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Sample records for nonmalignant cells nih-3t3

  1. Cytotoxicity of Alpinia galanga Rhizome Crude Extract on NIH-3T3 Cells

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    Ferry Sandra

    2017-04-01

    Full Text Available BACKGROUND: Alpinia galanga (A. galanga was reported as a potential medicinal source due to its wide effect. A. galanga rhizome crude extract (ARCE was reported to have high cytotoxic effect in cancer cells, but low in normal cells. However half maximal inhibitory concentration (IC50 of ARCE is not clearly known yet. Hence, current study was conducted to investigate the IC50 of ARCE in normal standard fibroblast cell line, NIH-3T3 cells. METHODS: Rhizomes of A. galanga were collected, peeled, dried, milled and weighed. Extraction was performed using maceration method, then filtered and evaporated. ARCE with various concentrations were applied in NIH-3T3 cells for 24 or 48 hours. Cells were documented and counted with 3-(4,5-dimethylthiazol-2-yl-2,5-Diphenyltetrazolium bromide (MTT assay. RESULTS: Five hundreds grams of simplicia were macerated with ethanol and evaporated, 1 mg/mL crude extract with total volume of 114 mL was obtained. By addition of ARCE in NIH-3T3 cell culture, number of NIH-3T3 cells were shown less when treated with higher concentration of ARCE. Cell numbers of 0, 3.125, 6.25, 12.5, 25 and 50% ARCE treatment for 24 hours are 11,531, 11,352, 10,920, 10,365, 9,471, 8,360, respectively, meanwhile for 48 hours are 13,219, 12,686, 12,278, 11,390, 10,279, 8,390, respectively. CONCLUSION: IC50 of ARCE in 24 hours treatment was 620.5 mg/mL, while in 48 hours treatment was 666.6 mg/mL. Hence, ARCE is suggested to have low cytotoxic effect in NIH-3T3 cells. KEYWORDS: Alpinia galanga, ginger, extract, cytotoxic, MTT, NIH-3T3

  2. Isolation and characterization of NIH 3T3 cells expressing polyomavirus small T antigen

    International Nuclear Information System (INIS)

    Noda, T.; Satake, M.; Robins, T.; Ito, Y.

    1986-01-01

    The polyomavirus small T-antigen gene, together with the polyomavirus promoter, was inserted into retrovirus vector pGV16 which contains the Moloney sarcoma virus long terminal repeat and neomycin resistance gene driven by the simian virus 40 promoter. This expression vector, pGVST, was packaged into retrovirus particles by transfection of PSI2 cells which harbor packaging-defective murine retrovirus genome. NIH 3T3 cells were infected by this replication-defective retrovirus containing pGVST. Of the 15 G418-resistant cell clones, 8 express small T antigen at various levels as revealed by immunoprecipitation. A cellular protein with an apparent molecular weight of about 32,000 coprecipitates with small T antigen. Immunofluorescent staining shows that small T antigen is mainly present in the nuclei. Morphologically, cells expressing small T antigen are indistinguishable from parental NIH 3T3 cells and have a microfilament pattern similar to that in parental NIH 3T3 cells. Cells expressing small T antigen form a flat monolayer but continue to grow beyond the saturation density observed for parental NIH 3T3 cells and eventually come off the culture plate as a result of overconfluency. There is some correlation between the level of expression of small T antigen and the growth rate of the cells. Small T-antigen-expressing cells form small colonies in soft agar. However, the proportion of cells which form these small colonies is rather small. A clone of these cells tested did not form tumors in nude mice within 3 months after inoculation of 10 6 cells per animal. Thus, present studies establish that the small T antigen of polyomavirus is a second nucleus-localized transforming gene product of the virus (the first one being large T antigen) and by itself has a function which is to stimulate the growth of NIH 3T3 cells beyond their saturation density in monolayer culture

  3. Expression of Nanog gene promotes NIH3T3 cell proliferation

    International Nuclear Information System (INIS)

    Zhang Jingyu; Wang Xia; Chen Bing; Suo Guangli; Zhao Yanhong; Duan Ziyuan; Dai Jianwu

    2005-01-01

    Cells are the functional elements in tissue engineering and regenerative medicine. A large number of cells are usually needed for these purposes. However, there are numbers of limitations for in vitro cell proliferation. Nanog is an important self-renewal determinant in embryonic stem cells. However, it remains unknown whether Nanog will influence the cell cycle and cell proliferation of mature cells. In this study, we expressed Nanog in NIH3T3 cells and showed that expression of Nanog in NIH3T3 promoted cells to enter into S phase and enhanced cell proliferation. This suggests that Nanog gene might function in a similar fashion in mature cells as in ES cells. In addition, it may provide an approach for in vitro cell expansion

  4. Drinking water disinfection byproduct iodoacetic acid induces tumorigenic transformation of NIH3T3 cells.

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    Wei, Xiao; Wang, Shu; Zheng, Weiwei; Wang, Xia; Liu, Xiaolin; Jiang, Songhui; Pi, Jingbo; Zheng, Yuxin; He, Gengsheng; Qu, Weidong

    2013-06-04

    Iodoacetic acid (IAA) and iodoform (IF) are unregulated iodinated disinfection byproducts (DBPs) found in drinking water. Their presence in the drinking water of China has not been documented. Recently, the carcinogenic potential of IAA and IF has been a concern because of their mutagenicity in bacteria and genotoxicity in mammalian cells. Therefore, we measured their concentrations in Shanghai drinking water and assessed their cytotoxicity, genotoxicity, and ability to transform NIH3T3 cells to tumorigenic lines. The concentrations of IAA and IF in Shanghai drinking water varied between summer and winter with maximum winter levels of 2.18 μg/L IAA and 0.86 μg/L IF. IAA with a lethal concentration 50 (LC50) of 2.77 μM exhibited more potent cytotoxicity in NIH3T3 cells than IF (LC50 = 83.37 μM). IAA, but not IF, induced a concentration-dependent DNA damage measured by γ-H2AX staining and increased tail moment in single-cell gel electrophoresis. Neither IAA nor IF increased micronucleus frequency. Prolonged exposure of NIH3T3 cells to IAA increased the frequencies of transformed cells with anchorage-independent growth and agglutination with concanavalin A. IAA-transformed cells formed aggressive fibrosarcomas after inoculation into Balb/c nude mice. This study demonstrated that IAA has a biological activity that is consistent with a carcinogen and human exposure should be of concern.

  5. Oxidative changes and apoptosis induced by 1800-MHz electromagnetic radiation in NIH/3T3 cells.

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    Hou, Qingxia; Wang, Minglian; Wu, Shuicai; Ma, Xuemei; An, Guangzhou; Liu, Huan; Xie, Fei

    2015-03-01

    To investigate the potential adverse effects of mobile phone radiation, we studied reactive oxygen species (ROS), DNA damage and apoptosis in mouse embryonic fibroblasts (NIH/3T3) after intermittent exposure (5 min on/10 min off, for various durations from 0.5 to 8 h) to an 1800-MHz GSM-talk mode electromagnetic radiation (EMR) at an average specific absorption rate of 2 W/kg. A 2',7'-dichlorofluorescin diacetate fluorescence probe was used to detect intracellular ROS levels, immunofluorescence was used to detect γH2AX foci as a marker for DNA damage, and flow cytometry was used to measure apoptosis. Our results showed a significant increase in intracellular ROS levels after EMR exposure and it reached the highest level at an exposure time of 1 h (p < 0.05) followed by a slight decrease when the exposure continued for as long as 8 h. No significant effect on the number of γH2AX was detected after EMR exposure. The percentage of late-apoptotic cells in the EMR-exposed group was significantly higher than that in the sham-exposed groups (p < 0.05). These results indicate that an 1800-MHz EMR enhances ROS formation and promotes apoptosis in NIH/3T3 cells.

  6. Lunasin-aspirin combination against NIH/3T3 cells transformation induced by chemical carcinogens.

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    Hsieh, Chia-Chien; Hernández-Ledesma, Blanca; de Lumen, Ben O

    2011-06-01

    Carcinogenesis is a multistage process involving a number of molecular pathways sensitive to intervention. Chemoprevention is defined as the use of natural and/or synthetic substances to block, reverse, or retard the process of carcinogenesis. To achieve greater inhibitory effects on cancer cells, combination of two or more chemopreventive agents is commonly considered as a better preventive and/or therapeutic strategy. Lunasin is a promising cancer preventive peptide identified in soybean and other seeds. Its efficacy has been demonstrated by both in vitro and in vivo models. This peptide has been found to inhibit human breast cancer MDA-MB-231 cells proliferation, suppressing cell cycle progress and inducing cell apoptosis. Moreover, lunasin potentiates the effects on these cells of different synthetic and natural compounds, such as aspirin and anacardic acid. This study explored the role of lunasin, alone and in combination with aspirin and anacardic acid on cell proliferation and foci formation of transformed NIH/3T3 cells induced by chemical carcinogens 7,12-dimethylbenz[a]anthracene or 3-methylcholanthrene. The results revealed that lunasin, acting as a single agent, inhibits cell proliferation and foci formation. When combined with aspirin, these effects were significantly increased, indicating that this combination might be a promising strategy to prevent/treat cancer induced by chemical carcinogens.

  7. A Nanodot Array Modulates Cell Adhesion and Induces an Apoptosis-Like Abnormality in NIH-3T3 Cells

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    Hung Yao-Ching

    2009-01-01

    Full Text Available Abstract Micro-structures that mimic the extracellular substratum promote cell growth and differentiation, while the cellular reaction to a nanostructure is poorly defined. To evaluate the cellular response to a nanoscaled surface, NIH 3T3 cells were grown on nanodot arrays with dot diameters ranging from 10 to 200 nm. The nanodot arrays were fabricated by AAO processing on TaN-coated wafers. A thin layer of platinum, 5 nm in thickness, was sputtered onto the structure to improve biocompatibility. The cells grew normally on the 10-nm array and on flat surfaces. However, 50-nm, 100-nm, and 200-nm nanodot arrays induced apoptosis-like events. Abnormality was triggered after as few as 24 h of incubation on a 200-nm dot array. For cells grown on the 50-nm array, the abnormality started after 72 h of incubation. The number of filopodia extended from the cell bodies was lower for the abnormal cells. Immunostaining using antibodies against vinculin and actin filament was performed. Both the number of focal adhesions and the amount of cytoskeleton were decreased in cells grown on the 100-nm and 200-nm arrays. Pre-coatings of fibronectin (FN or type I collagen promoted cellular anchorage and prevented the nanotopography-induced programed cell death. In summary, nanotopography, in the form of nanodot arrays, induced an apoptosis-like abnormality for cultured NIH 3T3 cells. The occurrence of the abnormality was mediated by the formation of focal adhesions.

  8. High-level expression of human insulin receptor cDNA in mouse NIH 3T3 cells

    International Nuclear Information System (INIS)

    Whittaker, J.; Okamoto, A.K.; Thys, R.; Bell, G.I.; Steiner, D.F.; Hofmann, C.A.

    1987-01-01

    In order to develop a simple, efficient system for the high-level expression of human insulin receptors in eukaryotic cells, a full-length human kidney insulin receptor cDNA was inserted into a bovine papilloma virus vector under the control of the mouse metallothionein promoter. After transfection of mouse NIH 3T3 cells with this construct, seven cell lines expressing insulin receptors were isolated; two cell lines had more than 10 6 receptors per cell. The cell line with the highest 125 I-insulin binding (NIH 3T3 HIR3.5) had 6 x 10 6 receptors with a K/sub d/ of 10 -9 M. This level was not dependent on exposure to metals but could be increased further to 2 x 10 7 receptors per cell by addition of sodium butyrate to the culture medium. The α and β subunits had apparent molecular weights of 147,000 and 105,000, respectively (compared to 135,000 and 95,000 in IM-9 human lymphocytes), values identical to those of the α and β subunits of the insulin receptors of nontransformed NIH 3T3 cells. This size difference was due to altered carbohydrate composition, as N-glycanase digestion reduced the apparent receptor subunit size of the transfected cells and IM-9 lymphocytes to identical values. The alteration in N-linked oligosaccharide composition could not be ascribed to differences in the kinetics of posttranslational processing of the insulin receptors, which was comparable to that of other cells studied. The basal rate of glycogen synthesis in the cells overexpressing insulin receptors was increased 4- to 5-fold compared with controls. Low levels of added insulin (0.1 nM) caused a 50% increase in the rate of glycogen synthesis

  9. DNA from radiation resistant human tumor cells transfers resistance to NIH/3T3 cells with varying degrees of penetrance

    International Nuclear Information System (INIS)

    Kasid, U.; Dritschilo, A.; Weichselbaum, R.

    1987-01-01

    Experimental evidence suggests that clinical radiation resistance may correlate with in vitro radiation survival parameters. Specifically, they isolated several cell lines from radioresistant head and neck carcinomas with D/sub 0/ values greater than 2 Gy. The authors co-transfected DNA from cell line SQ2OB (D/sub 0/ = 2.4 Gy) with the rhoSVNeO plasmid into NIH/3T3 cells (D/sub 0/ = 1.7 Gy). Antibiotic G418 resistant, transformed clones were isolated and confirmed by Southern blotting to contain human alu, as well as rhoSVNeO sequences. Screening for radiation resistance with 8Gy (Cs-137) revealed that 3 of 4 tested hybrid clones show a radiation survival intermediate between NIH/3T3 and SQ2OB. This suggests that radiation resistance is a dominant, transfectable phenotype of mammalian cells and can be expressed in more sensitive cells. Karyotyping of resistant hybrid clones shows the presence of double minute chromosomes. Secondary transfection results and experiments to clone the genetic factors responsible for radiation resistance are in progress and results will be reported

  10. Spindlin1, a novel nuclear protein with a role in the transformation of NIH3T3 cells

    International Nuclear Information System (INIS)

    Gao Yanhong; Yue Wen; Zhang Peng; Li Li; Xie Xiaoyan; Yuan Hongfeng; Chen Lin; Liu Daqing; Yan Fang; Pei Xuetao

    2005-01-01

    spindlin1, a novel human gene recently isolated by our laboratory, is highly homologous to mouse spindlin gene. In this study, we cloned cDNA full-length of this novel gene and send it to GenBank database as spindlin1 (Homo sapiens spindlin1) with Accession No. AF317228. In order to investigate the function of spindlin1, we studied further the subcellular localization of Spindlin1 protein and the effects of spindlin1 overexpression in NIH3T3 cells. The results showed that the fusion protein pEGFP-N1-spindlin1 was located in the nucleus and the C-terminal is correlated with nuclear localization of Spindlin1 protein. NIH3T3 cells which could stably express spindlin1 as a result of RT-PCR analysis compared with the control cells displayed a complete morphological change; made cell growth faster; and increased the percentage of cells in G 2 /M and S phase. Furthermore, overexpressed spindlin1 cells formed colonies in soft agar in vitro and formed tumors in nude mice. Our findings provide direct evidence that spindlin1 gene may contribute to tumorigenesis

  11. Genotoxicity of a Low-Dose Nitrosamine Mixture as Drinking Water Disinfection Byproducts in NIH3T3 Cells.

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    Wang, Hai-Yan; Qin, Ming; Dong, Lei; Lv, Jia-Ying; Wang, Xia

    2017-01-01

    N - nitrosamines (NAms), which can arise as byproducts of disinfection agents, are reportedly found in drinking water, and their potential carcinogenicity is a concern; however, little research exists regarding the genotoxicity or carcinogenicity of NAms exposure as a low-dose mixture. The three most common NAms components in China's drinking water are N -nitrosodimethylamine (NDMA), N -nitrosodiethylamine (NDEA) and N -nitrosomethylethylamine (NMEA). Thus, we measured the genotoxic and carcinogenic potential of these compounds and measured the cell cycle and gene expression. The data show that exposure to the NAms-mixture doubled the revertants in the TA98 and TA100 S. typhimurium strains and increased the DNA double-strand breaks and the micronuclear frequency in the NIH3T3 cells compared to a single exposure. After long-term NAms mixture exposure, a malignant transformation of NIH3T3 and a significantly increased G2/M distribution were observed. Furthermore, P53, CDK1, P38, CDC25A and CyclinB expressions were down-regulated in the NAms-mixture exposure group; however, P21 and GADD45A genes were up-regulated. Interestingly, the CHK1/CHK2 and CDC25A genes had two responses, depending on the NAms concentrations. Thus, we observed mutagenic, genotoxic and carcinogenic effects after a low-dose NAms-mixture exposure in drinking water, and DNA repair and apoptosis pathways may contribute to these adverse effects.

  12. Panax ginseng total protein promotes proliferation and secretion of collagen in NIH/3T3 cells by activating extracellular signal-related kinase pathway

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    Xuenan Chen

    2017-07-01

    Conclusion: Our studies suggest that GTP promoted proliferation and secretion of collagen in NIH/3T3 cells by activating the ERK signal pathway, which shed light on a potential function of GTP in promoting wound healing.

  13. High expression of the circadian gene mPer2 diminishes the radiosensitivity of NIH 3T3 cells

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    Chang, L.; Liu, Y.Y.; Zhu, B.; Li, Y.; Hua, H.; Wang, Y.H.; Zhang, J.; Jiang, Z.; Wang, Z.R. [Sichuan University, Chengdu (China). West China Medical Center. Health Ministry Key Lab. of Chronobiology], e-mail: wangzhengrong@126.com

    2009-10-15

    Period2 is a core circadian gene, which not only maintains the circadian rhythm of cells but also regulates some organic functions. We investigated the effects of mPeriod2 (mPer2) expression on radiosensitivity in normal mouse cells exposed to {sup 60}Co-{gamma}-rays. NIH 3T3 cells were treated with 12-O-tetradecanoyl phorbol-13-acetate (TPA) to induce endogenous mPer2 expression or transfected with pcDNA3.1(+)-mPer2 and irradiated with {sup 6}0Co-{gamma}-rays, and then analyzed by several methods such as flow cytometry, colony formation assay, RT-PCR, and immunohistochemistry. Flow cytometry and colony formation assay revealed that irradiated NIH 3T3 cells expressing high levels of mPer2 showed a lower death rate (TPA: 24 h 4.3% vs 12 h 6.8% and control 9.4%; transfection: pcDNA3.1-mPer2 3.7% vs pcDNA3.1 11.3% and control 8.2%), more proliferation and clonogenic survival (TPA: 121.7 {+-} 6.51 vs 66.0 {+-} 3.51 and 67.7 {+-} 7.37; transfection: 121.7 {+-} 6.50 vs 65.3 {+-} 3.51 and 69.0 {+-} 4.58) both when treated with TPA and transfected with mPer2. RT-PCR analysis showed an increased expression of bax, bcl-2, p53, cmyc, mre11, and nbs1, and an increased proportionality of bcl-2/bax in the irradiated cells at peak mPer2 expression compared with cells at trough mPer2 expression and control cells. However, no significant difference in rad50 expression was observed among the three groups of cells. Immunohistochemistry also showed increased protein levels of P53, BAX and proliferating cell nuclear antigen in irradiated cells with peak mPer2 levels. Thus, high expression of the circadian gene mPer2 may reduce the radiosensitivity of NIH 3T3 cells. For this effect, mPer2 may directly or indirectly regulate the expressions of cell proliferation- and apoptosis-related genes and DNA repair-related genes. (author)

  14. High expression of the circadian gene mPer2 diminishes the radiosensitivity of NIH 3T3 cells

    International Nuclear Information System (INIS)

    Chang, L.; Liu, Y.Y.; Zhu, B.; Li, Y.; Hua, H.; Wang, Y.H.; Zhang, J.; Jiang, Z.; Wang, Z.R.

    2009-01-01

    Period2 is a core circadian gene, which not only maintains the circadian rhythm of cells but also regulates some organic functions. We investigated the effects of mPeriod2 (mPer2) expression on radiosensitivity in normal mouse cells exposed to 60 Co-γ-rays. NIH 3T3 cells were treated with 12-O-tetradecanoyl phorbol-13-acetate (TPA) to induce endogenous mPer2 expression or transfected with pcDNA3.1(+)-mPer2 and irradiated with 6 0Co-γ-rays, and then analyzed by several methods such as flow cytometry, colony formation assay, RT-PCR, and immunohistochemistry. Flow cytometry and colony formation assay revealed that irradiated NIH 3T3 cells expressing high levels of mPer2 showed a lower death rate (TPA: 24 h 4.3% vs 12 h 6.8% and control 9.4%; transfection: pcDNA3.1-mPer2 3.7% vs pcDNA3.1 11.3% and control 8.2%), more proliferation and clonogenic survival (TPA: 121.7 ± 6.51 vs 66.0 ± 3.51 and 67.7 ± 7.37; transfection: 121.7 ± 6.50 vs 65.3 ± 3.51 and 69.0 ± 4.58) both when treated with TPA and transfected with mPer2. RT-PCR analysis showed an increased expression of bax, bcl-2, p53, cmyc, mre11, and nbs1, and an increased proportionality of bcl-2/bax in the irradiated cells at peak mPer2 expression compared with cells at trough mPer2 expression and control cells. However, no significant difference in rad50 expression was observed among the three groups of cells. Immunohistochemistry also showed increased protein levels of P53, BAX and proliferating cell nuclear antigen in irradiated cells with peak mPer2 levels. Thus, high expression of the circadian gene mPer2 may reduce the radiosensitivity of NIH 3T3 cells. For this effect, mPer2 may directly or indirectly regulate the expressions of cell proliferation- and apoptosis-related genes and DNA repair-related genes. (author)

  15. Tunable swelling of polyelectrolyte multilayers in cell culture media for modulating NIH-3T3 cells adhesion.

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    Qi, Wei; Cai, Peng; Yuan, Wenjing; Wang, Hua

    2014-11-01

    For polyelectrolyte multilayers (PEMs) assembled by the layer-by-layer (LbL) assembly technique, their nanostructure and properties can be governed by many parameters during the building process. Here, it was demonstrated that the swelling of the PEMs containing poly(diallyldimethylammonium chloride) (PDDA) and poly(sodium 4-styrenesulfonate) (PSS) in cell culture media could be tuned with changing supporting salt solutions during the assembly process. Importantly, the influence of the PEMs assembled in different salt solutions on NIH-3T3 cell adhesion was observable. Specifically, the cells could possess a higher affinity for the films assembled in low salt concentration (i.e. 0.15M NaCl) or no salt, the poorly swelling films in cell culture media, which was manifested by the large cell spreading area and focal adhesions. In contrast, those were assembled in higher salt concentration, highly swelling films in cell culture media, were less attractive for the fibroblasts. As a result, the cell adhesion behaviors may be manipulated by tailoring the physicochemical properties of the films, which could be performed by changing the assembly conditions such as supporting salt concentration. Such a finding might promise a great potential in designing desired biomaterials for tissue engineering and regenerative medicine. © 2014 Wiley Periodicals, Inc.

  16. Cell shrinkage as a signal to apoptosis in NIH 3T3 fibroblasts

    DEFF Research Database (Denmark)

    Friis, Martin B; Friborg, Christel R; Schneider, Linda

    2005-01-01

    Cell shrinkage is a hallmark of the apoptotic mode of programmed cell death, but it is as yet unclear whether a reduction in cell volume is a primary activation signal of apoptosis. Here we studied the effect of an acute elevation of osmolarity (NaCl or sucrose additions, final osmolarity 687...... accelerated in fibroblasts overexpressing Rac. Conversely, the activation of the extracellular signal-regulated kinase (Erk1/2) was initially significantly decreased. Subsequent to activation of p38, p53 was activated through serine-15 phosphorylation, and active p53 was translocated from the cytosol......: cellular shrinkage activates Rac, with activation of p38, followed by phosphorylation and nuclear translocation of p53, resulting in permeability increases and caspase-3 activation....

  17. Retroviral-mediated gene transfer of human phenylalanine hydroxylase into NIH 3T3 and hepatoma cells

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    Ledley, F.D.; Grenett, H.E.; McGinnis-Shelnutt, M.; Woo, S.L.C.

    1986-01-01

    Phenylketonuria (PKU) is caused by deficiency of the hepatic enzyme phenylalanine hydroxylase (PAH). A full-length human PAH cDNA sequence has been inserted into pzip-neoSV(X), which is a retroviral vector containing the bacterial neo gene. The recombinant has been transfected into Psi2 cells, which provide synthesis of the retroviral capsid. Recombinant virus was detected in the culture medium of the transfected Psi2 cells, which is capable of transmitting the human PAH gene into mouse NIH 3T3 cells by infection leading to stable incorporation of the recombinant provirus. Infected cells express PAH mRNA, immunoreactive PAH protein, and exhibit pterin-dependent phenylaline hydroxylase activity. The recombinant virus is also capable of infecting a mouse hepatoma cell line that does not normal synthesize PAH. PAH activity is present in the cellular extracts and the entire hydroxylation system is reconstituted in the hepatoma cells infected with the recombinant viruses. Thus, recombinant viruses containing human PAH cDNA provide a means for introducing functional PAH into mammalian cells of hepatic origin and can potentially be introduced into whole animals as a model for somatic gene therapy for PKU.

  18. Construction of pEgr.p-TNFα and its expression in NIH3T3 cells induced by ionizing irradiation

    International Nuclear Information System (INIS)

    Wu Congmei; Li Xiuyi; Liu Shuzheng

    2001-01-01

    Objective: To isolate and amplify Egr-1 promoter, construct pEgr.p-TNFα and study its response to different doses of ionizing radiation. Methods: Egr-1 promote was isolated from genomic DNA by PCR to construct pEgr.p-TNFα expression plasmid. Plasmids were transfected into NIH3T3 cells with liposome and the expression level of TNFα was detected by ELISA after irradiation with different doses of X-ray. Results: The sequence of Egr-1 promoter obtained was essentially same as reported. Egr-1 promoter and TNF α cDNA were inserted into expression vector correctly. Eight hours after irradiation with different doses of X-rays, the expression level of TNFα was higher than that of nonirradiated group (P< 0.05-0.001). Conclusion: Egr-1 promoter obtained can be activated by ionizing irradiation and regulate the expression of downstream gene. Low dose irradiation is for the first time found to be able to induce the expression of the downstream gene. the observation may be of potential significance in tumor therapy

  19. Effect of polysaccharides of ganoderma lucidum (PGL) on cell cycle and proliferation of NIH3T3 fibroblast exposed to γ-rays

    International Nuclear Information System (INIS)

    Ji Xiuqing; Wu Shiling; Zhou Yinghui; Xu Lan

    2001-01-01

    Objective: To study the effects of γ-irradiation on cell cycle and proliferation of fibroblasts and the radioprotective effect of PGL in term of cellular level. Methods: The cells in the control group and PGL (50 mg/L. 100 mg/L. 150 mg/L) groups were irradiated with 15 Gy γ-rays. The cell cycle and proliferation of NIH3T3 cells were determined by flow cytometry after irradiation within 12 h. Results: The percentage of G 0 -G 1 phase was 52.76% in the positive control group. Compared with the negative control group (90.9%), the proliferation of NIH3T3 was promoted significantly (P 0 -G 1 phases in the GPL (50 mg/L, 100 mg/L, 150 mg/L) groups were 66.17%, 71.73%, 76.10% respectively, the proliferation of NIH3T3 cells was inhibited by adding GPL. compared with the positive control group, in the 50 mg/L group P < 0.05, in the 100 mg/L and 150 mg/L groups P < 0.01. It was found that the apoptosis of NIH3T3 cells had little change after comparing non-irradiated group with the irradiated groups. Conclusion: Radiation with γ-rays at a dose of 15 Gy, can significantly stimulate proliferation of NIH3T3 cells, but no induced apoptosis can not be achieve within 12 h after irradiation. PGL can inhibit cell proliferation, so PGL has radioprotective activity on cell levels

  20. Antiproliferative activity of flower hexane extract obtained from Mentha spicata associated with Mentha rotundifolia against the MCF7, KB, and NIH/3T3 cell lines.

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    Nedel, Fernanda; Begnini, Karine; Carvalho, Pedro Henrique de Azambuja; Lund, Rafael Guerra; Beira, Fátima T A; Del Pino, Francisco Augusto B

    2012-11-01

    This study assessed the antiproliferative effect in vitro of the flower hexane extract obtained from Mentha spicata associated with Mentha rotundifolia against the human breast adenocarcinoma (MCF-7), human mouth epidermal carcinoma (KB), and mouse embryonic fibroblast (NIH 3T3) cell lines, using sulforhodamine B (SRB) assay. A cell density of 2×10(4)/well was seeded in 96-well plates, and samples at different concentrations ranging from 10 to 500 mg/mL were tested. The optical density was determined in an ELISA multiplate reader (Thermo Plate TP-Reader). Results demonstrated that the hexane extract presented antiproliferative activity against both the tumor cell lines KB and MCF-7, presenting a GI(50) (MCF-7=13.09 mg/mL), TGI (KB=37.76 mg/mL), and IL(50) (KB=291.07 mg/mL). Also, the hexane extract presented antiproliferative activity toward NIH 3T3 cells GI(50) (183.65 mg/mL), TGI (280.54 mg/mL), and IL(50) (384.59 mg/mL). The results indicate that the flower hexane extract obtained from M. spicata associated with M. rotundifolia presents an antineoplastic activity against KB and MCF-7, although an antiproliferative effect at a high concentration of the extract was observed toward NIH 3T3.

  1. Phenotypic and genotypic characteristics of novel mouse cell line (NIH/3T3-adapted human enterovirus 71 strains (EV71:TLLm and EV71:TLLmv.

