Using novel media formulations, it has been demonstrated that human placenta and umbilical cord blood-derived CD34+ cells can be expanded and differentiated into erythroid cells with high efficiency. However, obtaining mature and functional erythrocytes from the immature cell cultures with high purity and in an efficient manner remains a significant challenge. A distinguishing feature of a reticulocyte and maturing erythrocyte is the increasing concentration of hemoglobin and decreasing cell volume that results in increased cell magnetophoretic mobility (MM) when exposed to high magnetic fields and gradients, under anoxic conditions. Taking advantage of these initial observations, we studied a noninvasive (label-free) magnetic separation and analysis process to enrich and identify cultured functional erythrocytes. In addition to the magnetic cell separation and cell motion analysis in the magnetic field, the cell cultures were characterized for cell sedimentation rate, cell volume distributions using differential interference microscopy, immunophenotyping (glycophorin A), hemoglobin concentration and shear-induced deformability (elongation index, EI, by ektacytometry) to test for mature erythrocyte attributes. A commercial, packed column high-gradient magnetic separator (HGMS) was used for magnetic separation. The magnetically enriched fraction comprised 80% of the maturing cells (predominantly reticulocytes) that showed near 70% overlap of EI with the reference cord blood-derived RBC and over 50% overlap with the adult donor RBCs. The results demonstrate feasibility of label-free magnetic enrichment of erythrocyte fraction of CD34+ progenitor-derived cultures based on the presence of paramagnetic hemoglobin in the maturing erythrocytes. PMID:22952572

Anticancer activity of 7-epiclusianone, a benzophenone from Garcinia brasiliensis, in glioblastoma.

PubMed

Sales, Leilane; Pezuk, Julia Alejandra; Borges, Kleiton Silva; Brassesco, María Sol; Scrideli, Carlos Alberto; Tone, Luiz Gonzaga; dos Santos, Marcelo Henrique; Ionta, Marisa; de Oliveira, Jaqueline Carvalho

2015-10-30

Glioblastoma is the most common tumor of the central nervous system and one of the hardest tumors to treat. Consequently, the search for novel therapeutic options is imperative. 7-epiclusianone, a tetraprenylated benzophenone isolated from the epicarp of the native plant Garcinia brasiliensis, exhibits a range of biological activities but its prospect anticancer activity is underexplored. Thus, the aim of the present study was to evaluate the influence of 7-epiclusianone on proliferation, clonogenic capacity, cell cycle progression and induction of apoptosis in two glioblastoma cell lines (U251MG and U138MG). Cell viability was measured by the MTS assay; for the clonogenic assay, colonies were stained with Giemsa and counted by direct visual inspection; For cell cycle analysis, cells were stained with propidium iodide and analyzed by cytometry; Cyclin A expression was determined by immunoblotting; Apoptotic cell death was determined by annexin V fluorescein isothiocyanate labeling and Caspase-3 activity in living cells. Viability of both cell lines was drastically inhibited; moreover, the colony formation capacity was significantly reduced, demonstrating long-term effects even after removal of the drug. 7-epiclusianone treatment at low concentrations also altered cell cycle progression, decreased the S and G2/M populations and at higher concentrations increased the number of cells at sub-G1, in concordance with the increase of apoptotic cells. The present study demonstrates for the first time the anticancer potential of 7-epiclusianone against glioblastoma cells, thus meriting its further investigation as a potential therapeutic agent.

Antibiotic tigecycline enhances cisplatin activity against human hepatocellular carcinoma through inducing mitochondrial dysfunction and oxidative damage

DOE Office of Scientific and Technical Information (OSTI.GOV)

Tan, Jun; Song, Meijun; Zhou, Mi

Targeting mitochondrial metabolism has been recently demonstrated to be a promising therapeutic strategy for the treatment of various cancer. In this work, we demonstrate that antibiotic tigecycline is selectively against hepatocellular carcinoma (HCC) through inducing mitochondrial dysfunction and oxidative damage. Tigecycline is more effective in inhibiting proliferation and inducing apoptosis of HCC than normal liver cells. Importantly, tigecycline significantly enhances the inhibitory effects of chemotherapeutic drug cisplatin in HCC in vitro and in vivo. Mechanistically, tigecycline specifically inhibits mitochondrial translation as shown by the decreased protein levels of Cox-1 and -2 but not Cox-4 or Grp78, and increased mRNA levels of Cox-1more » and -2 but not Cox-4 in HCC cells exposed to tigecycline. In addition, tigecycline significantly induces mitochondrial dysfunction in HCC cells via decreasing mitochondrial membrane potential, complex I and IV activities, mitochondrial respiration and ATP levels. Tigecycline also increases levels of mitochondrial superoxide, hydrogen peroxide and ROS levels. Consistent with oxidative stress, oxidative damage on DNA, protein and lipid are also observed in tigecycline-treated cells. Importantly, antioxidant N-acetyl-L-cysteine (NAC) reverses the effects of tigecycline, suggesting that oxidative stress is required for the action of tigecycline in HCC cells. We further show that HCC cells have higher level of mitochondrial biogenesis than normal liver cells which might explain the different sensitivity to tigecycline between HCC and normal liver cells. Our work is the first to demonstrate that tigecycline is a promising candidate for HCC treatment and highlight the therapeutic value of targeting mitochondrial metabolism in HCC. - Highlights: • Tigecycline selectively targets HCC in vitro and in vivo. • Tigecycline enhances HCC cell response to chemotherapeutic drug. • Tigecycline inhibits mitochondrial translation and functions in HCC cells. • Tigecycline induces oxidative stress and damage in HCC cells. • Mitochondrial biogenesis and respiration is higher in HCC than normal liver cells.« less

Characterization of a Distinct Population of Circulating Human Non-Adherent Endothelial Forming Cells and Their Recruitment via Intercellular Adhesion Molecule-3

PubMed Central

Thompson, Emma J.; Barrett, Jeffrey M.; Tooley, Katie; Sen, Shaundeep; Sun, Wai Yan; Grose, Randall; Nicholson, Ian; Levina, Vitalina; Cooke, Ira; Talbo, Gert; Lopez, Angel F.; Bonder, Claudine S.

2012-01-01

Circulating vascular progenitor cells contribute to the pathological vasculogenesis of cancer whilst on the other hand offer much promise in therapeutic revascularization in post-occlusion intervention in cardiovascular disease. However, their characterization has been hampered by the many variables to produce them as well as their described phenotypic and functional heterogeneity. Herein we have isolated, enriched for and then characterized a human umbilical cord blood derived CD133+ population of non-adherent endothelial forming cells (naEFCs) which expressed the hematopoietic progenitor cell markers (CD133, CD34, CD117, CD90 and CD38) together with mature endothelial cell markers (VEGFR2, CD144 and CD31). These cells also expressed low levels of CD45 but did not express the lymphoid markers (CD3, CD4, CD8) or myeloid markers (CD11b and CD14) which distinguishes them from ‘early’ endothelial progenitor cells (EPCs). Functional studies demonstrated that these naEFCs (i) bound Ulex europaeus lectin, (ii) demonstrated acetylated-low density lipoprotein uptake, (iii) increased vascular cell adhesion molecule (VCAM-1) surface expression in response to tumor necrosis factor and (iv) in co-culture with mature endothelial cells increased the number of tubes, tubule branching and loops in a 3-dimensional in vitro matrix. More importantly, naEFCs placed in vivo generated new lumen containing vasculature lined by CD144 expressing human endothelial cells (ECs). Extensive genomic and proteomic analyses of the naEFCs showed that intercellular adhesion molecule (ICAM)-3 is expressed on their cell surface but not on mature endothelial cells. Furthermore, functional analysis demonstrated that ICAM-3 mediated the rolling and adhesive events of the naEFCs under shear stress. We suggest that the distinct population of naEFCs identified and characterized here represents a new valuable therapeutic target to control aberrant vasculogenesis. PMID:23144795

Proinflammatory Cytokine Tumor Necrosis Factor (TNF)-like Weak Inducer of Apoptosis (TWEAK) Suppresses Satellite Cell Self-renewal through Inversely Modulating Notch and NF-κB Signaling Pathways*

PubMed Central

Ogura, Yuji; Mishra, Vivek; Hindi, Sajedah M.; Kuang, Shihuan; Kumar, Ashok

2013-01-01

Satellite cell self-renewal is an essential process to maintaining the robustness of skeletal muscle regenerative capacity. However, extrinsic factors that regulate self-renewal of satellite cells are not well understood. Here, we demonstrate that TWEAK cytokine reduces the proportion of Pax7+/MyoD− cells (an index of self-renewal) on myofiber explants and represses multiple components of Notch signaling in satellite cell cultures. The number of Pax7+ cells is significantly increased in skeletal muscle of TWEAK knock-out (KO) mice compared with wild-type in response to injury. Furthermore, Notch signaling is significantly elevated in cultured satellite cells and in regenerating myofibers of TWEAK-KO mice. Forced activation of Notch signaling through overexpression of the Notch1 intracellular domain (N1ICD) rescued the TWEAK-mediated inhibition of satellite cell self-renewal. TWEAK also activates the NF-κB transcription factor in satellite cells and inhibition of NF-κB significantly improved the number of Pax7+ cells in TWEAK-treated cultures. Furthermore, our results demonstrate that a reciprocal interaction between NF-κB and Notch signaling governs the inhibitory effect of TWEAK on satellite cell self-renewal. Collectively, our study demonstrates that TWEAK suppresses satellite cell self-renewal through activating NF-κB and repressing Notch signaling. PMID:24151074

Proinflammatory cytokine tumor necrosis factor (TNF)-like weak inducer of apoptosis (TWEAK) suppresses satellite cell self-renewal through inversely modulating Notch and NF-κB signaling pathways.

PubMed

Ogura, Yuji; Mishra, Vivek; Hindi, Sajedah M; Kuang, Shihuan; Kumar, Ashok

2013-12-06

Satellite cell self-renewal is an essential process to maintaining the robustness of skeletal muscle regenerative capacity. However, extrinsic factors that regulate self-renewal of satellite cells are not well understood. Here, we demonstrate that TWEAK cytokine reduces the proportion of Pax7(+)/MyoD(-) cells (an index of self-renewal) on myofiber explants and represses multiple components of Notch signaling in satellite cell cultures. The number of Pax7(+) cells is significantly increased in skeletal muscle of TWEAK knock-out (KO) mice compared with wild-type in response to injury. Furthermore, Notch signaling is significantly elevated in cultured satellite cells and in regenerating myofibers of TWEAK-KO mice. Forced activation of Notch signaling through overexpression of the Notch1 intracellular domain (N1ICD) rescued the TWEAK-mediated inhibition of satellite cell self-renewal. TWEAK also activates the NF-κB transcription factor in satellite cells and inhibition of NF-κB significantly improved the number of Pax7(+) cells in TWEAK-treated cultures. Furthermore, our results demonstrate that a reciprocal interaction between NF-κB and Notch signaling governs the inhibitory effect of TWEAK on satellite cell self-renewal. Collectively, our study demonstrates that TWEAK suppresses satellite cell self-renewal through activating NF-κB and repressing Notch signaling.

Crocidolite asbestos causes an induction of p53 and apoptosis in cultured A-549 lung carcinoma cells.

PubMed

Pääkkö, P; Rämet, M; Vähäkangas, K; Korpela, N; Soini, Y; Turunen, S; Jaworska, M; Gillissen, A

1998-01-01

A number of genotoxic chemicals and agents, such as benzo(a)pyrene and ultraviolet light, are able to induce nuclear accumulation of p53 protein. Usually, this response is transient and a consequence of stabilization of the wild-type p53 protein. After withdrawal of the exposure, the amount of p53 protein returns to a normal level within hours or a few days. We have studied the p53 response to the exposure of crocidolite asbestos in A-549 lung carcinoma cells using three different methods, i.e., p53 immunohistochemistry, Western blotting and metabolic labelling followed by p53 immunoprecipitation. With these techniques we demonstrate a dose-dependent p53 nuclear response to crocidolite exposure. The half-life of p53 protein in A-549 lung carcinoma cells cultured in serum-free media increased from 30 up to 80 min, and the protein reacted with a wild-type specific antibody suggesting that it was in a wild-type conformation. In situ 3'-end labelling of A-549 cells demonstrated a dose-dependent increase in apoptotic activity. Our data support the idea that increased apoptotic activity, induced by crocidolite, is mediated by p53.

Human mesenchymal stromal cells are mechanosensitive to vibration stimuli.

PubMed

Kim, I S; Song, Y M; Lee, B; Hwang, S J

2012-12-01

Low-magnitude high-frequency (LMHF) vibrations have the ability to stimulate bone formation and reduce bone loss. However, the anabolic mechanisms that are mediated by vibration in human bone cells at the cellular level remain unclear. We hypothesized that human mesenchymal stromal cells (hMSCs) display direct osteoblastic responses to LMHF vibration signals. Daily exposure to vibrations increased the proliferation of hMSCs, with the highest efficiency occurring at a peak acceleration of 0.3 g and vibrations at 30 to 40 Hz. Specifically, these conditions promoted osteoblast differentiation through an increase in alkaline phosphatase activity and in vitro matrix mineralization. The effect of vibration on the expression of osteogenesis-related factors differed depending on culture method. hMSCs that underwent vibration in a monolayer culture did not exhibit any changes in the expressions of these genes, while cells in three-dimensional culture showed increased expression of type I collagen, osteoprotegerin, or VEGF, and VEGF induction appeared in 2 different hMSC lines. These results are among the first to demonstrate a dose-response effect upon LMHF stimulation, thereby demonstrating that hMSCs are mechanosensitive to LMHF vibration signals such that they could facilitate the osteogenic process.