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    Carla Bianca Luena Victorio

    Full Text Available Since its identification in 1969, Enterovirus 71 (EV71 has been causing periodic outbreaks of infection in children worldwide and most prominently in the Asia-Pacific Region. Understanding the pathogenesis of Enterovirus 71 (EV71 is hampered by the virus's inability to infect small animals and replicate in their derived in vitro cultured cells. This manuscript describes the phenotypic and genotypic characteristics of two selected EV71 strains (EV71:TLLm and EV71:TLLmv, which have been adapted to replicate in mouse-derived NIH/3T3 cells, in contrast to the original parental virus which is only able to replicate in primate cell lines. The EV71:TLLm strain exhibited productive infection in all primate and rodent cell lines tested, while EV71:TLLmv exhibited greater preference for mouse cell lines. EV71:TLLmv displayed higher degree of adaptation and temperature adaptability in NIH/3T3 cells than in Vero cells, suggesting much higher fitness in NIH/3T3 cells. In comparison with the parental EV71:BS strain, the adapted strains accumulated multiple adaptive mutations in the genome resulting in amino acid substitutions, most notably in the capsid-encoding region (P1 and viral RNA-dependent RNA polymerase (3D. Two mutations, E167D and L169F, were mapped to the VP1 canyon that binds the SCARB2 receptor on host cells. Another two mutations, S135T and K140I, were located in the VP2 neutralization epitope spanning amino acids 136-150. This is the first report of human EV71 with the ability to productively infect rodent cell lines in vitro.

  2. Riboflavin Depletion Promotes Tumorigenesis in HEK293T and NIH3T3 Cells by Sustaining Cell Proliferation and Regulating Cell Cycle-Related Gene Transcription.

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    Long, Lin; He, Jian-Zhong; Chen, Ye; Xu, Xiu-E; Liao, Lian-Di; Xie, Yang-Min; Li, En-Min; Xu, Li-Yan

    2018-05-07

    Riboflavin is an essential component of the human diet and its derivative cofactors play an established role in oxidative metabolism. Riboflavin deficiency has been linked with various human diseases. The objective of this study was to identify whether riboflavin depletion promotes tumorigenesis. HEK293T and NIH3T3 cells were cultured in riboflavin-deficient or riboflavin-sufficient medium and passaged every 48 h. Cells were collected every 5 generations and plate colony formation assays were performed to observe cell proliferation. Subcutaneous tumorigenicity assays in NU/NU mice were used to observe tumorigenicity of riboflavin-depleted HEK293T cells. Mechanistically, gene expression profiling and gene ontology analysis were used to identify abnormally expressed genes induced by riboflavin depletion. Western blot analyses, cell cycle analyses, and chromatin immunoprecipitation were used to validate the expression of cell cycle-related genes. Plate colony formation of NIH3T3 and HEK293T cell lines was enhanced >2-fold when cultured in riboflavin-deficient medium for 10-20 generations. Moreover, we observed enhanced subcutaneous tumorigenicity in NU/NU mice following injection of riboflavin-depleted compared with normal HEK293T cells (55.6% compared with 0.0% tumor formation, respectively). Gene expression profiling and gene ontology analysis revealed that riboflavin depletion induced the expression of cell cycle-related genes. Validation experiments also found that riboflavin depletion decreased p21 and p27 protein levels by ∼20%, and increased cell cycle-related and expression-elevated protein in tumor (CREPT) protein expression >2-fold, resulting in cyclin D1 and CDK4 levels being increased ∼1.5-fold, and cell cycle acceleration. We also observed that riboflavin depletion decreased intracellular riboflavin levels by 20% and upregulated expression of riboflavin transporter genes, particularly SLC52A3, and that the changes in CREPT and SLC52A3 correlated with

  3. Revelation of Different Nanoparticle-Uptake Behavior in Two Standard Cell Lines NIH/3T3 and A549 by Flow Cytometry and Time-Lapse Imaging

    Directory of Open Access Journals (Sweden)

    André Jochums

    2017-07-01

    Full Text Available The uptake of nanomaterials into different cell types is a central pharmacological issue for the determination of nanotoxicity as well as for the development of drug delivery strategies. Most responses of the cells depend on their intracellular interactions with nanoparticles (NPs. Uptake behavior can be precisely investigated in vitro, with sensitive high throughput methods such as flow cytometry. In this study, we investigated two different standard cell lines, human lung carcinoma (A549 and mouse fibroblast (NIH/3T3 cells, regarding their uptake behavior of titanium dioxide NPs. Cells were incubated with different concentrations of TiO2 NPs and samples were taken at certain time points to compare the uptake kinetics of both cell lines. Samples were analyzed with the help of flow cytometry by studying changes in the side and forward scattering signal. To additionally enable a detection via fluorescence, NPs were labeled with the fluorescent dye fluorescein isothiocyanate (FITC and propidium iodide (PI. We found that NIH/3T3 cells take up the studied NPs more efficiently than A549 cells. These findings were supported by time-lapse microscopic imaging of the cells incubated with TiO2 NPs. Our results confirm that the uptake behavior of individual cell types has to be considered before interpreting any results of nanomaterial studies.

  4. Activation and inactivation of the volume-sensitive taurine leak pathway in NIH3T3 fibroblasts and Ehrlich Lettre ascites cells

    DEFF Research Database (Denmark)

    Lambert, Ian Henry

    2007-01-01

    Hypotonic exposure provokes the mobilization of arachidonic acid, production of ROS, and a transient increase in taurine release in Ehrlich Lettre cells. The taurine release is potentiated by H(2)O(2) and the tyrosine phosphatase inhibitor vanadate and reduced by the phospholipase A(2) (PLA(2......)) inhibitors bromoenol lactone (BEL) and manoalide, the 5-lipoxygenase (5-LO) inhibitor ETH-615139, the NADPH oxidase inhibitor diphenyl iodonium (DPI), and antioxidants. Thus, swelling-induced taurine efflux in Ehrlich Lettre cells involves Ca(2+)-independent (iPLA(2))/secretory PLA(2) (sPLA(2)) plus 5-LO...... activity and modulation by ROS. Vanadate and H(2)O(2) stimulate arachidonic acid mobilization and vanadate potentiates ROS production in Ehrlich Lettre cells and NIH3T3 fibroblasts under hypotonic conditions. However, vanadate-induced potentiation of the volume-sensitive taurine efflux is, in both cell...

  5. ArF excimer laser modulation of TNF-alpha and gelatinase B in NIH 3T3 cells

    International Nuclear Information System (INIS)

    Naudy-Vives, C.; Courant, D.; Perot, J.C.; Garcia, J.; Fretier, P.; Court, L.; Dormont, D.

    1995-01-01

    The effects on TNF-alpha and gelatinase B activity in mammalian cells induced by 193 nm argon fluoride excimer laser have been investigated. The data show that a secretion of 92 kDa type IV collagenase and TNF-alpha were increased in cell culture supernatants. Moreover, the 193 nm laser radiation produces a decrease of cell proliferation and an increase of cell activation 8 hours after irradiation. The total protein amount increases with the delivered dose. Same, but less effects were obtained after exposure to a conventional UV lamp at 254 nm. (author)

  6. Stimulation of p38 (HOG1) kinase pathway by ionizing radiation results in downstream modulation of ATF/CREB transcription factor activity in NIH-3T3 cells

    International Nuclear Information System (INIS)

    Stevenson, Mary Ann; Yao Jin

    1997-01-01

    Purpose/Objective:p38 kinase, a member of the MAP kinase family, is activated in response to stresses such as high osmolarity and UV irradiation as well exposure to cytokines such as IL1β and TNFα. The kinase is part of a signal transduction pathway that leads from receptor activation through a three kinase cascade resulting in the activation of p38. p38 activation then leads to the phosphorylation of target proteins that include transcription factors such as nuclear factor of interleukin 6 and members of the activating transcription factor (ATF) family, and in addition, the stress protein, HSP27, via activation of MAPKAP2 kinase. In the present report, we have investigated the potential role of p38 in the response of NIH-3T3 cells to ionizing radiation. Materials and Methods:NIH-3T3 cells were grown to confluence in DMEM+10%CS and then serum deprived for 24 hours in DMEM+0.1%CS. Radiation exposures were delivered using a Philips RT250 (250Kvp X-ray tube). Activated forms of p38 kinase and ATF/CREB transcription factors were identified using immunoblotting techniques employing activation specific antibodies raised against the phosphorylated forms of the kinases/transcription factors. Kinase activity was directly measured using immunokinase assays. DNA binding of transcription factors to their respective consensus sequences was assayed by EMSA. Results:We found that p38 becomes rapidly phosphorylated and activated by exposure to ionizing radiation. Significantly, p38 is activated to a similar degree and with a similar time course by serum derpviation and entry of cells into a non-proliferating G 0 state, suggesting a causal role for p38 in quiescence. Phosphorylation of p38 directly correlated with phosphorylation and activation of ATF/CREB family members as well as DNA binding by these activated factors. Conclusion:Activation of p38 kinase and downstream transcription factors may play an important role in the response of cells to ionizing radiation. We are

  7. Tiamulin inhibits human CYP3A4 activity in an NIH/3T3 cell line stably expressing CYP3A4 cDNA.

    Science.gov (United States)

    De Groene, E M; Nijmeijer, S M; Horbach, G J; Witkamp, R F

    1995-09-07

    Tiamulin is an antibiotic frequently used in veterinary medicine. The drug has been shown to produce clinically important interactions with other compounds that are administered simultaneously. An NIH/3T3 cell line, stably expressing human cytochrome P450 (EC 1.14.14.1) cDNA (CYP3A4), was used to study the effect of tiamulin on CYP3A4 activity. The 6 beta-hydroxylation activity of testosterone, which is increased in CYP3A4-expressing cells compared to vector-transfected cells, showed reduced activity after incubation with 1 microM tiamulin and was completely reduced to background level after incubation with 2, 5 and 10 microM tiamulin. The CYP3A4-expressing cell line was used in combination with a shuttle vector containing the bacterial lacZ' gene to study the effect of tiamulin on CYP3A4-mediated mutagenicity of aflatoxin B1. The mutation frequency of aflatoxin B1 could be completely inhibited by tiamulin in CYP3A4-expressing cells, but no effect was observed on the mutation frequency of the direct mutagen ethylmethanesulphonate. Western blotting of homogenates of the CYP3A4-expressing cell line showed stabilization of CYP3A4 protein after incubation with tiamulin, supporting the hypothesis that the mechanism of inhibition is by binding of tiamulin to the cytochrome.

  8. Calcium-Induced Activation of a Mutant G-Protein-Coupled Receptor Causes In Vitro Transformation of NIH/3T3 Cells

    Directory of Open Access Journals (Sweden)

    Ana O. Hoff

    1999-12-01

    Full Text Available The calcium-sensing receptor (CaR is a G-proteincoupled receptor that is widely expressed, has tissuespecific functions, regulates cell growth. Activating mutations of this receptor cause autosomal dominant hypocalcemia, a syndrome characterized by hypocalcemia and hypercalciuria. The identification of a family with an activating mutation of the CaR (Thr151 Met in which hypocalcemia cosegregates with several unusual neoplasms led us to examine the transforming effects of this mutant receptor. Transfection of NIH/3T3 cells with the mutant but not the normal receptor supported colony formation in soft agar at subphysiologic calcium concentrations. The mutant CaR causes a calcium-dependent activation of the extracellular signal-regulated protein kinase (ERK 1/2 and Jun-N-terminal kinase/stress-activated (JNK/ SAPK pathways, but not P38 MAP kinase. These findings contribute to a growing body of information suggesting that this receptor plays a role in the regulation of cellular proliferation, that aberrant activation of the mutant receptor in this family may play a role in the unusual neoplastic manifestations.

  9. Oncogenic Ras-Induced Morphologic Change Is through MEK/ERK Signaling Pathway to Downregulate Stat3 at a Posttranslational Level in NIH3T3 Cells

    Directory of Open Access Journals (Sweden)

    Hsuan-Heng Yeh

    2008-01-01

    Full Text Available Ras is a key regulator of the MAP kinase-signaling cascade and may cause morphologic change of Ras-transformed cells. Signal transducer and activator of transcription 3 (Stat3 can be activated by cytokine stimulation. In this study, we unravel that Ha-rasV12 overexpression can downregulate the expression of Stat3 protein at a posttranslational level in NIH3T3 cells. Furthermore, we demonstrate that Stat3 expression downregulated by Ha-rasV12 overexpression is through proteosome degradation and not through a mTOR/p70S6K-related signaling pathway. The suppression of Stat3 accompanied by the morphologic change induced by Ha-rasV12 was through mitogen extracellular kinase (MEK/extracellular-regulated kinase (ERK signaling pathway. Microtubule disruption is involved in Ha-rasV12-induced morphologic change, which could be reversed by overexpression of Stat3. Taken together, we are the first to demonstrate that Stat3 protein plays a critical role in Ha-rasV12-induced morphologic change. Oncogenic Ras-triggered morphologic change is through the activation of MEK/ERK to posttranslationally downregulate Stat3 expression. Our finding may shed light on developing novel therapeutic strategies against Ras-related tumorigenesis.

  10. Preparation of Proper Immunogen by Cloning and Stable Expression of cDNA coding for Human Hematopoietic Stem Cell Marker CD34 in NIH-3T3 Mouse Fibroblast Cell Line

    Science.gov (United States)

    Shafaghat, Farzaneh; Abbasi-Kenarsari, Hajar; Majidi, Jafar; Movassaghpour, Ali Akbar; Shanehbandi, Dariush; Kazemi, Tohid

    2015-01-01

    Purpose: Transmembrane CD34 glycoprotein is the most important marker for identification, isolation and enumeration of hematopoietic stem cells (HSCs). We aimed in this study to clone the cDNA coding for human CD34 from KG1a cell line and stably express in mouse fibroblast cell line NIH-3T3. Such artificial cell line could be useful as proper immunogen for production of mouse monoclonal antibodies. Methods: CD34 cDNA was cloned from KG1a cell line after total RNA extraction and cDNA synthesis. Pfu DNA polymerase-amplified specific band was ligated to pGEMT-easy TA-cloning vector and sub-cloned in pCMV6-Neo expression vector. After transfection of NIH-3T3 cells using 3 μg of recombinant construct and 6 μl of JetPEI transfection reagent, stable expression was obtained by selection of cells by G418 antibiotic and confirmed by surface flow cytometry. Results: 1158 bp specific band was aligned completely to reference sequence in NCBI database corresponding to long isoform of human CD34. Transient and stable expression of human CD34 on transfected NIH-3T3 mouse fibroblast cells was achieved (25% and 95%, respectively) as shown by flow cytometry. Conclusion: Cloning and stable expression of human CD34 cDNA was successfully performed and validated by standard flow cytometric analysis. Due to murine origin of NIH-3T3 cell line, CD34-expressing NIH-3T3 cells could be useful as immunogen in production of diagnostic monoclonal antibodies against human CD34. This approach could bypass the need for purification of recombinant proteins produced in eukaryotic expression systems. PMID:25789221

  11. Insulin receptor substrate-1 prevents autophagy-dependent cell death caused by oxidative stress in mouse NIH/3T3 cells

    Directory of Open Access Journals (Sweden)

    Chan Shih-Hung

    2012-07-01

    Full Text Available Abstract Background Insulin receptor substrate (IRS-1 is associated with tumorigenesis; its levels are elevated in several human cancers. IRS-1 protein binds to several oncogene proteins. Oxidative stress and reactive oxygen species (ROS are involved in the initiation and progression of cancers. Cancer cells produce greater levels of ROS than normal cells do because of increased metabolic stresses. However, excessive production of ROS kills cancer cells. Autophagy usually serves as a survival mechanism in response to stress conditions, but excessive induction of autophagy results in cell death. In addition to inducing necrosis and apoptosis, ROS induces autophagic cell death. ROS inactivates IRS-1 mediated signaling and reduces intracellular IRS-1 concentrations. Thus, there is a complex relationship between IRS-1, ROS, autophagy, and cancer. It is not fully understood how cancer cells grow rapidly and survive in the presence of high ROS levels. Methods and results In this study, we established mouse NIH/3T3 cells that overexpressed IRS-1, so mimicking cancers with increased IRS-1 expression levels; we found that the IRS-1 overexpressing cells grow more rapidly than control cells do. Treatment of cells with glucose oxidase (GO provided a continuous source of ROS; low dosages of GO promoted cell growth, while high doses induced cell death. Evidence for GO induced autophagy includes increased levels of isoform B-II microtubule-associated protein 1 light chain 3 (LC3, aggregation of green fluorescence protein-tagged LC3, and increased numbers of autophagic vacuoles in cells. Overexpression of IRS-1 resulted in inhibition of basal autophagy, and reduced oxidative stress-induced autophagy and cell death. ROS decreased the mammalian target of rapamycin (mTOR/p70 ribosomal protein S6 kinase signaling, while overexpression of IRS-1 attenuated this inhibition. Knockdown of autophagy-related gene 5 inhibited basal autophagy and diminished oxidative stress

  12. Characterization of mutagen-activated cellular oncogenes that confer anchorage independence to human fibroblasts and tumorigenicity to NIH 3T3 cells: Sequence analysis of an enzymatically amplified mutant HRAS allele

    International Nuclear Information System (INIS)

    Stevens, C.W.; Manoharan, T.H.; Fahl, W.E.

    1988-01-01

    Treatment of diploid human fibroblasts with an alkylating mutagen has been shown to induce stable, anchorage-independent cell populations at frequencies consistent with an activating mutation. After treatment of human foreskin fibroblasts with the mutagen benzo[α]pyrene (±)anti-7,8-dihydrodiol 9,10-epoxide and selection in soft agar, 17 anchorage-independent clones were isolated and expanded, and their cellular DNA was used to cotransfect NIH 3T3 cells along with pSV2neo. DNA from 11 of the 17 clones induced multiple NIH 3T3 cell tumors in recipient nude mice. Southern blot analyses showed the presence of human Alu repetitive sequences in all of the NIH 3T3 tumor cell DNAs. Intact, human HRAS sequences were observed in 2 of the 11 tumor groups, whereas no hybridization was detected when human KRAS or NRAS probes were used. Slow-migrating ras p21 proteins, consistent with codon 12 mutations, were observed in the same two NIH 3T3 tumor cell groups that contained the human HRAS bands. Genomic DNA from one of these two human anchorage-independent cell populations (clone 21A) was used to enzymatically amplify a portion of exon 1 of the HRAS gene. The results demonstrate that exposure of normal human cells to a common environmental mutagen yields HRAS GC → TA codon 12 transversions that have been commonly observed in human tumors

  13. Characterization of Nrf2 activation and heme oxygenase-1 expression in NIH3T3 cells exposed to aqueous extracts of cigarette smoke.

    Science.gov (United States)

    Knörr-Wittmann, Constanze; Hengstermann, Arnd; Gebel, Stephan; Alam, Jawed; Müller, Thomas

    2005-12-01

    Cigarette smoke (CS) is a complex chemical mixture estimated to be composed of up to 5000 different chemicals, many of which are prooxidant. Here we show that, at least in vitro, the cellular response designed to combat oxidative stress resulting from CS exposure is primarily controlled by the transcription factor Nrf2, a principal inducer of antioxidant and phase II-related genes. The prominent role of Nrf2 in the cellular response to CS is substantiated by the following observations: In NIH3T3 cells exposed to aqueous extracts of CS (i) Nrf2 is strongly stabilized and becomes detectable in nuclear extracts. (ii) Nuclear localization of Nrf2 coincides with increased DNA binding of a putative Nrf2/MafK heterodimer to its cognate cis-regulatory site, i.e., the antioxidant-responsive element (ARE). (iii) Studies on the regulatory elements of the oxidative stress-inducible gene heme oxygenase-1 (hmox1) using various hmox1 promoter/luciferase reporter constructs revealed that the strong CS-dependent expression of this gene is primarily governed by the distal enhancers 1 ("E1") and 2 ("E2"), which both contain three canonical ARE-like stress-responsive elements (StREs). Notably, depletion of Nrf2 levels caused by RNA interference significantly compromised CS-induced hmox1 promoter activation, based on the distinct Nrf2 sensitivity exhibited by E1 and E2. Finally, (iv) siRNA-dependent knock-down of Nrf2 completely abrogated CS-induced expression of phase II-related genes. Taken together, these results confirm the outstanding role of Nrf2 both in sensing (oxidant) stress and in orchestrating an efficient transcriptional response aimed at resolving the stressing conditions.

  14. 2-Methoxy-4-vinylphenol can induce cell cycle arrest by blocking the hyper-phosphorylation of retinoblastoma protein in benzo[a]pyrene-treated NIH3T3 cells

    International Nuclear Information System (INIS)

    Jeong, Jin Boo; Jeong, Hyung Jin

    2010-01-01

    Research highlights: → 2M4VP activated the expression of p21 and p15 protein, and down-regulated the expression of cyclin D1 and cyclin E. → 2M4VP inhibited hyper-phosphorylation of Rb protein. → 2M4VP induced cell cycle arrest from G1 to S. → 2M4VP inhibited hyper-proliferation of the cells in BaP-treated cells. → 2M4VP induces growth arrest of BaP-treated cells by blocking hyper-phosphorylation of Rb via regulating the expression of cell cycle-related proteins. -- Abstract: Benzo[a]pyrene (BaP) is an environment carcinogen that can enhance cell proliferation by disturbing the signal transduction pathways in cell cycle regulation. In this study, the effects of 2M4VP on cell proliferation, cell cycle and cell cycle regulatory proteins were studied in BaP-treated NIH 3T3 cells to establish the molecular mechanisms of 2M4VP as anti-proliferative agents. 2M4VP exerted a dose-dependent inhibitory effect on cell growth correlated with a G1 arrest. Analysis of G1 cell cycle regulators expression revealed 2M4VP increased expression of CDK inhibitor, p21Waf1/Cip1 and p15 INK4b, decreased expression of cyclin D1 and cyclin E, and inhibited kinase activities of CDK4 and CDK2. However, 2M4VP did not affect the expression of CDK4 and CDK2. Also, 2M4VP inhibited the hyper-phosphorylation of Rb induced by BaP. Our results suggest that 2M4VP induce growth arrest of BaP-treated NIH 3T3 cells by blocking the hyper-phosphorylation of Rb via regulating the expression of cell cycle-related proteins.

  15. 2-Methoxy-4-vinylphenol can induce cell cycle arrest by blocking the hyper-phosphorylation of retinoblastoma protein in benzo[a]pyrene-treated NIH3T3 cells

    Energy Technology Data Exchange (ETDEWEB)

    Jeong, Jin Boo [Bioresource Sciences, Andong National University, Andong 760749 (Korea, Republic of); Jeong, Hyung Jin, E-mail: jhj@andong.ac.kr [Bioresource Sciences, Andong National University, Andong 760749 (Korea, Republic of)

    2010-10-01

    Research highlights: {yields} 2M4VP activated the expression of p21 and p15 protein, and down-regulated the expression of cyclin D1 and cyclin E. {yields} 2M4VP inhibited hyper-phosphorylation of Rb protein. {yields} 2M4VP induced cell cycle arrest from G1 to S. {yields} 2M4VP inhibited hyper-proliferation of the cells in BaP-treated cells. {yields} 2M4VP induces growth arrest of BaP-treated cells by blocking hyper-phosphorylation of Rb via regulating the expression of cell cycle-related proteins. -- Abstract: Benzo[a]pyrene (BaP) is an environment carcinogen that can enhance cell proliferation by disturbing the signal transduction pathways in cell cycle regulation. In this study, the effects of 2M4VP on cell proliferation, cell cycle and cell cycle regulatory proteins were studied in BaP-treated NIH 3T3 cells to establish the molecular mechanisms of 2M4VP as anti-proliferative agents. 2M4VP exerted a dose-dependent inhibitory effect on cell growth correlated with a G1 arrest. Analysis of G1 cell cycle regulators expression revealed 2M4VP increased expression of CDK inhibitor, p21Waf1/Cip1 and p15 INK4b, decreased expression of cyclin D1 and cyclin E, and inhibited kinase activities of CDK4 and CDK2. However, 2M4VP did not affect the expression of CDK4 and CDK2. Also, 2M4VP inhibited the hyper-phosphorylation of Rb induced by BaP. Our results suggest that 2M4VP induce growth arrest of BaP-treated NIH 3T3 cells by blocking the hyper-phosphorylation of Rb via regulating the expression of cell cycle-related proteins.

  16. Does the intracellular ionic concentration or the cell water content (cell volume) determine the activity of TonEBP in NIH3T3 cells?

    DEFF Research Database (Denmark)

    Rødgaard, Tina; Schou, Kenneth; Friis, Martin Barfred

    2008-01-01

    of the present investigation was to investigate whether cell shrinkage or high intracellular ionic concentration induced the activation of TonEBP. We designed a model system for isotonically shrinking cells over a prolonged period of time. Cells swelled in hypotonic medium and performed a regulatory volume...... decrease (RVD). Upon return to the original isotonic medium, cells shrank initially followed by a regulatory volume increase (RVI). To maintain cell shrinkage, the RVI process was inhibited as follows: Ethyl-isopropyl-amiloride (EIPA) inhibited the Na(+)/H(+) antiport, Bumetanide inhibited the Na(+)/K(+)/2......Cl(-) co-transporter, and Gadolinium inhibited shrinkage-activated Na(+) channels. Cells remained shrunken for at least 4 hours (isotonically shrunken cells). The activity of TonEBP was investigated with a Luciferase assay after isotonic shrinkage and after shrinkage in a high NaCl hypertonic medium...

  17. Chemical composition of the essential oil from basil (Ocimum basilicum Linn.) and its in vitro cytotoxicity against HeLa and HEp-2 human cancer cell lines and NIH 3T3 mouse embryonic fibroblasts.

    Science.gov (United States)

    Kathirvel, Poonkodi; Ravi, Subban

    2012-01-01

    This study examines the chemical composition and in vitro anticancer activity of the essential oil from Ocimum basilicum Linn. (Lamiaceae), cultivated in the Western Ghats of South India. The chemical compositions of basil fresh leaves were identified by GC-MS: 11 components were identified. The major constituents were found to be methyl cinnamate (70.1%), linalool (17.5%), β-elemene (2.6%) and camphor (1.52%). The results revealed that this plant may belong to the methyl cinnamate and linalool chemotype. A methyl thiazol tetrazolium assay was used for in vitro cytotoxicity screening against the human cervical cancer cell line (HeLa), human laryngeal epithelial carcinoma cell line (HEp-2) and NIH 3T3 mouse embryonic fibroblasts. The IC(50) values obtained were 90.5 and 96.3 µg mL(-1), respectively, and the results revealed that basil oil has potent cytotoxicity.

  18. Involvement of up-regulated Necl-5/Tage4/PVR/CD155 in the loss of contact inhibition in transformed NIH3T3 cells

    International Nuclear Information System (INIS)

    Minami, Yukiko; Ikeda, Wataru; Kajita, Mihoko; Fujito, Tsutomu; Monden, Morito; Takai, Yoshimi

    2007-01-01

    Normal cells show contact inhibition of cell movement and proliferation, but this is lost following transformation. We found that Necl-5, originally identified as a poliovirus receptor and up-regulated in many cancer cells, enhances growth factor-induced cell movement and proliferation. We showed that when cells contact other cells, Necl-5 interacts in trans with nectin-3 and is removed by endocytosis from the cell surface, resulting in a reduction of cell movement and proliferation. We show here that up-regulation of the gene encoding Necl-5 by the oncogene V12-Ki-Ras causes enhanced cell movement and proliferation. Upon cell-cell contact, de novo synthesis of Necl-5 exceeds the rate of Necl-5 endocytosis, eventually resulting in a net increase in the amount of Necl-5 at the cell surface. In addition, expression of the gene encoding nectin-3 is markedly reduced in transformed cells. Thus, up-regulation of Necl-5 following transformation contributes to the loss of contact inhibition in transformed cells

  19. Suppression of cadmium-induced JNK/p38 activation and HSP70 family gene expression by LL-Z1640-2 in NIH3T3 cells

    International Nuclear Information System (INIS)

    Sugisawa, Nobusuke; Matsuoka, Masato; Okuno, Takeo; Igisu, Hideki

    2004-01-01

    When NIH3T3 cells were exposed to CdCl 2 , the three major mitogen-activated protein kinases (MAPKs), extracellular signal-regulated protein kinase (ERK), c-Jun NH 2 -terminal kinase (JNK), and p38, were phosphorylated in a time (1-9 h)- and dose (1-20 μM)-dependent manner. Treatment with a macrocyclic nonaketide compound, LL-Z1640-2 (10-100 ng/ml), suppressed the phosphorylation of MAPKs without affecting the total protein level in cells exposed to 10 μM CdCl 2 for 6 h. CdCl 2 -induced phosphorylation of c-Jun on Ser63 and that on Ser73, and resultant accumulation of total c-Jun protein were also suppressed by LL-Z1640-2 treatment. The in vitro kinase assays also showed significant inhibitory effects of LL-Z1640-2 (at 10 or 25 ng/ml) on JNK and p38 but less markedly. In contrast to JNK and p38, ERK activity was inhibited moderately only at 50 or 100 ng/ml LL-Z1640-2. On the other hand, other JNK inhibitors, SP600125 and L-JNKI1, failed to suppress CdCl 2 -induced activation of the JNK pathway. Among the mouse stress response genes upregulated in response to CdCl 2 exposure, the expressions of hsp68 (encoding for heat shock 70 kDa protein 1; Hsp70-1) and grp78 (encoding for 78 kDa glucose-regulated protein; Grp78) genes were suppressed by treatment with 25 ng/ml LL-Z1640-2. Thus, LL-Z1640-2 could suppress CdCl 2 -induced activation of JNK/p38 pathways and expression of HSP70 family genes in NIH3T3 cells. LL-Z1640-2 seems to be useful to analyze functions of toxic metal-induced JNK/p38 activation

  20. Preparation of Preproinsulin Gene Construct Containing the Metallothionein2A (pBINDMTChIns and Its Expression in NIH3T3 Cell Line and Muscle Tissue of Alloxan Diabetic Rabbits

    Directory of Open Access Journals (Sweden)

    Piri

    2014-08-01

    Full Text Available Background Diabetes mellitus type 1, formerly called insulin-dependent diabetes, is one of the autoimmune diseases where insulin-producing cells are destroyed by autoimmune response via T cells. The new approaches in treatment of diabetes are using the stem cells, cell transplantation of islet β cell, gene transfer by virus based plasmids, and non-viral gene constructs. Objectives The purpose of this study was to construct glucose inducible insulin gene plasmid and use it in the muscle tissue of the rabbit. Materials and Methods To achieve this goal, the preproinsulin, metallothionein2A promoter and the response element to carbohydrate genes were cloned into pBIND plasmid by standard cloning methods, to construct pBINDMTChIns. The gene cloning products were confirmed by the polymerase chain reaction (PCR and restriction enzyme digestion template. The recombinant plasmid, containing the preproinsulin gene, was transferred into NIH3T3 cells and insulin gene expression was evaluated by reverse transcriptase PCR and western blotting techniques. Plasmid naked DNA containing the preproinsulin gene was injected into the rabbits’ thigh muscles, and its expression was confirmed by western blotting method. Results This study shows the prepared gene construct is inducible by glucose. Gene expression of preproinsulin was observed in muscle tissue of rabbits. Conclusions These finding indicated that research in diabetes mellitus gene therapy could be performed on larger animals.

  1. Molecularly characterized solvent extracts and saponins from Polygonum hydropiper L show high anti-angiogenic, anti-tumor, brine shrimp and fibroblast NIH/3T3 cell line cytotoxicity

    Directory of Open Access Journals (Sweden)

    Muhammad eAyaz

    2016-03-01

    Full Text Available Polygonum hydropiper is used as anti-cancer and anti-rheumatic agent in folk medicine. This study was designed to investigate the anti-angiogenic, anti-tumor and cytotoxic potentials of different solvent extracts and isolated saponins. Samples were analyzed using GC, GC-MS to identify major and bioactive compounds. Quantitation of antiangiogenesis for the plant's samples including methanolic extract (Ph.Cr, its subsequent fractions; n-hexane (Ph.Hex, chloroform (Ph.Chf, ethyl acetate (Ph.EtAc, n-Butanol (Ph.Bt, aqueous (Ph.Aq, saponins (Ph.Sp were performed using the chick embryo chorioallantoic membrane (CAM assay. Potato disc anti-tumor assay was performed on Agrobacterium tumefaciens containing tumor inducing plasmid. Cytotoxicity was performed on Artemia salina and mouse embryonic fibroblast NIH/3T3 cell line using brine shrimps and MTT cells viability assays. The GC-MS analysis of Ph.Cr, Ph.Hex, Ph.Chf, Ph.Bt and Ph.EtAc identified 126, 124, 153, 131 and 164 compounds respectively. In anti-angiogenic assay, Ph.Chf, Ph.Sp, Ph.EtAc and Ph.Cr exhibited highest activity with IC50 of 28.65, 19.21, 88.75 and 461.53 µg/ml respectively. In anti-tumor assay, Ph.Sp, Ph.Chf, Ph.EtAc and Ph.Cr were most potent with IC50 of 18.39, 73.81, 217.19 and 342.53 µg/ml respectively. In MTT cells viability assay, Ph.Chf, Ph.EtAc, Ph.Sp were most active causing 79.00, 72.50 and 71.50% cytotoxicity respectively at 1000 µg/ml with the LD50 of 140, 160 and 175 µg/ml respectively. In overall study, Ph.Chf and Ph.Sp have shown overwhelming results which signifies their potentials as sources of therapeutic agents against cancer.

  2. ArF excimer laser modulation of TNF-alpha and gelatinase B in NIH 3T3 cells; Modulation de l`expression du TNF-alpha et de la gelatinase B, apres irradiation de fibroblastes NIH 3T3 par un laser a excimeres a 193 NM

    Energy Technology Data Exchange (ETDEWEB)

    Naudy-Vives, C.; Courant, D.; Perot, J.C.; Garcia, J.; Fretier, P.; Court, L.; Dormont, D.

    1995-12-31

    The effects on TNF-alpha and gelatinase B activity in mammalian cells induced by 193 nm argon fluoride excimer laser have been investigated. The data show that a secretion of 92 kDa type IV collagenase and TNF-alpha were increased in cell culture supernatants. Moreover, the 193 nm laser radiation produces a decrease of cell proliferation and an increase of cell activation 8 hours after irradiation. The total protein amount increases with the delivered dose. Same, but less effects were obtained after exposure to a conventional UV lamp at 254 nm. (author). 8 refs.

  3. ROS activate KCl cotransport in nonadherent Ehrlich ascites cells but K+ and Cl- channels in adherent Ehrlich Lettré and NIH3T3 cells

    DEFF Research Database (Denmark)

    Lambert, Ian Henry; Klausen, Thomas Kjær; Bergdahl, Andreas

    2009-01-01

    Addition of H2O2 (0.5 mM) to Ehrlich ascites tumor cells under isotonic conditions results within 25 min in a substantial (22 +/- 1 %) reduction in cell volume. The cell shrinkage is paralleled by net loss of K(+), which was significant within 8 min, whereas no concomitant increase in the K......(+) or Cl(-) conductances could be observed. The H2O2-induced cell shrinkage was unaffected by the presence of clofilium and clotrimazole, that block volume-sensitive and Ca(2+)-activated K(+) channels, respectively, and unaffected by a raise in extracellular K(+) concentration to a value which eliminates...... the electrochemical driving force for K(+). On the other hand, the H2O2-induced cell shrinkage was impaired in the presence of the KCl cotransport inhibitor DIOA, following substitution of NO3(-) for Cl(-), and when the driving force for KCl cotransport was omitted. It is suggested that H2O2 activates electro neutral...

  4. Using quantitative PCR to Identify Kinesin-3 Genes that are Upregulated During Growth Arrest in MouseNIH3T3 Cells

    DEFF Research Database (Denmark)

    Thorsteinsson, Rikke; Christensen, Søren Tvorup; Pedersen, Lotte Bang

    2009-01-01

    Most cells in our body form a single primary cilium when entering growth arrest. During the past decade, a number of studies have revealed a key role for primary cilia in coordinating a variety of signaling pathways that control important cellular and developmental processes. Consequently, signif...

  5. Irradiation of Polystyrene and Polypropylene to study NIH 3T3 fibroblasts adhesion

    International Nuclear Information System (INIS)

    Arbeitman, C.R.; Grosso, M.F. del; Ibanez, I.; Garcia Bermudez, G.; Duran, H.; Chappa, V.C.; Mazzei, R.; Behar, M.

    2010-01-01

    When polymers are irradiated with heavy ions new chemical groups are created in a few microns of the material. The irradiation changed the polarity and wettability on the surface so that could enhance the biocompatibility of the modified polymer. The study of chemistry and nanoscale topography of the biomaterial is important in determining its potential applications in medicine and biotechnology, because their strong influence on cell function, adhesion and proliferation. In this study, thin films of Polystyrene and Polypropylene samples were modified by irradiation with low energy ion beams (30-150 keV) and swift heavy ions both with various fluences and energies. The changes were evaluated with different methods. Adhesion of NIH 3T3 fibroblasts onto unirradiated and irradiated surfaces has been studied by in vitro techniques. The correlations between physicochemical properties as a function of different irradiations parameters were compared with cell adhesion on the modified polymer surface.

  6. Casein kinase 2 regulates the active uptake of the organic osmolyte taurine in NIH3T3 mouse fibroblasts

    DEFF Research Database (Denmark)

    Jacobsen, Jack H; Clement, Christian A; Friis, Martin B

    2008-01-01

    Inhibition of the constitutively active casein kinase 2 (CK2) with 2-dimethyl-amino-4,5,6,7-tetrabromo-1H-benzimidasole stimulates the Na(+)-dependent taurine influx via the taurine transporter TauT in NIH3T3 cells. CK2 inhibition reduces the TauT mRNA level and increases the localization of TauT...

  7. PLA2 - a major regulator of volume-sensitive taurine release in NIH3T3 fibroblasts

    DEFF Research Database (Denmark)

    Lambert, I. H.

    2006-01-01

    -lipoxygenase (5-LO) system is prevented by the 5-LO inhibitor ETH 615-139 and is reduced under hypertonic conditions. Exposure to the amphiphilic bee venom peptide melittin, which has no effect on the kinetic properties of PLA2 but promotes substrate replenishment, induces release of arachidonic acid...... conditions but has only a minor effect on the melittin-induced taurine efflux under hypertonic conditions. Bromoenol lactone and manoalide, known inhibitors of Ca2+-independent phospholipase A2 (iPLA2) and secretory phospholipase A2 (sPLA2), respectively, reduce arachidonic acid and taurine release from NIH3......T3 cells under hypotonic conditions and following addition of melittin. It is suggested that iPLA2/sPLA2 activity is responsible for the volume-sensitivity of taurine release in NIH3T3 mouse fibroblasts....

  8. Rho family GTP binding proteins are involved in the regulatory volume decrease process in NIH3T3 mouse fibroblasts

    DEFF Research Database (Denmark)

    Pedersen, Stine F; Beisner, Kristine H; Willumsen, Berthe M

    2002-01-01

    The role of Rho GTPases in the regulatory volume decrease (RVD) process following osmotic cell swelling is controversial and has so far only been investigated for the swelling-activated Cl- efflux. We investigated the involvement of RhoA in the RVD process in NIH3T3 mouse fibroblasts, using wild......-type cells and three clones expressing constitutively active RhoA (RhoAV14). RhoAV14 expression resulted in an up to fourfold increase in the rate of RVD, measured by large-angle light scattering. The increase in RVD rate correlated with RhoAV14 expression. RVD in wild-type cells was unaffected by the Rho...

  9. Roles of phospholipase A2 isoforms in swelling- and melittin-induced arachidonic acid release and taurine efflux in NIH3T3 fibroblasts

    DEFF Research Database (Denmark)

    Pedersen, Stine Helene Falsig; Poulsen, Kristian Arild; Lambert, Ian H.

    2006-01-01

    Osmotic swelling of NIH3T3 mouse fibroblasts activates a bromoenol lactone (BEL)-sensitive taurine efflux, pointing to the involvement of a Ca2+-independent phospholipase A2 (iPLA2) (Lambert IH. J Membr Biol 192: 19-32, 2003). We report that taurine efflux from NIH3T3 cells was not only increased...... by cell swelling but also decreased by cell shrinkage. Arachidonic acid release to the cell exterior was similarly decreased by shrinkage yet not detectably increased by swelling. NIH3T3 cells were found to express cytosolic calcium-dependent cPLA2-IVA, cPLA2-IVB, cPLA2-IVC, iPLA2-VIA, iPLA2-VIB......, and secretory sPLA2-V. Arachidonic acid release from swollen cells was partially inhibited by BEL and by the sPLA2-inhibitor manoalide. Cell swelling elicited BEL-sensitive arachidonic acid release from the nucleus, to which iPLA2-VIA localized. Exposure to the bee venom peptide melittin, to increase PLA2...

  10. Volume-sensitive NADPH oxidase activity and taurine efflux in NIH3T3 mouse fibroblasts

    DEFF Research Database (Denmark)

    Friis, Martin Barfred; Vorum, Katrine Gribel; Lambert, Ian Henry

    2008-01-01

    Reactive oxygen species (ROS) are produced in NIH3T3 fibroblasts during hypotonic stress, and H(2)O(2) potentiates the concomitant release of the organic osmolyte taurine (Lambert IH. J Membr Biol 192: 19-32, 2003). The increase in ROS production [5-(and-6)-carboxy-2', 7'-dichlorodihydrofluorescein......+-mobilizing agonist ATP (10 microM) potentiates the release of taurine but has no effect on ROS production under hypotonic conditions. On the other hand, addition of the protein kinase C (PKC) activator phorbol 12-myristate 13-acetate (PMA, 100 nM) or the lipid messenger lysophosphatidic acid (LPA, 10 n......M) potentiates the swelling-induced taurine release as well as the ROS production. Overexpression of Rac1 or p47 phox or p47 phox knockdown [small interfering (si)RNA] had no effect on the swelling-induced ROS production or taurine release. NOX4 knockdown (siRNA) impairs the increase in the ROS production...

  11. Carnosine retards tumor growth in vivo in an NIH3T3-HER2/neu mouse model

    Directory of Open Access Journals (Sweden)

    Meixensberger Jürgen

    2010-01-01

    Full Text Available Abstract Background It was previously demonstrated that the dipeptide carnosine inhibits growth of cultured cells isolated from patients with malignant glioma. In the present work we investigated whether carnosine also affects tumor growth in vivo and may therefore be considered for human cancer therapy. Results A mouse model was used to investigate whether tumor growth in vivo can be inhibited by carnosine. Therefore, NIH3T3 fibroblasts, conditionally expressing the human epidermal growth factor receptor 2 (HER2/neu, were implanted into the dorsal skin of nude mice, and tumor growth in treated animals was compared to control mice. In two independent experiments nude mice that received tumor cells received a daily intra peritoneal injection of 500 μl of 1 M carnosine solution. Measurable tumors were detected 12 days after injection. Aggressive tumor growth in control animals, that received a daily intra peritoneal injection of NaCl solution started at day 16 whereas aggressive growth in mice treated with carnosine was delayed, starting around day 19. A significant effect of carnosine on tumor growth was observed up to day 24. Although carnosine was not able to completely prevent tumor growth, a microscopic examination of tumors revealed that those from carnosine treated animals had a significant lower number of mitosis (p Conclusion As a naturally occurring substance with a high potential to inhibit growth of malignant cells in vivo, carnosine should be considered as a potential anti-cancer drug. Further experiments should be performed in order to understand how carnosine acts at the molecular level.

  12. Carnosine retards tumor growth in vivo in an NIH3T3-HER2/neu mouse model.

    Science.gov (United States)

    Renner, Christof; Zemitzsch, Nadine; Fuchs, Beate; Geiger, Kathrin D; Hermes, Matthias; Hengstler, Jan; Gebhardt, Rolf; Meixensberger, Jürgen; Gaunitz, Frank

    2010-01-06

    It was previously demonstrated that the dipeptide carnosine inhibits growth of cultured cells isolated from patients with malignant glioma. In the present work we investigated whether carnosine also affects tumor growth in vivo and may therefore be considered for human cancer therapy. A mouse model was used to investigate whether tumor growth in vivo can be inhibited by carnosine. Therefore, NIH3T3 fibroblasts, conditionally expressing the human epidermal growth factor receptor 2 (HER2/neu), were implanted into the dorsal skin of nude mice, and tumor growth in treated animals was compared to control mice. In two independent experiments nude mice that received tumor cells received a daily intra peritoneal injection of 500 microl of 1 M carnosine solution. Measurable tumors were detected 12 days after injection. Aggressive tumor growth in control animals, that received a daily intra peritoneal injection of NaCl solution started at day 16 whereas aggressive growth in mice treated with carnosine was delayed, starting around day 19. A significant effect of carnosine on tumor growth was observed up to day 24. Although carnosine was not able to completely prevent tumor growth, a microscopic examination of tumors revealed that those from carnosine treated animals had a significant lower number of mitosis (p < 0.0003) than untreated animals, confirming that carnosine affects proliferation in vivo. As a naturally occurring substance with a high potential to inhibit growth of malignant cells in vivo, carnosine should be considered as a potential anti-cancer drug. Further experiments should be performed in order to understand how carnosine acts at the molecular level.

  13. Downregulation of the taurine transporter TauT during hypo-osmotic stress in NIH3T3 mouse fibroblasts

    DEFF Research Database (Denmark)

    Hansen, Daniel Bloch; Friis, Martin Barfred; Hoffmann, Else Kay

    2012-01-01

    The present work was initiated to investigate regulation of the taurine transporter TauT by reactive oxygen species (ROS) and the tonicity-responsive enhancer binding protein (TonEBP) in NIH3T3 mouse fibroblasts during acute and long-term (4 h) exposure to low-sodium/hypo-osmotic stress. Taurine...... are significantly increased following hyperosmotic exposure. Swelling-induced ROS production in NIH3T3 fibroblasts is generated by NOX4 and by increasing total ROS, by either exogenous application of H(2)O(2) or overexpressing NOX4, we demonstrate that TonEBP activity and taurine influx are regulated negatively...... by ROS under hypo-osmotic, low-sodium conditions, whereas the TauT mRNA level is unaffected. Acute exposure to ROS reduces taurine uptake as a result of modulated TauT transport kinetics. Thus, swelling-induced ROS production could account for the reduced taurine uptake under low...

  14. Effects of osmotic stress on the activity of MAPKs and PDGFR-beta-mediated signal transduction in NIH-3T3 fibroblasts

    DEFF Research Database (Denmark)

    Nielsen, M-B; Christensen, Søren Tvorup; Hoffmann, E K

    2008-01-01

    Signaling in cell proliferation, cell migration, and apoptosis is highly affected by osmotic stress and changes in cell volume, although the mechanisms underlying the significance of cell volume as a signal in cell growth and death are poorly understood. In this study, we used NIH-3T3 fibroblasts...... in a serum- and nutrient-free inorganic medium (300 mosM) to analyze the effects of osmotic stress on MAPK activity and PDGF receptor (PDGFR)-beta-mediated signal transduction. We found that hypoosmolarity (cell swelling at 211 mosM) induced the phosphorylation and nuclear translocation of ERK1/2, most...... likely via a pathway independent of PDGFR-beta and MEK1/2. Conversely, hyperosmolarity (cell shrinkage at 582 mosM) moved nuclear and phosphorylated ERK1/2 to the cytoplasm and induced the phosphorylation and nuclear translocation of p38 and phosphorylation of JNK1/2. In a series of parallel experiments...

  15. A homeopathic remedy from arnica, marigold, St. John's wort and comfrey accelerates in vitro wound scratch closure of NIH 3T3 fibroblasts.

    Science.gov (United States)

    Hostanska, Katarina; Rostock, Matthias; Melzer, Joerg; Baumgartner, Stephan; Saller, Reinhard

    2012-07-18

    Drugs of plant origin such as Arnica montana, Calendula officinalis or Hypericum perforatum have been frequently used to promote wound healing. While their effect on wound healing using preparations at pharmacological concentrations was supported by several in vitro and clinical studies, investigations of herbal homeopathic remedies on wound healing process are rare. The objective of this study was to investigate the effect of a commercial low potency homeopathic remedy Similasan® Arnica plus Spray on wound closure in a controlled, blind trial in vitro. We investigated the effect of an ethanolic preparation composed of equal parts of Arnica montana 4x, Calendula officinalis 4x, Hypericum perforatum 4x and Symphytum officinale 6x (0712-2), its succussed hydroalcoholic solvent (0712-1) and unsuccussed solvent (0712-3) on NIH 3T3 fibroblasts. Cell viability was determined by WST-1 assay, cell growth using BrdU uptake, cell migration by chemotaxis assay and wound closure by CytoSelect ™Wound Healing Assay Kit which generated a defined "wound field". All assays were performed in three independent controlled experiments. None of the three substances affected cell viability and none showed a stimulating effect on cell proliferation. Preparation (0712-2) exerted a stimulating effect on fibroblast migration (31.9%) vs 14.7% with succussed solvent (0712-1) at 1:100 dilutions (p 0.05). Preparation (0712-2) at a dilution of 1:100 promoted in vitro wound closure by 59.5% and differed significantly (p < 0.001) from succussed solvent (0712-1), which caused 22.1% wound closure. Results of this study showed that the low potency homeopathic remedy (0712-2) exerted in vitro wound closure potential in NIH 3T3 fibroblasts. This effect resulted from stimulation of fibroblasts motility rather than of their mitosis.

  16. A homeopathic remedy from arnica, marigold, St. John’s wort and comfrey accelerates in vitro wound scratch closure of NIH 3T3 fibroblasts

    Directory of Open Access Journals (Sweden)

    Hostanska Katarina

    2012-07-01

    Full Text Available Abstract Background Drugs of plant origin such as Arnica montana, Calendula officinalis or Hypericum perforatum have been frequently used to promote wound healing. While their effect on wound healing using preparations at pharmacological concentrations was supported by several in vitro and clinical studies, investigations of herbal homeopathic remedies on wound healing process are rare. The objective of this study was to investigate the effect of a commercial low potency homeopathic remedy Similasan® Arnica plus Spray on wound closure in a controlled, blind trial in vitro. Methods We investigated the effect of an ethanolic preparation composed of equal parts of Arnica montana 4x, Calendula officinalis 4x, Hypericum perforatum 4x and Symphytum officinale 6x (0712–2, its succussed hydroalcoholic solvent (0712–1 and unsuccussed solvent (0712–3 on NIH 3T3 fibroblasts. Cell viability was determined by WST-1 assay, cell growth using BrdU uptake, cell migration by chemotaxis assay and wound closure by CytoSelect ™Wound Healing Assay Kit which generated a defined “wound field”. All assays were performed in three independent controlled experiments. Results None of the three substances affected cell viability and none showed a stimulating effect on cell proliferation. Preparation (0712–2 exerted a stimulating effect on fibroblast migration (31.9% vs 14.7% with succussed solvent (0712–1 at 1:100 dilutions (p  0.05. Preparation (0712–2 at a dilution of 1:100 promoted in vitro wound closure by 59.5% and differed significantly (p  Conclusion Results of this study showed that the low potency homeopathic remedy (0712–2 exerted in vitro wound closure potential in NIH 3T3 fibroblasts. This effect resulted from stimulation of fibroblasts motility rather than of their mitosis.

  17. A homeopathic remedy from arnica, marigold, St. John’s wort and comfrey accelerates in vitro wound scratch closure of NIH 3T3 fibroblasts

    Science.gov (United States)

    2012-01-01

    Background Drugs of plant origin such as Arnica montana, Calendula officinalis or Hypericum perforatum have been frequently used to promote wound healing. While their effect on wound healing using preparations at pharmacological concentrations was supported by several in vitro and clinical studies, investigations of herbal homeopathic remedies on wound healing process are rare. The objective of this study was to investigate the effect of a commercial low potency homeopathic remedy Similasan® Arnica plus Spray on wound closure in a controlled, blind trial in vitro. Methods We investigated the effect of an ethanolic preparation composed of equal parts of Arnica montana 4x, Calendula officinalis 4x, Hypericum perforatum 4x and Symphytum officinale 6x (0712–2), its succussed hydroalcoholic solvent (0712–1) and unsuccussed solvent (0712–3) on NIH 3T3 fibroblasts. Cell viability was determined by WST-1 assay, cell growth using BrdU uptake, cell migration by chemotaxis assay and wound closure by CytoSelect ™Wound Healing Assay Kit which generated a defined “wound field”. All assays were performed in three independent controlled experiments. Results None of the three substances affected cell viability and none showed a stimulating effect on cell proliferation. Preparation (0712–2) exerted a stimulating effect on fibroblast migration (31.9%) vs 14.7% with succussed solvent (0712–1) at 1:100 dilutions (p  0.05). Preparation (0712–2) at a dilution of 1:100 promoted in vitro wound closure by 59.5% and differed significantly (p < 0.001) from succussed solvent (0712–1), which caused 22.1% wound closure. Conclusion Results of this study showed that the low potency homeopathic remedy (0712–2) exerted in vitro wound closure potential in NIH 3T3 fibroblasts. This effect resulted from stimulation of fibroblasts motility rather than of their mitosis. PMID:22809174

  18. The nucleus is an intracellular propagator of tensile forces in NIH 3T3 fibroblasts

    Science.gov (United States)

    Alam, Samer G.; Lovett, David; Kim, Dae In; Roux, Kyle J.; Dickinson, Richard B.; Lele, Tanmay P.

    2015-01-01

    ABSTRACT Nuclear positioning is a crucial cell function, but how a migrating cell positions its nucleus is not understood. Using traction-force microscopy, we found that the position of the nucleus in migrating fibroblasts closely coincided with the center point of the traction-force balance, called the point of maximum tension (PMT). Positioning of the nucleus close to the PMT required nucleus–cytoskeleton connections through linker of nucleoskeleton-to-cytoskeleton (LINC) complexes. Although the nucleus briefly lagged behind the PMT following spontaneous detachment of the uropod during migration, the nucleus quickly repositioned to the PMT within a few minutes. Moreover, traction-generating spontaneous protrusions deformed the nearby nucleus surface to pull the nuclear centroid toward the new PMT, and subsequent retraction of these protrusions relaxed the nuclear deformation and restored the nucleus to its original position. We propose that the protruding or retracting cell boundary transmits a force to the surface of the nucleus through the intervening cytoskeletal network connected by the LINC complexes, and that these forces help to position the nucleus centrally and allow the nucleus to efficiently propagate traction forces across the length of the cell during migration. PMID:25908852

  19. A proteomic analysis of the functional effects of fatty acids in NIH 3T3 fibroblasts

    LENUS (Irish Health Repository)

    Magdalon, Juliana

    2011-11-24

    Abstract Previous studies have demonstrated that long chain fatty acids influence fibroblast function at sub-lethal concentrations. This study is the first to assess the effects of oleic, linoleic or palmitic acids on protein expression of fibroblasts, as determined by standard proteomic techniques. The fatty acids were not cytotoxic at the concentration used in this work as assessed by membrane integrity, DNA fragmentation and the MTT assay but significantly increased cell proliferation. Subsequently, a proteomic analysis was performed using two dimensional difference gel electrophoresis (2D-DIGE) and MS based identification. Cells treated with 50 μM oleic, linoleic or palmitic acid for 24 h were associated with 24, 22, 16 spots differentially expressed, respectively. Among the identified proteins, α-enolase and far upstream element binding protein 1 (FBP-1) are of importance due to their function in fibroblast-associated diseases. However, modulation of α-enolase and FBP-1 expression by fatty acids was not validated by the Western blot technique.

  20. Expression of human choline kinase in NIH 3T3 fibroblasts increases the mitogenic potential of insulin and insulin-like growth factor I.

    Science.gov (United States)

    Chung, T; Huang, J S; Mukherjee, J J; Crilly, K S; Kiss, Z

    2000-05-01

    In mammalian cells, growth factors, oncogenes, and carcinogens stimulate phosphocholine (PCho) synthesis by choline kinase (CK), suggesting that PCho may regulate cell growth. To validate the role of PCho in mitogenesis, we determined the effects of insulin, insulin-like growth factor I (IGF-I), and other growth factors on DNA synthesis in NIH 3T3 fibroblast sublines highly expressing human choline kinase (CK) without increasing phosphatidylcholine synthesis. In serum-starved CK expressor cells, insulin and IGF-I stimulated DNA synthesis, p70 S6 kinase (p70 S6K) activity, phosphatidylinositol 3-kinase (PI3K) activity, and activating phosphorylation of p42/p44 mitogen-activated protein kinases (MAPK) to greater extents than in the corresponding vector control cells. Furthermore, the CK inhibitor hemicholinium-3 (HC-3) inhibited insulin- and IGF-I-induced DNA synthesis in the CK overexpressors, but not in the vector control cells. The results indicate that high cellular levels of PCho potentiate insulin- and IGF-I-induced DNA synthesis by MAPK- and p70 S6K-regulated mechanisms.

  1. Iminolactones as Tools for Inversion of the Absolute Configuration of alpha-Amino Acids and as Inhibitors of Cancer Cell Proliferation

    DEFF Research Database (Denmark)

    Jensen, Christina Mernøe; Chow, Hsiao-Qing; Chen, Ming

    2016-01-01

    -proliferative effects toward three cancer cell lines (EL4, MCF7, PC3) with insignificant effect on non-malign cell lines (McCoy, MCF10A, NIH3T3). Thus, iminolactones appear to be potential lead structures for preparation of drugs selectively affecting proliferation of malign cell lines....

  2. High expression of the taurine transporter TauT in primary cilia of NIH3T3 fibroblasts

    DEFF Research Database (Denmark)

    Christensen, Søren Tvorup; Voss, Jesper W.; Teilmann, Stefan C.

    2005-01-01

    Taurine, present in high concentrations in various mammalian cells, is essential for regulation of cell volume, cellular oxidative status as well as the cellular Ca2+ homeostasis. Cellular taurine content is a balance between active uptake through the saturable, Na+-dependent taurine transporter...... TauT expression and (iii) long-term exposure to hypertonic taurine medium, i.e., growth medium supplemented with 100 mM taurine, reduces ciliary TauT expression. These results point to an important role of taurine in the regulation of physiological processes located to the primary cilium....

  3. Magnetic Beads Enhance Adhesion of NIH 3T3 Fibroblasts: A Proof-of-Principle In Vitro Study for Implant-Mediated Long-Term Drug Delivery to the Inner Ear.

    Science.gov (United States)

    Aliuos, Pooyan; Schulze, Jennifer; Schomaker, Markus; Reuter, Günter; Stolle, Stefan R O; Werner, Darja; Ripken, Tammo; Lenarz, Thomas; Warnecke, Athanasia

    2016-01-01

    Long-term drug delivery to the inner ear may be achieved by functionalizing cochlear implant (CI) electrodes with cells providing neuroprotective factors. However, effective strategies in order to coat implant surfaces with cells need to be developed. Our vision is to make benefit of electromagnetic field attracting forces generated by CI electrodes to bind BDNF-secreting cells that are labelled with magnetic beads (MB) onto the electrode surfaces. Thus, the effect of MB-labelling on cell viability and BDNF production were investigated. Murine NIH 3T3 fibroblasts-genetically modified to produce BDNF-were labelled with MB. Atomic force and bright field microscopy illustrated the internalization of MB by fibroblasts after 24 h of cultivation. Labelling cells with MB did not expose cytotoxic effects on fibroblasts and allowed adhesion on magnetic surfaces with sufficient BDNF release. Our data demonstrate a novel approach for mediating enhanced long-term adhesion of BDNF-secreting fibroblasts on model electrode surfaces for cell-based drug delivery applications in vitro and in vivo. This therapeutic strategy, once transferred to cells suitable for clinical application, may allow the biological modifications of CI surfaces with cells releasing neurotrophic or other factors of interest.