Progranulin Is a Chemoattractant for Microglia and Stimulates Their Endocytic Activity

PubMed Central

Pickford, Fiona; Marcus, Jacob; Camargo, Luiz Miguel; Xiao, Qiurong; Graham, Danielle; Mo, Jan-Rung; Burkhardt, Matthew; Kulkarni, Vinayak; Crispino, Jamie; Hering, Heike; Hutton, Michael

2011-01-01

Mutations resulting in progranulin haploinsufficiency cause disease in patients with a subset of frontotemporal lobar degeneration; however, the biological functions of progranulin in the brain remain unknown. To address this subject, the present study initially assessed changes in gene expression and cytokine secretion in rat primary cortical neurons treated with progranulin. Molecular pathways enriched in the progranulin gene set included cell adhesion and cell motility pathways and pathways involved in growth and development. Secretion of cytokines and several chemokines linked to chemoattraction but not inflammation were also increased from progranulin-treated primary neurons. Therefore, whether progranulin is involved in recruitment of immune cells in the brain was investigated. Localized lentiviral expression of progranulin in C57BL/6 mice resulted in an increase of Iba1-positive microglia around the injection site. Moreover, progranulin alone was sufficient to promote migration of primary mouse microglia in vitro. Primary microglia and C4B8 cells demonstrated more endocytosis of amyloid β1-42 when treated with progranulin. These data demonstrate that progranulin acts as a chemoattractant in the brain to recruit or activate microglia and can increase endocytosis of extracellular peptides such as amyloid β. PMID:21224065

The influence of prostatic anatomy and neurotrophins on basal prostate epithelial progenitor cells.

PubMed

Höfner, Thomas; Klein, Corinna; Eisen, Christian; Rigo-Watermeier, Teresa; Haferkamp, Axel; Trumpp, Andreas; Sprick, Martin R

2016-01-01

Based on findings of surface marker, protein screens as well as the postulated near-urethral location of the prostate stem cell niche, we were interested whether androgen ablation, distinct anatomic regions within the prostate or neurotrophins have an influence on basal prostate epithelial progenitor cells (PESCs). Microdissection of the prostate, enzymatic digestion, and preparation of single cells was performed from murine and human prostates. Adult PESC marker expressions were compared between a group of C57BL/6 mice and a separate group of castrated C57BL/6 mice. Surface markers CD13/CD271 on human prostate epithelial progenitor cells were evaluated by FACS analyses in cells cultured under novel stem cell conditions. The effect of neurotrophins NGF, NT3, and BDNF were evaluated with respect to their influence on proliferation and activation of human basal PESCs in vitro. We demonstrate the highest percentage of CD49f+ and Trop2+ expressing cells in the urethra near prostatic regions of WT mice (Trop2+ proximal: 10% vs. distal to the urethra: 3%, P < 0.001). While a marked increase of Trop2 expressing cells can be measured both in the proximal and distal prostatic regions after castration, the most prominent increase in Trop2+ cells can be measured in the prostatic tissue distant to the urethra. Furthermore, we demonstrate that the proportion of syndecan-1 expressing cells greatly increases in the regions proximal to the urethra after castration (WT: 5% vs. castrated: 40%). We identified heterogeneous CD13 and nerve growth factor receptor (p75(NGFR), CD271) expression on CD49f(+)/TROP2(high) human basal PESCs. Addition of the neurotrophins NT3, BDNF, and NGF to the stem cell media led to a marked temporary increase in the proliferation of human basal PESCs. Our results in mice support the model, in which the proximal urethral region contains the prostate stem cell niche while a stronger androgen-dependent regulation of adult prostate stem cells can be found in the peripheral prostatic tissue. Neutrophin signaling via nerve growth factor receptor is possibly involved in human prostate stem cell homeostasis. © 2015 Wiley Periodicals, Inc.

Tissue Factor promotes breast cancer stem cell activity in vitro.

PubMed

Shaker, Hudhaifah; Harrison, Hannah; Clarke, Robert; Landberg, Goran; Bundred, Nigel J; Versteeg, Henri H; Kirwan, Cliona C

2017-04-18

Cancer stem cells (CSCs) are a subpopulation of cells that can self-renew and initiate tumours. The clotting-initiating protein Tissue Factor (TF) promotes metastasis and may be overexpressed in cancer cells with increased CSC activity. We sought to determine whether TF promotes breast CSC activity in vitro using human breast cancer cell lines. TF expression was compared in anoikis-resistant (CSC-enriched) and unselected cells. In cells sorted into of TF-expressing and TF-negative (FACS), and in cells transfected to knockdown TF (siRNA) and overexpress TF (cDNA), CSC activity was compared by (i) mammosphere forming efficiency (MFE) (ii) holoclone colony formation (Hc) and (iii) ALDH1 activity. TF expression was increased in anoikis-resistant and high ALDH1-activity T47D cells compared to unselected cells. FACS sorted TF-expressing T47Ds and TF-overexpressing MCF7s had increased CSC activity compared to TF-low cells. TF siRNA cells (MDAMB231,T47D) had reduced CSC activity compared to control cells. FVIIa increased MFE and ALDH1 in a dose-dependent manner (MDAMB231, T47D). The effects of FVIIa on MFE were abrogated by TF siRNA (T47D). Breast CSCs (in vitro) demonstrate increased activity when selected for high TF expression, when induced to overexpress TF, and when stimulated (with FVIIa). Targeting the TF pathway in vivo may abrogate CSC activity.

Functional Erythropoietin Autocrine Loop in Melanoma

PubMed Central

Kumar, Suresh M.; Acs, Geza; Fang, Dong; Herlyn, Meenhard; Elder, David E.; Xu, Xiaowei

2005-01-01

Although erythropoietin (Epo) is a known stimulator of erythropoiesis, recent evidence suggests that its biological functions are not confined to hematopoietic cells. To elucidate the role of Epo and erythropoietin receptor (EpoR) in melanoma, we examined the expression and function of these proteins in melanocytes and melanoma cells. We found increased expression of Epo in melanoma cells compared to melanocyte in vitro. EpoR was also strongly expressed in all of the melanoma cell lines and two of the three melanocyte cell lines examined. Epo expression was significantly higher in melanoma than in benign nevi as determined by immunohistochemistry. Although melanoma cells secreted Epo in normoxic condition in vitro, hypoxia and CoCl2 treatment increased Epo secretion. EpoR in melanoma cells was functional, because exogenous Epo increased melanoma resistance to hypoxic stress, pretreatment of melanoma cells with Epo significantly increased resistance to dacarbazine treatment, and Epo increased the phosphorylation of EpoR, RAF, and MEK. In conclusion, we demonstrated constitutive expression of Epo and EpoR as well as autonomous secretion of Epo by melanoma cells, indicating a novel autocrine loop of Epo in melanoma. The results suggest that the autocrine and paracrine functions of Epo might play a role in malignant transformation of melanocytes and in the survival of melanoma cells in hypoxia and other adverse conditions. PMID:15743794

CARs in Chronic Lymphocytic Leukemia – Ready to Drive

PubMed Central

Wierda, William; Jena, Bipulendu; Cooper, Laurence J. N.; Shpall, Elizabeth

2013-01-01

Adoptive transfer of antigen-specific T cells has been adapted by investigators for treatment of chronic lymphocytic leukemia (CLL). To overcome issues of immune tolerance which limits the endogenous adaptive immune response to tumor-associated antigens (TAAs), robust systems for the genetic modification and characterization of T cells expressing chimeric antigen receptors (CARs) to redirect specificity have been produced. Refinements with regards to persistence and trafficking of the genetically modified T cells are underway to help improve potency. Clinical trials utilizing this technology demonstrate feasibility, and increasingly, these early-phase trials are demonstrating impressive anti-tumor effects, particularly for CLL patients, paving the way for multi-center trials to establish the efficacy of CAR+ T cell therapy. PMID:23225251

The timing and location of glial cell line-derived neurotrophic factor expression determine enteric nervous system structure and function.

PubMed

Wang, Hongtao; Hughes, Inna; Planer, William; Parsadanian, Alexander; Grider, John R; Vohra, Bhupinder P S; Keller-Peck, Cynthia; Heuckeroth, Robert O

2010-01-27

Ret signaling is critical for formation of the enteric nervous system (ENS) because Ret activation promotes ENS precursor survival, proliferation, and migration and provides trophic support for mature enteric neurons. Although these roles are well established, we now provide evidence that increasing levels of the Ret ligand glial cell line-derived neurotrophic factor (GDNF) in mice causes alterations in ENS structure and function that are critically dependent on the time and location of increased GDNF availability. This is demonstrated using two different strains of transgenic mice and by injecting newborn mice with GDNF. Furthermore, because different subclasses of ENS precursors withdraw from the cell cycle at different times during development, increases in GDNF at specific times alter the ratio of neuronal subclasses in the mature ENS. In addition, we confirm that esophageal neurons are GDNF responsive and demonstrate that the location of GDNF production influences neuronal process projection for NADPH diaphorase-expressing, but not acetylcholinesterase-, choline acetyltransferase-, or tryptophan hydroxylase-expressing, small bowel myenteric neurons. We further demonstrate that changes in GDNF availability influence intestinal function in vitro and in vivo. Thus, changes in GDNF expression can create a wide variety of alterations in ENS structure and function and may in part contribute to human motility disorders.

High-performance ternary blend polymer solar cells involving both energy transfer and hole relay processes.

PubMed

Lu, Luyao; Chen, Wei; Xu, Tao; Yu, Luping

2015-06-04

The integration of multiple materials with complementary absorptions into a single junction device is regarded as an efficient way to enhance the power conversion efficiency (PCE) of organic solar cells (OSCs). However, because of increased complexity with one more component, only limited high-performance ternary systems have been demonstrated previously. Here we report an efficient ternary blend OSC with a PCE of 9.2%. We show that the third component can reduce surface trap densities in the ternary blend. Detailed studies unravel that the improved performance results from synergistic effects of enlarged open circuit voltage, suppressed trap-assisted recombination, enhanced light absorption, increased hole extraction, efficient energy transfer and better morphology. The working mechanism and high device performance demonstrate new insights and design guidelines for high-performance ternary blend solar cells and suggest that ternary structure is a promising platform to boost the efficiency of OSCs.

Leptin regulates glutamate and glucose transporters in hypothalamic astrocytes

PubMed Central

Fuente-Martín, Esther; García-Cáceres, Cristina; Granado, Miriam; de Ceballos, María L.; Sánchez-Garrido, Miguel Ángel; Sarman, Beatrix; Liu, Zhong-Wu; Dietrich, Marcelo O.; Tena-Sempere, Manuel; Argente-Arizón, Pilar; Díaz, Francisca; Argente, Jesús; Horvath, Tamas L.; Chowen, Julie A.

2012-01-01

Glial cells perform critical functions that alter the metabolism and activity of neurons, and there is increasing interest in their role in appetite and energy balance. Leptin, a key regulator of appetite and metabolism, has previously been reported to influence glial structural proteins and morphology. Here, we demonstrate that metabolic status and leptin also modify astrocyte-specific glutamate and glucose transporters, indicating that metabolic signals influence synaptic efficacy and glucose uptake and, ultimately, neuronal function. We found that basal and glucose-stimulated electrical activity of hypothalamic proopiomelanocortin (POMC) neurons in mice were altered in the offspring of mothers fed a high-fat diet. In adulthood, increased body weight and fasting also altered the expression of glucose and glutamate transporters. These results demonstrate that whole-organism metabolism alters hypothalamic glial cell activity and suggest that these cells play an important role in the pathology of obesity. PMID:23064363

Testing of resveratrol microemulsion photostability and protective effect against UV induced oxidative stress.

PubMed

Juškaitė, Vaida; Ramanauskienė, Kristina; Briedis, Vitalis

2017-06-27

Resveratrol is well known for its antioxidant activity and susceptibility to ultraviolet radiation. Development of formulations providing improved stability and relevant drug delivery of resveratrol is still a challenging task. The aim of this study was to determine protective characteristics of formulated microemulsions by evaluating photoisomerization of resveratrol and to investigate the effects of resveratrol on human keratinocyte cells under oxidative stress caused by ultraviolet radiation. Incorporation of resveratrol into microemulsions resulted in increased photostability of active compounds and the results demonstrated that photodegradation of resveratrol was significantly delayed. Results of biopharmaceutical evaluation in vitro demonstrated that up to 60 % of resveratrol was released from microemulsions within 6 hours under a constant release rate profile. In vivo biological testing confirmed the ability of resveratrol to protect cells from oxidative stress and to increase cell viability. It was concluded that microemulsions might be considered in the development of UV light sensitive compounds.

Experimentally-induced immune activation in natural hosts of SIV induces significant increases in viral replication and CD4+ T cell depletion

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ribeiro, Ruy M

2008-01-01

Chronically SIVagm-infected African green monkeys (AGMs) have a remarkably stable non-pathogenic disease course, with levels of immune activation in chronic SIVagm infection similar to those observed in uninfected monkeys and stable viral loads (VLs) for long periods of time. In vivo administration of lipopolysaccharide (LPS) or an IL-2/diphtheria toxin fusion protein (Ontak) to chronically SIVagm-infected AGMs triggered increases in immune activation and subsequently of viral replication and depletion of intestinal CD4{sup +} T cells. Our study indicates that circulating microbial products can increase viral replication by inducing immune activation and increasing the number of viral target cells, thus demonstrating thatmore » immune activation and T cell prolifeation are key factors in AIDS pathogenesis.« less

YAP-dependent Mechanotransduction is Required for Proliferation and Migration on Native-like Substrate Topography

PubMed Central

Mascharak, Shamik; Benitez, Patrick L.; Proctor, Amy C.; Madl, Christopher M.; Hu, Kenneth H.; Dewi, Ruby E.; Butte, Manish J.; Heilshorn, Sarah C.