  4. Magnetic Beads Enhance Adhesion of NIH 3T3 Fibroblasts: A Proof-of-Principle In Vitro Study for Implant-Mediated Long-Term Drug Delivery to the Inner Ear.

    Directory of Open Access Journals (Sweden)

    Pooyan Aliuos

    Full Text Available Long-term drug delivery to the inner ear may be achieved by functionalizing cochlear implant (CI electrodes with cells providing neuroprotective factors. However, effective strategies in order to coat implant surfaces with cells need to be developed. Our vision is to make benefit of electromagnetic field attracting forces generated by CI electrodes to bind BDNF-secreting cells that are labelled with magnetic beads (MB onto the electrode surfaces. Thus, the effect of MB-labelling on cell viability and BDNF production were investigated.Murine NIH 3T3 fibroblasts-genetically modified to produce BDNF-were labelled with MB.Atomic force and bright field microscopy illustrated the internalization of MB by fibroblasts after 24 h of cultivation. Labelling cells with MB did not expose cytotoxic effects on fibroblasts and allowed adhesion on magnetic surfaces with sufficient BDNF release.Our data demonstrate a novel approach for mediating enhanced long-term adhesion of BDNF-secreting fibroblasts on model electrode surfaces for cell-based drug delivery applications in vitro and in vivo. This therapeutic strategy, once transferred to cells suitable for clinical application, may allow the biological modifications of CI surfaces with cells releasing neurotrophic or other factors of interest.

  5. Lysophosphatidylcholine-induced taurine release in HeLa cells involves protein kinase activity

    DEFF Research Database (Denmark)

    Lambert, Ian H.; Falktoft, Birgitte

    2001-01-01

    Calmodulin; CaMKII; Ehrlich ascites tumor cells; Ischemia; Lysophospholipids; NIH/3T3; Chelerythrine; Reactive oxygen species......Calmodulin; CaMKII; Ehrlich ascites tumor cells; Ischemia; Lysophospholipids; NIH/3T3; Chelerythrine; Reactive oxygen species...

  6. Nonmalignant T cells stimulate growth of T-cell lymphoma cells in the presence of bacterial toxins

    DEFF Research Database (Denmark)

    Woetmann, Anders; Lovato, Paola; Eriksen, Karsten W

    2007-01-01

    Bacterial toxins including staphylococcal enterotoxins (SEs) have been implicated in the pathogenesis of cutaneous T-cell lymphomas (CTCLs). Here, we investigate SE-mediated interactions between nonmalignant T cells and malignant T-cell lines established from skin and blood of CTCL patients....... The malignant CTCL cells express MHC class II molecules that are high-affinity receptors for SE. Although treatment with SE has no direct effect on the growth of the malignant CTCL cells, the SE-treated CTCL cells induce vigorous proliferation of the SE-responsive nonmalignant T cells. In turn, the nonmalignant...... T cells enhance proliferation of the malignant cells in an SE- and MHC class II-dependent manner. Furthermore, SE and, in addition, alloantigen presentation by malignant CTCL cells to irradiated nonmalignant CD4(+) T-cell lines also enhance proliferation of the malignant cells. The growth...

  7. Different Cytokine and Chemokine Expression Patterns in Malignant Compared to Those in Nonmalignant Renal Cells

    Directory of Open Access Journals (Sweden)

    Nadine Gelbrich

    2017-01-01

    Full Text Available Objective. Cytokines and chemokines are widely involved in cancer cell progression and thus represent promising candidate factors for new biomarkers. Methods. Four renal cell cancer (RCC cell lines (Caki-1, 786-O, RCC4, and A498 and a nonmalignant renal cell line (RC-124 were examined with respect to their proliferation. The cytokine and chemokine expression pattern was examined by a DNA array (Human Cytokines & Chemokines RT2 Profiler PCR Array; Qiagen, Hilden, Germany, and expression profiles were compared. Results. Caki-1 and 786-O cells exhibited significantly increased proliferation rates, whereas RCC4 and A498 cells demonstrated attenuated proliferation, compared to nonmalignant RC-124 cells. Expression analysis revealed 52 cytokines and chemokines primarily involved in proliferation and inflammation and differentially expressed not only in malignant and nonmalignant renal cells but also in the four RCC cell lines. Conclusion. This is the first study examining the expression of 84 cytokines and chemokines in four RCC cell lines compared to that in a nonmalignant renal cell line. VEGFA, NODAL, and BMP6 correlated with RCC cell line proliferation and, thus, may represent putative clinical biomarkers for RCC progression as well as for RCC diagnosis and prognosis.

  8. Extracellular vesicle-mediated phenotype switching in malignant and non-malignant colon cells

    International Nuclear Information System (INIS)

    Mulvey, Hillary E.; Chang, Audrey; Adler, Jason; Del Tatto, Michael; Perez, Kimberly; Quesenberry, Peter J.; Chatterjee, Devasis

    2015-01-01

    Extracellular vesicles (EVs) are secreted from many cells, carrying cargoes including proteins and nucleic acids. Research has shown that EVs play a role in a variety of biological processes including immunity, bone formation and recently they have been implicated in promotion of a metastatic phenotype. EVs were isolated from HCT116 colon cancer cells, 1459 non-malignant colon fibroblast cells, and tumor and normal colon tissue from a patient sample. Co-cultures were performed with 1459 cells and malignant vesicles, as well as HCT116 cells and non-malignant vesicles. Malignant phenotype was measured using soft agar colony formation assay. Co-cultures were also analyzed for protein levels using mass spectrometry. The importance of 14-3-3 zeta/delta in transfer of malignant phenotype was explored using siRNA. Additionally, luciferase reporter assay was used to measure the transcriptional activity of NF-κB. This study demonstrates the ability of EVs derived from malignant colon cancer cell line and malignant patient tissue to induce the malignant phenotype in non-malignant colon cells. Similarly, EVs derived from non-malignant colon cell lines and normal patient tissue reversed the malignant phenotype of HCT116 cells. Cells expressing an EV-induced malignant phenotype showed increased transcriptional activity of NF-κB which was inhibited by the NF--κB inhibitor, BAY117082. We also demonstrate that knock down of 14-3-3 zeta/delta reduced anchorage-independent growth of HCT116 cells and 1459 cells co-cultured with HCT derived EVs. Evidence of EV-mediated induction of malignant phenotype, and reversal of malignant phenotype, provides rational basis for further study of the role of EVs in tumorigenesis. Identification of 14-3-3 zeta/delta as up-regulated in malignancy suggests its potential as a putative drug target for the treatment of colorectal cancer

  9. Lack of evidence for AT1R/B2R heterodimerization in COS-7, HEK293, and NIH3T3 cells: how common is the AT1R/B2R heterodimer?

    DEFF Research Database (Denmark)

    Hansen, Jakob L; Hansen, Jonas T; Speerschneider, Tobias

    2008-01-01

    dimerization using bioluminescence resonance energy transfer and regulated secretion/aggregation technology. However, although both the AT1Rs and B2Rs were functional in our systems and the systems were fine tuned to detect small changes in receptor function, we failed to detect any functional modulation...

  10. Bone marrow-derived CD13+ cells sustain tumor progression: A potential non-malignant target for anticancer therapy.

    Science.gov (United States)

    Dondossola, Eleonora; Corti, Angelo; Sidman, Richard L; Arap, Wadih; Pasqualini, Renata

    2014-01-01

    Non-malignant cells found within neoplastic lesions express alanyl (membrane) aminopeptidase (ANPEP, best known as CD13), and CD13-null mice exhibit limited tumor growth and angiogenesis. We have recently demonstrated that a subset of bone marrow-derived CD11b + CD13 + myeloid cells accumulate within neoplastic lesions in several murine models of transplantable cancer to promote angiogenesis. If these findings were confirmed in clinical settings, CD11b + CD13 + myeloid cells could become a non-malignant target for the development of novel anticancer regimens.

  11. Cooperative effects of aminopeptidase N (CD13) expressed by nonmalignant and cancer cells within the tumor microenvironment.

    Science.gov (United States)

    Guzman-Rojas, Liliana; Rangel, Roberto; Salameh, Ahmad; Edwards, Julianna K; Dondossola, Eleonora; Kim, Yun-Gon; Saghatelian, Alan; Giordano, Ricardo J; Kolonin, Mikhail G; Staquicini, Fernanda I; Koivunen, Erkki; Sidman, Richard L; Arap, Wadih; Pasqualini, Renata

    2012-01-31

    Processes that promote cancer progression such as angiogenesis require a functional interplay between malignant and nonmalignant cells in the tumor microenvironment. The metalloprotease aminopeptidase N (APN; CD13) is often overexpressed in tumor cells and has been implicated in angiogenesis and cancer progression. Our previous studies of APN-null mice revealed impaired neoangiogenesis in model systems without cancer cells and suggested the hypothesis that APN expressed by nonmalignant cells might promote tumor growth. We tested this hypothesis by comparing the effects of APN deficiency in allografted malignant (tumor) and nonmalignant (host) cells on tumor growth and metastasis in APN-null mice. In two independent tumor graft models, APN activity in both the tumors and the host cells cooperate to promote tumor vascularization and growth. Loss of APN expression by the host and/or the malignant cells also impaired lung metastasis in experimental mouse models. Thus, cooperation in APN expression by both cancer cells and nonmalignant stromal cells within the tumor microenvironment promotes angiogenesis, tumor growth, and metastasis.

  12. CD47 limits antibody dependent phagocytosis against non-malignant B cells.

    Science.gov (United States)

    Gallagher, Sandra; Turman, Sean; Lekstrom, Kristen; Wilson, Susan; Herbst, Ronald; Wang, Yue

    2017-05-01

    Recent studies have demonstrated the importance of CD47 in protecting malignant B cells from antibody dependent cellular phagocytosis (ADCP). Combined treatment of anti-CD47 and -CD20 antibodies synergistically augment elimination of tumor B cells in xenograft mouse models. This has led to the development of novel reagents that can potentially enhance killing of malignant B cells in patients. B cell depleting therapy is also a promising treatment for autoimmune patients. In the current study, we aimed to investigate whether or not CD47 protects non-malignant B cells from ADCP. We show that CD47 is expressed on all B cells in mice, with the highest level on plasma cells in bone marrow and spleen. Although its expression is dispensable for B cell development in mice, CD47 on B cells limits antibody mediated phagocytosis. B cell depletion following in vivo anti-CD19 treatment is more efficient in CD47-/- mice than in wild type mice. In vitro, both naïve and activated B cells from CD47-/- mice are more sensitive to ADCP than wild type B cells. Lastly, we show in an ADCP assay that blocking CD47 can enhance anti-CD19 antibody mediated phagocytosis of wild type B cells. These results suggest that in addition to its already demonstrated benefit in cancer, targeting CD47 may be used as an adjunct in combination with B cell depletion antibodies for treatment of autoimmune diseases. Copyright © 2017 Elsevier Ltd. All rights reserved.

  13. Telomerase-immortalized non-malignant human prostate epithelial cells retain the properties of multipotent stem cells

    International Nuclear Information System (INIS)

    Li Hongzhen; Zhou Jianjun; Miki, Jun; Furusato, Bungo; Gu Yongpeng; Srivastava, Shiv; McLeod, David G.; Vogel, Jonathan C.; Rhim, Johng S.

    2008-01-01

    Understanding prostate stem cells may provide insight into the origin of prostate cancer. Primary cells have been cultured from human prostate tissue but they usually survive only 15-20 population doublings before undergoing senescence. We report here that RC-170N/h/clone 7 cells, a clonal cell line from hTERT-immortalized primary non-malignant tissue-derived human prostate epithelial cell line (RC170N/h), retain multipotent stem cell properties. The RC-170N/h/clone 7 cells expressed a human embryonic stem cell marker, Oct-4, and potential prostate epithelial stem cell markers, CD133, integrin α2β1 hi and CD44. The RC-170N/h/clone 7 cells proliferated in KGM and Dulbecco's Modified Eagle Medium with 10% fetal bovine serum and 5 μg/ml insulin (DMEM + 10% FBS + Ins.) medium, and differentiated into epithelial stem cells that expressed epithelial cell markers, including CK5/14, CD44, p63 and cytokeratin 18 (CK18); as well as the mesenchymal cell markers, vimentin, desmin; the neuron and neuroendocrine cell marker, chromogranin A. Furthermore the RC170 N/h/clone 7 cells differentiated into multi tissues when transplanted into the sub-renal capsule and subcutaneously of NOD-SCID mice. The results indicate that RC170N/h/clone 7 cells retain the properties of multipotent stem cells and will be useful as a novel cell model for studying the mechanisms of human prostate stem cell differentiation and transformation

  14. In vitro Rb-1 gene transfer to retinoblastoma cell lines

    International Nuclear Information System (INIS)

    Choi, Sang Wook; Ham, Yong Hoh; Kim, Mee Heui

    1994-04-01

    After transfection of Rb-vector to packaging cell line (CRIP) by Ca-P precipitation method, we could select nineteen colonies of G-418 resistant clone by ring cloning. Each colony was transduced to NIH3T3 cells to select the one which produces high titer virus. After NIH3T3 cells transduction, we could get 28 colony counts for the high, 127 for the middle, and 6 for the low viral titer. With the supernatant of the high viral titer colony (CRIPRb 2-5). We transduct retinoblastoma cell lines. 5 figs, 11 refs. (Author)

  15. CD34+ (Non-Malignant) Stem Cell Selection for Patients Receiving Allogeneic Stem Cell Transplantation

    Science.gov (United States)

    2017-07-13

    Bone Marrow Failure Syndrome; Severe Aplastic Anemia; Severe Congenital Neutropenia; Amegakaryocytic Thrombocytopenia; Diamond-Blackfan Anemia; Schwachman Diamond Syndrome; Primary Immunodeficiency Syndromes; Acquired Immunodeficiency Syndromes; Histiocytic Syndrome; Familial Hemophagocytic Lymphocytosis; Lymphohistiocytosis; Macrophage Activation Syndrome; Langerhans Cell Histiocytosis (LCH); Hemoglobinopathies; Sickle Cell Disease; Sickle Cell-beta-thalassemia

  16. Ionizing radiation predisposes non-malignant human mammaryepithelial cells to undergo TGF beta-induced epithelial to mesenchymaltransition

    Energy Technology Data Exchange (ETDEWEB)

    Andarawewa, Kumari L.; Erickson, Anna C.; Chou, William S.; Costes, Sylvain; Gascard, Philippe; Mott, Joni D.; Bissell, Mina J.; Barcellos-Hoff, Mary Helen

    2007-04-06

    Transforming growth factor {beta}1 (TGF{beta}) is a tumor suppressor during the initial stage of tumorigenesis, but it can switch to a tumor promoter during neoplastic progression. Ionizing radiation (IR), both a carcinogen and a therapeutic agent, induces TGF{beta}, activation in vivo. We now show that IR sensitizes human mammary epithelial cells (HMEC) to undergo TGF{beta}-mediated epithelial to mesenchymal transition (EMT). Non-malignant HMEC (MCF10A, HMT3522 S1 and 184v) were irradiated with 2 Gy shortly after attachment in monolayer culture, or treated with a low concentration of TGF{beta} (0.4 ng/ml), or double-treated. All double-treated (IR+TGF{beta}) HMEC underwent a morphological shift from cuboidal to spindle-shaped. This phenotype was accompanied by decreased expression of epithelial markers E-cadherin, {beta}-catenin and ZO-1, remodeling of the actin cytoskeleton, and increased expression of mesenchymal markers N-cadherin, fibronectin and vimentin. Furthermore, double-treatment increased cell motility, promoted invasion and disrupted acinar morphogenesis of cells subsequently plated in Matrigel{trademark}. Neither radiation nor TGF{beta} alone elicited EMT, even though IR increased chronic TGF{beta} signaling and activity. Gene expression profiling revealed that double treated cells exhibit a specific 10-gene signature associated with Erk/MAPK signaling. We hypothesized that IR-induced MAPK activation primes non-malignant HMEC to undergo TGF{beta}-mediated EMT. Consistent with this, Erk phosphorylation were transiently induced by irradiation, persisted in irradiated cells treated with TGF{beta}, and treatment with U0126, a Mek inhibitor, blocked the EMT phenotype. Together, these data demonstrate that the interactions between radiation-induced signaling pathways elicit heritable phenotypes that could contribute to neoplastic progression.

  17. Identification of the zinc finger 216 (ZNF216) in human carcinoma cells: a potential regulator of EGFR activity

    Science.gov (United States)

    Mincione, Gabriella; Di Marcantonio, Maria Carmela; Tarantelli, Chiara; Savino, Luca; Ponti, Donatella; Marchisio, Marco; Lanuti, Paola; Sancilio, Silvia; Calogero, Antonella; Di Pietro, Roberta; Muraro, Raffaella

    2016-01-01

    Epidermal Growth Factor Receptor (EGFR), a member of the ErbB family of receptor tyrosine kinase (RTK) proteins, is aberrantly expressed or deregulated in tumors and plays pivotal roles in cancer onset and metastatic progression. ZNF216 gene has been identified as one of Immediate Early Genes (IEGs) induced by RTKs. Overexpression of ZNF216 protein sensitizes 293 cell line to TNF-α induced apoptosis. However, ZNF216 overexpression has been reported in medulloblastomas and metastatic nasopharyngeal carcinomas. Thus, the role of this protein is still not clearly understood. In this study, the inverse correlation between EGFR and ZNF216 expression was confirmed in various human cancer cell lines differently expressing EGFR. EGF treatment of NIH3T3 cells overexpressing both EGFR and ZNF216 (NIH3T3-EGFR/ZNF216), induced a long lasting activation of EGFR in the cytosolic fraction and an accumulation of phosphorylated EGFR (pEGFR) more in the nuclear than in the cytosolic fraction compared to NIH3T3-EGFR cells. Moreover, EGF was able to stimulate an increased expression of ZNF216 in the cytosolic compartment and its nuclear translocation in a time-dependent manner in NIH3T3-EGFR/ZNF216. A similar trend was observed in A431 cells endogenously expressing the EGFR and transfected with Znf216. The increased levels of pEGFR and ZNF216 in the nuclear fraction of NIH3T3-EGFR/ZNF216 cells were paralleled by increased levels of phospho-MAPK and phospho-Akt. Surprisingly, EGF treatment of NIH3T3-EGFR/ZNF216 cells induced a significant increase of apoptosis thus indicating that ZNF216 could sensitize cells to EGF-induced apoptosis and suggesting that it may be involved in the regulation and effects of EGFR signaling. PMID:27732953

  18. High levels of the type III inorganic phosphate transporter PiT1 (SLC20A1) can confer faster cell adhesion

    OpenAIRE

    Kongsfelt, Iben Boutrup; Byskov, Kristina; Pedersen, Lasse Ebdrup; Pedersen, Lene

    2014-01-01

    The inorganic phosphate transporter PiT1 (SLC20A1) is ubiquitously expressed in mammalian cells. We recently showed that overexpression of human PiT1 was sufficient to increase proliferation of two strict density-inhibited cell lines, murine fibroblastic NIH3T3 and pre-osteoblastic MC3T3-E1 cells, and allowed the cultures to grow to higher cell densities. In addition, upon transformation NIH3T3 cells showed increased ability to form colonies in soft agar. The cellular regulation of PiT1 expre...

  19. The effects of erythropoietin signaling on telomerase regulation in non-erythroid malignant and non-malignant cells

    International Nuclear Information System (INIS)

    Uziel, Orit; Kanfer, Gil; Beery, Einat; Yelin, Dana; Shepshelovich, Daniel; Bakhanashvili, Mary; Nordenberg, Jardena; Lahav, Meir

    2014-01-01

    Highlights: • We assumed that some of erythropoietin adverse effects may be mediated by telomerase activity. • EPO administration increased telomerase activity, cells proliferation and migration. • The inhibition of telomerase modestly repressed the proliferative effect of erythropoietin. • Telomere shortening caused by long term inhibition of the enzyme totally abolished that effect. • This effect was mediated via the Lyn–AKT axis and not by the canonical JAK2–STAT pathway. - Abstract: Treatment with erythropoietin (EPO) in several cancers is associated with decreased survival due to cancer progression. Due to the major importance of telomerase in cancer biology we hypothesized that some of these effects may be mediated through EPO effect on telomerase. For this aim we explored the possible effects of EPO on telomerase regulation, cell migration and chemosensitivity in non-erythroid malignant and non-malignant cells. Cell proliferation, telomerase activity (TA) and cell migration increased in response to EPO. EPO had no effect on cancer cells sensitivity to cisplatinum and on the cell cycle status. The inhibition of telomerase modestly repressed the proliferative effect of EPO. Telomere shortening caused by long term inhibition of the enzyme abolished the effect of EPO, suggesting that EPO effects on cancer cells are related to telomere dynamics. TA was correlated with the levels of Epo-R. The increase in TA was mediated post-translationally through the Lyn-Src and not the canonical JAK2 pathway

  20. The effects of erythropoietin signaling on telomerase regulation in non-erythroid malignant and non-malignant cells

    Energy Technology Data Exchange (ETDEWEB)

    Uziel, Orit, E-mail: Oritu@clalit.org.il [Felsenstein Medical Research Center, Sackler School of Medicine, Tel-Aviv University, Ramat-Aviv (Israel); Kanfer, Gil [Felsenstein Medical Research Center, Sackler School of Medicine, Tel-Aviv University, Ramat-Aviv (Israel); Dep. of Human Molecular Genetics and Biochemistry, Sackler School of Medicine, Tel-Aviv University, Ramat-Aviv (Israel); Beery, Einat [Felsenstein Medical Research Center, Sackler School of Medicine, Tel-Aviv University, Ramat-Aviv (Israel); Yelin, Dana; Shepshelovich, Daniel [Medicine A, Sackler School of Medicine, Tel-Aviv University, Ramat-Aviv (Israel); Bakhanashvili, Mary [Unit of Infectious Diseases, Sheba Medical Center, Tel-Hashomer (Israel); Nordenberg, Jardena [Felsenstein Medical Research Center, Sackler School of Medicine, Tel-Aviv University, Ramat-Aviv (Israel); Dep. of Human Molecular Genetics and Biochemistry, Sackler School of Medicine, Tel-Aviv University, Ramat-Aviv (Israel); Endocrinology Laboratory, Beilinson Medical Center, Petah-Tikva (Israel); Lahav, Meir [Felsenstein Medical Research Center, Sackler School of Medicine, Tel-Aviv University, Ramat-Aviv (Israel); Medicine A, Sackler School of Medicine, Tel-Aviv University, Ramat-Aviv (Israel)

    2014-07-18

    Highlights: • We assumed that some of erythropoietin adverse effects may be mediated by telomerase activity. • EPO administration increased telomerase activity, cells proliferation and migration. • The inhibition of telomerase modestly repressed the proliferative effect of erythropoietin. • Telomere shortening caused by long term inhibition of the enzyme totally abolished that effect. • This effect was mediated via the Lyn–AKT axis and not by the canonical JAK2–STAT pathway. - Abstract: Treatment with erythropoietin (EPO) in several cancers is associated with decreased survival due to cancer progression. Due to the major importance of telomerase in cancer biology we hypothesized that some of these effects may be mediated through EPO effect on telomerase. For this aim we explored the possible effects of EPO on telomerase regulation, cell migration and chemosensitivity in non-erythroid malignant and non-malignant cells. Cell proliferation, telomerase activity (TA) and cell migration increased in response to EPO. EPO had no effect on cancer cells sensitivity to cisplatinum and on the cell cycle status. The inhibition of telomerase modestly repressed the proliferative effect of EPO. Telomere shortening caused by long term inhibition of the enzyme abolished the effect of EPO, suggesting that EPO effects on cancer cells are related to telomere dynamics. TA was correlated with the levels of Epo-R. The increase in TA was mediated post-translationally through the Lyn-Src and not the canonical JAK2 pathway.

  1. Non-myeloablative allogeneic stem cell transplantation focusing on immunotherapy of life-threatening malignant and non-malignant diseases.

    Science.gov (United States)

    Slavin, S; Nagler, A; Shapira, M; Panigrahi, S; Samuel, S; Or, A

    2001-01-01

    Allogeneic bone marrow transplantation (BMT) represents an important therapeutic tool for treatment of otherwise incurable malignant and non-malignant diseases. Until recently, myeloablative regimens were considered mandatory for eradication of all undesirable host-derived hematopoietic elements. Our preclinical and ongoing clinical studies indicated that much more effective eradication of host immunohematopoietic system cells could be achieved by adoptive allogeneic cell therapy with donor lymphocyte infusion (DLI) following BMT. Thus, eradication of blood cancer cells, especially in patients with CML can be frequently accomplished despite complete resistance of such tumor cells to maximally tolerated doses of chemoradiotherapy. Our cumulative experience suggested that graft versus leukemia (GVL) effects might be a useful tool for eradication of otherwise resistant tumor cells of host origin. The latter working hypothesis suggested that effective BMT procedures may be accomplished without lethal conditioning of the host, using new well tolerated non-myeloablative regimen, thus possibly minimizing immediate and late side effects related to myeloablative procedures considered until recently mandatory for conditioning of BMT recipients. Recent clinical data that will be presented suggests that safe non-myeloablative stem cell transplantation (NST), with no major toxicity can replace the conventional BMT. Thus, NST may provide an option for cure for a large spectrum of clinical indications in children and elderly individuals without lower or upper age limit, while minimizing procedure-related toxicity and mortality.

  2. Monoamine oxidase inhibitors l-deprenyl and clorgyline protect nonmalignant human cells from ionising radiation and chemotherapy toxicity.

    LENUS (Irish Health Repository)

    Seymour, C B

    2003-11-17

    l-Deprenyl (R-(-)-deprenyl, selegiline) is an inhibitor of monoamine oxidase-B (MAO-B) that is known to protect nerve cells from a variety of chemical and physical insults. As apoptosis is a common mechanism of radiation-induced cell death, the effect of l-deprenyl on the survival of cultured cells and tissue explants was studied following exposure to gamma radiation. The results obtained were compared with the effects of the less-selective MAO-B inhibitor pargyline and the MAO-A inhibitor clorgyline. l-Deprenyl at a concentration of 10(-9) M protected the nontumorigenic cell line (HaCaT) and normal human urothelial explants from the effects of cobalt-60 gamma radiation, but did not protect tumorigenic human cell lines HaCaT-ras, HPV-transfected human keratinocytes (HPV-G cells), or PC3. Human bladder carcinoma explants were not protected. Clorgyline showed a smaller protective effect of normal cells, whereas pargyline had no effect. Radiation-induced delayed effects (genomic instability measured as delayed cell death) were prevented in normal cells by l-deprenyl but, interestingly, deprenyl appeared to increase the amount of delayed death in the tumorigenic cell lines. Studies using l-deprenyl prior to the exposure of nonmalignant cells to cisplatin showed that cell death due to this agent was also reduced. Treatment of cultures of nontumorigenic cells with l-deprenyl or clorgyline significantly increased the levels of the protein Bcl-2 following irradiation, but there was no such effect on the already-elevated levels of this protein in the tumour samples. Since the Bcl-2 has been shown to be an inhibitor of apoptosis or programmed cell death, this would imply that the protective effects of l-deprenyl and clorgyline involve activation of antiapoptotic pathways within the normal cell. This hypothesis is supported by data showing reduced levels of apoptosis in HaCAT cells and in normal bladder explant cultures following treatment with l-deprenyl.

  3. Growth-inhibitory effects of the red alga Gelidium amansii on cultured cells.

    Science.gov (United States)

    Chen, Yue-Hwa; Tu, Ching-Jung; Wu, Hsiao-Ting

    2004-02-01

    The objective of this study was to investigate the effects of Gelidium amansii, an edible red agar cultivated off the northeast coast of Taiwan, on the growth of two lines of cancer cells, murine hepatoma (Hepa-1) and human leukemia (HL-60) cells, as well as a normal cell line, murine embryo fibroblast cells (NIH-3T3). The potential role of G. amansii on the induction of apoptosis was also examined. The results indicated that all extracts from G. amansii, including phosphate-buffered saline (PBS) and methanol extracts from dried algae as well as the dimethyl sulfoxide (DMSO) extract from freeze-dried G. amansii agar, inhibited the growth of Hepa-1 and NIH-3T3 cells, but not the growth of HL-60 cells. Annexin V-positive cells were observed in methanol and DMSO extract-treated, but not PBS extract-treated Hepa-1 and NIH-3T3 cells, suggesting that the lipid-soluble extracts of G. amansii induced apoptosis. In summary, extracts of G. amansii from various preparations exhibited antiproliferative effects on Hepa-1 and NIH-3T3 cells, and apoptosis may play a role in the methanol and DMSO extract-induced inhibitory effects. However, the antiproliferative effects of PBS extracts was not through apoptosis. Moreover, the growth-inhibitory effects of G. amansii were not specific to cancer cells.

  4. Cytotoxic Activity of Selected Iranian Traditional Medicinal Plants on Colon, Colorectal and Breast Cancer Cell Lines

    Directory of Open Access Journals (Sweden)

    Leila Mohammad Taghizadeh Kashani

    2014-11-01

    Full Text Available Background: Many natural products from plants have been recognized to exert anticancer activity. In this study, ethanolic extracts of selected medicinal herbs from Iranian flora including Alyssum homolocarpum Fisch. (from seeds, Urtica dioica L. (from aerial parts, Cichorium intybus L. (from roots and Solanum nigrum L. (from fruits, were evaluated for their cytotoxic effect on different cell lines.Methods: Cytotoxic effect of these extracts was studied on three different cancer cell lines; colon carcinoma (HT-29, colorectal adenocarcinoma (Caco-2 and breast ductal carcinoma (T47D. In addition, Swiss mouse embryo fibroblasts (NIH 3T3 were used as normal nonmalignant cells. MTT assay (3-(4,5-dimethylthiazol-2-yl-2,5-diphenyltetrazolium bromide was utilized for calculating the cytotoxicity of extracts on cell lines.Results: Results showed the potent cytotoxic activity of U. dioica ethanolic extract against T47D cell line with IC50 value of 46.14±4.55 µg/ml. Other extracts showed poor activity with IC50>100 µg/ml.Conclusions: Cytotoxic activity recorded in the present study revealed high potential antiproliferative activity of U. dioica ethanolic extract against T47D cell line. The real IC50 values of this extract may be considerably lower than the IC50 measured in our study if its pharmacological active compounds become pure. The results emphasize the importance of studies on U. dioica ethanolic extract to characterize potential components as cytotoxic natural medicines.