2017-01-01

Native vascular extracellular matrices (vECM) consist of elastic fibers that impart varied topographical properties, yet most in vitro models designed to study the effects of topography on cell behavior are not representative of native architecture. Here, we engineer an electrospun elastin-like protein (ELP) system with independently tunable, vECM-mimetic topography and demonstrate that increasing topographical variation causes loss of endothelial cell-cell junction organization. This loss of VE-cadherin signaling and increased cytoskeletal contractility on more topographically varied ELP substrates in turn promote YAP activation and nuclear translocation, resulting in significantly increased endothelial cell migration and proliferation. Our findings identify YAP as a required signaling factor through which fibrous substrate topography influences cell behavior and highlights topography as a key design parameter for engineered biomaterials. PMID:27889666

Lectin-like oxidized low-density lipoprotein receptor (LOX-1) in sickle cell disease vasculopathy

PubMed Central

Chen, Mingyi; Qiu, Hong; Lin, Xin; Nam, David; Ogbu-Nwobodo, Lucy; Archibald, Hannah; Joslin, Amelia; Wun, Ted; Sawamura, Tatsuya; Green, Ralph

2017-01-01

Lectin-like oxidized low-density lipoprotein (LDL) receptor-1 (LOX-1) is an endothelial receptor for oxidized LDL. Increased expression of LOX-1 has been demonstrated in atherosclerotic lesions and diabetic vasculopathy. In this study, we investigate the expression of LOX-1 receptor in sickle cell disease (SCD) vasculopathy. Expression of LOX-1 in brain vascular endothelium is markedly increased and LOX-1 gene expression is upregulated in cultured human brain microvascular endothelial cells by incubation with SCD erythrocytes. Also, the level of circulating soluble LOX-1 concentration is elevated in the plasma of SCD patients. Increased LOX-1 expression in endothelial cells is potentially involved in the pathogenesis of SCD vasculopathy. Soluble LOX-1 concentration in SCD may provide a novel biomarker for risk stratification of sickle cell vascular complications. PMID:27519944

Protease Activated Receptor-2 Expression and Function in Asthmatic Bronchial Smooth Muscle

PubMed Central

Gilbert, Guillaume; Carvalho, Gabrielle; Trian, Thomas; Ozier, Annaig; Gillibert-Duplantier, Jennifer; Ousova, Olga; Maurat, Elise; Thumerel, Matthieu; Quignard, Jean-François; Girodet, Pierre-Olivier; Marthan, Roger; Berger, Patrick

2014-01-01

Asthmatic bronchial smooth muscle (BSM) is characterized by structural remodeling associated with mast cell infiltration displaying features of chronic degranulation. Mast cell-derived tryptase can activate protease activated receptor type-2 (PAR-2) of BSM cells. The aims of the present study were (i) to evaluate the expression of PAR-2 in both asthmatic and non asthmatic BSM cells and, (ii) to analyze the effect of prolonged stimulation of PAR-2 in asthmatic BSM cells on cell signaling and proliferation. BSM cells were obtained from both 33 control subjects and 22 asthmatic patients. PAR-2 expression was assessed by flow cytometry, western blot and quantitative RT-PCR. Calcium response, transduction pathways and proliferation were evaluated before and following PAR-2 stimulation by SLIGKV-NH2 or trypsin for 1 to 3 days. Asthmatic BSM cells expressed higher basal levels of functional PAR-2 compared to controls in terms of mRNA, protein expression and calcium response. When PAR-2 expression was increased by means of lentivirus in control BSM cells to a level similar to that of asthmatic cells, PAR-2-induced calcium response was then similar in both types of cell. However, repeated PAR-2 stimulations increased the proliferation of asthmatic BSM cells but not that of control BSM cells even following lentiviral over-expression of PAR-2. Such an increased proliferation was related to an increased phosphorylation of ERK in asthmatic BSM cells. In conclusion, we have demonstrated that asthmatic BSM cells express increased baseline levels of functional PAR-2. This higher basal level of PAR-2 accounts for the increased calcium response to PAR-2 stimulation, whereas the increased proliferation to repeated PAR-2 stimulation is related to increased ERK phosphorylation. PMID:24551046

Inactivation of Mirk/Dyrk1b Kinase Targets Quiescent Pancreatic Cancer Cells *

PubMed Central

Ewton, Daina Z.; Hu, Jing; Vilenchik, Maria; Deng, Xiaobing; Luk, Kin-chun; Polonskaia, Ann; Hoffman, Ann F.; Zipf, Karen; Boylan, John F.; Friedman, Eileen A.

2011-01-01

A major problem in the treatment of cancer arises from quiescent cancer cells that are relatively insensitive to most chemotherapeutic drugs and radiation. Such residual cancer cells can cause tumor regrowth or recurrence when they re-enter the cell cycle. Earlier studies demonstrated that levels of the serine/theronine kinase Mirk/dyrk1B are elevated up to 10-fold in quiescent G0 tumor cells, that Mirk uses several mechanisms to block cell cycling, and that Mirk increases expression of antioxidant genes which lower ROS levels and increase quiescent cell viability. We now show that a novel small molecule Mirk kinase inhibitor blocked tumor cells from undergoing reversible arrest in a quiescent G0 state and enabled some cells to exit quiescence. The inhibitor increased cycling in Panc1, AsPc1 and SW620 cells that expressed Mirk, but not in HCT116 cells that did not. Mirk kinase inhibition elevated ROS levels and DNA damage detected by increased phosphorylation of the histone protein H2AX and by S phase checkpoints. The Mirk kinase inhibitor increased cleavage of the apoptotic proteins PARP and caspase 3, and increased tumor cell kill several-fold by gemcitabine and cisplatin. A phenocopy of these effects occurred following Mirk depletion, showing drug specificity. In prior studies Mirk knockout or depletion had no detectable effect on normal tissue, suggesting that the Mirk kinase inhibitor could have a selective effect on cancer cells expressing elevated levels of Mirk kinase. PMID:21878655

Homologous recombination mediates S-phase-dependent radioresistance in cells deficient in DNA polymerase eta

PubMed Central

Sharma, Ricky A.

2012-01-01

DNA polymerase eta (pol η) is the only DNA polymerase causally linked to carcinogenesis in humans. Inherited deficiency of pol η in the variant form of xeroderma pigmentosum (XPV) predisposes to UV-light-induced skin cancer. Pol η-deficient cells demonstrate increased sensitivity to cisplatin and oxaliplatin chemotherapy. We have found that XP30R0 fibroblasts derived from a patient with XPV are more resistant to cell kill by ionising radiation (IR) than the same cells complemented with wild-type pol η. This phenomenon has been confirmed in Burkitt’s lymphoma cells, which either expressed wild-type pol η or harboured a pol η deletion. Pol η deficiency was associated with accumulation of cells in S-phase, which persisted after IR. Cells deficient in pol η demonstrated increased homologous recombination (HR)-directed repair of double strand breaks created by IR. Depletion of the HR protein, X-ray repair cross-complementing protein 3 (XRCC3), abrogated the radioresistance observed in pol η-deficient cells as compared with pol η-complemented cells. These findings suggest that HR mediates S-phase-dependent radioresistance associated with pol η deficiency. We propose that pol η protein levels in tumours may potentially be used to identify patients who require treatment with chemo-radiotherapy rather than radiotherapy alone for adequate tumour control. PMID:22822095

CXCL5 knockdown expression inhibits human bladder cancer T24 cells proliferation and migration

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zheng, Jiajia; Zhu, Xi; Zhang, Jie, E-mail: zhangjiebjmu@163.com

2014-03-28

Highlights: • We first demonstrated CXCL5 is highly expressed in human bladder tumor tissues and cells. • CXCL5 knockdown inhibits proliferation, migration and promotes apoptosis in T24 cells. • CXCL5 knockdown inhibits Snail, PI3K-AKT and ERK1/2 signaling pathways in T24 cells. • CXCL5 is critical for bladder tumor growth and progression. - Abstract: CXCL5 (epithelial neutrophil activating peptide-78) which acts as a potent chemoattractant and activator of neutrophil function was reported to play a multifaceted role in tumorigenesis. To investigate the role of CXCL5 in bladder cancer progression, we examined the CXCL5 expression in bladder cancer tissues by real-time PCRmore » and Western blot, additionally, we used shRNA-mediated silencing to generate stable CXCL5 silenced bladder cancer T24 cells and defined its biological functions. Our results demonstrated that mRNA and protein of CXCL5 is increased in human bladder tumor tissues and cell lines, down-regulation of CXCL5 in T24 cells resulted in significantly decreased cell proliferation, migration and increased cell apoptosis in vitro through Snail, PI3K-AKT and ERK1/2 signaling pathways. These data suggest that CXCL5 is critical for bladder tumor growth and progression, it may represent a potential application in cancer diagnosis and therapy.« less

Resistin modulates glucose uptake and glucose transporter-1 (GLUT-1) expression in trophoblast cells.

PubMed

Di Simone, Nicoletta; Di Nicuolo, Fiorella; Marzioni, Daniela; Castellucci, Mario; Sanguinetti, Maurizio; D'lppolito, Silvia; Caruso, Alessandro

2009-02-01

The adipocytokine resistin impairs glucose tolerance and insulin sensitivity. Here, we examine the effect of resistin on glucose uptake in human trophoblast cells and we demonstrate that transplacental glucose transport is mediated by glucose transporter (GLUT)-1. Furthermore, we evaluate the type of signal transduction induced by resistin in GLUT-1 regulation. BeWo choriocarcinoma cells and primary cytotrophoblast cells were cultured with increasing resistin concentrations for 24 hrs. The main outcome measures include glucose transport assay using [(3)H]-2-deoxy glucose, GLUT-1 protein expression by Western blot analysis and GLUT-1 mRNA detection by quantitative real-time RT-PCR. Quantitative determination of phospho(p)-ERK1/2 in cell lysates was performed by an Enzyme Immunometric Assay and Western blot analysis. Our data demonstrate a direct effect of resistin on normal cytotrophoblastic and on BeWo cells: resistin modulates glucose uptake, GLUT-1 messenger ribonucleic acid (mRNA) and protein expression in placental cells. We suggest that ERK1/2 phosphorylation is involved in the GLUT-1 regulation induced by resistin. In conclusion, resistin causes activation of both the ERK1 and 2 pathway in trophoblast cells. ERK1 and 2 activation stimulated GLUT-1 synthesis and resulted in increase of placental glucose uptake. High resistin levels (50-100 ng/ml) seem able to affect glucose-uptake, presumably by decreasing the cell surface glucose transporter.

Role of high-mobility group box 1 in methamphetamine-induced activation and migration of astrocytes.

PubMed

Zhang, Yuan; Zhu, Tiebing; Zhang, Xiaotian; Chao, Jie; Hu, Gang; Yao, Honghong

2015-09-04

Mounting evidence has indicated that high-mobility group box 1 (HMGB1) is involved in cell activation and migration. Our previous study demonstrated that methamphetamine mediates activation of astrocytes via sigma-1 receptor (σ-1R). However, the elements downstream of σ-1R in this process remain poorly understood. Thus, we examined the molecular mechanisms involved in astrocyte activation and migration induced by methamphetamine. The expression of HMGB1, σ-1R, and glial fibrillary acidic protein (GFAP) was examined by western blot and immunofluorescent staining. The phosphorylation of cell signaling pathways was detected by western blot, and cell migration was examined using a wound-healing assay in rat C6 astroglia-like cells transfected with lentivirus containing red fluorescent protein (LV-RFP) as well as in primary human astrocytes. The role of HMGB1 in astrocyte activation and migration was validated using a siRNA approach. Exposure of C6 cells to methamphetamine increased the expression of HMGB1 via the activation of σ-1R, Src, ERK mitogen-activated protein kinase, and downstream NF-κB p65 pathways. Moreover, methamphetamine treatment resulted in increased cell activation and migration in C6 cells and primary human astrocytes. Knockdown of HMGB1 in astrocytes transfected with HMGB1 siRNA attenuated the increased cell activation and migration induced by methamphetamine, thereby implicating the role of HMGB1 in the activation and migration of C6 cells and primary human astrocytes. This study demonstrated that methamphetamine-mediated activation and migration of astrocytes involved HMGB1 up-regulation through an autocrine mechanism. Targeting HMGB1 could provide insights into the development of a potential therapeutic approach for alleviation of cell activation and migration of astrocytes induced by methamphetamine.

DNA from Porphyromonas gingivalis and Tannerella forsythia induce cytokine production in human monocytic cell lines.

PubMed

Sahingur, S E; Xia, X-J; Alamgir, S; Honma, K; Sharma, A; Schenkein, H A

2010-04-01

Toll-like receptor 9 (TLR9) expression is increased in periodontally diseased tissues compared with healthy sites indicating a possible role of TLR9 and its ligand, bacterial DNA (bDNA), in periodontal disease pathology. Here, we determine the immunostimulatory effects of periodontal bDNA in human monocytic cells (THP-1). THP-1 cells were stimulated with DNA of two putative periodontal pathogens: Porphyromonas gingivalis and Tannerella forsythia. The role of TLR9 in periodontal bDNA-initiated cytokine production was determined either by blocking TLR9 signaling in THP-1 cells with chloroquine or by measuring IL-8 production and nuclear factor-kappaB (NF-kappaB) activation in HEK293 cells stably transfected with human TLR9. Cytokine production (IL-1beta, IL-6, and TNF-alpha) was increased significantly in bDNA-stimulated cells compared with controls. Chloroquine treatment of THP-1 cells decreased cytokine production, suggesting that TLR9-mediated signaling pathways are operant in the recognition of DNA from periodontal pathogens. Compared with native HEK293 cells, TLR9-transfected cells demonstrated significantly increased IL-8 production (P < 0.001) and NF-kappaB activation in response to bDNA, further confirming the role of TLR9 in periodontal bDNA recognition. The results of PCR arrays demonstrated upregulation of proinflammatory cytokine and NF-kappaB genes in response to periodontal bDNA in THP-1 cells, suggesting that cytokine induction is through NF-kappaB activation. Hence, immune responses triggered by periodontal bacterial nucleic acids may contribute to periodontal disease pathology by inducing proinflammatory cytokine production through the TLR9 signaling pathway.