  5. Fabrication of Nanostructured Poly-ε-caprolactone 3D Scaffolds for 3D Cell Culture Technology

    KAUST Repository

    Schipani, Rossana

    2015-01-01

    . The suitability of these devices as cell culture technology supports was evaluated by plating NIH/3T3 mouse embryonic fibroblasts and human Neural Stem Cells (hNSC) on them. Scanning Electron Microscopy (SEM) analysis was carried out in order to examine the micro

  6. Regulation of p53 in NIH3T3 mouse fibroblasts following hyperosmotic stress

    DEFF Research Database (Denmark)

    Lambert, Ian Henry; Enghoff, Maria Stine; Brandi, Marie-Luise

    2015-01-01

    regulating proteins p38 MAP kinase and the ubiquitin ligase MDM2 were studied as a function of increasing osmolarity. MDM2 protein expression was unchanged at all osmolarities, whereas MDM2 phosphorylation (Ser(166)) increased at osmolarities up to 537 mOsm and remained constant at higher osmolarities...

  7. Enantioselective cellular localisation of europium(iii) coordination complexes† †Electronic supplementary information (ESI) available: Complex synthesis and characterisation, selected cell images showing correspondence with commercial lysosomal or mitochondrial stains and spectral imaging profiles. See DOI: 10.1039/c7sc04422d

    Science.gov (United States)

    Frawley, Andrew T.; Linford, Holly V.; Starck, Matthieu; Pal, Robert

    2017-01-01

    The selective mitochondrial localisation of the Λ enantiomer of three different emissive europium(iii) complexes in NIH 3T3 and MCF7 cells contrasts with the behaviour of the Δ enantiomer, for which a predominant lysosomal localisation was observed by confocal microscopy. In each case, cell uptake occurs via macropinocytosis. PMID:29675151

  8. Biochemical studies on the conversion of tumor cells to their counterpart normal cells(II)

    International Nuclear Information System (INIS)

    Yun, Taik Koo; Lim, In Kyoung; Lee, Kee Ho; Lee, Do Jong

    1986-12-01

    We describe here the expression of c-fos oncogene in the growth stimulated cells e.g., mouse prenatal tissues, FCS treated NIH-3T3 fibroblast and A549 human lung cancer cells. Total cellular RNA was isolated from A/J and C57BL/5J mouse embryos at the 13 and 17 day of pregnancy. NIH-3T3 mouse fibroblast and A549 lung cancer cells were stimulated using the 15% FCS after serum depletion for 2-3 days. During the serum stimulation, c-fos expression was detected at time intervals by the dot blot analysis using the 32 P-labelled c-fos DNA probe. RNAs isolated from mouse prenatal tissues were strongly hybridized with c-fos. c-fos induction was detected from 30 minutes after serum stimulation in NIH-3T3 cells, and turned off after 2hrs. On the other hand, c-fos in the A549 lung cancer cells was independent on the serum stimulation and very strongly expressed even in the serum depleted codition. These results indicate the possible implication of growth control by the turning off the oncogene expression. (Author)

  9. Model-based investigation of the circadian clock and cell cycle coupling in mouse embryonic fibroblasts: Prediction of RevErb-α up-regulation during mitosis

    OpenAIRE

    Traynard , Pauline; Feillet , Céline; Soliman , Sylvain; Delaunay , Franck; Fages , François

    2016-01-01

    International audience; Experimental observations have put in evidence autonomous self-sustained circadian oscillators in most mammalian cells, and proved the existence of molecular links between the circadian clock and the cell cycle. Some mathematical models have also been built to assess conditions of control of the cell cycle by the circadian clock. However, recent studies in individual NIH3T3 fibroblasts have shown an unexpected acceleration of the circadian clock together with the cell ...

  10. In vitro extracellular matrix model to evaluate stroma cell response to transvaginal mesh.

    Science.gov (United States)

    Wu, Ming-Ping; Huang, Kuan-Hui; Long, Cheng-Yu; Yang, Chau-Chen; Tong, Yat-Ching

    2014-04-01

    The use of surgical mesh for female pelvic floor reconstruction has increased in recent years. However, there is paucity of information about the biological responses of host stroma cells to different meshes. This study was aimed to establish an in vitro experimental model to study the micro-environment of extracellular matrix (ECM) with embedded mesh and the stroma cell behaviors to different synthetic meshes. Matrigel multi-cellular co-culture system with embedded mesh was used to evaluate the interaction of stroma cells and synthetic mesh in a simulated ECM environment. Human umbilical vein endothelial cells (HUVEC) and NIH3T3 fibroblasts were inoculated in the system. The established multi-cellular Matrigel co-culture system was used to detect stroma cell recruitment and tube formation ability for different synthetic meshes. HUVEC and NIH3T3 cells were recruited into the mesh interstices and organized into tube-like structures in type I mesh material from Perigee, Marlex and Prolift 24 hr after cell inoculation. On the contrary, there was little recruitment of HUVEC and NIH3T3 cells into the type III mesh of intra-vaginal sling (IVS). The Matrigel multi-cellular co-culture system with embedded mesh offers a useful in vitro model to study the biological behaviors of stroma cells in response to different types of synthetic meshes. The system can help to select ideal mesh candidates before actual implantation into the human body. © 2013 Wiley Periodicals, Inc.

  11. Phenotypic characterization of telomerase-immortalized primary non-malignant and malignant tumor-derived human prostate epithelial cell lines

    International Nuclear Information System (INIS)

    Gu Yongpeng; Li Hongzhen; Miki, Jun; Kim, Kee-Hong; Furusato, Bungo; Sesterhenn, Isabell A.; Chu, Wei-Sing; McLeod, David G.; Srivastava, Shiv; Ewing, Charles M.; Isaacs, William B.; Rhim, Johng S.

    2006-01-01

    In vitro human prostate cell culture models are critical for clarifying the mechanism of prostate cancer progression and for testing preventive and therapeutic agents. Cell lines ideal for the study of human primary prostate tumors would be those derived from spontaneously immortalized tumor cells; unfortunately, explanted primary prostate cells survive only short-term in culture, and rarely immortalize spontaneously. Therefore, we recently have generated five immortal human prostate epithelial cell cultures derived from both the benign and malignant tissues of prostate cancer patients with telomerase, a gene that prevents cellular senescence. Examination of these cell lines for their morphologies and proliferative capacities, their abilities to grow in low serum, to respond to androgen stimulation, to grow above the agar layer, to form tumors in SCID mice, suggests that they may serve as valid, useful tools for the elucidation of early events in prostate tumorigenesis. Furthermore, the chromosome alterations observed in these immortalized cell lines expressing aspects of the malignant phenotypes imply that these cell lines accurately recapitulate the genetic composition of primary tumors. These novel in vitro models may offer unique models for the study of prostate carcinogenesis and also provide the means for testing both chemopreventive and chemotherapeutic agents

  12. Experimental control of neoplastic progression in cell populations: Foulds' rules revisited.

    OpenAIRE

    Rubin, H

    1994-01-01

    Foulds introduced six rules of tumor progression based on his observations of spontaneous mammary cancer in mice and generalized them to all forms of neoplasia [Foulds, L. (1954) Cancer Res. 14, 327-339 and Foulds, L. (1969) Neoplastic Development (Academic, New York), Vol. 1, preface and pp. 72-74.] Rules III, IV, and V are considered controversial, and research in animals seems inadequate to resolve the controversies. A subline of NIH 3T3 cells undergoes progressive transformation to produc...

  13. The Flaxseed-Derived Lignan Phenolic Secoisolariciresinol Diglucoside (SDG) Protects Non-Malignant Lung Cells from Radiation Damage.

    Science.gov (United States)

    Velalopoulou, Anastasia; Tyagi, Sonia; Pietrofesa, Ralph A; Arguiri, Evguenia; Christofidou-Solomidou, Melpo

    2015-12-22

    Plant phenolic compounds are common dietary antioxidants that possess antioxidant and anti-inflammatory properties. Flaxseed (FS) has been reported to be radioprotective in murine models of oxidative lung damage. Flaxseed's protective properties are attributed to its main biphenolic lignan, secoisolariciresinol diglucoside (SDG). SDG is a free radical scavenger, shown in cell free systems to protect DNA from radiation-induced damage. The objective of this study was to investigate the in vitro radioprotective efficacy of SDG in murine lung cells. Protection against irradiation (IR)-induced DNA double and single strand breaks was assessed by γ-H2AX labeling and alkaline comet assay, respectively. The role of SDG in modulating the levels of cytoprotective enzymes was evaluated by qPCR and confirmed by Western blotting. Additionally, effects of SDG on clonogenic survival of irradiated cells were evaluated. SDG protected cells from IR-induced death and ameliorated DNA damage by reducing mean comet tail length and percentage of γ-H2AX positive cells. Importantly, SDG significantly increased gene and protein levels of antioxidant HO-1, GSTM1 and NQO1. Our results identify the potent radioprotective properties of the synthetic biphenolic SDG, preventing DNA damage and enhancing the antioxidant capacity of normal lung cells; thus, rendering SDG a potential radioprotector against radiation exposure.

  14. The Flaxseed-Derived Lignan Phenolic Secoisolariciresinol Diglucoside (SDG Protects Non-Malignant Lung Cells from Radiation Damage

    Directory of Open Access Journals (Sweden)

    Anastasia Velalopoulou

    2015-12-01

    Full Text Available Plant phenolic compounds are common dietary antioxidants that possess antioxidant and anti-inflammatory properties. Flaxseed (FS has been reported to be radioprotective in murine models of oxidative lung damage. Flaxseed’s protective properties are attributed to its main biphenolic lignan, secoisolariciresinol diglucoside (SDG. SDG is a free radical scavenger, shown in cell free systems to protect DNA from radiation-induced damage. The objective of this study was to investigate the in vitro radioprotective efficacy of SDG in murine lung cells. Protection against irradiation (IR-induced DNA double and single strand breaks was assessed by γ-H2AX labeling and alkaline comet assay, respectively. The role of SDG in modulating the levels of cytoprotective enzymes was evaluated by qPCR and confirmed by Western blotting. Additionally, effects of SDG on clonogenic survival of irradiated cells were evaluated. SDG protected cells from IR-induced death and ameliorated DNA damage by reducing mean comet tail length and percentage of γ-H2AX positive cells. Importantly, SDG significantly increased gene and protein levels of antioxidant HO-1, GSTM1 and NQO1. Our results identify the potent radioprotective properties of the synthetic biphenolic SDG, preventing DNA damage and enhancing the antioxidant capacity of normal lung cells; thus, rendering SDG a potential radioprotector against radiation exposure.

  15. In vitro selection of high-infectious, leukemogenic virus from low-infectious, non-leukemogenic type C virus from a malignant ST/a mouse cell line

    DEFF Research Database (Denmark)

    Willumsen, B M

    1979-01-01

    ; however, virus was detected in supernatant fluids only after two to four subcultures of the infected cells. The virus thus produced was XC(+) and a large plaque former. The virus released from infected SC-1 cells was N-tropic, whereas the viruses from infected NIH/3T3 and BALB/3T3 cells were NB...... nanogram of p30, was 30- to 60-fold lower for the virus released from the ST-L1 cell line than that of the viruses after passage in SC-1, NIH/3T3, and BALB/3T3 cells. None of the viruses could infect rabbit or mink cells. Inoculation of the viruses into newborn mice showed that the ST-L1 virus was non......-leukemogenic, whereas the NB-tropic virus selected from this after passage in BALB/3T3 or NIH/3T3 cells was highly leukemogenic. Viruses isolated from leukemic animals were indistinguishable with respect to host range and protein mobilities in SDS gels from the ones with which the mice were inoculated. Although the SC...

  16. Accumulation and Toxicity of Superparamagnetic Iron Oxide Nanoparticles in Cells and Experimental Animals.

    Science.gov (United States)

    Jarockyte, Greta; Daugelaite, Egle; Stasys, Marius; Statkute, Urte; Poderys, Vilius; Tseng, Ting-Chen; Hsu, Shan-Hui; Karabanovas, Vitalijus; Rotomskis, Ricardas

    2016-08-19

    The uptake and distribution of negatively charged superparamagnetic iron oxide (Fe₃O₄) nanoparticles (SPIONs) in mouse embryonic fibroblasts NIH3T3, and magnetic resonance imaging (MRI) signal influenced by SPIONs injected into experimental animals, were visualized and investigated. Cellular uptake and distribution of the SPIONs in NIH3T3 after staining with Prussian Blue were investigated by a bright-field microscope equipped with digital color camera. SPIONs were localized in vesicles, mostly placed near the nucleus. Toxicity of SPION nanoparticles tested with cell viability assay (XTT) was estimated. The viability of NIH3T3 cells remains approximately 95% within 3-24 h of incubation, and only a slight decrease of viability was observed after 48 h of incubation. MRI studies on Wistar rats using a clinical 1.5 T MRI scanner were showing that SPIONs give a negative contrast in the MRI. The dynamic MRI measurements of the SPION clearance from the injection site shows that SPIONs slowly disappear from injection sites and only a low concentration of nanoparticles was completely eliminated within three weeks. No functionalized SPIONs accumulate in cells by endocytic mechanism, none accumulate in the nucleus, and none are toxic at a desirable concentration. Therefore, they could be used as a dual imaging agent: as contrast agents for MRI and for traditional optical biopsy by using Prussian Blue staining.

  17. [Regulation of [12Asp]K-ras4B on transcriptional activity of estrogen receptor in endometrial carcinoma HEC-1A cell lines].

    Science.gov (United States)

    Gui, Li-ming; Wei, Li-hui; Xu, Ming-xu; Wang, Jian-liu; Zhong, Ying-cheng; Li, Xiao-ping; Tu, Zheng; Sun, Peng-ming; Ma, Da-long

    2004-01-01

    To investigate the effect of mutant-type [(12)Asp]K-ras4B gene on the expression of estrogen receptor (ER) alpha and beta and their transcriptional activity as a transcription factor in endometrial carcinoma HEC-1A cell line. (1) Effect of [(12)Asp]K-ras4B on the expression of ER alpha and beta were determined using Western blot assay. (2) Eukaryotic expression plasmid pGL3-luciferase-ERE containing luciferase report gene and estrogen receptor element (ERE) was constructed, and co-transfected into NIH3T3 and HEC-1A cell lines with pEGFP-N1 to examine the effect of [(12)Asp]K-ras4B on ER transcription that is regulated by estradiol. In addition, they were transfected into pSV5-HER0 (containing full length wide type ERalpha cDNA) and pCMV-rafS621A (inhibiting raf kinase) plasmids to test the effect of [(12)Asp]K-ras4B/raf signal pathway on transcriptional activity of ER proteins. (1) Protein level of ERs expressed in pcDI transfected control cells was low while it was increased for 3.6-fold (97 +/- 25, 349 +/- 67, P ras4B NIH3T3 cells after transfection. (2) In pcDI-[(12)Asp]K-ras4B NIH3T3 cells, the ratios for ERalpha and and ERbeta levels before transfection of rafS621A plasmids to that after the transfection, were 2.4:1 (724 +/- 45, 310 +/- 46, P HEC-1A cells, these ratios were 2.1:1 (566 +/- 22, 279 +/- 30, P ras4B NIH3T3 cells, 19-fold (141 +/- 39, 2644 +/- 331, P HEC-1A cells, respectively, when compared with those in the absence of E(2). (4) In pSV5-HER0 transfected pcDI-[(12)Asp] K-ras4B NIH3T3 cells and HEC-1A cells, compared to the untransfected cells, the ER transcriptional activity in the transfected cells increased markedly. The luciferase activity was increased for 8-fold (1048 +/- 91, 8099 +/- 452, P HEC-1A cells and pcDI-[(12)Asp]K-ras4B NIH3T3 cells. The ratio of luciferase activities in pcDI-[(12)Asp]K-ras4B NIH3T3 and HEC-1A cells, before and after transfection was 7.8:1 (1184 +/- 168, 152 +/- 27, P ras4B can enhance the expression of ERalpha and

  18. Unirradiated cells rescue cells exposed to ionizing radiation: Activation of NF-κB pathway in irradiated cells

    Energy Technology Data Exchange (ETDEWEB)

    Lam, R.K.K. [Department of Physics and Materials Science, City University of Hong Kong (Hong Kong); Han, Wei [Center of Medical Physics and Technology, Hefei Institutes of Physical Science, Chinese Academy of Sciences, Hefei 230031 (China); Yu, K.N., E-mail: peter.yu@cityu.edu.hk [Department of Physics and Materials Science, City University of Hong Kong (Hong Kong); State Key Laboratory in Marine Pollution, City University of Hong Kong (Hong Kong)

    2015-12-15

    Highlights: • Rescue effect was observed in both irradiated and HeLa and NIH/3T3 cells. • Novel setup and procedures to separate the rescue signals and the bystander signals. • Confirmed activation of NF-κB pathway in rescue effect using activation inhibitor. • Confirmed activation of NF-κB pathway in rescue effect using anti-NF-κB p65 antibody. - Abstract: We studied the involvement of NF-κB pathway activation in the rescue effect in HeLa and NIH/3T3 cells irradiated by α particles. Firstly, upon irradiation by 5 cGy of α particles, for both cell lines, the numbers of 53BP1 foci/cell at 12 h post-irradiation were significantly smaller when only 2.5% of the cell population was irradiated as compared to 100% irradiation, which demonstrated the rescue effect. Secondly, we studied the effect of NF-κB on the rescue effect through the use of the NF-κB activation inhibitor BAY-11-7082. Novel experimental setup and procedures were designed to prepare the medium (CM) which had conditioned the bystander cells previously partnered with irradiated cells, to ensure physical separation between rescue and bystander signals. BAY-11-7082 itself did not inflict DNA damages in the cells or have effects on activation of the NF-κB response pathway in the irradiated cells through direct irradiation. The rescue effect was induced in both cell lines by the CM, which was abrogated if BAY-11-7082 was added to the CM. Thirdly, we studied the effect of NF-κB on the rescue effect through staining for phosphorylated NF-κB (p-NF-κB) expression using the anti-NF-κB p65 (phospho S536) antibody. When the fraction of irradiated cells dropped from 100% to 2.5%, the p-NF-κB expression in the cell nuclei of irradiated NIH/3T3 cells increased significantly, while that in the cell nuclei of irradiated HeLa cells also increased although not significantly. Moreover, the p-NF-κB expression in the cell nuclei of irradiated HeLa cells and NIH/3T3 cells treated with CM also increased

  19. Study of cell cycle and apoptosis after radiation with electron linear accelerator injury

    International Nuclear Information System (INIS)

    Xu Lan; Zhou Yinghui; Shi Ning; Peng Miao; Wu Shiliang

    2002-01-01

    Purpose: To determine the cell cycle and apoptosis of the injured cells after radiation with the electron linear accelerator. Methods: NIH 3T3 cells were irradiated by the radiation with the electron linear accelerator. In the experiment the condition of the cell cycle and apoptosis of the injured cells were measured. The expression of p53 was also tested. Results: After exposure to radiation, the number of apoptotic cells as well as the expression of p53 increased. Conclusion: The electron linear accelerator radiation injury can induce cell apoptosis

  20. Use of electroporation to study the cytotoxic effects of fluorodeoxyuridylate in intact cells.

    Science.gov (United States)

    Jastreboff, M M; Sokoloski, J A; Bertino, J R; Narayanan, R

    1987-04-15

    The introduction of 2'-deoxyuridine 5'-monophosphate and its analog, 5-fluoro-2'-deoxyuridine 5'-monophosphate, into intact CCRF-CEM and NIH3T3 cells was achieved by electroporation. Following electroporation, cells were shown to be fully functional as monitored by the incorporation of deoxyuridylate, after conversion to thymidylate, into DNA. Pretreatment of cells with fluorodeoxyuridine completely abolished this effect. In contrast, introduction of the fluoro analog into cells by electroporation markedly inhibited both DNA synthesis and cell growth in a time-dependent manner. Thus, electroporation offers a powerful tool to permeabilize cells to a variety of cellular metabolites and antimetabolites.

  1. The anti-apoptotic activity associated with phosphatidylinositol transfer protein α activates the MAPK and Akt/PKB pathway

    NARCIS (Netherlands)

    Schenning, M.; Goedhart, J.; Gadella (jr.), T.W.J.; Avram, D.; Wirtz, K.W.A.; Snoek, G.T.

    2008-01-01

    The conditioned medium (CM) from mouse NIH3T3 fibroblast cells overexpressing phosphatidylinositol transfer protein α (PI-TPα; SPIα cells) demonstrates an increased anti-apoptotic activity compared with CM from wild type NIH3T3 (wtNIH3T3) cells. As previously shown, the anti-apoptotic activity acts

  2. The hazards of using ionising radiation for non-malignant conditions - a case study

    International Nuclear Information System (INIS)

    Morton, Tracy

    1989-01-01

    A case study is presented in which a patient received low dose radiotherapy at seven months for a non-malignant cavernous haemangioma on the upper chest. She subsequently developed three separate malignancies before the age of 40 years: a thyroid carcinoma, a breast carcinoma and a basal cell carcinoma of the skin on the upper chest. This case illustrates the hazards of using ionizing radiation for the treatment of non-malignant conditions. (U.K.)

  3. Assessment of Membrane Fluidity Fluctuations during Cellular Development Reveals Time and Cell Type Specificity

    KAUST Repository

    Noutsi, Bakiza Kamal; Gratton, Enrico; Chaieb, Saharoui

    2016-01-01

    Cell membrane is made up of a complex structure of lipids and proteins that diffuse laterally giving rise to what we call membrane fluidity. During cellular development, such as differentiation cell membranes undergo dramatic fluidity changes induced by proteins such as ARC and Cofilin among others. In this study we used the generalized polarization (GP) property of fluorescent probe Laurdan using two-photon microscopy to determine membrane fluidity as a function of time and for various cell lines. A low GP value corresponds to a higher fluidity and a higher GP value is associated with a more rigid membrane. Four different cell lines were monitored such as hN2, NIH3T3, HEK293 and L6 cells. Membrane fluidity was measured at 12h, 72h and 92 h. Our results show significant changes in membrane fluidity among all cell types at different time points. GP values tend to increase significantly within 92 h in hN2 cells and 72 h in NIH3T3 cells and only at 92 h in HEK293 cells. L6 showed a marked decrease in membrane fluidity at 72 h and starts to increase at 92 h. As expected, NIH3T3 cells have more rigid membrane at earlier time points. On the other hand, neurons tend to have the highest membrane fluidity at early time points emphasizing its correlation with plasticity and the need for this malleability during differentiation. This study sheds light on the involvement of membrane fluidity during neuronal differentiation and development of other cell lines.

  4. Assessment of Membrane Fluidity Fluctuations during Cellular Development Reveals Time and Cell Type Specificity

    KAUST Repository

    Noutsi, Bakiza Kamal

    2016-06-30

    Cell membrane is made up of a complex structure of lipids and proteins that diffuse laterally giving rise to what we call membrane fluidity. During cellular development, such as differentiation cell membranes undergo dramatic fluidity changes induced by proteins such as ARC and Cofilin among others. In this study we used the generalized polarization (GP) property of fluorescent probe Laurdan using two-photon microscopy to determine membrane fluidity as a function of time and for various cell lines. A low GP value corresponds to a higher fluidity and a higher GP value is associated with a more rigid membrane. Four different cell lines were monitored such as hN2, NIH3T3, HEK293 and L6 cells. Membrane fluidity was measured at 12h, 72h and 92 h. Our results show significant changes in membrane fluidity among all cell types at different time points. GP values tend to increase significantly within 92 h in hN2 cells and 72 h in NIH3T3 cells and only at 92 h in HEK293 cells. L6 showed a marked decrease in membrane fluidity at 72 h and starts to increase at 92 h. As expected, NIH3T3 cells have more rigid membrane at earlier time points. On the other hand, neurons tend to have the highest membrane fluidity at early time points emphasizing its correlation with plasticity and the need for this malleability during differentiation. This study sheds light on the involvement of membrane fluidity during neuronal differentiation and development of other cell lines.

  5. Cytotoxicity, Antiproliferative Effects, and Apoptosis Induction of Methanolic Extract of Cynometra cauliflora Linn. Whole Fruit on Human Promyelocytic Leukemia HL-60 Cells

    Directory of Open Access Journals (Sweden)

    T-Johari S. A. Tajudin

    2012-01-01

    Full Text Available Methanolic extract of Cynometra cauliflora whole fruit was assayed for cytotoxicity against the human promyelocytic leukemia HL-60 and the normal mouse fibroblast NIH/3T3 cell lines by using the MTT assay. The CD50 of the extract for 72 hours was 0.9 μg/mL whereas the value for the cytotoxic drug vincristine was 0.2 μg/mL. The viability of the NIH/3T3 cells was at 80.0% when treated at 15.0 μg/mL. The extract inhibited HL-60 cell proliferation with dose dependence. AO/PI staining of HL-60 cells treated with the extract revealed that majority of cells were in the apoptotic cell death mode. Flow cytometry analysis of HL-60 cells treated at CD50 of the extract showed that the early apoptotic cells were 31.0, 26.3 and 19.9% at 24, 48, and 72 hours treatment, respectively. The percentage of late apoptotic cells was increased from 62.0 at 24 hours to 64.1 and 70.2 at 48 and 72 hours, respectively. Meanwhile, percent of necrotic cells were 4.9, 6.6, and 8.5 at 24, 48, and 72 hours, respectively. This study has shown that the methanolic extract of C. cauliflora whole fruit was cytotoxic towards HL-60 cells and induced the cells into apoptotic cell death mode, but less cytotoxic towards NIH/3T3 cells.

  6. Enhancement of cell growth on honeycomb-structured polylactide surface using atmospheric-pressure plasma jet modification

    Energy Technology Data Exchange (ETDEWEB)

    Cheng, Kuang-Yao; Chang, Chia-Hsing; Yang, Yi-Wei; Liao, Guo-Chun; Liu, Chih-Tung; Wu, Jong-Shinn, E-mail: chongsin@faculty.nctu.edu.tw

    2017-02-01

    Graphical abstract: Atmospheric-pressure plasma enhances cell growth on two different pore sizes of honeycomb pattern on polylactide surface. - Highlights: • Different pore sizes of honeycomb pattern on PLA film are created. • The two-step plasma treatment provided the oxygen- and nitrogen-containing functional groups that had a major impact on cell cultivation. • The plasma treatment had a significant effect for cell proliferation. • The surface structures are the main influence on cell cultivation, while plasma treatment can indeed improve the growth environment. - Abstract: In this paper, we compare the cell growth results of NIH-3T3 and Neuro-2A cells over 72 h on flat and honeycomb structured PLA films without and with a two-step atmospheric-pressure nitrogen-based plasma jet treatment. We developed a fabrication system used for forming of a uniform honeycomb structure on PLA surface, which can produce two different pore sizes, 3–4 μm and 7–8 μm, of honeycomb pattern. We applied a previously developed nitrogen-based atmospheric-pressure dielectric barrier discharge (DBD) jet system to treat the PLA film without and with honeycomb structure. NIH-3T3 and a much smaller Neuro-2A cells were cultivated on the films under various surface conditions. The results show that the two-step plasma treatment in combination with a honeycomb structure can enhance cell growth on PLA film, should the cell size be not too smaller than the pore size of honeycomb structure, e.g., NIH-3T3. Otherwise, cell growth would be better on flat PLA film, e.g., Neuro-2A.

  7. 1800MHz Microwave Induces p53 and p53-Mediated Caspase-3 Activation Leading to Cell Apoptosis In Vitro.

    Directory of Open Access Journals (Sweden)

    Fuqiang Xing

    Full Text Available Recent studies have reported that exposure of mammalian cells to microwave radiation may have adverse effects such as induction of cell apoptosis. However, the molecular mechanisms underlying microwave induced mammalian cell apoptosis are not fully understood. Here, we report a novel mechanism: exposure to 1800MHz microwave radiation induces p53-dependent cell apoptosis through cytochrome c-mediated caspase-3 activation pathway. We first measured intensity of microwave radiation from several electronic devices with an irradiation detector. Mouse NIH/3T3 and human U-87 MG cells were then used as receivers of 1800MHz electromagnetic radiation (EMR at a power density of 1209 mW/m2. Following EMR exposure, cells were analyzed for viability, intracellular reactive oxygen species (ROS generation, DNA damage, p53 expression, and caspase-3 activity. Our analysis revealed that EMR exposure significantly decreased viability of NIH/3T3 and U-87 MG cells, and increased caspase-3 activity. ROS burst was observed at 6 h and 48 h in NIH/3T3 cells, while at 3 h in U-87 MG cells. Hoechst 33258 staining and in situ TUNEL assay detected that EMR exposure increased DNA damage, which was significantly restrained in the presence of N-acetyl-L-cysteine (NAC, an antioxidant. Moreover, EMR exposure increased the levels of p53 protein and p53 target gene expression, promoted cytochrome c release from mitochondrion, and increased caspase-3 activity. These events were inhibited by pretreatment with NAC, pifithrin-α (a p53 inhibitor and caspase inhibitor. Collectively, our findings demonstrate, for the first time, that 1800MHz EMR induces apoptosis-related events such as ROS burst and more oxidative DNA damage, which in turn promote p53-dependent caspase-3 activation through release of cytochrome c from mitochondrion. These findings thus provide new insights into physiological mechanisms underlying microwave-induced cell apoptosis.