Invasive ability of human renal cell carcinoma cell line Caki-2 is accelerated by gamma-aminobutyric acid, via sustained activation of ERK1/2 inducible matrix metalloproteinases.

PubMed

Inamoto, Teruo; Azuma, Haruhito; Sakamoto, Takeshi; Kiyama, Satoshi; Ubai, Takanobu; Kotake, Yatsugu; Watanabe, Masahito; Katsuoka, Yoji

2007-10-01

Gamma-aminobutyric acid (GABA) was first discovered as an inhibitory neurotransmitter in the central nervous system (CNS) and has been reported to have a variety of functions, including regulation of cell division, cell differentiation and maturation, and to be involved in the development of certain cancers outside the CNS. In the present study, using the human renal cell carcinoma cell line Caki-2, we demonstrated that GABA stimulation significantly increased the expression of MMP-2 and -9 and subsequently increased the invasive activity of the cancer cells. Because MAPK signaling is one of the key regulators of MMP expression, we further evaluated MAPK signaling after stimulation with GABA. It was found that GABA stimulation promoted the phosphorylation of MAPKs, including ERK1/2, JNK, and p38. ERK1/2 phosphorylation was sustained for up to 12 h, while phosphorylation of JNK and p38 returned to the endogenous level by 30 min. It was noteworthy that the ras/raf/MEK/ERK pathway inhibitor PD98059 attenuated GABA-induced MMP-9 expression and that both PD98059 and MMP inhibitors attenuated the GABA-induced invasive activity of Caki-2 cells. Moreover, data obtained by depletion of the MEK/ERK pathway using interfering RNA transfection of Caki-2 cells clearly corroborated the above results, as both MMP-9 expression and GABA-induced invasive ability were decreased significantly. We also demonstrated that the GABA-induced increase in invasive ability via ERK1/2 up-regulation was mediated mainly through the GABA-B receptor. These results indicate that GABA stimulation promotes cancer cell invasion and that the effect is partly due to ERK1/2-dependent up-regulation of MMPs.

Cell cycle progression in irradiated endothelial cells cultured from bovine aorta

DOE Office of Scientific and Technical Information (OSTI.GOV)

Rubin, D.B.; Drab, E.A.; Ward, W.F.

1988-11-01

Logarithmically growing endothelial cells from bovine aortas were exposed to single doses of 0-10 Gy of 60Co gamma rays, and cell cycle phase distribution and progression were examined by flow cytometry and autoradiography. In some experiments, cells were synchronized in the cell cycle with hydroxyurea (1 mM). Cell number in sham-irradiated control cultures doubled in approximately 24 h. Estimated cycle stage times for control cells were 14.4 h for G1 phase, 7.2 h for S phase, and 2.4 h for G2 + M phase. Irradiated cells demonstrated a reduced distribution at the G1/S phase border at 4 h, and anmore » increased distribution in G2 + M phase at 24 h postirradiation. Autoradiographs of irradiated cells after continuous (3H)thymidine labeling indicated a block in G1 phase or at the G1/S-phase border. The duration of the block was dose dependent (2-3 min/cGy). Progression of the endothelial cells through S phase after removal of the hydroxyurea block also was retarded by irradiation, as demonstrated by increased distribution in early S phase and decreased distribution in late S phase. These results indicate that progression of asynchronous cultured bovine aortic endothelial cells through the DNA synthetic cycle is susceptible to radiation inhibition at specific sites in the cycle, resulting in redistribution and partial synchronization of the population. Thus aortic endothelial cells, diploid cells from a normal tissue, resemble many immortal cell types that have been examined in this regard in vitro.« less

A microfluidic fuel cell with flow-through porous electrodes.

PubMed

Kjeang, Erik; Michel, Raphaelle; Harrington, David A; Djilali, Ned; Sinton, David

2008-03-26

A microfluidic fuel cell architecture incorporating flow-through porous electrodes is demonstrated. The design is based on cross-flow of aqueous vanadium redox species through the electrodes into an orthogonally arranged co-laminar exit channel, where the waste solutions provide ionic charge transfer in a membraneless configuration. This flow-through architecture enables improved utilization of the three-dimensional active area inside the porous electrodes and provides enhanced rates of convective/diffusive transport without increasing the parasitic loss required to drive the flow. Prototype fuel cells are fabricated by rapid prototyping with total material cost estimated at 2 USD/unit. Improved performance as compared to previous microfluidic fuel cells is demonstrated, including power densities at room temperature up to 131 mW cm-2. In addition, high overall energy conversion efficiency is obtained through a combination of relatively high levels of fuel utilization and cell voltage. When operated at 1 microL min-1 flow rate, the fuel cell produced 20 mW cm-2 at 0.8 V combined with an active fuel utilization of 94%. Finally, we demonstrate in situ fuel and oxidant regeneration by running the flow-through architecture fuel cell in reverse.

Hydrogen-Oxygen PEM Regenerative Fuel Cell Development at NASA Glenn Research Center

NASA Technical Reports Server (NTRS)

Bents, David J.; Scullin, Vincent J.; Chang, B. J.; Johnson, Donald W.; Garcia, Christopher P.; Jakupca, Ian J.

2006-01-01

The closed-cycle hydrogen-oxygen PEM regenerative fuel cell (RFC) at NASA Glenn Research Center has demonstrated multiple back to back contiguous cycles at rated power, and round trip efficiencies up to 52 percent. It is the first fully closed cycle regenerative fuel cell ever demonstrated (entire system is sealed: nothing enters or escapes the system other than electrical power and heat). During FY2006 the system has undergone numerous modifications and internal improvements aimed at reducing parasitic power, heat loss and noise signature, increasing its functionality as an unattended automated energy storage device, and in-service reliability. It also serves as testbed towards development of a 600 W-hr/kg flight configuration, through the successful demonstration of lightweight fuel cell and electrolyser stacks and supporting components. The RFC has demonstrated its potential as an energy storage device for aerospace solar power systems such as solar electric aircraft, lunar and planetary surface installations; any airless environment where minimum system weight is critical. Its development process continues on a path of risk reduction for the flight system NASA will eventually need for the manned lunar outpost.

Alpha-actinin binding kinetics modulate cellular dynamics and force generation

PubMed Central

Ehrlicher, Allen J.; Krishnan, Ramaswamy; Guo, Ming; Bidan, Cécile M.; Weitz, David A.; Pollak, Martin R.

2015-01-01

The actin cytoskeleton is a key element of cell structure and movement whose properties are determined by a host of accessory proteins. Actin cross-linking proteins create a connected network from individual actin filaments, and though the mechanical effects of cross-linker binding affinity on actin networks have been investigated in reconstituted systems, their impact on cellular forces is unknown. Here we show that the binding affinity of the actin cross-linker α-actinin 4 (ACTN4) in cells modulates cytoplasmic mobility, cellular movement, and traction forces. Using fluorescence recovery after photobleaching, we show that an ACTN4 mutation that causes human kidney disease roughly triples the wild-type binding affinity of ACTN4 to F-actin in cells, increasing the dissociation time from 29 ± 13 to 86 ± 29 s. This increased affinity creates a less dynamic cytoplasm, as demonstrated by reduced intracellular microsphere movement, and an approximate halving of cell speed. Surprisingly, these less motile cells generate larger forces. Using traction force microscopy, we show that increased binding affinity of ACTN4 increases the average contractile stress (from 1.8 ± 0.7 to 4.7 ± 0.5 kPa), and the average strain energy (0.4 ± 0.2 to 2.1 ± 0.4 pJ). We speculate that these changes may be explained by an increased solid-like nature of the cytoskeleton, where myosin activity is more partitioned into tension and less is dissipated through filament sliding. These findings demonstrate the impact of cross-linker point mutations on cell dynamics and forces, and suggest mechanisms by which such physical defects lead to human disease. PMID:25918384

Anti-myeloma activity of a multi targeted kinase inhibitor, AT9283, via potent Aurora Kinase and STAT3 inhibition either alone or in combination with lenalidomide

PubMed Central

Santo, Loredana; Hideshima, Teru; Cirstea, Diana; Bandi, Madhavi; Nelson, Erik A.; Gorgun, Gullu; Rodig, Scott; Vallet, Sonia; Pozzi, Samantha; Patel, Kishan; Unitt, Christine; Squires, Matt; Hu, Yiguo; Chauhan, Dharminder; Mahindra, Anuj; Munshi, Nikhil C.; Anderson, Kenneth C.; Raje, Noopur

2014-01-01

Purpose Aurora Kinases, whose expression is linked to genetic instability and cellular proliferation, are under investigation as novel therapeutic targets in multiple myeloma (MM). Here, we investigated the preclinical activity of a small molecule–multi-targeted kinase inhibitor, AT9283, with potent activity against Aurora kinase A (AURKA), Aurora kinase B (AURKB) and Janus Kinase 2/3. Experimental design We evaluated the in vitro anti myeloma activity of AT9283 alone and in combination with lenalidomide and the in vivo efficacy by using a Xenograft mouse model of human MM. Results Our data demonstrated AT9283 induced cell growth inhibition and apoptosis in MM. Studying the apoptosis mechanism of AT9283 in MM, we observed features consistent with both AURKA and AURKB inhibition, e.g increase of cells with polyploid DNA content, decrease in phospho-Histone H3, and decrease of phospho-Aurora A. Importantly, AT9283 also inhibited STAT3 tyrosine phosphorylation in MM cells. Genetic depletion of STAT3, AURKA or AURKB showed growth inhibition of MM cells, suggesting a role of AT9283-induced inhibition of these molecules in the underlying mechanism of MM cell death. In vivo studies demonstrated decreased MM cell growth and prolonged survival in AT9283-treated mice compared to controls. Importantly, combination studies of AT9283 with lenalidomide showed significant synergistic cytotoxicity in MM cells, even in the presence of bone marrow stromal cells (BMSCs). Enhanced cytotoxicity was associated with increased inhibition of pSTAT3 and pERK. Conclusions Demonstration of in vitro and in vivo anti-MM activity of AT9283 provides the rationale for the clinical evaluation of AT9283 as monotherapy and in combination in patients with MM. PMID:21430070

Compressive elasticity of three-dimensional nanofiber matrix directs mesenchymal stem cell differentiation to vascular cells with endothelial or smooth muscle cell markers.

PubMed

Wingate, K; Bonani, W; Tan, Y; Bryant, S J; Tan, W

2012-04-01

The importance of mesenchymal stem cells (MSC) in vascular regeneration is becoming increasingly recognized. However, few in vitro studies have been performed to identify the effects of environmental elasticity on the differentiation of MSC into vascular cell types. Electrospinning and photopolymerization techniques were used to fabricate a three-dimensional (3-D) polyethylene glycol dimethacrylate nanofiber hydrogel matrix with tunable elasticity for use as a cellular substrate. Compression testing demonstrated that the elastic modulus of the hydrated 3-D matrices ranged from 2 to 15 kPa, similar to the in vivo elasticity of the intima basement membrane and media layer. MSC seeded on rigid matrices (8-15 kPa) showed an increase in cell area compared with those seeded on soft matrices (2-5 kPa). Furthermore, the matrix elasticity guided the cells to express different vascular-specific phenotypes with high differentiation efficiency. Around 95% of MSC seeded on the 3-D matrices with an elasticity of 3 kPa showed Flk-1 endothelial markers within 24h, while only 20% of MSC seeded on the matrices with elasticity >8 kPa demonstrated Flk-1 marker. In contrast, ∼80% of MSC seeded on 3-D matrices with elasticity >8 kPa demonstrated smooth muscle α-actin marker within 24h, while fewer than 10% of MSC seeded on 3-D matrices with elasticity <5 kPa showed α-actin markers. The ability to control MSC differentiation into either endothelial or smooth muscle-like cells based purely on the local elasticity of the substrate could be a powerful tool for vascular tissue regeneration. Copyright © 2012 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Survival of tissue-resident memory T cells requires exogenous lipid uptake and metabolism

PubMed Central

Pan, Youdong; Tian, Tian; Park, Chang Ook; Lofftus, Serena Y.; Mei, Shenglin; Liu, Xing; Luo, Chi; O’Malley, John T.; Gehad, Ahmed; Teague, Jessica E.; Divito, Sherrie J.; Fuhlbrigge, Robert; Puigserver, Pere; Krueger, James G.; Hotamisligil, Gökhan S.; Clark, Rachael A.; Kupper, Thomas S.

2017-01-01

Tissue-resident memory T (TRM) cells persist indefinitely in epithelial barrier tissues and protect the host against pathogens1–4. However, the biological pathways that enable the long-term survival of TRM cells are obscure4,5. Here we show that mouse CD8+ TRM cells generated by viral infection of the skin differentially express high levels of several molecules that mediate lipid uptake and intracellular transport, including fatty-acid-binding proteins 4 and 5 (FABP4 and FABP5). We further show that T-cell-specific deficiency of Fabp4 and Fabp5 (Fabp4/Fabp5) impairs exogenous free fatty acid (FFA) uptake by CD8+ TRM cells and greatly reduces their long-term survival in vivo, while having no effect on the survival of central memory T (TCM) cells in lymph nodes. In vitro, CD8+ TRM cells, but not CD8+ TCM, demonstrated increased mitochondrial oxidative metabolism in the presence of exogenous FFAs; this increase was not seen in Fabp4/Fabp5 double-knockout CD8+ TRM cells. The persistence of CD8+ TRM cells in the skin was strongly diminished by inhibition of mitochondrial FFA β-oxidation in vivo. Moreover, skin CD8+ TRM cells that lacked Fabp4/Fabp5 were less effective at protecting mice from cutaneous viral infection, and lung Fabp4/Fabp5 double-knockout CD8+ TRM cells generated by skin vaccinia virus (VACV) infection were less effective at protecting mice from a lethal pulmonary challenge with VACV. Consistent with the mouse data, increased FABP4 and FABP5 expression and enhanced extracellular FFA uptake were also demonstrated in human CD8+ TRM cells in normal and psoriatic skin. These results suggest that FABP4 and FABP5 have a critical role in the maintenance, longevity and function of CD8+ TRM cells, and suggest that CD8+ TRM cells use exogenous FFAs and their oxidative metabolism to persist in tissue and to mediate protective immunity. PMID:28219080

Particle-size-dependent cytokine responses and cell damage induced by silica particles and macrophages-derived mediators in endothelial cell.

PubMed

Rong, Yi; Zhou, Ting; Cheng, Wenjuan; Guo, Jiali; Cui, Xiuqing; Liu, Yuewei; Chen, Weihong

2013-11-01

Epidemiological evidence reports silica dust exposure has been associated with increased risk of cardiovascular diseases, but the mechanisms are largely unknown. In this study, endothelial cells were exposed to increasing concentrations of two sizes silica particles and the soluble mediators released by macrophages treated with the same particles for 24 h. Expression and release of cytokines (IL-1β, TNF-α and IL-6) were measured by using ELISA. Cytotoxicity was measured by MTT assay and LDH release. We show that both ways induced increases in cell toxicity and cytokines in a dose-dependent manner. For smaller particles, the soluble mediators are more capable of increasing cytokines compared with the effect of particles directly. For larger particles, evaluating results of these two ways are similar. Either way, smaller particles make the increasing action of cell toxicity and cytokines more remarkable. Our results indicate both silica particle and macrophage-derived mediators can induce endothelial cell injury and inflammation and demonstrate the potential importance of the particle sizes in this effect. Copyright © 2013. Published by Elsevier B.V.

Inhibition of the proliferation and acceleration of migration of vascular endothelial cells by increased cysteine-rich motor neuron 1

DOE Office of Scientific and Technical Information (OSTI.GOV)

Nakashima, Yukiko; Morimoto, Mayuka; Toda, Ken-ichi

2015-07-03

Cysteine-rich motor neuron 1 (CRIM1) is upregulated only in extracellular matrix gels by angiogenic factors such as vascular endothelial growth factor (VEGF). It then plays a critical role in the tube formation of endothelial cells. In the present study, we investigated the effects of increased CRIM1 on other endothelial functions such as proliferation and migration. Knock down of CRIM1 had no effect on VEGF-induced proliferation or migration of human umbilical vein endothelial cells (HUVECs), indicating that basal CRIM1 is not involved in the proliferation or migration of endothelial cells. Stable CRIM1-overexpressing endothelial F-2 cells, termed CR1 and CR2, were constructed,more » because it was difficult to prepare monolayer HUVECs that expressed high levels of CRIM1. Proliferation was reduced and migration was accelerated in both CR1 and CR2 cells, compared with normal F-2 cells. Furthermore, the transient overexpression of CRIM1 resulted in decreased proliferation and increased migration of bovine aortic endothelial cells. In contrast, neither proliferation nor migration of COS-7 cells were changed by the overexpression of CRIM1. These results demonstrate that increased CRIM1 reduces the proliferation and accelerates the migration of endothelial cells. These CRIM1 effects might contribute to tube formation of endothelial cells. CRIM1 induced by angiogenic factors may serve as a regulator in endothelial cells to switch from proliferating cells to morphological differentiation. - Highlights: • CRIM1 was upregulated only in tubular endothelial cells, but not in monolayers. • Increased CRIM1 reduced the proliferation of endothelial cells. • Increased CRIM1 accelerated the migration of endothelial cells. • Increased CRIM1 had no effect on the proliferation or migration of COS-7 cells.« less

Reactive Oxygen Species/Hypoxia-Inducible Factor-1α/Platelet-Derived Growth Factor-BB Autocrine Loop Contributes to Cocaine-Mediated Alveolar Epithelial Barrier Damage

PubMed Central

Yang, Lu; Chen, Xufeng; Simet, Samantha M.; Hu, Guoku; Cai, Yu; Niu, Fang; Kook, Yeonhee

2016-01-01

Abuse of psychostimulants, such as cocaine, has been shown to be closely associated with complications of the lung, such as pulmonary hypertension, edema, increased inflammation, and infection. However, the mechanism by which cocaine mediates impairment of alveolar epithelial barrier integrity that underlies various pulmonary complications has not been well determined. Herein, we investigate the role of cocaine in disrupting the alveolar epithelial barrier function and the associated signaling cascade. Using the combinatorial electric cell–substrate impedance sensing and FITC-dextran permeability assays, we demonstrated cocaine-mediated disruption of the alveolar epithelial barrier, as evidenced by increased epithelial monolayer permeability with a concomitant loss of the tight junction protein zonula occludens-1 (Zo-1) in both mouse primary alveolar epithelial cells and the alveolar epithelial cell line, L2 cells. To dissect the signaling pathways involved in this process, we demonstrated that cocaine-mediated induction of permeability factors, platelet-derived growth factor (PDGF-BB) and vascular endothelial growth factor, involved reactive oxygen species (ROS)-dependent induction of hypoxia-inducible factor (HIF)-1α. Interestingly, we demonstrated that ROS-dependent induction of another transcription factor, nuclear factor erythroid-2–related factor-2, that did not play a role in cocaine-mediated barrier dysfunction. Importantly, this study identifies, for the first time, that ROS/HIF-1α/PDGF-BB autocrine loop contributes to cocaine-mediated barrier disruption via amplification of oxidative stress and downstream signaling. Corroboration of these cell culture findings in vivo demonstrated increased permeability of the alveolar epithelial barrier, loss of expression of Zo-1, and a concomitantly increased expression of both HIF-1α and PDGF-BB. Pharmacological blocking of HIF-1α significantly abrogated cocaine-mediated loss of Zo-1. Understanding the mechanism(s) by which cocaine mediates barrier dysfunction could provide insights into the development of potential therapeutic targets for cocaine-mediated pulmonary hypertension. PMID:27391108

Conditionally reprogrammed normal and primary tumor prostate epithelial cells: a novel patient-derived cell model for studies of human prostate cancer

PubMed Central

Timofeeva, Olga A.; Palechor-Ceron, Nancy; Li, Guanglei; Yuan, Hang; Krawczyk, Ewa; Zhong, Xiaogang; Liu, Geng; Upadhyay, Geeta; Dakic, Aleksandra; Yu, Songtao; Fang, Shuang; Choudhury, Sujata; Zhang, Xueping; Ju, Andrew; Lee, Myeong-Seon; Dan, Han C.; Ji, Youngmi; Hou, Yong; Zheng, Yun-Ling; Albanese, Chris; Rhim, Johng; Schlegel, Richard; Dritschilo, Anatoly; Liu, Xuefeng

2017-01-01

Our previous study demonstrated that conditional reprogramming (CR) allows the establishment of patient-derived normal and tumor epithelial cell cultures from a variety of tissue types including breast, lung, colon and prostate. Using CR, we have established matched normal and tumor cultures, GUMC-29 and GUMC-30 respectively, from a patient's prostatectomy specimen. These CR cells proliferate indefinitely in vitro and retain stable karyotypes. Most importantly, only tumor-derived CR cells (GUMC-30) produced tumors in xenografted SCID mice, demonstrating maintenance of the critical tumor phenotype. Characterization of cells with DNA fingerprinting demonstrated identical patterns in normal and tumor CR cells as well as in xenografted tumors. By flow cytometry, both normal and tumor CR cells expressed basal, luminal, and stem cell markers, with the majority of the normal and tumor CR cells expressing prostate basal cell markers, CD44 and Trop2, as well as luminal marker, CD13, suggesting a transit-amplifying phenotype. Consistent with this phenotype, real time RT-PCR analyses demonstrated that CR cells predominantly expressed high levels of basal cell markers (KRT5, KRT14 and p63), and low levels of luminal markers. When the CR tumor cells were injected into SCID mice, the expression of luminal markers (AR, NKX3.1) increased significantly, while basal cell markers dramatically decreased. These data suggest that CR cells maintain high levels of proliferation and low levels of differentiation in the presence of feeder cells and ROCK inhibitor, but undergo differentiation once injected into SCID mice. Genomic analyses, including SNP and INDEL, identified genes mutated in tumor cells, including components of apoptosis, cell attachment, and hypoxia pathways. The use of matched patient-derived cells provides a unique in vitro model for studies of early prostate cancer. PMID:28009986

External beam radiation therapy enhances mesenchymal stem cell-mediated sodium iodide symporter gene delivery.

PubMed

Schug, Christina; Sievert, Wolfgang; Urnauer, Sarah; Müller, Andrea Maria; Schmohl, Kathrin Alexandra; Wechselberger, Alexandra; Schwenk, Nathalie; Lauber, Kirsten; Schwaiger, Markus; Multhoff, Gabriele; Wagner, Ernst; Nelson, Peter J; Spitzweg, Christine

2018-05-04

The tumor-homing properties of mesenchymal stem cells (MSC) have led to their development as delivery vehicles for the targeted delivery of therapeutic genes such as the sodium iodide symporter (NIS) to solid tumors. External beam radiation therapy (EBRT) may represent an ideal setting for the application of engineered MSC-based gene therapy as tumor irradiation may enhance MSC recruitment into irradiated tumors through the increased production of select factors linked to MSC migration. In the present study, the irradiation of human liver cancer cells (HuH7) (1-10 Gy) showed a strong dose-dependent increase in steady state mRNA levels of CXCL8, CXCL12/SDF-1, FGF2, PDGFβ, TGFβ1, TSP-1 and VEGF (0-48 h), which was verified for most factors at the protein level (after 48 h). Radiation effects on directed MSC migration was tested in vitro using a live cell tracking migration assay and supernatants from control and irradiated HuH7 cells. A robust increase in mean forward migration index (yFMI), mean center of mass (yCoM) and mean directionality of MSCs towards supernatants was seen from irradiated as compared to nonirradiated tumor cells. Transferability of this effect to other tumor sources was demonstrated using the human breast adenocarcinoma cell line (MDA-MB-231), which showed a similar behavior to radiation as seen with HuH7 cells in qPCR and migration assay. To evaluate this in a more physiologic in vivo setting, subcutaneously growing HuH7 xenograft tumors were irradiated with 0, 2 or 5 Gy followed by CMV-NIS-MSC application 24 h later. Tumoral iodide uptake was monitored using ¹²³I-scintigraphy. The results showed increased tumor-specific dose-dependent accumulation of radioiodide in irradiated tumors. Our results demonstrate that EBRT enhances the migratory capacity of MSCs and may thus increase the therapeutic efficacy of MSC-mediated NIS radionuclide therapy.

Nanoscale Light Manipulation for Improved Organic Solar Cells

NASA Astrophysics Data System (ADS)

Fisher, Brett

Organic Solar Cells can be made to be flexible, semi-transparent, and low-cost making them ideal for novel energy harvesting applications such as in greenhouses. However, the main disadvantage of this technology is its low energy conversion efficiency (<15%); mostly due to high recombination rates, compared with other higher performing technologies, such as thinfilm GaAs (>30% Efficiency), and Si-based (>20% Efficiency), solar cells, where recombination within these technologies is much less than Organic Solar Cells. There are still many challenges to overcome to improve the efficiency of Organic Solar Cells. Some of these challenges include: Maximising the absorption of the solar spectrum; improving the charge dynamics; and increasing the lifetime of the devices. One method to address some of these challenges is to include plasmonic nanoparticles into the devices, which has been shown to increase the absorption through scattering, and improve the charge dynamic through localised surface plasmon resonance effects. However, including nanoparticles into Organic Solar Cells has shown to adversely affect the performance of the devices in other ways, such as increasing the recombination of excitons. To address this, an additional (insulating) coating around the nanoparticles supresses this increase, and has shown to be able to increase the performance of the solar cells. In this work, we demonstrate the use of our all-inclusive optical model in the design and optimisation of bespoke colour-specific windows (i.e. Red, Green, and Blue), where the solar cells can be made to have a specific transparency and colour, whilst maximizing their efficiency. For example, we could specify that we wish the colour to be red, with 50% transmissivity; the model will then maximise the Power Conversion Efficiency. We also demonstrate how our extension to Mie theory can simulate nanoparticle systems and can be used to tune the plasmon resonance utilising different coatings, and configurations thereof.

Diacetyl Induces Amphiregulin Shedding in Pulmonary Epithelial Cells and in Experimental Bronchiolitis Obliterans

PubMed Central

Sun, Jesse; Fischer, Bernard M.; Voynow, Judith A.; Kummarapurugu, Apparao B.; Zhang, Helen L.; Nugent, Julia L.; Beasley, Robert F.; Martinu, Tereza; Gwinn, William M.; Morgan, Daniel L.; Palmer, Scott M.