  8. Kaposi's sarcoma-associated herpesvirus latency-associated nuclear antigen prolongs the life span of primary human umbilical vein endothelial cells.

    Science.gov (United States)

    Watanabe, Takahiro; Sugaya, Makoto; Atkins, April M; Aquilino, Elisabeth A; Yang, Aparche; Borris, Debra L; Brady, John; Blauvelt, Andrew

    2003-06-01

    Tumor spindle cells in all clinical types of Kaposi's sarcoma (KS) are infected with Kaposi's sarcoma-associated herpesvirus (KSHV). Although KSHV contains more than 80 genes, only a few are expressed in tumor spindle cells, including latency-associated nuclear antigen (LANA) and k-cyclin (kCYC). To assess the oncogenic potential of LANA and kCYC, primary human umbilical vein endothelial cells (HUVEC) and murine NIH 3T3 cells were stably transduced by using recombinant retroviruses expressing these genes or the known viral oncogene simian virus 40 large T antigen (LTAg). Interestingly, LANA-transduced HUVEC proliferated faster and demonstrated a greatly prolonged life span (mean +/- standard deviation, 38.3 +/- 11.0 passages) than untransduced cells and vector-transduced cells (<20 passages). By contrast, kCYC-transduced HUVEC did not proliferate faster or live longer than control cells. LANA- and kCYC-transduced HUVEC, but not LTAg-transduced HUVEC, retained the ability to form normal vessel-like structures in an in vitro model of angiogenesis. In cellular assays of transformation, LANA- and kCYC-transduced NIH 3T3 cells demonstrated minimal or no anchorage-independent growth in soft agar and no tumorigenicity when injected into nude mice, unlike LTAg-transduced NIH 3T3 cells. Lastly, gene expression profiling revealed down-regulation, or silencing, of a number of genes within LANA-transduced HUVEC. Taken together, these results suggest that KSHV LANA is capable of inducing prolonged life span, but not transformation, in primary human cells. These findings may explain why LANA-expressing spindle cells proliferate within KS tumors, yet most often do not demonstrate biologic characteristics of transformation or true malignant conversion.

  9. In vivo T-cell depletion using alemtuzumab in family and unrelated donor transplantation for pediatric non-malignant disease achieves engraftment with low incidence of graft vs. host disease.

    Science.gov (United States)

    Saif, M A; Borrill, R; Bigger, B W; Lee, H; Logan, A; Poulton, K; Hughes, S; Turner, A J; Bonney, D K; Wynn, R F

    2015-03-01

    In vivo T-cell depletion, using alemtuzumab therapy prior to SCT, can reduce the incidence of GVHD. This treatment has a potential to delay immune reconstitution resulting in increased morbidity due to viral illnesses. We retrospectively analyzed data on all pediatric patients with non-malignant disorders who received alemtuzumab-based conditioning regimens in our center over the last 10 yr (n = 91). Our data show an OS of 91.2%. The incidence of acute (grade 2-4) GVHD was 18.7% and that of chronic GVHD 5.5%. Viremia due to adenovirus, EBV and CMV was seen in 19.8%, 64.8% and 39.6% patients, respectively, with only two deaths attributed to viral infection (adenovirus). Chimerism level at three month was predictive of graft outcome. Nine patients, who had graft failure after first SCT, were salvaged with a second SCT using RIC and same donor (if available). Based on these results, we conclude that the use of in vivo T-cell depletion is safe, achieves good chimerism and does not lead to increased morbidity and mortality due to viral infections. It is associated with a reduced incidence of chronic GVHD. © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  10. Mammalian knock out cells reveal prominent roles for atlastin GTPases in ER network morphology

    Energy Technology Data Exchange (ETDEWEB)

    Zhao, Guohua; Zhu, Peng-Peng; Renvoisé, Benoît; Maldonado-Báez, Lymarie; Park, Seong Hee; Blackstone, Craig, E-mail: blackstc@ninds.nih.gov

    2016-11-15

    Atlastins are large, membrane-bound GTPases that participate in the fusion of endoplasmic reticulum (ER) tubules to generate the polygonal ER network in eukaryotes. They also regulate lipid droplet size and inhibit bone morphogenetic protein (BMP) signaling, though mechanisms remain unclear. Humans have three atlastins (ATL1, ATL2, and ATL3), and ATL1 and ATL3 are mutated in autosomal dominant hereditary spastic paraplegia and hereditary sensory neuropathies. Cellular investigations of atlastin orthologs in most yeast, plants, flies and worms are facilitated by the presence of a single or predominant isoform, but loss-of-function studies in mammalian cells are complicated by multiple, broadly-expressed paralogs. We have generated mouse NIH-3T3 cells lacking all three mammalian atlastins (Atl1/2/3) using CRISPR/Cas9-mediated gene knockout (KO). ER morphology is markedly disrupted in these triple KO cells, with prominent impairment in formation of three-way ER tubule junctions. This phenotype can be rescued by expression of distant orthologs from Saccharomyces cerevisiae (Sey1p) and Arabidopsis (ROOT HAIR DEFECTIVE3) as well as any one of the three human atlastins. Minimal, if any, changes are observed in the morphology of mitochondria and the Golgi apparatus. Alterations in BMP signaling and increased sensitivity to ER stress are also noted, though effects appear more modest. Finally, atlastins appear required for the proper differentiation of NIH-3T3 cells into an adipocyte-like phenotype. These findings have important implications for the pathogenesis of hereditary spastic paraplegias and sensory neuropathies associated with atlastin mutations. - Highlights: • NIH-3T3 cells lacking all three atlastin paralogs were generated using CRISPR/Cas9. • Cells lacking all atlastin GTPases exhibit far fewer 3-way ER tubule junctions. • ER morphology defects in atlastin knockout cells are rescued by distant plant and yeast orthologs. • Atlastin knock out cells also

  11. Mammalian knock out cells reveal prominent roles for atlastin GTPases in ER network morphology

    International Nuclear Information System (INIS)

    Zhao, Guohua; Zhu, Peng-Peng; Renvoisé, Benoît; Maldonado-Báez, Lymarie; Park, Seong Hee; Blackstone, Craig

    2016-01-01

    Atlastins are large, membrane-bound GTPases that participate in the fusion of endoplasmic reticulum (ER) tubules to generate the polygonal ER network in eukaryotes. They also regulate lipid droplet size and inhibit bone morphogenetic protein (BMP) signaling, though mechanisms remain unclear. Humans have three atlastins (ATL1, ATL2, and ATL3), and ATL1 and ATL3 are mutated in autosomal dominant hereditary spastic paraplegia and hereditary sensory neuropathies. Cellular investigations of atlastin orthologs in most yeast, plants, flies and worms are facilitated by the presence of a single or predominant isoform, but loss-of-function studies in mammalian cells are complicated by multiple, broadly-expressed paralogs. We have generated mouse NIH-3T3 cells lacking all three mammalian atlastins (Atl1/2/3) using CRISPR/Cas9-mediated gene knockout (KO). ER morphology is markedly disrupted in these triple KO cells, with prominent impairment in formation of three-way ER tubule junctions. This phenotype can be rescued by expression of distant orthologs from Saccharomyces cerevisiae (Sey1p) and Arabidopsis (ROOT HAIR DEFECTIVE3) as well as any one of the three human atlastins. Minimal, if any, changes are observed in the morphology of mitochondria and the Golgi apparatus. Alterations in BMP signaling and increased sensitivity to ER stress are also noted, though effects appear more modest. Finally, atlastins appear required for the proper differentiation of NIH-3T3 cells into an adipocyte-like phenotype. These findings have important implications for the pathogenesis of hereditary spastic paraplegias and sensory neuropathies associated with atlastin mutations. - Highlights: • NIH-3T3 cells lacking all three atlastin paralogs were generated using CRISPR/Cas9. • Cells lacking all atlastin GTPases exhibit far fewer 3-way ER tubule junctions. • ER morphology defects in atlastin knockout cells are rescued by distant plant and yeast orthologs. • Atlastin knock out cells also

  12. Identification of BC005512 as a DNA damage responsive murine endogenous retrovirus of GLN family involved in cell growth regulation.

    Directory of Open Access Journals (Sweden)

    Yuanfeng Wu

    Full Text Available Genotoxicity assessment is of great significance in drug safety evaluation, and microarray is a useful tool widely used to identify genotoxic stress responsive genes. In the present work, by using oligonucleotide microarray in an in vivo model, we identified an unknown gene BC005512 (abbreviated as BC, official full name: cDNA sequence BC005512, whose expression in mouse liver was specifically induced by seven well-known genotoxins (GTXs, but not by non-genotoxins (NGTXs. Bioinformatics revealed that BC was a member of the GLN family of murine endogenous retrovirus (ERV. However, the relationship to genotoxicity and the cellular function of GLN are largely unknown. Using NIH/3T3 cells as an in vitro model system and quantitative real-time PCR, BC expression was specifically induced by another seven GTXs, covering diverse genotoxicity mechanisms. Additionally, dose-response and linear regression analysis showed that expression level of BC in NIH/3T3 cells strongly correlated with DNA damage, measured using the alkaline comet assay,. While in p53 deficient L5178Y cells, GTXs could not induce BC expression. Further functional studies using RNA interference revealed that down-regulation of BC expression induced G1/S phase arrest, inhibited cell proliferation and thus suppressed cell growth in NIH/3T3 cells. Together, our results provide the first evidence that BC005512, a member from GLN family of murine ERV, was responsive to DNA damage and involved in cell growth regulation. These findings could be of great value in genotoxicity predictions and contribute to a deeper understanding of GLN biological functions.

  13. Following the Dynamics of pH in Endosomes of Live Cells with SERS Nanosensors

    DEFF Research Database (Denmark)

    Kneipp, J.; Kneipp, Harald; Wittig, B.

    2010-01-01

    The surface enhanced Raman scattering (SERS) spectrum of a reporter molecule attached to gold or silver nanostructures, which is pH-sensitive, can deliver information on the local pH in the environment of the nanostructure. Here, we demonstrate the use of a mobile SERS nanosensor made from gold...... nanaoaggregates and 4-mercaptobenzoic acid (pMBA) attached as a reporter for monitoring changes in local pH of the cellular compartments of living NIH/3T3 cells. We show that SERS nanosensors enable the dynamics of local pH in individual live cells to be followed at subendosomal resolution in a timeline...

  14. Titanium-hydroxyapatite composites sintered at low temperature for tissue engineering: in vitro cell support and biocompatibility.

    Science.gov (United States)

    Comín, Romina; Cid, Mariana P; Grinschpun, Luciano; Oldani, Carlos; Salvatierra, Nancy A

    2017-04-26

    In clinical orthopedics, a critical problem is the bone tissue loss produced by a disease or injury. The use of composites from titanium and hydroxyapatite for biomedical applications has increased due to the resulting advantageous combination of hydroxyapatite bioactivity and favorable mechanical properties of titanium. Powder metallurgy is a simple and lower-cost method that uses powder from titanium and hydroxyapatite to obtain composites having hydroxyapatite phases in a metallic matrix. However, this method has certain limitations arising from thermal decomposition of hydroxyapatite in the titanium-hydroxyapatite system above 800°C. We obtained a composite from titanium and bovine hydroxyapatite powders sintered at 800°C and evaluated its bioactivity and cytocompatibility according to the ISO 10993 standard. Surface analysis and bioactivity of the composite was evaluated by X-ray diffraction and SEM. MTT assay was carried out to assess cytotoxicity on Vero and NIH3T3 cells. Cell morphology and cell adhesion on the composite surface were analyzed using fluorescence and SEM. We obtained a porous composite with hydroxyapatite particles well integrated in titanium matrix which presented excellent bioactivity. Our data did not reveal any toxicity of titanium-hydroxyapatite composite on Vero or NIH3T3 cells. Moreover, extracts from composite did not affect cell morphology or density. Finally, NIH3T3 cells were capable of adhering to and proliferating on the composite surface. The composite obtained displayed promising biomedical applications through the simple method of powder metallurgy. Additionally, these findings provide an in vitro proof for adequate biocompatibility of titanium-hydroxyapatite composite sintered at 800°C.

  15. Avidin-biotin-based approach to forming heterotypic cell clusters and cell sheets on a gas-permeable membrane

    Energy Technology Data Exchange (ETDEWEB)

    Hamon, M; Ozawa, T; Montagne, K; Kojima, N; Ishii, R; Sakai, Y [Institute of Industrial Science (IIS), University of Tokyo, 4-6-1 Komaba, Meguro-ku, Tokyo 153-8505 (Japan); Yamaguchi, S; Nagamune, T [Department of Bioengineering, Graduate School of Engineering, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656 (Japan); Ushida, T, E-mail: mzh0026@auburn.edu [Center for Disease Biology and Integrative Medicine, Graduate School of Medicine, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033 (Japan)

    2011-09-15

    Implantation of sheet-like liver tissues is a promising method in hepatocyte-based therapies, because angiogenesis is expected to occur upon implantation from the surrounding tissues. In this context, we introduce here a new methodology for the formation of a functional thick hepatic tissue usable for cell sheet technology. First, we report the formation of composite tissue elements in suspension culture. Composite elements were composed of human hepatoma Hep G2 cells and mouse NIH/3T3 fibroblasts which are important modulators for thick-tissue formation. To overcome the very low attachment and organization capability between different cells in suspension, we synthesized a new cell-to-cell binding molecule based on the avidin-biotin binding system that we previously applied to attach hepatocytes on artificial substrata. This newly synthesized biotin-conjugated biocompatible anchoring molecule was inserted in the plasma membrane of both cell types. NIH/3T3 cells were further conjugated with avidin and incubated with biotin-presenting Hep G2 cells to form highly composite tissue elements. Then, we seeded those elements on highly gas-permeable membranes at their closest packing density to induce the formation of a thick, composite, functional hepatic tissue without any perfusion. This methodology could open a new way to engineer implantable thick liver tissue sheets where different cell types are spatially organized and well supplied with oxygen.

  16. CXCL17 expression by tumor cells recruits CD11b+Gr1 high F4/80- cells and promotes tumor progression.

    Directory of Open Access Journals (Sweden)

    Aya Matsui

    Full Text Available BACKGROUND: Chemokines are involved in multiple aspects of pathogenesis and cellular trafficking in tumorigenesis. In this study, we report that the latest member of the C-X-C-type chemokines, CXCL17 (DMC/VCC-1, recruits immature myeloid-derived cells and enhances early tumor progression. METHODOLOGY/PRINCIPAL FINDINGS: CXCL17 was preferentially expressed in some aggressive types of gastrointestinal, breast, and lung cancer cells. CXCL17 expression did not impart NIH3T3 cells with oncogenic potential in vitro, but CXCL17-expressing NIH3T3 cells could form vasculature-rich tumors in immunodeficient mice. Our data showed that CXCL17-expressing tumor cells increased immature CD11b(+Gr1(+ myeloid-derived cells at tumor sites in mice and promoted CD31(+ tumor angiogenesis. Extensive chemotactic assays proved that CXCL17-responding cells were CD11b(+Gr1(highF4/80(- cells (≈ 90% with a neutrophil-like morphology in vitro. Although CXCL17 expression could not increase the number of CD11b(+Gr1(+ cells in tumor-burdened SCID mice or promote metastases of low metastatic colon cancer cells, the existence of CXCL17-responding myeloid-derived cells caused a striking enhancement of xenograft tumor formation. CONCLUSIONS/SIGNIFICANCE: These results suggest that aberrant expression of CXCL17 in tumor cells recruits immature myeloid-derived cells and promotes tumor progression through angiogenesis.

  17. Correlation between membrane fluidity cellular development and stem cell differentiation

    KAUST Repository

    Noutsi, Pakiza

    2016-12-01

    Cell membranes are made up of a complex structure of lipids and proteins that diffuse laterally giving rise to what we call membrane fluidity. During cellular development, such as neuronal differentiation, cell membranes undergo dramatic structural changes induced by proteins such as ARC and Cofilin among others in the case of synaptic modification. In this study we used the generalized polarization (GP) property of fluorescent probe Laurdan using two-photon microscopy to determine membrane fluidity as a function of time and for various cell lines. A low GP value corresponds to a higher fluidity and a higher GP value is associated with a more rigid membrane. Four different cell lines were monitored such as hN2, NIH3T3, HEK293 and L6 cells. As expected, NIH3T3 cells have more rigid membrane at earlier stages of their development. On the other hand neurons tend to have the highest membrane fluidity early in their development emphasizing its correlation with plasticity and the need for this malleability during differentiation. This study sheds light on the involvement of membrane fluidity during neuronal differentiation and development of other cell lines.

  18. The effects of selenium on glutathione peroxidase activity and radioprotection in mammalian cells

    International Nuclear Information System (INIS)

    Diamond, A.M.; Murray, J.L.; Dale, P.; Tritz, R.; Grdina, D.J.

    1995-01-01

    The media of representative mammalian cell lines were supplemented with low levels of selenium in the form of sodium selenite in order to investigate the effects of selenium on mammalian cells. Following incubation in 30 nM sodium selenite, these cells were assayed for changes in glutathione peroxidase (GPx) activity. The cells examined included NIH 3T3 mouse fibroblasts, PC12 rat sympathetic precursor cells, SupT-1 human lymphocytes, MCF-7 adr human breast carcinoma cells and AA8 Chinese hamster ovary cells. Selenium supplementation resulted in a marginal increase in GPx activity for the NIH 3T3, MCF-7 adr and Supt-1 cells but stimulated GPx activity approximately 5-fold in PC12 and AA8 cells. AA8 cells were selected to evaluate whether selenium supplementation was radioprotective against 60 cobalt gamma irradiation. Protection against radiation-induced mutation was measured by evaluating mutation frequency at the hprt locus. In this assay, preincubation of AA8 CHO cells significantly protected these cells from exposure to 8 Gy

  19. Clonal proliferation of cultured nonmalignant and malignant human breast epithelia

    International Nuclear Information System (INIS)

    Smith, H.S.; Lan, S.; Ceriani, R.; Hackett, A.J.; Stampfer, M.R.

    1981-01-01

    We have developed a method for clonal growth of human mammary epithelial cells of both nonmalignant and malignant origin. Plating efficiencies of 1 to 50% were obtained by seeding second-passage mammary epithelial cells on fibroblast feeder layers in an enriched medium composed of various hormones and growth factors, as well as conditioned media from three specific human cell lines. Single mammary epithelial cells seeded sparsely onto the fibroblasts underwent at least eight population doublings to form large, readily visible colonies. Optimal colony formation required both feeder cells and the enriched medium. Epithelial colonies containing at least 16 cells were visible 5 days postseeding, and these colonies continued to grow progressively. Plating efficiency and colony size were similar on ultraviolet-irradiated or nonirradiated fibroblasts. The number of colonies formed was proportional to the number of epithelial cells plated. The colonies were identified as epithelial by the presence of human mammary epithelial antigens

  20. BRAFV600E Negatively Regulates the AKT Pathway in Melanoma Cell Lines

    OpenAIRE

    Chen, Brenden; Tardell, Christine; Higgins, Brian; Packman, Kathryn; Boylan, John F.; Niu, Huifeng

    2012-01-01

    Cross-feedback activation of MAPK and AKT pathways is implicated as a resistance mechanism for cancer therapeutic agents targeting either RAF/MEK or PI3K/AKT/mTOR. It is thus important to have a better understanding of the molecular resistance mechanisms to improve patient survival benefit from these agents. Here we show that BRAFV600E is a negative regulator of the AKT pathway. Expression of BRAFV600E in NIH3T3 cells significantly suppresses MEK inhibitor (RG7167) or mTORC1 inhibitor (rapamy...

  1. Bacterial Infections Following Splenectomy for Malignant and Nonmalignant Hematologic Diseases

    Science.gov (United States)

    Leone, Giuseppe; Pizzigallo, Eligio

    2015-01-01

    Splenectomy, while often necessary in otherwise healthy patients after major trauma, finds its primary indication for patients with underlying malignant or nonmalignant hematologic diseases. Indications of splenectomy for hematologic diseases have been reducing in the last few years, due to improved diagnostic and therapeutic tools. In high-income countries, there is a clear decrease over calendar time in the incidence of all indication splenectomy except nonmalignant hematologic diseases. However, splenectomy, even if with different modalities including laparoscopic splenectomy and partial splenectomy, continue to be a current surgical practice both in nonmalignant hematologic diseases, such as Immune Thrombocytopenic Purpura (ITP), Autoimmune Hemolytic Anemia (AIHA), Congenital Hemolytic Anemia such as Spherocytosis, Sickle Cell Anemia and Thalassemia and Malignant Hematological Disease, such as lymphoma. Today millions of people in the world are splenectomized. Splenectomy, independently of its cause, induces an early and late increase in the incidence of venous thromboembolism and infections. Infections remain the most dangerous complication of splenectomy. After splenectomy, the levels of antibody are preserved but there is a loss of memory B cells against pneumococcus and tetanus, and the loss of marginal zone monocytes deputed to immunological defense from capsulated bacteria. Commonly, the infections strictly correlated to the absence of the spleen or a decreased or absent splenic function are due to encapsulated bacteria that are the most virulent pathogens in this set of patients. Vaccination with polysaccharide and conjugate vaccines again Streptococcus pneumoniae, Haemophilus influenzae, and Neisseria meningitidis should be performed before the splenectomy. This practice reduces but does not eliminate the occurrence of overwhelming infections due to capsulated bacteria. At present, most of infections found in splenectomized patients are due to Gram

  2. Chemical sporulation and germination: cytoprotective nanocoating of individual mammalian cells with a degradable tannic acid-FeIII complex

    Science.gov (United States)

    Lee, Juno; Cho, Hyeoncheol; Choi, Jinsu; Kim, Doyeon; Hong, Daewha; Park, Ji Hun; Yang, Sung Ho; Choi, Insung S.

    2015-11-01

    Individual mammalian cells were coated with cytoprotective and degradable films by cytocompatible processes maintaining the cell viability. Three types of mammalian cells (HeLa, NIH 3T3, and Jurkat cells) were coated with a metal-organic complex of tannic acid (TA) and ferric ion, and the TA-FeIII nanocoat effectively protected the coated mammalian cells against UV-C irradiation and a toxic compound. More importantly, the cell proliferation was controlled by programmed formation and degradation of the TA-FeIII nanocoat, mimicking the sporulation and germination processes found in nature.Individual mammalian cells were coated with cytoprotective and degradable films by cytocompatible processes maintaining the cell viability. Three types of mammalian cells (HeLa, NIH 3T3, and Jurkat cells) were coated with a metal-organic complex of tannic acid (TA) and ferric ion, and the TA-FeIII nanocoat effectively protected the coated mammalian cells against UV-C irradiation and a toxic compound. More importantly, the cell proliferation was controlled by programmed formation and degradation of the TA-FeIII nanocoat, mimicking the sporulation and germination processes found in nature. Electronic supplementary information (ESI) available: Experimental details, LSCM images, and SEM and TEM images. See DOI: 10.1039/c5nr05573c

  3. Controlling cell adhesion via replication of laser micro/nano-textured surfaces on polymers

    Energy Technology Data Exchange (ETDEWEB)

    Koufaki, Niki; Ranella, Anthi; Barberoglou, Marios; Psycharakis, Stylianos; Fotakis, Costas; Stratakis, Emmanuel [Institute of Electronic Structure and Laser (IESL), Foundation for Research and Technology-Hellas (FORTH), 711 10, Heraklion, Crete (Greece); Aifantis, Katerina E, E-mail: stratak@iesl.forth.gr [Lab of Mechanics and Materials, Aristotle University of Thessaloniki, Thessaloniki (Greece)

    2011-12-15

    The aim of this study is to investigate cell adhesion and viability on highly rough polymeric surfaces with gradient roughness ratios and wettabilities prepared by microreplication of laser micro/nano-textured Si surfaces. Negative replicas on polydimethylsiloxane as well as positive ones on a photocurable (organically modified ceramic) and a biodegradable (poly(lactide-co-glycolide)) polymer have been successfully reproduced. The final culture substrates comprised from forests of micron-sized conical spikes exhibiting a range of roughness ratios and wettabilities, was achieved by changing the laser fluence used to fabricate the original template surfaces. Cell culture experiments were performed with the fibroblast NIH/3T3 and PC12 neuronal cell lines in order to investigate how these surfaces are capable of modulating different types of cellular responses including, viability, adhesion and morphology. The results showed a preferential adhesion of both cell types on the microstructured surfaces compared to the unstructured ones. In particular, the fibroblast NIH/3T3 cells show optimal adhesion for small roughness ratios, independent of the surface wettability and polymer type, indicating a non-monotonic dependence of cell adhesion on surface energy. In contrast, the PC12 cells were observed to adhere well to the patterned surfaces independent of the roughness ratio and wettability. These experimental findings are correlated with micromechanical measurements performed on the unstructured and replicated surfaces and discussed on the basis of previous observations describing the relation of cell response to surface energy and rigidity.

  4. Controlling cell adhesion via replication of laser micro/nano-textured surfaces on polymers

    International Nuclear Information System (INIS)

    Koufaki, Niki; Ranella, Anthi; Barberoglou, Marios; Psycharakis, Stylianos; Fotakis, Costas; Stratakis, Emmanuel; Aifantis, Katerina E

    2011-01-01

    The aim of this study is to investigate cell adhesion and viability on highly rough polymeric surfaces with gradient roughness ratios and wettabilities prepared by microreplication of laser micro/nano-textured Si surfaces. Negative replicas on polydimethylsiloxane as well as positive ones on a photocurable (organically modified ceramic) and a biodegradable (poly(lactide-co-glycolide)) polymer have been successfully reproduced. The final culture substrates comprised from forests of micron-sized conical spikes exhibiting a range of roughness ratios and wettabilities, was achieved by changing the laser fluence used to fabricate the original template surfaces. Cell culture experiments were performed with the fibroblast NIH/3T3 and PC12 neuronal cell lines in order to investigate how these surfaces are capable of modulating different types of cellular responses including, viability, adhesion and morphology. The results showed a preferential adhesion of both cell types on the microstructured surfaces compared to the unstructured ones. In particular, the fibroblast NIH/3T3 cells show optimal adhesion for small roughness ratios, independent of the surface wettability and polymer type, indicating a non-monotonic dependence of cell adhesion on surface energy. In contrast, the PC12 cells were observed to adhere well to the patterned surfaces independent of the roughness ratio and wettability. These experimental findings are correlated with micromechanical measurements performed on the unstructured and replicated surfaces and discussed on the basis of previous observations describing the relation of cell response to surface energy and rigidity.

  5. High levels of the type III inorganic phosphate transporter PiT1 (SLC20A1) can confer faster cell adhesion

    DEFF Research Database (Denmark)

    Kongsfelt, Iben Boutrup; Byskov, Kristina; Pedersen, Lasse Ebdrup

    2014-01-01

    overexpression led to faster cell spreading. The final total numbers of attached cells did, however, not differ between cultures of PiT1 overexpressing cells and control cells of neither cell type. We suggest that the PiT1-mediated fast adhesion potentials allow the cells to go faster out of G0/G1 and thereby......The inorganic phosphate transporter PiT1 (SLC20A1) is ubiquitously expressed in mammalian cells. We recently showed that overexpression of human PiT1 was sufficient to increase proliferation of two strict density-inhibited cell lines, murine fibroblastic NIH3T3 and pre-osteoblastic MC3T3-E1 cells......, and allowed the cultures to grow to higher cell densities. In addition, upon transformation NIH3T3 cells showed increased ability to form colonies in soft agar. The cellular regulation of PiT1 expression supports that cells utilize the PiT1 levels to control proliferation, with non-proliferating cells showing...

  6. A common multiple cloning site in a set of vectors for expression of eukaryotic genes in mammalian, insect and bacterial cells

    DEFF Research Database (Denmark)

    Pallisgaard, N; Pedersen, FS; Birkelund, Svend

    1994-01-01

    a start Met codon was included in the same reading frame as in lambda gt11Sfi-Not to support expression of partial cDNA clones. Thus a cDNA insert of lambda gt11Sfi-Not could be shuttled among the new vectors for expression. The other set of vectors without a start codon were suitable for expression of c......DNA carrying their own start Met codon. By Western blot analysis and by transactivation of a reporter plasmid in co-transfections we show that cDNA is very efficiently expressed in NIH 3T3 cells under control of the elongation factor 1 alpha promoter....

  7. Value of bioimpedance analysis and anthropometry for complication prediction in children with malignant and non-malignant diseases after hematopoietic stem cells transplantation

    Directory of Open Access Journals (Sweden)

    G. Ya. Tseytlin

    2013-01-01

    Full Text Available Hematopoietic stem cell transplantation (HSCT is widely used in the treatment of malignant and autoimmune diseases. Various complications often develop during the post-transplantation period that can significantly impair the clinical outcomes, so the ability to predict therisk of severe complications is of great practical importance. Predictive value of some anthropometric indices and bioimpedance analysis(BIA measured before conditioning to assess the risks of serious complications and graft hypofunction in the early post-transplant period(100 days were analyzed. Anthropometry and BIA used in a comprehensive assessment of nutritional status in order to optimize the nutritional support of these patients. 101 patients were examined before conditioning and at different times during the early post-transplant period: 50 children (5–17 years of age were examined using BIA and anthropometry, 61 children (6 months – 4 years of age – using only anthropometry without BIA due to age restrictions. The prognostic value of the phase angle (FA, ratio of the active cell mass to lean body mass (ACM/LBM and shoulder muscle circumference (SMC was shown. Thus, in patients with FA ≤ 4, ACM/LBM < 0.45 and SMC ≤ 10th percentile before conditioning risk of severe complications during early post-transplant period was significantly higher (p < 0.05. Also, in patients with FA ≤ 4 and ACM/LBM < 0.45 a significantly higher risk of graft hypofunction developing was observed (p < 0.05.

  8. Value of bioimpedance analysis and anthropometry for complication prediction in children with malignant and non-malignant diseases after hematopoietic stem cells transplantation

    Directory of Open Access Journals (Sweden)

    G. Ya. Tseytlin

    2014-07-01

    Full Text Available Hematopoietic stem cell transplantation (HSCT is widely used in the treatment of malignant and autoimmune diseases. Various complications often develop during the post-transplantation period that can significantly impair the clinical outcomes, so the ability to predict therisk of severe complications is of great practical importance. Predictive value of some anthropometric indices and bioimpedance analysis(BIA measured before conditioning to assess the risks of serious complications and graft hypofunction in the early post-transplant period(100 days were analyzed. Anthropometry and BIA used in a comprehensive assessment of nutritional status in order to optimize the nutritional support of these patients. 101 patients were examined before conditioning and at different times during the early post-transplant period: 50 children (5–17 years of age were examined using BIA and anthropometry, 61 children (6 months – 4 years of age – using only anthropometry without BIA due to age restrictions. The prognostic value of the phase angle (FA, ratio of the active cell mass to lean body mass (ACM/LBM and shoulder muscle circumference (SMC was shown. Thus, in patients with FA ≤ 4, ACM/LBM < 0.45 and SMC ≤ 10th percentile before conditioning risk of severe complications during early post-transplant period was significantly higher (p < 0.05. Also, in patients with FA ≤ 4 and ACM/LBM < 0.45 a significantly higher risk of graft hypofunction developing was observed (p < 0.05.

  9. Rhodamine B conjugates of triterpenoic acids are cytotoxic mitocans even at nanomolar concentrations.

    Science.gov (United States)

    Sommerwerk, Sven; Heller, Lucie; Kerzig, Christoph; Kramell, Annemarie E; Csuk, René

    2017-02-15

    Triterpenoic acids 1-6 exhibited very low or no cytotoxicity at all, but their corresponding 2,3-di-O-acetyl-piperazinyl amides 13-18 showed low EC 50 values for several human tumor cell lines. Their cytotoxicity, however, was also high for the non-malignant mouse fibroblasts NIH 3T3. A significant improvement was achieved by preparing the rhodamine B derivatives 19-24. While rhodamine B is not cytotoxic (up to a concentration of 30μM - cut-off of the assay), the triterpenoid piperazine-spacered rhodamine B derivatives were cytotoxic in nano-molar concentration. Compound 24 (a diacetylated maslinic acid derivative) was most toxic for several human tumor cell lines but less toxic for mouse fibroblasts NIH 3T3. Staining and double-staining experiments revealed 24 to act as a mitocan. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

  10. Antisense oligonucleotides suppress cell-volume-induced activation of chloride channels.

    Science.gov (United States)

    Gschwentner, M; Nagl, U O; Wöll, E; Schmarda, A; Ritter, M; Paulmichl, M

    1995-08-01

    Cell volume regulation is an essential feature of most cells. After swelling in hypotonic media, the simultaneous activation of potassium and chloride channels is believed to be the initial, time-determining step in cell volume regulation. The activation of both pathways is functionally linked and enables the cells to lose ions and water, subsequently leading to cell shrinkage and readjustment of the initial volume. NIH 3T3 fibroblasts efficiently regulate their volume after swelling and bear chloride channels that are activated by decreasing extracellular osmolarity. The chloride current elicited in these cells after swelling is reminiscent of the current found in oocytes expressing an outwardly rectifying chloride current termed ICln. Introduction of antisense oligodeoxynucleotides complementary to the first 30 nucleotides of the coding region of the ICln channel into NIH 3T3 fibroblasts suppresses the activation of the swelling-induced chloride current. The experiments directly demonstrate an unambiguous link between a volume-activated chloride current and a cloned protein involved in chloride transport.

  11. Early-stage apoptosis is associated with DNA-damage-independent ATM phosphorylation and chromatin decondensation in NIH3T3 fibroblasts

    DEFF Research Database (Denmark)

    Schou, Kenneth Bødtker; Schneider, Linda; Christensen, Søren Tvorup

    2008-01-01

    Chromatin condensation and degradation of DNA into internucleosomal DNA fragments are key hallmarks of apoptosis. The phosphorylation of protein kinase ataxia telangiectasia mutated (ATM) and histone H2A.X was recently shown to occur concurrently with apoptotic DNA fragmentation. We have used...... necrosis factor-alpha mixed with cycloheximide (TNF-alpha/CHX). In extension to previous findings, ATM phosphorylation was associated with chromatin decondensation, i.e., by loss of dense foci of constitutive heterochromatin. These results suggest that chromatin is decondensed and that ATM is activated...

  12. Ultrasound-mediated structural changes in cells revealed by FTIR spectroscopy: A contribution to the optimization of gene and drug delivery

    Science.gov (United States)

    Grimaldi, Paola; Di Giambattista, Lucia; Giordani, Serena; Udroiu, Ion; Pozzi, Deleana; Gaudenzi, Silvia; Bedini, Angelico; Giliberti, Claudia; Palomba, Raffaele; Congiu Castellano, Agostina

    2011-12-01

    Ultrasound effects on biological samples are gaining a growing interest concerning in particular, the intracellular delivery of drugs and genes in a safe and in a efficient way. Future progress in this field will require a better understanding of how ultrasound and acoustic cavitation affect the biological system properties. The morphological changes of cells due to ultrasound (US) exposure have been extensively studied, while little attention has been given to the cells structural changes. We have exposed two different cell lines to 1 MHz frequency ultrasound currently used in therapy, Jurkat T-lymphocytes and NIH-3T3 fibroblasts, both employed as models respectively in the apoptosis and in the gene therapy studies. The Fourier Transform Infrared (FTIR) Spectroscopy was used as probe to reveal the structural changes in particular molecular groups belonging to the main biological systems. The genotoxic damage of cells exposed to ultrasound was ascertained by the Cytokinesis-Block Micronucleus (CBMN) assay. The FTIR spectroscopy results, combined with multivariate statistical analysis, regarding all cellular components (lipids, proteins, nucleic acids) of the two cell lines, show that Jurkat cells are more sensitive to therapeutic ultrasound in the lipid and protein regions, whereas the NIH-3T3 cells are more sensitive in the nucleic acids region; a meaningful genotoxic effect is present in both cell lines only for long sonication times while in the Jurkat cells also a significant cytotoxic effect is revealed for long times of exposure to ultrasound.

  13. Cell fusion through a microslit between adhered cells and observation of their nuclear behavior.

    Science.gov (United States)

    Wada, Ken-Ichi; Hosokawa, Kazuo; Kondo, Eitaro; Ito, Yoshihiro; Maeda, Mizuo

    2014-07-01

    This paper describes a novel cell fusion method which induces cell fusion between adhered cells through a microslit for preventing nuclear mixing. For this purpose, a microfluidic device which had ∼ 100 cell pairing structures (CPSs) making cell pairs through microslits with 2.1 ± 0.3 µm width was fabricated. After trapping NIH3T3 cells with hydrodynamic forces at the CPSs, the cells were fused through the microslit by the Sendai virus envelope method. With following timelapse observation, we discovered that the spread cells were much less susceptible to nuclear migration passing through the microslit compared with round cells, and that cytoplasmic fraction containing mitochondria was transferred through the microslit without nuclear mixing. These findings will provide an effective method for cell fusion without nuclear mixing, and will lead to an efficient method for reprograming and transdifferentiation of target cells toward regenerative medicine. © 2014 Wiley Periodicals, Inc.

  14. Induction of G2/M arrest and apoptosis through mitochondria pathway by a dimer sesquiterpene lactone from Smallanthus sonchifolius in HeLa cells.

    Science.gov (United States)

    Kitai, Yurika; Zhang, Xia; Hayashida, Yushi; Kakehi, Yoshiyuki; Tamura, Hirotoshi

    2017-07-01

    Dimer sesquiterpene lactones (SLs), uvedafolin and enhydrofolin, against four monomer SLs isolated from yacon, Smallanthus sonchifolius, leaf were the most cytotoxic substances on HeLa cells (IC 50 values 2.96-3.17 μM at 24 hours). However, the cytotoxic mechanism of dimer SL has not been elucidated yet. Therefore, in this study, we clarified the in vitro cytotoxic mechanism of uvedafolin on the HeLa cells, and evaluated the cytotoxicity against NIH/3T3 cells which were used as normal cells. In consequence, the dimer SLs had low toxicity for the NIH/3T3 cells (IC 50 4.81-4.98 μM at 24 hours) and then the uvedafolin mediated cell cycle arrest at the G 2 /M phase and induced apoptosis on the HeLa cells evidenced by appearance of a subG1 peak. Uvedafolin induced apoptosis was attributed to caspase-9 and caspase-3/7 activities. An effectively induced apoptosis pathway was demonstrated from mitochondria membrane potential change and cytochrome c release to cytosol. These results reveal that uvedafolin induced apoptosis via the mitochondria pathway. The present results indicate the potential of uvedafolin as a leading compound of new anticancer agents. Copyright © 2016. Published by Elsevier B.V.

  15. Cytotoxic Activity of Kenaf Seed Oils from Supercritical Carbon Dioxide Fluid Extraction towards Human Colorectal Cancer (HT29 Cell Lines

    Directory of Open Access Journals (Sweden)

    Siti Aisyah Abd Ghafar

    2013-01-01

    Full Text Available Kenaf (Hibiscus cannabinus from the family Malvaceae, is a valuable fiber plant native to India and Africa and is currently planted as the fourth commercial crop in Malaysia. Kenaf seed oil contains alpha-linolenic acid, phytosterol such as β-sitosterol, vitamin E, and other antioxidants with chemopreventive properties. Kenaf seeds oil (KSO was from supercritical carbon dioxide extraction fluid (SFE at 9 different permutations of parameters based on range of pressures from 200 to 600 bars and temperature from 40 to 80°C. They were 200/40, 200/60, 200/80, 400/40, 400/60, 400/80, 600/40, 600/60, and 600/80. Extraction from 9 parameters of KSO-SFE was screened for cytotoxicity towards human colorectal cancer cell lines (HT29 and mouse embryonic fibroblast (NIH/3T3 cell lines using MTS assay. KSO-SFE at 600/40 showed the strongest cytotoxicity towards HT29 with IC50 of 200 µg/mL. The IC50 for NIH/3T3 was not detected even at highest concentration employed. Cell cycle analysis showed a significant increase in the accumulation of KSO-SFE-treated cells at sub-G1 phase, indicating the induction of apoptosis by KSO-SFE. Further apoptosis induction was confirmed by Annexin V/PI and AO/PI staining.

  16. Cytotoxic Activity of Kenaf Seed Oils from Supercritical Carbon Dioxide Fluid Extraction towards Human Colorectal Cancer (HT29) Cell Lines.

    Science.gov (United States)

    Abd Ghafar, Siti Aisyah; Ismail, Maznah; Saiful Yazan, Latifah; Fakurazi, Sharida; Ismail, Norsharina; Chan, Kim Wei; Md Tahir, Paridah

    2013-01-01

    Kenaf (Hibiscus cannabinus) from the family Malvaceae, is a valuable fiber plant native to India and Africa and is currently planted as the fourth commercial crop in Malaysia. Kenaf seed oil contains alpha-linolenic acid, phytosterol such as β -sitosterol, vitamin E, and other antioxidants with chemopreventive properties. Kenaf seeds oil (KSO) was from supercritical carbon dioxide extraction fluid (SFE) at 9 different permutations of parameters based on range of pressures from 200 to 600 bars and temperature from 40 to 80°C. They were 200/40, 200/60, 200/80, 400/40, 400/60, 400/80, 600/40, 600/60, and 600/80. Extraction from 9 parameters of KSO-SFE was screened for cytotoxicity towards human colorectal cancer cell lines (HT29) and mouse embryonic fibroblast (NIH/3T3) cell lines using MTS assay. KSO-SFE at 600/40 showed the strongest cytotoxicity towards HT29 with IC50 of 200 µg/mL. The IC50 for NIH/3T3 was not detected even at highest concentration employed. Cell cycle analysis showed a significant increase in the accumulation of KSO-SFE-treated cells at sub-G1 phase, indicating the induction of apoptosis by KSO-SFE. Further apoptosis induction was confirmed by Annexin V/PI and AO/PI staining.

  17. Opioid Therapy for Chronic Nonmalignant Pain

    Directory of Open Access Journals (Sweden)

    Russell K Portenoy

    1996-01-01

    Full Text Available Long term administration of an opioid drug for chronic nonmalignant pain continues to be controversial, but is no longer uniformly rejected by pain specialists. This is true despite concerns that the regulatory agencies that oversee physician prescribing of opioid drugs continue to stigmatize the practice. The changing clinical perspective has been driven, in part, by widespread acknowledgement of the remarkably favourable outcomes achieved during opioid treatment of cancer pain. These outcomes contrast starkly with popular teaching about chronic opioid therapy and affirm the potential for prolonged efficacy, tolerable side effects, enhanced function associated with improved comfort and minimal risk of aberrant drug-related behaviours consistent with addiction. A large anecdotal experience in populations with nonmalignant pain suggests that these patients are more heterogeneous and that opioid therapy will greatly benefit some and will contribute to negative outcomes for others. The few controlled clinical trials that have been performed support the safety and efficacy of opioid therapy, but have been too limited to ensure generalization to the clinical setting. A critical review of the medical literature pertaining to chronic pain, opioid pharmacology and addiction medicine can clarify misconceptions about opioid therapy and provide a foundation for patient selection and drug administration. The available data support the view that opioids are no panacea for chronic pain, but should be considered in carefully selected patients using clinically derived guidelines that stress a structured approach and ongoing monitoring of efficacy, adverse effects, functional outcomes and the occurrence of aberrant drug-related behaviours.

  18. Radiotherapy for non-malignant disorders

    International Nuclear Information System (INIS)

    Seegenschmiedt, Michael Heinrich; Makoski, Hans-Bruno; Trott, Klaus-Ruediger; Brady, Luther W.

    2008-01-01

    This volume discusses the general background, radiobiology, radiophysics and clinical applications of radiation therapy in the treatment of non-malignant diseases. Within 39 chapters, it documents the rationale and indications for the use of state-of-the-art radiotherapy for various non-malignant disorders of the CNS, head and neck, eye, skin and soft tissues, bone and joints, and the vascular system. In so doing, it draws attention to and elucidates the scope for application of radiotherapy beyond the treatment of malignancies. Both the risks and the benefits of such treatment are fully considered, the former ranging from minor clinical problems to life-threatening diseases. With the assistance of many tables and colored figures, the extensive data from clinical studies are presented in a well-structured and informative way. Each chapter concludes with a list of key points, allowing the reader to quickly comprehend the main facts. Since this approach offers an interdisciplinary perspective, this book will be of interest not only to radiotherapists but also to many other practitioners and medical specialists, for example orthopedists, surgeons, and ophthalmologists. (orig.)

  19. Helicobacter pylori and non-malignant diseases.

    Science.gov (United States)

    Furuta, Takahisa; Delchier, Jean-Charles

    2009-09-01

    It is well known that Helicobacter pylori infection is associated with many nonmalignant disorders such as gastritis, peptic ulcer, gastroesophageal reflux disease (GERD), gastric polyp, nonsteroidal anti-inflammatory drug (NSAID)/aspirin-induced gastric injury, and functional dyspepsia. In 2008, interesting articles on the association of H. pylori infection with these disorders were presented, some of which intended to reveal the mechanisms of inter-individual differences in response to H. pylori infection, and have demonstrated that genetic differences in host and bacterial factors as well as environmental factors account for these differences. A decline in the occurrence of peptic ulcer related to H. pylori was confirmed. An inverse relationship between H. pylori infection and GERD was also confirmed but the impact of gastric atrophy on the prevention of GERD remained debatable. For NSAID-induced gastric injury, eradication of H. pylori infection has been recommended. During this year, eradication of H. pylori infection was recommended for patients treated with antiplatelet therapy as well as aspirin and NSAID. It was also reported that for patients with functional dyspepsia, eradication of H. pylori offers a modest but significant benefit.

  20. Isolation of Lysosomes from Mammalian Tissues and Cultured Cells.

    Science.gov (United States)

    Aguado, Carmen; Pérez-Jiménez, Eva; Lahuerta, Marcos; Knecht, Erwin

    2016-01-01

    Lysosomes participate within the cells in the degradation of organelles, macromolecules, and a wide variety of substrates. In any study on specific roles of lysosomes, both under physiological and pathological conditions, it is advisable to include methods that allow their reproducible and reliable isolation. However, purification of lysosomes is a difficult task, particularly in the case of cultured cells. This is mainly because of the heterogeneity of these organelles, along with their low number and high fragility. Also, isolation methods, while disrupting plasma membranes, have to preserve the integrity of lysosomes, as the breakdown of their membranes releases enzymes that could damage all cell organelles, including themselves. The protocols described below have been routinely used in our laboratory for the specific isolation of lysosomes from rat liver, NIH/3T3, and other cultured cells, but can be adapted to other mammalian tissues or cell lines.

  1. Effect of chronic nonmalignant pain on highway driving performance.

    Science.gov (United States)

    Veldhuijzen, D S; van Wijck, A J M; Wille, F; Verster, J C; Kenemans, J L; Kalkman, C J; Olivier, B; Volkerts, E R

    2006-05-01

    Most pain patients are treated in an outpatient setting and are engaged in daily activities including driving. Since several studies showed that cognitive functioning may be impaired in chronic nonmalignant pain, the question arises whether or not chronic nonmalignant pain affects driving performance. Therefore, the objective of the present study was to determine the effects of chronic nonmalignant pain on actual highway driving performance during normal traffic. Fourteen patients with chronic nonmalignant pain and 14 healthy controls, matched on age, educational level, and driving experience, participated in the study. Participants performed a standardized on-the-road driving test during normal traffic, on a primary highway. The primary parameter of the driving test is the Standard Deviation of Lateral Position (SDLP). In addition, driving-related skills (tracking, divided attention, and memory) were examined in the laboratory. Subjective assessments, such as pain intensity, and subjective driving quality, were rated on visual analogue scales. The results demonstrated that a subset of chronic nonmalignant pain patients had SDLPs that were higher than the matched healthy controls, indicating worse highway driving performance. Overall, there was a statistically significant difference in highway driving performance between the groups. Further, chronic nonmalignant pain patients rated their subjective driving quality to be normal, although their ratings were significantly lower than those of the healthy controls. No significant effects were found on the laboratory tests.

  2. [Distribution diversity of integrins and calcium channels on major human and mouse host cells of Leptospira species].

    Science.gov (United States)

    Li, Cheng-xue; Zhao, Xin; Qian, Jing; Yan, Jie

    2012-07-01

    To determine the distribution of integrins and calcium channels on major human and mouse host cells of Leptospira species. The expression of β1, β2 and β3 integrins was detected with immunofluorescence assay on the surface of human monocyte line THP-1, mouse mononuclear-macrophage-like cell line J774A.1, human vascular endothelial cell line HUVEC, mouse vascular endothelial cell EOMA, human hepatocyte line L-02, mouse hepatocyte line Hepa1-6, human renal tubular epithelial cell line HEK-293, mouse glomerular membrane epithelial cell line SV40-MES13, mouse collagen blast line NIH/3T3, human and mouse platelets. The distribution of voltage gate control calcium channels Cav3.1, Cav3.2, Cav3.3 and Cav2.3, and receptor gate calcium channels P(2)X(1), P(2)2X(2), P(2)X(3), P(2)X(4), P(2)X(5), P(2)X(6) and P(2)X(7) were determined with Western blot assay. β1 integrin proteins were positively expressed on the membrane surface of J774A.1, THP-1, HUVEC, EOMA, L-02, Hepa1-6 and HEK-239 cells as well as human and mouse platelets. β2 integrin proteins were expressed on the membrane surface of J774A.1, THP-1, HUVEC, EOMA, and NIH/3T3 cells. β3 integrin proteins were expressed on the membrane surface of J774A.1, THP-1, HUVEC, EOMA, Hepa1-6, HEK-239 and NIH/3T3 cells as well as human and mouse platelets. P(2)X(1) receptor gate calcium channel was expressed on the membrane surface of human and mouse platelets, while P(2)X(5) receptor gate calcium channel was expressed on the membrane surface of J774A.1, THP-1, L-02, Hepa1-6, HEK-239 and HUVEC cells. However, the other calcium channels were not detected on the tested cell lines or platelets. There is a large distribution diversity of integrins and calcium channel proteins on the major human and mouse host cells of Leptospira species, which may be associated with the differences of leptospira-induced injury in different host cells.

  3. Bone marrow-derived cultured mast cells and peritoneal mast cells as targets of a growth activity secreted by BALB/3T3 fibroblasts

    International Nuclear Information System (INIS)

    Jozaki, K.; Kuriu, A.; Hirota, S.; Onoue, H.; Ebi, Y.; Adachi, S.; Ma, J.Y.; Tarui, S.; Kitamura, Y.

    1991-01-01

    When fibroblast cell lines were cultured in contact with bone marrow-derived cultured mast cells (CMC), both NIH/3T3 and BALB/3T3 cell lines supported the proliferation of CMC. In contrast, when contact between fibroblasts and CMC was prohibited by Biopore membranes or soft agar, only BALB/3T3 fibroblasts supported CMC proliferation, suggesting that BALB/3T3 but not NIH/3T3 cells secreted a significant amount of a mast cell growth activity. Moreover, the BALB/3T3-derived growth activity induced the incorporation of [3H]thymidine by CMC and the clonal growth of peritoneal mast cells in methylcellulose. The mast cell growth activity appeared to be different from interleukin 3 (IL-3) and interleukin 4 (IL-4), because mRNAs for these interleukins were not detectable in BALB/3T3 fibroblasts. Although mast cells are genetically deficient in tissues of W/Wv mice, CMC did develop when bone marrow cells of W/Wv mice were cultured with pokeweed mitogen-stimulated spleen cell-conditioned medium. Because BALB/3T3 fibroblast-conditioned medium (BALB-FCM) did not induce the incorporation of [3H]thymidine by W/Wv CMC, the growth activity in BALB-FCM appeared to be a ligand for the receptor encoded by the W (c-kit) locus. Because CMC and peritoneal mast cells are obtained as homogeneous suspensions rather easily, these cells may be potentially useful as targets for the fibroblast-derived mast cell growth activity

  4. Lipid peroxidation and antioxidants status in human malignant and non-malignant thyroid tumours.

    Science.gov (United States)

    Stanley, J A; Neelamohan, R; Suthagar, E; Vengatesh, G; Jayakumar, J; Chandrasekaran, M; Banu, S K; Aruldhas, M M

    2016-06-01

    Thyroid epithelial cells produce moderate amounts of reactive oxygen species that are physiologically required for thyroid hormone synthesis. Nevertheless, when they are produced in excessive amounts, they may become toxic. The present study is aimed to compare the lipid peroxidation (LPO), antioxidant enzymes - superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx) and non-protein thiols (reduced glutathione (GSH)) in human thyroid tissues with malignant and non-malignant disorders. The study used human thyroid tissues and blood samples from 157 women (147 diseased and 10 normal). Thyroid hormones, oxidative stress markers and antioxidants were estimated by standard methods. LPO significantly increased in most of the papillary thyroid carcinoma (PTC: 82.9%) and follicular thyroid adenoma (FTA: 72.9%) tissues, whilst in a majority of nodular goitre (69.2%) and Hashimoto's thyroiditis (HT: 73.7%) thyroid tissues, it remained unaltered. GSH increased in PTC (55.3%), remained unaltered in FTA (97.3%) and all other goiter samples studied. SOD increased in PTC (51.1%) and all other malignant thyroid tissues studied. CAT remained unaltered in PTC (95.7%), FTA (97.3%) and all other non-malignant samples (HT, MNG, TMNG) studied. GPx increased in PTC (63.8%), all other malignant thyroid tissues and remained unaltered in many of the FTA (91.9%) tissues and all other non-malignant samples (HT, MNG, TMNG) studied. In the case of non-malignant thyroid tumours, the oxidant-antioxidant balance was undisturbed, whilst in malignant tumours the balance was altered, and the change in r value observed in the LPO and SOD pairs between normal and PTC tissues and also in many pairs with multi-nodular goitre (MNG)/toxic MNG tissues may be used as a marker to differentiate/detect different malignant/non-malignant thyroid tumours. © The Author(s) 2015.

  5. Evaluation of cell behavior on modified polypropylene with swift heavy ion irradiation

    International Nuclear Information System (INIS)

    Arbeitman, Claudia R.; Ibañez, Irene L.; García Bermúdez, Gerardo; Durán, Hebe; Grosso, Mariela F. del; Salguero, Noelia; Mazzei, Rubén

    2012-01-01

    Ion beam irradiation is a well known means to change the physico-chemical properties of polymers, and induced bio and citocompatibility in controlled conditions and in selected areas of surface. However, the enhancement of cell adhesion on a modified substrate does not mean that the surface is adequate for functional cells. The purpose of the present work is to study proliferation, changes in cytoskeleton and cell morphology on substrates as a function of irradiation parameters. We irradiated polypropylene with sulfur (S) ion-beam at energies of 110 MeV with fluences between 1 × 10 6 and 2 × 10 10 ions cm −2 . NIH 3T3 cells were cultured on each sample. Cell morphology was observed using phase contrast microscopy and cytoskeleton proteins with fluorescence microscopy. The analysis show different cellular responses as a functions of irradiation parameter, strongly suggests that different underlying substratum can result in distinct types of cytoskeleton reorganization.

  6. Drug-induced in vitro inhibition of neutrophil-endothelial cell adhesion.

    Science.gov (United States)

    Pellegatta, F.; Lu, Y.; Radaelli, A.; Zocchi, M. R.; Ferrero, E.; Chierchia, S.; Gaja, G.; Ferrero, M. E.

    1996-01-01

    1. Leukocyte-endothelial cell interactions play an important role during ischaemia-reperfusion events. Adhesion molecules are specifically implicated in this interaction process. 2. Since defibrotide has been shown to be an efficient drug in reducing damage due to ischaemia-reperfusion in many experimental models, we analysed the effect of defibrotide in vitro on leukocyte adhesion to endothelial cells in basal conditions and after their stimulation. 3. In basal conditions, defibrotide (1000 micrograms ml-1) partially inhibited leukocyte adhesion to endothelial cells by 17.3% +/- 3.6 (P defibrotide. 5. This result was confirmed in NIH/3T3-ICAM-1 transfected cells. 6. We conclude that defibrotide is able to interfere with leukocyte adhesion to endothelial cells mainly in activated conditions and that the ICAM-1/LFA-1 adhesion system is involved in the defibrotide mechanism of action. PMID:8762067

  7. Transforming capacity of two novel genes JS-1 and JS-2 located in chromosome 5p and their overexpression in human esophageal squamous cell carcinoma.

    Science.gov (United States)

    Fatima, Sarwat; Chui, Chung H; Tang, Wing K; Hui, Kin S; Au, Ho W; Li, Wing Y; Wong, Mei M; Cheung, Filly; Tsao, S W; Lam, King Y; Beh, Philip S L; Wong, John; Law, Simon; Srivastava, Gopesh; Ho, Kwok P; Chan, Albert S C; Tang, Johnny C O

    2006-01-01

    Esophageal squamous cell carcinoma (ESCC) has a high mortality rate and geographic differences in incidence. Previous studies of comparative genomic hybridization (CGH) showed that chromosomal 5p is frequently amplified in cell lines and primary ESCC of Hong Kong Chinese origin. In this report, attempt was made to study two novel genes, named as JS-1 and JS-2, which are located in chromosome 5p15.2 and are 5' upstream to delta catenin for their roles in molecular pathogenesis of ESCC. Eleven cell lines, 27 primary ESCC cases and multiple human tissue cDNA panels (MTC) of digestive system were studied for the expression level of JS-1 and JS-2 by RT-PCR. The full-length cDNA sequences of JS-1 and JS-2 were determined from a non-tumor esophageal epithelial cell line by 3' and 5' rapid amplification of cDNA ends (RACE). The transforming capacity of JS-1 and JS-2 was also investigated by transfecting NIH 3T3 cells with the expression vector pcDNA3.1(-) cloned with the full coding sequences and it was followed by the study of foci formation of the transfected cells under confluence growth and the anchorage-independent growth in soft agar. Forty-five percent (5/11) and 18% (2/11) of the ESCC cell lines showed overexpression of JS-1 and JS-2 respectively, while 55% (15/27) and 14% (3/22) primary ESCC cases showed overexpression of JS-1 and JS-2 respectively. JS-1 overexpression was most common in patients with stage II ESCC (6/27; 22%) whereas JS-2 was only overexpressed in a dysplastic lesion (1/22; 4%) and stage III tumors (2/22; 9%). The expression levels of JS-1 and JS-2 are both low in normal esophageal tissues. Overexpression of JS-1 in NIH 3T3 cells caused foci formation in confluence growth and colony formation in soft agar but not for JS-2. A high grade sarcoma was formed in the athymic nude mice when NIH 3T3 cells overexpressing JS-1 were injected subcutaneously. Our results thus indicate that the frequent overexpression of JS-1 in ESCC and its transforming

  8. Inhibition of clathrin by pitstop 2 activates the spindle assembly checkpoint and induces cell death in dividing HeLa cancer cells

    Directory of Open Access Journals (Sweden)

    Smith Charlotte M

    2013-01-01

    Full Text Available Abstract Background During metaphase clathrin stabilises the mitotic spindle kinetochore(K-fibres. Many anti-mitotic compounds target microtubule dynamics. Pitstop 2™ is the first small molecule inhibitor of clathrin terminal domain and inhibits clathrin-mediated endocytosis. We investigated its effects on a second function for clathrin in mitosis. Results Pitstop 2 did not impair clathrin recruitment to the spindle but disrupted its function once stationed there. Pitstop 2 trapped HeLa cells in metaphase through loss of mitotic spindle integrity and activation of the spindle assembly checkpoint, phenocopying clathrin depletion and aurora A kinase inhibition. Conclusions Pitstop 2 is therefore a new tool for investigating clathrin spindle dynamics. Pitstop 2 reduced viability in dividing HeLa cells, without affecting dividing non-cancerous NIH3T3 cells, suggesting that clathrin is a possible novel anti-mitotic drug target.