2014-01-01

Diacetyl (DA), a component of artificial butter flavoring, has been linked to the development of bronchiolitis obliterans (BO), a disease of airway epithelial injury and airway fibrosis. The epidermal growth factor receptor ligand, amphiregulin (AREG), has been implicated in other types of epithelial injury and lung fibrosis. We investigated the effects of DA directly on the pulmonary epithelium, and we hypothesized that DA exposure would result in epithelial cell shedding of AREG. Consistent with this hypothesis, we demonstrate that DA increases AREG by the pulmonary epithelial cell line NCI-H292 and by multiple independent primary human airway epithelial donors grown under physiologically relevant conditions at the air–liquid interface. Furthermore, we demonstrate that AREG shedding occurs through a TNF-α–converting enzyme (TACE)-dependent mechanism via inhibition of TACE activity in epithelial cells using the small molecule inhibitor, TNF-α protease inhibitor-1, as well as TACE-specific small inhibitor RNA. Finally, we demonstrate supportive in vivo results showing increased AREG transcript and protein levels in the lungs of rodents with DA-induced BO. In summary, our novel in vitro and in vivo observations suggest that further study of AREG is warranted in the pathogenesis of DA-induced BO. PMID:24816162

Priming Dental Pulp Stem Cells With Fibroblast Growth Factor-2 Increases Angiogenesis of Implanted Tissue-Engineered Constructs Through Hepatocyte Growth Factor and Vascular Endothelial Growth Factor Secretion

PubMed Central

Gorin, Caroline; Rochefort, Gael Y.; Bascetin, Rumeyza; Ying, Hanru; Lesieur, Julie; Sadoine, Jérémy; Beckouche, Nathan; Berndt, Sarah; Novais, Anita; Lesage, Matthieu; Hosten, Benoit; Vercellino, Laetitia; Merlet, Pascal; Le-Denmat, Dominique; Marchiol, Carmen; Letourneur, Didier; Nicoletti, Antonino; Vital, Sibylle Opsahl; Poliard, Anne; Salmon, Benjamin; Germain, Stéphane

2016-01-01

Tissue engineering strategies based on implanting cellularized biomaterials are promising therapeutic approaches for the reconstruction of large tissue defects. A major hurdle for the reliable establishment of such therapeutic approaches is the lack of rapid blood perfusion of the tissue construct to provide oxygen and nutrients. Numerous sources of mesenchymal stem cells (MSCs) displaying angiogenic potential have been characterized in the past years, including the adult dental pulp. Establishment of efficient strategies for improving angiogenesis in tissue constructs is nevertheless still an important challenge. Hypoxia was proposed as a priming treatment owing to its capacity to enhance the angiogenic potential of stem cells through vascular endothelial growth factor (VEGF) release. The present study aimed to characterize additional key factors regulating the angiogenic capacity of such MSCs, namely, dental pulp stem cells derived from deciduous teeth (SHED). We identified fibroblast growth factor-2 (FGF-2) as a potent inducer of the release of VEGF and hepatocyte growth factor (HGF) by SHED. We found that FGF-2 limited hypoxia-induced downregulation of HGF release. Using three-dimensional culture models of angiogenesis, we demonstrated that VEGF and HGF were both responsible for the high angiogenic potential of SHED through direct targeting of endothelial cells. In addition, FGF-2 treatment increased the fraction of Stro-1+/CD146+ progenitor cells. We then applied in vitro FGF-2 priming to SHED before encapsulation in hydrogels and in vivo subcutaneous implantation. Our results showed that FGF-2 priming is more efficient than hypoxia at increasing SHED-induced vascularization compared with nonprimed controls. Altogether, these data demonstrate that FGF-2 priming enhances the angiogenic potential of SHED through the secretion of both HGF and VEGF. Significance The results from the present study show that fibroblast growth factor-2 (FGF-2) priming is more efficient than hypoxia at increasing dental pulp stem cells derived from deciduous teeth (SHED)-induced vascularization compared with nonprimed controls. Together, these data demonstrate that FGF-2 priming enhances the angiogenic potential of SHED through the secretion of both hepatocyte growth factor and vascular endothelial growth factor. PMID:26798059

Pleural mesothelial cells express both BLT2 and PPARalpha and mount an integrated response to pleural leukotriene B4.

PubMed

Pace, Elisabetta; Ferraro, Maria; Mody, Christopher H; Melis, Mario R; Scafidi, Valeria; Bonanno, Anna; Profita, Mirella; Giarratano, Antonino; Gjomarkaj, Mark

2008-11-15

Leukotriene B(4) (LTB(4)) plays a crucial role in the recruitment of neutrophils into the pleural space. We identified for the first time the mechanisms by which LTB(4) interacts with mesothelial cells and recruits neutrophils in the pleural compartment. Primary pleural mesothelial cells express both the proinflammatory receptor for LTB(4) BLT2, and the anti-inflammatory receptor for LTB(4), PPARalpha. Parapneumonic pleural effusions highly increase BLT2 expression and, via BLT2 activation, increase the adhesion between mesothelial cells and neutrophils and the expression of ICAM-1 on mesothelial cells. The block of PPARalpha further increases both cell adhesion and ICAM-1 expression. BLT2 activation promotes the activation, on mesothelial cells, of STAT-1 but not the activation of NF-kappaB transcription factor. The increase of ICAM-1 expression is achieved via increased tyrosine phosphorylation activity since herbimycin, a tyrosine kinase inhibitor, reduces and since Na orthovanadate, a tyrosine phosphatase inhibitor, further increases ICAM-1 expression. This study demonstrates that pleural mesothelial cells, expressing both proinflammatory and anti-inflammatory LTB(4) receptors, are able to mount an integrated response to LTB(4) with a prevalence of BLT2 activities in the presence of an inflammatory milieu within the pleura.

Live Cell Characterization of DNA Aggregation Delivered through Lipofection

PubMed Central

Mieruszynski, Stephen; Briggs, Candida; Digman, Michelle A.; Gratton, Enrico; Jones, Mark R

2015-01-01

DNA trafficking phenomena, such as information on where and to what extent DNA aggregation occurs, have yet to be fully characterised in the live cell. Here we characterise the aggregation of DNA when delivered through lipofection by applying the Number and Brightness (N&B) approach. The N&B analysis demonstrates extensive aggregation throughout the live cell with DNA clusters in the extremity of the cell and peri-nuclear areas. Once within the nucleus aggregation had decreased 3-fold. In addition, we show that increasing serum concentration of cell media results in greater cytoplasmic aggregation. Further, the effects of the DNA fragment size on aggregation was explored, where larger DNA constructs exhibited less aggregation. This study demonstrates the first quantification of DNA aggregation when delivered through lipofection in live cells. In addition, this study has presents a model for alternative uses of this imaging approach, which was originally developed to study protein oligomerization and aggregation. PMID:26013547

In vivo time-lapse imaging of cell proliferation and differentiation in the optic tectum of Xenopus laevis tadpoles

PubMed Central

Bestman, Jennifer E.; Lee-Osbourne, Jane; Cline, Hollis T.

2012-01-01

We analyzed the function of neural progenitors in the developing CNS of Xenopus laevis tadpoles using in vivo time-lapse confocal microscopy to collect images through the tectum at intervals of 2 to 24 hours over 3 days. Neural progenitor cells were labeled with fluorescent protein reporters based on expression of endogenous Sox2 transcription factor. With this construct, we identified Sox2-expressing cells as radial glia and as a component of the progenitor pool of cells in the developing tectum that gives rise to neurons and other radial glia. Lineage analysis of individual radial glia and their progeny demonstrated that less than 10% of radial glia undergo symmetric divisions resulting in two radial glia, while the majority of radial glia divide asymmetrically to generate neurons and radial glia. Time-lapse imaging revealed the direct differentiation of radial glia into neurons. Although radial glia may guide axons as they navigate to superficial tectum, we find no evidence that radial glia function as a scaffold for neuronal migration at early stages of tectal development. Over three days, the number of labeled cells increased 20%, as the fraction of radial glia dropped and the proportion of neuronal progeny increased to approximately 60% of the labeled cells. Tadpoles provided with short-term visual enhancement generated significantly more neurons, with a corresponding decrease in cell proliferation. Together these results demonstrate that radial glial cells are neural progenitors in the developing optic tectum and reveal that visual experience increases the proportion of neurons generated in an intact animal. PMID:22113462

Increasing Stem Cell Dose Promotes Posttransplant Immune Reconstitution.

PubMed

Xu, Ning; Shen, Sylvie; Dolnikov, Alla

2017-04-01

Umbilical cord blood (UCB) transplantation can provide a successful therapeutic option for patients that have no suitable related donor. UCB transplantation is often limited by the relatively small hematopoietic stem cell (HSC) numbers in UCB especially for adult recipients. Early neutrophil and platelet engraftment correlates with the stem cell numbers in UCB transplant. Compared to other HSC sources, immune reconstitution following UCB transplant is slower and complicated by increased frequency of opportunistic infections. The effect of HSC numbers in UCB transplant on immune reconstitution was not thoroughly examined. Using immunocompromised mice transplanted with purified UCB CD34+ stem cells, we have demonstrated that increasing the numbers of CD34+ cells in the transplant promotes hematopoietic and immune reconstitution. At early stages posttransplant, high stem cell dose generated relatively more B cells, while lower dose generated more myeloid and T cells. Thus, the size of the stem cell graft appears to modulate the differentiation potential of infused stem cells. In addition, increasing stem cell dose in the transplant improved CD8+ T cell development and delayed late memory T cell skewing in expense of naive T cells highlighting the importance of HSC dose to maintain the pool of naive T cells able to develop strong immune responses. Transplantation of ex vivo expanded CD34+ cells did not promote, but rather delayed immune reconstitution suggesting the loss of primitive lymphoid precursor cells during ex vivo expansion.

Mirna biogenesis pathway is differentially regulated during adipose derived stromal/stem cell differentiation.

PubMed

Martin, E C; Qureshi, A T; Llamas, C B; Burow, M E; King, A G; Lee, O C; Dasa, V; Freitas, M A; Forsberg, J A; Elster, E A; Davis, T A; Gimble, J M

2018-02-07

Stromal/stem cell differentiation is controlled by a vast array of regulatory mechanisms. Included within these are methods of mRNA gene regulation that occur at the level of epigenetic, transcriptional, and/or posttranscriptional modifications. Current studies that evaluate the posttranscriptional regulation of mRNA demonstrate microRNAs (miRNAs) as key mediators of stem cell differentiation through the inhibition of mRNA translation. miRNA expression is enhanced during both adipogenic and osteogenic differentiation; however, the mechanism by which miRNA expression is altered during stem cell differentiation is less understood. Here we demonstrate for the first time that adipose-derived stromal/stem cells (ASCs) induced to an adipogenic or osteogenic lineage have differences in strand preference (-3p and -5p) for miRNAs originating from the same primary transcript. Furthermore, evaluation of miRNA expression in ASCs demonstrates alterations in both miRNA strand preference and 5'seed site heterogeneity. Additionally, we show that during stem cell differentiation there are alterations in expression of genes associated with the miRNA biogenesis pathway. Quantitative RT-PCR demonstrated changes in the Argonautes (AGO1-4), Drosha, and Dicer at intervals of ASC adipogenic and osteogenic differentiation compared to untreated ASCs. Specifically, we demonstrated altered expression of the AGOs occurring during both adipogenesis and osteogenesis, with osteogenesis increasing AGO1-4 expression and adipogenesis decreasing AGO1 gene and protein expression. These data demonstrate changes to components of the miRNA biogenesis pathway during stromal/stem cell differentiation. Identifying regulatory mechanisms for miRNA processing during ASC differentiation may lead to novel mechanisms for the manipulation of lineage differentiation of the ASC through the global regulation of miRNA as opposed to singular regulatory mechanisms.

IGF-1 Regulates Cyr61 Induced Breast Cancer Cell Proliferation and Invasion

PubMed Central

Sarkissyan, Suren; Sarkissyan, Marianna; Wu, Yanyuan; Cardenas, Jessica; Koeffler, H. Phillip; Vadgama, Jaydutt V.

2014-01-01

Background Studies from our laboratory and others have shown that cysteine-rich 61 (Cyr61) may be involved in tumor proliferation and invasion. In earlier studies, we demonstrated increased insulin-like growth factor-I (IGF-1) is associated with breast tumor formation and poor clinical outcomes. In our current study we have investigated IGF-1 regulation of Cyr61 and whether targeting IGF-1 could inhibit Cyr61 induced tumor growth and proliferation. Methods Several ATCC derived normal and breast cancer cell lines were used in this study: MDA-MB231, BT474, MCF-7, and SKBR3. We also tested cells stably transfected in our laboratory with active Akt1 (pAkt; SKBR3/AA and MCF-7/AA) and dominant negative Akt1 (SKBR3/DN and MCF-7/DN). In addition, we used MCF-7 cells transfected with full length Cyr61 (CYA). Monolayer cultures treated with IGF-1 were analyzed for Cyr61 expression by RT-PCR and immunohistochemical staining. Migration assays and MTT based proliferation assays were used to determine invasive characteristics in response to IGF-1/Cyr61 activation. Results Cells with activated Akt have increased levels of Cyr61. Conversely, cells with inactive Akt have decreased levels of Cyr61. IGF-1 treatment increased Cyr61 expression significantly and cells with high level of Cyr61 demonstrate increased invasiveness and proliferation. Cyr61 overexpression and activation led to decrease in E-cadherin and decrease in FOXO1. Inhibition of the PI3K and MAPK pathways resulted in significant decrease in invasiveness and proliferation, most notably in the PI3K pathway inhibited cells. Conclusion The findings of this study show that IGF-1 upregulates Cyr61 primarily through activation of the Akt-PI3K pathway. IGF-1 induced MAPK plays a partial role. Increase in Cyr61 leads to increase in breast cancer cell growth and invasion. Hence, targeting Cyr61 and associated pathways may offer an opportunity to inhibit IGF-1 mediated Cyr61 induced breast cancer growth and invasion. PMID:25062088

The interaction between cytotrophoblasts and their derived tumor cells.

PubMed

Rachmilewitz, J; Goshen, R; Elkin, M; Gonik, B; Neaman, Z; Giloh, H; Strauss, B; Komitowsky, D; de Groot, N; Hochberg, A

1995-06-01

Previous experiments demonstrated that human cytotrophoblasts and cells of the choriocarcinoma cell line JAr interact in vitro. As a result of this interaction there is an increased synthesis of CG and hPL, probably as a result of the increased CG and hPL synthesis by the cytotrophoblasts. In the present investigation we studied this interaction in greater detail and found that both cytotrophoblasts and JAr cells undergo changes in their biological properties as a result of this interaction. JAr cells and cytotrophoblasts cocultured for 72 hr were fractionated according to their size by centrifugal elutriation. The number of cells in the fraction which contain the largest cells was very significantly increased as a result of the coculture. This increase was due to an increase in the number of cells of both cell types. This fraction was the most active one in the synthesis of CG and hPL. The synthesis of DNA by the JAr nuclei in this fraction of the cocultured cells was almost completely inhibited but in the parallel fraction of the JAr cells cultivated alone the level of DNA synthesis was equal to that of all other JAr cell fractions. Heterokaryons are formed in the coculture. In these heterokaryons a factor which inhibits DNA synthesis in the cytotrophoblasts may inhibit DNA synthesis in JAr nuclei and at least be partly responsible for the inhibition of DNA synthesis observed.

Enzymatic cell wall degradation of Chlorella vulgaris and other microalgae for biofuels production.

PubMed

Gerken, Henri G; Donohoe, Bryon; Knoshaug, Eric P

2013-01-01

Cell walls of microalgae consist of a polysaccharide and glycoprotein matrix providing the cells with a formidable defense against its environment. We characterized enzymes that can digest the cell wall and weaken this defense for the purpose of protoplasting or lipid extraction. A growth inhibition screen demonstrated that chitinase, lysozyme, pectinase, sulfatase, β-glucuronidase, and laminarinase had the broadest effect across the various Chlorella strains tested and also inhibited Nannochloropsis and Nannochloris strains. Chlorella is typically most sensitive to chitinases and lysozymes, both enzymes that degrade polymers containing N-acetylglucosamine. Using a fluorescent DNA stain, we developed rapid methodology to quantify changes in permeability in response to enzyme digestion and found that treatment with lysozyme in conjunction with other enzymes has a drastic effect on cell permeability. Transmission electron microscopy of enzymatically treated Chlorella vulgaris indicates that lysozyme degrades the outer surface of the cell wall and removes hair-like fibers protruding from the surface, which differs from the activity of chitinase. This action on the outer surface of the cell causes visible protuberances on the cell surface and presumably leads to the increased settling rate when cells are treated with lysozyme. We demonstrate radical ultrastructural changes to the cell wall in response to treatment with various enzyme combinations which, in some cases, causes a greater than twofold increase in the thickness of the cell wall. The enzymes characterized in this study should prove useful in the engineering and extraction of oils from microalgae.

Mcl1 regulates the terminal mitosis of neural precursor cells in the mammalian brain through p27Kip1.

PubMed

Hasan, S M Mahmudul; Sheen, Ashley D; Power, Angela M; Langevin, Lisa Marie; Xiong, Jieying; Furlong, Michael; Day, Kristine; Schuurmans, Carol; Opferman, Joseph T; Vanderluit, Jacqueline L

2013-08-01

Cortical development requires the precise timing of neural precursor cell (NPC) terminal mitosis. Although cell cycle proteins regulate terminal mitosis, the factors that influence the cell cycle machinery are incompletely understood. Here we show in mice that myeloid cell leukemia 1 (Mcl1), an anti-apoptotic Bcl-2 protein required for the survival of NPCs, also regulates their terminal differentiation through the cell cycle regulator p27(Kip1). A BrdU-Ki67 cell profiling assay revealed that in utero electroporation of Mcl1 into NPCs in the embryonic neocortex increased NPC cell cycle exit (the leaving fraction). This was further supported by a decrease in proliferating NPCs (Pax6(+) radial glial cells and Tbr2(+) neural progenitors) and an increase in differentiating cells (Dcx(+) neuroblasts and Tbr1(+) neurons). Similarly, BrdU birth dating demonstrated that Mcl1 promotes premature NPC terminal mitosis giving rise to neurons of the deeper cortical layers, confirming their earlier birthdate. Changes in Mcl1 expression within NPCs caused concomitant changes in the levels of p27(Kip1) protein, a key regulator of NPC differentiation. Furthermore, in the absence of p27(Kip1), Mcl1 failed to induce NPC cell cycle exit, demonstrating that p27(Kip1) is required for Mcl1-mediated NPC terminal mitosis. In summary, we have identified a novel physiological role for anti-apoptotic Mcl1 in regulating NPC terminal differentiation.

An alternatively spliced CXCL16 isoform expressed by dendritic cells is a secreted chemoattractant for CXCR6+ cells.

PubMed

van der Voort, Robbert; Verweij, Viviènne; de Witte, Theo M; Lasonder, Edwin; Adema, Gosse J; Dolstra, Harry

2010-06-01

DC are professional APCs that initiate and regulate adaptive immune responses by interacting with naïve and memory T cells. Chemokines released by DC play an essential role in T cell recruitment and in the maintenance of antigen-specific T cell-DC conjugates. Here, we characterized the expression of the T cell-attracting chemokine CXCL16 by murine DC. We demonstrate that through alternative RNA splicing, DC not only express the previously characterized transmembrane CXCL16 isoform, which can be cleaved from the cell surface, but also a novel isoform lacking the transmembrane and cytoplasmic domains. Transfection of HEK293 cells shows that this novel isoform, termed CXCL16v, is not expressed on the cell membrane but is secreted as a protein of approximately 10 kDa. Quantitative PCR demonstrates that CXCL16v is broadly expressed in lymphoid and nonlymphoid tissues resembling the tissue distribution of DC. Indeed, CXCL16v mRNA is expressed significantly by spleen DC and BM-DC. Moreover, we show that mature DC have increased CXCL16v mRNA levels and express transmembrane and soluble CXCL16 proteins. Finally, we show that CXCL16v specifically attracts cells expressing the chemokine receptor CXCR6. Our data demonstrate that mature DC express secreted, transmembrane, and cleaved CXCL16 isoforms to recruit and communicate efficiently with CXCR6(+) lymphoid cells.

Osteopontin in Systemic Sclerosis and its Role in Dermal Fibrosis

PubMed Central

Wu, Minghua; Schneider, Daniel J.; Mayes, Maureen D; Assassi, Shervin; Arnett, Frank C.; Tan, Filemon K.; Blackburn, Michael R.; Agarwal, Sandeep K.

2012-01-01

Osteopontin (OPN) is a matricellular protein with proinflammatory and profibrotic properties. Previous reports demonstrate a role for OPN in wound healing and pulmonary fibrosis. Herein, we determined if OPN levels are increased in a large cohort of systemic sclerosis (SSc) patients and if OPN contributes dermal fibrosis. Plasma OPN levels were increased in SSc patients, including patients with limited and diffuse disease, compared to healthy controls. Immunohistology demonstrated OPN on fibroblast-like and inflammatory cells in SSc skin and lesional skin from mice in the bleomycin-induced dermal fibrosis model. OPN deficient (OPN−/−) mice developed less dermal fibrosis compared to wild-type mice in the bleomycin-induced dermal fibrosis model. Additional in vivo studies demonstrated that lesional skin from OPN−/− mice had fewer Mac-3+ cells, fewer myofibroblasts, decreased TGF-beta (TGFβ) and genes in the TGFβ pathway and decreased numbers of cells expressing phosphorylated SMAD2 (pSMAD) and ERK. In vitro, OPN−/− dermal fibroblasts had decreased migratory capacity but similar phosphorylation of SMAD2 by TGFβ. Finally, TGFβ production by OPN deficient macrophages was reduced compared to wild type. These data demonstrate an important role for OPN in the development of dermal fibrosis and suggest that OPN may be a novel therapeutic target in SSc. PMID:22402440

[Signal transudation pathways in parietal cells of the gastric mucosa in experimental stomach ulcer].

PubMed

Ostapchenko, L I; Drobins'ka, O V; Chaĭka, V O; Bohun, L I; Bohdanova, O V; Kot, L I; Haĭda, L M

2009-01-01

The goal of the presented work was the research of signal transduction mechanism in the rat gastric parietal cells under stomach ulcer conditions. In these cells activation of adenylate cyclase (increase of cAMP level and proteinkinase A activity) and phosphoinositide (increases [Ca2+]i; cGMP and phoshatidylinocitole levels; proteinkinase C, proteinkinase G, and calmodulin-dependent-proteinkinase activity) of signals pathway was shown. An increase of plasma membrane phospholipids (PC, PS, PE, PI, LPC) level was shown. Under conditions of influence of the stress factor the membran enzymes activity (H+, K+ -ATPase, 5'-AMPase, Na+, K+ -ATPase, Ca2+, Mg2+ -ATPase and H+, K+ -ATPase) was considerably increased. The intensification of lipid peroxidation processes in rats was demonstrated.

Differential Regulation of Mouse B Cell Development by Transforming Growth Factor β1

PubMed Central

Kaminski, Denise A.; Letterio, John J.; Burrows, Peter D.

2002-01-01

Transforming growth factor β (TGFβ) can inhibit the in vitro proliferation, survival and differentiation of B cell progenitors, mature B lymphocytes and plasma cells. Here we demonstrate unexpected, age-dependent reductions in the bone marrow (BM) B cell progenitors and immature B cells in TGFβ1-/- mice. To evaluate TGFβ responsiveness during normal B lineage development, cells were cultured in interleukin 7 (IL7)±TGFβ. Picomolar doses of TGFβ1 reduced pro-B cell recoveries at every timepoint. By contrast, the pre-B cells were initially reduced in number, but subsequently increased compared to IL7 alone, resulting in a 4-fold increase in the growth rate for the pre-B cell population. Analysis of purified BM sub-populations indicated that pro-B cells and the earliest BP1- pre-B cells were sensitive to the inhibitory effects of TGFβ1. However, the large BP1+ pre-B cells, although initially reduced, were increased in number at days 5 and 7 of culture. These results indicate that TGFβ1 is important for normal B cell development in vivo, and that B cell progenitors are differentially affected by the cytokine according to their stage of differentiation. PMID:12739785

Experiments with suspended cells on the Space Shuttle

NASA Technical Reports Server (NTRS)

Morrison, D. R.; Chapes, S. K.; Guikema, J. A.; Spooner, B. S.; Lewis, M. L.

1992-01-01

Spaceflight experiments since 1981 have demonstrated that certain cell functions are altered by micro-g. Biophysical models suggest that cell membranes and organelles should not be affected directly by gravity, however, the chemical microenvironment surrounding the cell and molecular transport could be altered by reduced gravity. Most experiments have used suspended live cells in small chambers without stirring or medium exchange. Flight results include increased attachment of anchorage-dependent human cells to collagen coated microcarriers, reduced secretion of growth hormone from pituitary cells, decreased mitogenic response of lymphocytes, increased Interferon-alpha by lymphocytes, increased Interleukin-1 and Tumor Necrosis Factor secretion by macrophages. Related experiments on cells immediately postflight and on procaryotic cells have shown significant changes in secretory capacity, cell proliferation, differentiation and development. Postulated mechanism include altered cell-cell interactions, altered calcium ion transport, effects on cell cytoskeleton, transport of transmitters and interactions with receptors. The discussion includes use of new molecular methods, considerations for cell environmental control and a preview of several experiments planned for the Shuttle and Spacelab flights to study the basic effects of microgravity on cellular physiology and potential interactions of spaceflight with radiation damage and cellular repair mechanisms.

Dysregulation in microRNA Expression Is Associated with Alterations in Immune Functions in Combat Veterans with Post-Traumatic Stress Disorder

PubMed Central

Zhou, Juhua; Nagarkatti, Prakash; Zhong, Yin; Ginsberg, Jay P.; Singh, Narendra P.; Zhang, Jiajia; Nagarkatti, Mitzi

2014-01-01

While the immunological dysfunction in combat Veterans with post-traumatic stress disorder (PTSD) has been well documented, the precise mechanisms remain unclear. The current study evaluated the role of microRNA (miR) in immunological dysfunction associated with PTSD. The presence of peripheral blood mononuclear cells (PBMC) and various lymphocyte subsets in blood collected from PTSD patients were analyzed. Our studies demonstrated that the numbers of both PBMC and various lymphocyte subsets increased significantly in PTSD patients. When T cells were further analyzed, the percentage of Th1 cells and Th17 cells increased, regulatory T cells(Tregs) decreased, while Th2 cells remained unaltered in PTSD patients. These data correlated with increased plasma levels of IFN-γ and IL-17 while IL-4 showed no significant change. The increase in PBMC counts, Th1 and Th17 cells seen in PTSD patients correlated with the clinical scores. High-throughput analysis of PBMCs for 1163 miRs showed that the expression of a significant number of miRs was altered in PTSD patients. Pathway analysis of dysregulated miRs seen in PTSD patients revealed relationship between selected miRNAs and genes that showed direct/indirect role in immunological signaling pathways consistent with the immunological changes seen in these patients. Of interest was the down-regulation of miR-125a in PTSD, which specifically targeted IFN-γ production. Together, the current study demonstrates for the first time that PTSD was associated with significant alterations in miRNAs, which may promote pro-inflammatory cytokine profile. Such epigenetic events may provide useful tools to identify potential biomarkers for diagnosis, and facilitate therapy of PTSD. PMID:24759737

Dysregulation in microRNA expression is associated with alterations in immune functions in combat veterans with post-traumatic stress disorder.

PubMed

Zhou, Juhua; Nagarkatti, Prakash; Zhong, Yin; Ginsberg, Jay P; Singh, Narendra P; Zhang, Jiajia; Nagarkatti, Mitzi

2014-01-01

While the immunological dysfunction in combat Veterans with post-traumatic stress disorder (PTSD) has been well documented, the precise mechanisms remain unclear. The current study evaluated the role of microRNA (miR) in immunological dysfunction associated with PTSD. The presence of peripheral blood mononuclear cells (PBMC) and various lymphocyte subsets in blood collected from PTSD patients were analyzed. Our studies demonstrated that the numbers of both PBMC and various lymphocyte subsets increased significantly in PTSD patients. When T cells were further analyzed, the percentage of Th1 cells and Th17 cells increased, regulatory T cells(Tregs) decreased, while Th2 cells remained unaltered in PTSD patients. These data correlated with increased plasma levels of IFN-γ and IL-17 while IL-4 showed no significant change. The increase in PBMC counts, Th1 and Th17 cells seen in PTSD patients correlated with the clinical scores. High-throughput analysis of PBMCs for 1163 miRs showed that the expression of a significant number of miRs was altered in PTSD patients. Pathway analysis of dysregulated miRs seen in PTSD patients revealed relationship between selected miRNAs and genes that showed direct/indirect role in immunological signaling pathways consistent with the immunological changes seen in these patients. Of interest was the down-regulation of miR-125a in PTSD, which specifically targeted IFN-γ production. Together, the current study demonstrates for the first time that PTSD was associated with significant alterations in miRNAs, which may promote pro-inflammatory cytokine profile. Such epigenetic events may provide useful tools to identify potential biomarkers for diagnosis, and facilitate therapy of PTSD.

Inhibition of SIRT1 by a small molecule induces apoptosis in breast cancer cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kalle, Arunasree M., E-mail: arunasreemk@ilsresearch.org; Mallika, A.; Badiger, Jayasree

2010-10-08

Research highlights: {yields} Novel small molecule SIRT1 inhibitor better than sirtinol. {yields} IC{sub 50} 500 nM. {yields} Specific tumor cytotoxicity towards breast cancer cells. {yields} Restoration of H3K9 acetylation levels to baseline when co-treated with SIRT1 activator (Activator X) and inhibitor (ILS-JGB-1741). -- Abstract: Overexpression of SIRT1, a NAD{sup +}-dependent class III histone deacetylases (HDACs), is implicated in many cancers and therefore could become a promising antitumor target. Here we demonstrate a small molecule SIRT1 inhibitor, ILS-JGB-1741(JGB1741) with potent inhibitory effects on the proliferation of human metastatic breast cancer cells, MDA-MB 231. The molecule has been designed using medicinal chemistrymore » approach based on known SIRT1 inhibitor, sirtinol. The molecule showed a significant inhibition of SIRT1 activity compared to sirtinol. Studies on the antitumor effects of JGB on three different cancer cell lines, K562, HepG2 and MDA-MB 231 showed an IC{sub 50} of 1, 10 and 0.5 {mu}M, respectively. Further studies on MDA-MB 231 cells showed a dose-dependent increase in K9 and K382 acetylation of H3 and p53, respectively. Results also demonstrated that JGB1741-induced apoptosis is associated with increase in cytochrome c release, modulation in Bax/Bcl2 ratio and cleavage of PARP. Flowcytometric analysis showed increased percentage of apoptotic cells, decrease in mitochondrial membrane potential and increase in multicaspase activation. In conclusion, the present study indicates the potent apoptotic effects of JGB1741 in MDA-MB 231 cells.« less

Micro Solar Cells with Concentration and Light Trapping Optics

NASA Astrophysics Data System (ADS)

Li, Lanfang; Breuckner, Eric; Corcoran, Christopher; Yao, Yuan; Xu, Lu; Nuzzo, Ralph

2013-03-01

Compared with conventional bulk plate semiconductor solar cells, micro solar cells provide opportunity for novel design geometry and provide test bed for light trapping at the device level as well as module level. Surface recombination, however, will have to be addressed properly as the much increased surface area due to the reduced dimension is more prominent in these devices than conventional solar cells. In this poster, we present experimental demonstration of silicon micro solar cells with concentration and light trapping optics. Silicon micro solar cell with optimized surface passivation and doping profile that exhibit high efficiency is demonstrated. Effective incorporation of high quantum yield fluorescent centers in the polymer matrix into which micro solar cell was encapsulated was investigated for luminescent solar concentration application. Micro-cell on a semi-transparent, nanopatterned reflector formed by soft-imprint lithography was investigated for near field effect related solar conversion performance enhancement. This work is supported by the DOE `Light-Material Interactions in Energy Conversion' Energy Frontier Research Center under grant DE-SC0001293

Glucose deprivation activates a metabolic and signaling amplification loop leading to cell death

PubMed Central

Graham, Nicholas A; Tahmasian, Martik; Kohli, Bitika; Komisopoulou, Evangelia; Zhu, Maggie; Vivanco, Igor; Teitell, Michael A; Wu, Hong; Ribas, Antoni; Lo, Roger S; Mellinghoff, Ingo K; Mischel, Paul S; Graeber, Thomas G

2012-01-01

The altered metabolism of cancer can render cells dependent on the availability of metabolic substrates for viability. Investigating the signaling mechanisms underlying cell death in cells dependent upon glucose for survival, we demonstrate that glucose withdrawal rapidly induces supra-physiological levels of phospho-tyrosine signaling, even in cells expressing constitutively active tyrosine kinases. Using unbiased mass spectrometry-based phospho-proteomics, we show that glucose withdrawal initiates a unique signature of phospho-tyrosine activation that is associated with focal adhesions. Building upon this observation, we demonstrate that glucose withdrawal activates a positive feedback loop involving generation of reactive oxygen species (ROS) by NADPH oxidase and mitochondria, inhibition of protein tyrosine phosphatases by oxidation, and increased tyrosine kinase signaling. In cells dependent on glucose for survival, glucose withdrawal-induced ROS generation and tyrosine kinase signaling synergize to amplify ROS levels, ultimately resulting in ROS-mediated cell death. Taken together, these findings illustrate the systems-level cross-talk between metabolism and signaling in the maintenance of cancer cell homeostasis. PMID:22735335

Rim Pathway-Mediated Alterations in the Fungal Cell Wall Influence Immune Recognition and Inflammation.

PubMed

Ost, Kyla S; Esher, Shannon K; Leopold Wager, Chrissy M; Walker, Louise; Wagener, Jeanette; Munro, Carol; Wormley, Floyd L; Alspaugh, J Andrew

2017-01-31

Compared to other fungal pathogens, Cryptococcus neoformans is particularly adept at avoiding detection by innate immune cells. To explore fungal cellular features involved in immune avoidance, we characterized cell surface changes of the C. neoformans rim101Δ mutant, a strain that fails to organize and shield immunogenic epitopes from host detection. These cell surface changes are associated with an exaggerated, detrimental inflammatory response in mouse models of infection. We determined that the disorganized strain rim101Δ cell wall increases macrophage detection in a contact-dependent manner. Using biochemical and microscopy methods, we demonstrated that the rim101Δ strain shows a modest increase in the levels of both cell wall chitin and chitosan but that it shows a more dramatic increase in chito-oligomer exposure, as measured by wheat germ agglutinin staining. We also created a series of mutants with various levels of cell wall wheat germ agglutinin staining, and we demonstrated that the staining intensity correlates with the degree of macrophage activation in response to each strain. To explore the host receptors responsible for recognizing the rim101Δ mutant, we determined that both the MyD88 and CARD9 innate immune signaling proteins are involved. Finally, we characterized the immune response to the rim101Δ mutant in vivo, documenting a dramatic and sustained increase in Th1 and Th17 cytokine responses. These results suggest that the Rim101 transcription factor actively regulates the C. neoformans cell wall to prevent the exposure of immune stimulatory molecules within the host. These studies further explored the ways in which immune cells detect C. neoformans and other fungal pathogens by mechanisms that include sensing N-acetylglucosamine-containing structures, such as chitin and chitosan. Infectious microorganisms have developed many ways to avoid recognition by the host immune system. For example, pathogenic fungi alter their cell surfaces to mask immunogenic epitopes. We have created a fungal strain with a targeted mutation in a pH response pathway that is unable to properly organize its cell wall, resulting in a dramatic immune reaction during infection. This mutant cell wall is defective in hiding important cell wall components, such as the chito-oligomers chitin and chitosan. By creating a series of cell wall mutants, we demonstrated that the degree of chito-oligomer exposure correlates with the intensity of innate immune cell activation. This activation requires a combination of host receptors to recognize and respond to these infecting microorganisms. Therefore, these experiments explored host-pathogen interactions that determine the degree of the subsequent inflammatory response and the likely outcome of infection. Copyright © 2017 Ost et al.

Metabolic Adaptation to Chronic Inhibition of Mitochondrial Protein Synthesis in Acute Myeloid Leukemia Cells

PubMed Central

Jhas, Bozhena; Sriskanthadevan, Shrivani; Skrtic, Marko; Sukhai, Mahadeo A.; Voisin, Veronique; Jitkova, Yulia; Gronda, Marcela; Hurren, Rose; Laister, Rob C.; Bader, Gary D.; Minden, Mark D.; Schimmer, Aaron D.

2013-01-01

Recently, we demonstrated that the anti-bacterial agent tigecycline preferentially induces death in leukemia cells through the inhibition of mitochondrial protein synthesis. Here, we sought to understand mechanisms of resistance to tigecycline by establishing a leukemia cell line resistant to the drug. TEX leukemia cells were treated with increasing concentrations of tigecycline over 4 months and a population of cells resistant to tigecycline (RTEX+TIG) was selected. Compared to wild type cells, RTEX+TIG cells had undetectable levels of mitochondrially translated proteins Cox-1 and Cox-2, reduced oxygen consumption and increased rates of glycolysis. Moreover, RTEX+TIG cells were more sensitive to inhibitors of glycolysis and more resistant to hypoxia. By electron microscopy, RTEX+TIG cells had abnormally swollen mitochondria with irregular cristae structures. RNA sequencing demonstrated a significant over-representation of genes with binding sites for the HIF1α:HIF1β transcription factor complex in their promoters. Upregulation of HIF1α mRNA and protein in RTEX+TIG cells was confirmed by Q-RTPCR and immunoblotting. Strikingly, upon removal of tigecycline from RTEX+TIG cells, the cells re-established aerobic metabolism. Levels of Cox-1 and Cox-2, oxygen consumption, glycolysis, mitochondrial mass and mitochondrial membrane potential returned to wild type levels, but HIF1α remained elevated. However, upon re-treatment with tigecycline for 72 hours, the glycolytic phenotype was re-established. Thus, we have generated cells with a reversible metabolic phenotype by chronic treatment with an inhibitor of mitochondrial protein synthesis. These cells will provide insight into cellular adaptations used to cope with metabolic stress. PMID:23520503

Metabolic adaptation to chronic inhibition of mitochondrial protein synthesis in acute myeloid leukemia cells.

PubMed

Jhas, Bozhena; Sriskanthadevan, Shrivani; Skrtic, Marko; Sukhai, Mahadeo A; Voisin, Veronique; Jitkova, Yulia; Gronda, Marcela; Hurren, Rose; Laister, Rob C; Bader, Gary D; Minden, Mark D; Schimmer, Aaron D

2013-01-01

Recently, we demonstrated that the anti-bacterial agent tigecycline preferentially induces death in leukemia cells through the inhibition of mitochondrial protein synthesis. Here, we sought to understand mechanisms of resistance to tigecycline by establishing a leukemia cell line resistant to the drug. TEX leukemia cells were treated with increasing concentrations of tigecycline over 4 months and a population of cells resistant to tigecycline (RTEX+TIG) was selected. Compared to wild type cells, RTEX+TIG cells had undetectable levels of mitochondrially translated proteins Cox-1 and Cox-2, reduced oxygen consumption and increased rates of glycolysis. Moreover, RTEX+TIG cells were more sensitive to inhibitors of glycolysis and more resistant to hypoxia. By electron microscopy, RTEX+TIG cells had abnormally swollen mitochondria with irregular cristae structures. RNA sequencing demonstrated a significant over-representation of genes with binding sites for the HIF1α:HIF1β transcription factor complex in their promoters. Upregulation of HIF1α mRNA and protein in RTEX+TIG cells was confirmed by Q-RTPCR and immunoblotting. Strikingly, upon removal of tigecycline from RTEX+TIG cells, the cells re-established aerobic metabolism. Levels of Cox-1 and Cox-2, oxygen consumption, glycolysis, mitochondrial mass and mitochondrial membrane potential returned to wild type levels, but HIF1α remained elevated. However, upon re-treatment with tigecycline for 72 hours, the glycolytic phenotype was re-established. Thus, we have generated cells with a reversible metabolic phenotype by chronic treatment with an inhibitor of mitochondrial protein synthesis. These cells will provide insight into cellular adaptations used to cope with metabolic stress.

Three immobilized-cell columnar bioreactors for enhanced production of commodity chemicals

DOE Office of Scientific and Technical Information (OSTI.GOV)

Davison, B.H.; Scott, C.D.; Kaufman, E.N.

1993-07-01

Immobilized-cell fluidized-bed bioreactors (FBRS) can be used with a variety of fermentations to increase production of fuels, solvents, organic acids, and other fermentation products. Part of the increased rates and yields are due to the immobilization of the biocatalyst at high concentrations. This FBR system with immobilized Zymomonas mobiles increased ethanol productivity more than tenfold with 99% conversion and near stoichiometric yields. FBRs also offer several additional modes of operation for simultaneous fermentation and separation to further increase production by removing the inhibitory products directly from the continuous fermentation. The production of lactic acid by immobilized Lactobacillus was augmented withmore » the addition and removal of solid adsorbent particles to the FBR. An immiscible organic extractant also was used to extract butanol from the acetone-butanol fermentation by Clostridium acetobutylicum. Demonstrations with these FBR systems have already shown definite advantages by improved overall product yields (decreasing feed costs) and by increased rates (decreasing capital and operating costs). Further demonstration and scale-up continue.« less

Three immobilized-cell columnar bioreactors for enhanced production of commodity chemicals

DOE Office of Scientific and Technical Information (OSTI.GOV)

Davison, B.H.; Scott, C.D.; Kaufman, E.N.

1993-12-31

Immobilized-cell fluidized-bed bioreactors (FBRs) can be used with a variety of fermentations to increase production of fuels, solvents, organic acids, and other fermentation products. Part of the increased rates and yields are due to the immobilization of the biocatalyst at high concentrations. This FBR system with immobilized Zymomonas mobilis increased ethanol productivity more than tenfold with 99% conversion and near stoichiometric yields. FBRs also offer several additional modes of operation for simultaneous fermentation and separation to further increase production by removing the inhibitory products directly from the continuous fermentation. The production of lactic acid by immobilized Lactobacillus was augmented withmore » the addition and removal of solid adsorbent particles to the FBR. An immiscible organic extractant also was used to extract butanol from the acetone-butanol fermentation by Clostridium acetobutylicum. Demonstrations with these FBR systems have already shown definite advantages by improved overall product yields (decreasing feed costs) and by increased rates (decreasing capital and operating costs). Further demonstration and scale-up continue.« less