  9. Mustard Gas Surrogate, 2-Chloroethyl Ethylsulfide (2-CEES), Induces Centrosome Amplification and Aneuploidy in Human and Mouse Cells

    Science.gov (United States)

    2014-03-01

    increase in aneuploidy in treated  cells .      Methods and Materials    Cell   Culture     Saos2 (human  osteosarcoma ) and NIH3T3 (murine embryonic...fibroblasts)  cells  were obtained  from ATCC (HTB‐85 and CRL‐1658, respectively) and  cultured  in complete media:  Dulbecco’s  Modified Eagle Medium (DMEM...subconfluent  cultures .  After the 5 day incubation,  cells  were treated     with 0.5 μg/ml colcemid (Gibco) for 4 hours.   Cell  media was harvested and retained

  10. Test of critical steps towards a combined cell and gene therapy approach for the treatment of Duchenne muscular dystrophy

    DEFF Research Database (Denmark)

    Kajhøj, Tine Qvistgaard; Duch, Mogens R.; Pedersen, Finn Skou

    2015-01-01

    Background: Therapies for muscular dystrophies remain a major challenge in spite of advanced strategies using either cell or gene therapy. We here propose a combined approach of cell and gene therapy. As gene delivery vehicles with specific homing potential we have chosen mesoangioblasts which...... for myogenic properties in coculture. Survival and in situ myogenic differentiation were studied upon injection into degenerating M. gastrocnemius of athymic mice. In situ participation in muscle regeneration was confirmed on cryo-sections using EGFP fluorescence as marker. The ability of mesoangioblasts...... to serve as retroviral packaging cells was tested using the murine cell line NIH 3T3 fibroblasts as recipients in vitro and evaluation of transduction by fluorescence microscopy. Results: EGFP-MA retained the ability to differentiate into skeletal muscle myotubes upon co-culture with C2C12 cells. In vivo...

  11. Micropatterned co-culture of hepatocyte spheroids layered on non-parenchymal cells to understand heterotypic cellular interactions

    International Nuclear Information System (INIS)

    Otsuka, Hidenori; Sasaki, Kohei; Okimura, Saya; Nagamura, Masako; Nakasone, Yuichi

    2013-01-01

    Microfabrication and micropatterning techniques in tissue engineering offer great potential for creating and controlling cellular microenvironments including cell–matrix interactions, soluble stimuli and cell–cell interactions. Here, we present a novel approach to generate layered patterning of hepatocyte spheroids on micropatterned non-parenchymal feeder cells using microfabricated poly(ethylene glycol) (PEG) hydrogels. Micropatterned PEG-hydrogel-treated substrates with two-dimensional arrays of gelatin circular domains (ϕ = 100 μm) were prepared by photolithographic method. Only on the critical structure of PEG hydrogel with perfect protein rejection, hepatocytes were co-cultured with non-parenchymal cells to be led to enhanced hepatocyte functions. Then, we investigated the mechanism of the functional enhancement in co-culture with respect to the contributions of soluble factors and direct cell–cell interactions. In particular, to elucidate the influence of soluble factors on hepatocyte function, hepatocyte spheroids underlaid with fibroblasts (NIH/3T3 mouse fibroblasts) or endothelial cells (BAECs: bovine aortic endothelial cells) were compared with physically separated co-culture of hepatocyte monospheroids with NIH3T3 or BAEC using trans-well culture systems. Our results suggested that direct heterotypic cell-to-cell contact and soluble factors, both of these between hepatocytes and fibroblasts, significantly enhanced hepatocyte functions. In contrast, direct heterotypic cell-to-cell contact between hepatocytes and endothelial cells only contributed to enhance hepatocyte functions. This patterning technique can be a useful experimental tool for applications in basic science, drug screening and tissue engineering, as well as in the design of artificial liver devices. (paper)

  12. Expression and Purification of Recombinant Human Basic Fibroblast Growth Factor Fusion Proteins and Their Uses in Human Stem Cell Culture.

    Science.gov (United States)

    Imsoonthornruksa, Sumeth; Pruksananonda, Kamthorn; Parnpai, Rangsun; Rungsiwiwut, Ruttachuk; Ketudat-Cairns, Mariena

    2015-01-01

    To reduce the cost of cytokines and growth factors in stem cell research, a simple method for the production of soluble and biological active human basic fibroblast growth factor (hbFGF) fusion protein in Escherichia coli was established. Under optimal conditions, approximately 60-80 mg of >95% pure hbFGF fusion proteins (Trx-6xHis-hbFGF and 6xHis-hbFGF) were obtained from 1 liter of culture broth. The purified hbFGF proteins, both with and without the fusion tags, were biologically active, which was confirmed by their ability to stimulate proliferation of NIH3T3 cells. The fusion proteins also have the ability to support several culture passages of undifferentiated human embryonic stem cells and induce pluripotent stem cells. This paper describes a low-cost and uncomplicated method for the production and purification of biologically active hbFGF fusion proteins. © 2015 S. Karger AG, Basel.

  13. 28 CFR 79.65 - Proof of nonmalignant respiratory disease.

    Science.gov (United States)

    2010-07-01

    ... expiratory volume in one second (FEV1) and the forced vital capacity (FVC) administered and reported in... Program will treat as equivalent to a diagnosis of pulmonary fibrosis any diagnosis of “restrictive lung... that the claimant contracted a nonmalignant respiratory disease, including pulmonary fibrosis, fibrosis...

  14. Aberrant immunoglobulin and c-myc gene rearrangements in patients with nonmalignant monoclonal cryoglobulinemia

    International Nuclear Information System (INIS)

    Perl, A.; Wang, N.; Williams, J.M.; Hunt, M.J.; Rosenfeld, S.I.; Condemi, J.J.; Packman, C.H.; Abraham, G.N.

    1987-01-01

    The status of the immunoglobulin (Ig) genes was investigated in patients with idiopathic nonmalignant monoclonal IgG cryoglobulinemia (NCG). In NCG, monoclonal antibodies are synthesized at an accelerated rate by nonmalignant B lymphocytes. In order to determine whether this high production rate is related to a clonal B cell expansion, the rearrangement of the Ig genes was investigated by Southern blot analysis of genomic, 32 P-labelled, DNA extracted from the peripheral blood lymphocytes of four NCG patients. In three of four (VI, BR, and CH) clonal expansion of B cells was detected using probes specific for the genes. BamHI digestion of DNA from VI and BR produced three rearranged fragments which cohybridized with two of the probes. This finding suggested the presence of additional nonsecretory B cell clones and/or disruption of the gene segments spanned by and detected with the probes. In addition, the possibility of aberrant gene rearrangements was supported by noting the alteration of the c-myc gene locus in genomic DNA from peripheral blood leukocytes of VI and CH. Northern blot analysis of RNA isolated from peripheral blood B cells of VI and CH demonstrated aberrant transcripts of the c-myc gene, showing an active role of the altered c-myc locus. Detection of c-myc rearrangement in NCG patients clearly shows that this event may not be a final step in malignant B cell transformation

  15. A magnetic trap for living cells suspended in a paramagnetic buffer

    Science.gov (United States)

    Winkleman, Adam; Gudiksen, Katherine L.; Ryan, Declan; Whitesides, George M.; Greenfield, Derek; Prentiss, Mara

    2004-09-01

    This manuscript describes the fabrication and use of a three-dimensional magnetic trap for diamagnetic objects in an aqueous solution of paramagnetic ions; this trap uses permanent magnets. It demonstrates trapping of polystyrene spheres, and of various types of living cells: mouse fibroblast (NIH-3T3), yeast (Saccharomyces cerevisiae), and algae (Chlamydomonas reinhardtii). For a 40mM solution of gadolinium (III) diethylenetriaminepentaacetic acid (Gd .DTPA) in aqueous buffer, the smallest cell (particle) that could be trapped had a radius of ˜2.5μm. The trapped particle and location of the magnetic trap can be translated in three dimensions by independent manipulation of the permanent magnets. This letter a1so characterizes the biocompatibility of the trapping solution.

  16. Co-ordinate regulation of the cytoskeleton in 3T3 cells overexpressing thymosin-beta4.

    Science.gov (United States)

    Golla, R; Philp, N; Safer, D; Chintapalli, J; Hoffman, R; Collins, L; Nachmias, V T

    1997-01-01

    In several cell types, short-term increases in the concentration of the G-actin-sequestering peptide thymosin-beta4 (Tbeta4) cause the disassembly of F-actin bundles. To determine the extent of cell adaptability to these reductions in F-actin, we overexpressed Tbeta4 in NIH 3T3 cells. In cell lines with Tbeta4 levels twice those of vector controls, G-actin increased approximately twofold as expected. However, F-actin did not decrease as in short-term experiments but rather also increased approximately twofold so that the G-F ratio remained constant. Surprisingly, the cytoskeletal proteins myosin IIA, alpha-actinin, and tropomyosin also increased nearly twofold. These increases were specific; DNA, total protein, lactic dehydrogenase, profilin, and actin depolymerizing factor levels were unchanged in the overexpressing cells. The Tbeta4 lines spread more fully and adhered to the dish more strongly than vector controls; this altered phenotype correlated with a twofold increase in talin and alpha5-integrin and a nearly threefold increase in vinculin. Focal adhesions, detected by indirect immunofluorescence with antivinculin, were increased in size over the controls. Northern blotting showed that mRNAs for both beta-actin and vinculin were increased twofold in the overexpressing lines. We conclude that 1) NIH 3T3 cells adapt to increased levels of G-actin sequestered by increased Tbeta4 by increasing their total actin so that the F-actin/G-actin ratio remains constant; 2) these cells coordinately increase several cytoskeletal and adhesion plaque proteins; and 3) at least for actin and vinculin, this regulation is at the transcriptional level. We therefore propose that the proteins of this multimember interacting complex making up the actin-based cytoskeleton, are coordinately regulated by factors that control the expression of several proteins. The mechanism may bear similarities to the control of synthesis of another multimember interacting complex, the myofibril of

  17. 4-[125I]iodo-(2,5-dimethoxy)phenylisopropylamine and [3H]ketanserin labeling of 5-hydroxytryptamine2 (5HT2) receptors in mammalian cells transfected with a rat 5HT2 cDNA: Evidence for multiple states and not multiple 5HT2 receptor subtypes

    International Nuclear Information System (INIS)

    Teitler, M.; Leonhardt, S.; Weisberg, E.L.; Hoffman, B.J.

    1990-01-01

    Evidence has accumulated indicating that the radioactive hallucinogens 4-bromo-[3H](2,5-dimethoxy)phenylisopropylamine ([3H]DOB) and 4-[125I]iodo-(2,5-dimethoxy)phenylisopropylamine ([125I]DOI) label an agonist high affinity state of the 5-hydroxytryptamine2 (5HT2) receptor and [3H]ketanserin labels both agonist high and low affinity states. Recently, an alternative hypothesis has been put forward proposing that the radioactive hallucinogens are labeling a 5HT2 receptor subtype distinct from the receptor labeled by [3H]ketanserin. In order to provide definitive evidence as to which of these hypotheses is correct, the rat 5HT2 receptor gene was transfected into NIH-3T3 cells and COS cells. Neither nontransfected cell type expresses 5HT2 receptors; the transfected cells expressed high affinity binding sites for both [125I] DOI (KD = 0.8 nM and Bmax = 363 fmol/mg in NIH-3T3 cells; KD = 0.2 nM and Bmax = 26 fmol/mg in COS cells) and [3H]ketanserin (KD = 0.4 nM and Bmax = 5034 fmol/mg in NIH-3T3 cells; KD = 1.0 nM and Bmax = 432 fmol/mg in COS cells). The affinities of agonists and antagonists for the [125I]DOI-labeled receptor were significantly higher than for the [3H]ketanserin-labeled receptor. The affinities of agonists and antagonists for these binding sites were essentially identical to their affinities for the sites radiolabeled by these radioligands in mammalian brain homogenates. The [125I]DOI binding was guanyl nucleotide sensitive, indicating a coupling to a GTP-binding protein. These data indicate that the 5HT2 receptor gene product contains both the guanyl nucleotide-sensitive [125I]DOI binding site and the [3H]ketanserin binding site. Therefore, these data indicate that the 5HT2 receptor gene product can produce a high affinity binding site for the phenylisopropylamine hallucinogen agonists as well as for the 5HT2 receptor antagonists

  18. BMT Abatacept for Non-Malignant Diseases

    Science.gov (United States)

    2018-05-16

    Hurler Syndrome; Fanconi Anemia; Glanzmann Thrombasthenia; Wiskott-Aldrich Syndrome; Chronic Granulomatous Disease; Severe Congenital Neutropenia; Leukocyte Adhesion Deficiency; Shwachman-Diamond Syndrome; Diamond-Blackfan Anemia; Dyskeratosis-congenita; Chediak-Higashi Syndrome; Severe Aplastic Anemia; Thalassemia Major; Hemophagocytic Lymphohistiocytosis; Sickle Cell Disease

  19. Experiment list: SRX262781 [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available _name=NIH3T3_SRF_15 || cell line=NIH3T3 fibroblasts || genotype=normal || chip antibody=SRF || chip antibody vendor=Santa Cruz Biotec...hnology http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/e

  20. Experiment list: SRX262782 [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available echnology http://dbarchive.biosciencedbc.jp/kyushu-u/mm9...ce_name=NIH3T3_SRF_15 || cell line=NIH3T3 fibroblasts || genotype=normal || chip antibody=SRF || chip antibody vendor=Santa Cruz Biot

  1. Experiment list: SRX262780 [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available chnology http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/...e_name=NIH3T3_SRF_03 || cell line=NIH3T3 fibroblasts || genotype=normal || chip antibody=SRF || chip antibody vendor=Santa Cruz Biote

  2. A Girl with Autoimmune Cytopenias, Nonmalignant Lymphadenopathy, and Recurrent Infections

    Directory of Open Access Journals (Sweden)

    Marjolein A. C. Mattheij

    2012-01-01

    Full Text Available We describe a girl, now 9 years of age, with chronic idiopathic thrombocytopenic purpura, persistent nonmalignant lymphadenopathy, splenomegaly, recurrent infections, and autoimmune hemolytic anemia. Her symptoms partly fit the definitions of both autoimmune lymphoproliferative syndrome (ALPS and common variable immunodeficiency disorders (CVIDs. Genetic analysis showed no abnormalities in the ALPS-genes FAS, FASLG, and CASP10. The CVID-associated TACI gene showed a homozygous polymorphism (Pro251Leu, which is found also in healthy controls.

  3. Characterization of the growth of murine fibroblasts that express human insulin receptors. II. Interaction of insulin with other growth factors

    International Nuclear Information System (INIS)

    Randazzo, P.A.; Jarett, L.

    1990-01-01

    The effects of insulin-like growth factor-1 (IGF-1), epidermal growth factor (EGF), platelet-derived growth factor (PDGF), and insulin on DNA synthesis were studied in murine fibroblasts transfected with an expression vector containing human insulin receptor cDNA (NIH 3T3/HIR) and the parental NIH 3T3 cells. In NIH 3T3/HIR cells, individual growth factors in serum-free medium stimulated DNA synthesis with the following relative efficacies: insulin greater than or equal to 10% fetal calf serum greater than PDGF greater than IGF-1 much greater than EGF. In comparison, the relative efficacies of these factors in stimulating DNA synthesis by NIH 3T3 cells were 10% fetal calf serum greater than PDGF greater than EGF much greater than IGF-1 = insulin. In NIH 3T3/HIR cells, EGF was synergistic with 1-10 ng/ml insulin but not with 100 ng/ml insulin or more. Synergy of PDGF or IGF-1 with insulin was not detected. In the parental NIH 3T3 cells, insulin and IGF-1 were found to be synergistic with EGF (1 ng/ml), PDGF (100 ng/ml), and PDGF plus EGF. In NIH 3T3/HIR cells, the lack of interaction of insulin with other growth factors was also observed when the percentage of cells synthesizing DNA was examined. Despite insulin's inducing only 60% of NIH 3T3/HIR cells to incorporate thymidine, addition of PDGF, EGF, or PDGF plus EGF had no further effect. In contrast, combinations of growth factors resulted in 95% of the parental NIH 3T3 cells synthesizing DNA. The independence of insulin-stimulated DNA synthesis from other mitogens in the NIH 3T3/HIR cells is atypical for progression factor-stimulated DNA synthesis and is thought to be partly the result of insulin receptor expression in an inappropriate context or quantity

  4. Dual functional NaYF4:Yb3+, Er3+@NaYF4:Yb3+, Nd3+ core-shell nanoparticles for cell temperature sensing and imaging

    Science.gov (United States)

    Shi, Zengliang; Duan, Yue; Zhu, Xingjun; Wang, Qiwei; Li, DongDong; Hu, Ke; Feng, Wei; Li, Fuyou; Xu, Chunxiang

    2018-03-01

    Lanthanide-doped up-conversion nanoparticles (UCNPs) provide a remote temperature sensing approach to monitoring biological microenvironments. In this research, the UCNPs of NaYF4:Yb3+, Er3+@NaYF4:Yb3+, Nd3+ with hexagonal (β)-phase were synthesized and applied in cell temperature sensing as well as imaging after surface modification with meso-2, 3-dimercaptosuccinic acid. In the core-shell UCNPs, Yb3+ ions were introduced as energy transfer media between sensitizers of Nd3+ and activators of Er3+ to improve Er3+emission and prevent their quenching behavior due to multiple energy levels of Nd3+. Under the excitations of 808 nm and 980 nm lasers, the NaYF4:Yb3+, Er3+@NaYF4:Yb3+, Nd3+ nanoparticles exhibited an efficient green band with two emission peaks at 525 nm and 545 nm, respectively, which originated from the transitions of 2H11/2 → 4I15/2 and 4S3/2 → 4I15/2 for Er3+ ions. We demonstrate that an occurrence of good logarithmic linearity exists between the intensity ratio of these two emission peaks and the reciprocal of the inside or outside temperature of NIH-3T3 cells. A better thermal stability is proved through temperature-dependent spectra with a heating-cooling cycle. The obtained viability of NIH-3T3 cells is greater than 90% after incubations of about 12 and 24 (h), and they possess a lower cytotoxicity of UCNPs. This work provides a method for monitoring the cell temperature and its living state from multiple dimensions including temperature response, cell images and visual up-conversion fluorescent color.

  5. Factors Affecting Mortality in Elderly Patients Hospitalized for Nonmalignant Reasons

    Directory of Open Access Journals (Sweden)

    Teslime Ayaz

    2014-01-01

    Full Text Available Elderly population is hospitalized more frequently than young people, and they suffer from more severe diseases that are difficult to diagnose and treat. The present study aimed to investigate the factors affecting mortality in elderly patients hospitalized for nonmalignant reasons. Demographic data, reason for hospitalization, comorbidities, duration of hospital stay, and results of routine blood testing at the time of first hospitalization were obtained from the hospital records of the patients, who were over 65 years of age and hospitalized primarily for nonmalignant reasons. The mean age of 1012 patients included in the study was 77.8 ± 7.6. The most common reason for hospitalization was diabetes mellitus (18.3%. Of the patients, 90.3% had at least a single comorbidity. Whilst 927 (91.6% of the hospitalized patients were discharged, 85 (8.4% died. Comparison of the characteristics of the discharged and dead groups revealed that the dead group was older and had higher rates of poor general status and comorbidity. Differences were observed between the discharged and dead groups in most of the laboratory parameters. Hypoalbuminemia, hypertriglyceridemia, hypopotassemia, hypernatremia, hyperuricemia, and high TSH level were the predictors of mortality. In order to meet the health necessities of the elderly population, it is necessary to well define the patient profiles and to identify the risk factors.

  6. v-Src oncogene product increases sphingosine kinase 1 expression through mRNA stabilization: alteration of AU-rich element-binding proteins.

    Science.gov (United States)

    Sobue, S; Murakami, M; Banno, Y; Ito, H; Kimura, A; Gao, S; Furuhata, A; Takagi, A; Kojima, T; Suzuki, M; Nozawa, Y; Murate, T

    2008-10-09

    Sphingosine kinase 1 (SPHK1) is overexpressed in solid tumors and leukemia. However, the mechanism of SPHK1 overexpression by oncogenes has not been defined. We found that v-Src-transformed NIH3T3 cells showed a high SPHK1 mRNA, SPHK1 protein and SPHK enzyme activity. siRNA of SPHK1 inhibited the growth of v-Src-NIH3T3, suggesting the involvement of SPHK1 in v-Src-induced oncogenesis. v-Src-NIH3T3 showed activations of protein kinase C-alpha, signal transducers and activators of transcription 3 and c-Jun NH(2)-terminal kinase. Their inhibition suppressed SPHK1 expression in v-Src-NIH3T3, whereas their overexpression increased SPHK1 mRNA in NIH3T3. Unexpectedly, the nuclear run-on assay and the promoter analysis using 5'-promoter region of mouse SPHK1 did not show any significant difference between mock- and v-Src-NIH3T3. Furthermore, the half-life of SPHK1 mRNA in mock-NIH3T3 was nearly 15 min, whereas that of v-Src-NIH3T3 was much longer. Examination of two AU-rich region-binding proteins, AUF1 and HuR, that regulate mRNA decay reciprocally, showed decreased total AUF1 protein associated with increased tyrosine-phosphorylated form and increased serine-phosphorylated HuR protein in v-Src-NIH3T3. Modulation of AUF1 and HuR by their overexpression or siRNA revealed that SPHK1 mRNA in v-Src- and mock-NIH3T3 was regulated reciprocally by these factors. Our results showed, for the first time, a novel mechanism of v-Src-induced SPHK1 overexpression.

  7. Immunohistochemical and radioimmunological demonstration of alpha1-fetoprotein in nonmalignant changes of human gastric mucosa

    International Nuclear Information System (INIS)

    Falser, N.; Lederer, B.; Reissigl, H.; Innsbruck Univ.

    1977-01-01

    The occurence of α 1 fetoprotein in nonmalignant changes of the gastric mucosa was investigated by means of immunohistochemistry and radioimmunonoassay. The investigations were performed in tissue sections, cytological imprint preparations as well as in homogenized tissue samples (obtained by gastroscopy). α 1 fetoprotein could be demonstrated by immuno-histochemistry in about 90% of the samples originating from the surroundings of gastric ulcer and the region of gastrojejunostomy after B II-resection. The RIA was positive in about 75% of the tissue samples, whereas from gastric juice only 40% of positive results could be obtained. No α 1 fetoprotein-activity could be demonstrated in serum samples. These investigations indicate that α 1 fetoprotein is not exclusively synthesized by embryonic or neoplastic tissues and also can be synthesized also by regenerating cell-systems. It may be supposed that this synthesis represents an unspecific answer to growth-stimulation. (orig.) [de

  8. Characterization of the growth of murine fibroblasts that express human insulin receptors. I. The effect of insulin in the absence of other growth factors

    International Nuclear Information System (INIS)

    Randazzo, P.A.; Morey, V.A.; Polishook, A.K.; Jarett, L.

    1990-01-01

    The effect of insulin on the growth of murine fibroblasts transfected with an expression vector containing human insulin receptor cDNA (NIH 3T3/HIR) and the parental cells (NIH/3T3) was characterized. Insulin in the absence of other mitogens increased the rate of incorporation of thymidine into NIH 3T3/HIR cells with a half-maximal response occurring at an insulin concentration of 35 ng/ml and a maximal response that was equivalent to that elicited by 10% fetal calf serum. The thymidine incorporation rate was increased by 12 h, was maximal at approximately 16 h, and returned to basal rates at 24 h after the addition of insulin. Insulin induced a maximum of 65% of cells to incorporate thymidine. The increased DNA synthesis was accompanied by net growth. Addition of insulin to the NIH 3T3/HIR cells resulted in increased DNA content with a half-maximal response occurring at approximately 30 ng/ml insulin and a maximal response equivalent to that elicited by serum. An increase in cell number detected after the addition of insulin to the NIH 3T3/HIR suggests that the cells had progressed through mitosis. Insulin did not increase the rate of thymidine incorporation, DNA content, or number of the parental NIH 3T3 cells. These data show that insulin, in the absence of a second mitogen, is able to induce NIH 3T3/HIR fibroblasts to traverse the cell cycle

  9. Male Infertility and Risk of Nonmalignant Chronic Diseases

    DEFF Research Database (Denmark)

    Glazer, Clara Helene; Bonde, Jens Peter; Eisenberg, Michael L.

    2017-01-01

    The association between male infertility and increased risk of certain cancers is well studied. Less is known about the long-term risk of nonmalignant diseases in men with decreased fertility. A systemic literature review was performed on the epidemiologic evidence of male infertility...... as a precursor for increased risk of diabetes, cardiovascular diseases, and all-cause mortality. PubMed and Embase were searched from January 1, 1980, to September 1, 2016, to identify epidemiological studies reporting associations between male infertility and the outcomes of interest. Animal studies, case...... prospective (three on risk of mortality, one on risk of chronic diseases) and three were cross-sectional relating male infertility to the Charlson Comorbidity Index. The current epidemiological evidence is compatible with an association between male infertility and risk of chronic disease and mortality...

  10. MEMS-based dynamic cell-to-cell culture platforms using electrochemical surface modifications

    International Nuclear Information System (INIS)

    Chang, Jiyoung; Lin, Liwei; Yoon, Sang-Hee; Mofrad, Mohammad R K

    2011-01-01

    MEMS-based biological platforms with the capability of both spatial placements and time releases of living cells for cell-to-cell culture experiments have been designed and demonstrated utilizing electrochemical surface modification effects. The spatial placement is accomplished by electrochemical surface modification of substrate surfaces to be either adhesive or non-adhesive for living cells. The time control is achieved by the electrical activation of the selective indium tin oxide co-culture electrode to allow the migration of living cells onto the electrode to start the cell-to-cell culture studies. Prototype devices have a three-electrode design with an electrode size of 50 × 50 µm 2 and the separation gaps of 2 µm between them. An electrical voltage of −1.5 V has been used to activate the electrodes independently and sequentially to demonstrate the dynamic cell-to-cell culture experiments of NIH 3T3 fibroblast and Madin Darby canine kidney cells. As such, this MEMS platform could be a basic yet versatile tool to characterize transient cell-to-cell interactions

  11. Determinants of hematology-oncology trainees' postfellowship career pathways with a focus on nonmalignant hematology

    Science.gov (United States)

    Jenkins, Sarah; Mikhael, Joseph; Gitlin, Scott D.

    2018-01-01

    Nonmalignant hematologic conditions are extremely prevalent and contribute significantly to the global burden of disease. The US health care system may soon face a shortage of specialists in nonmalignant hematology. We sought to identify factors that lead hematology-oncology fellows to pursue (or not to pursue) careers in nonmalignant hematology. Cross-sectional, web-based survey distributed to 149 graduates of a hematology-oncology fellowship program at a large academic medical center between 1998 and 2016. Eighty-six out of 149 graduates responded (57.7%); most (59 [68.6%]) practice at an academic medical center. Respondents spend a mean of 61% of their time in clinical practice, 23.7% conducting research, 5.2% in education, and 5.2% in administration. Those in clinical practice spend a mean of 52.1% of their time in solid tumor oncology, 37.5% in hematologic malignancies, and 10% in nonmalignant hematology; only 1 spent >50% of time practicing nonmalignant hematology. Factors most significantly affecting choice of patient population included clinical experience during fellowship and intellectual stimulation of the patient population/disease type. Factors that could have most significantly influenced a decision to spend more time in nonmalignant hematology included increased exposure/access to role models and mentors and opportunities for better career growth/advancement. Fellowship graduates spend >50% of their time in clinical practice, but almost none spend a significant amount of time practicing nonmalignant hematology. Given the growing number of patients with nonmalignant hematologic conditions and a possible future provider shortage, medical trainees should be encouraged to pursue careers in nonmalignant hematology. PMID:29463548

  12. Manipulation of Cell Cycle and Chromatin Configuration by Means of Cell-Penetrating Geminin.

    Directory of Open Access Journals (Sweden)

    Yoshinori Ohno

    Full Text Available Geminin regulates chromatin remodeling and DNA replication licensing which play an important role in regulating cellular proliferation and differentiation. Transcription of the Geminin gene is regulated via an E2F-responsive region, while the protein is being closely regulated by the ubiquitin-proteasome system. Our objective was to directly transduce Geminin protein into cells. Recombinant cell-penetrating Geminin (CP-Geminin was generated by fusing Geminin with a membrane translocating motif from FGF4 and was efficiently incorporated into NIH 3T3 cells and mouse embryonic fibroblasts. The withdrawal study indicated that incorporated CP-Geminin was quickly reduced after removal from medium. We confirmed CP-Geminin was imported into the nucleus after incorporation and also that the incorporated CP-Geminin directly interacted with Cdt1 or Brahma/Brg1 as the same manner as Geminin. We further demonstrated that incorporated CP-Geminin suppressed S-phase progression of the cell cycle and reduced nuclease accessibility in the chromatin, probably through suppression of chromatin remodeling, indicating that CP-Geminin constitutes a novel tool for controlling chromatin configuration and the cell cycle. Since Geminin has been shown to be involved in regulation of stem cells and cancer cells, CP-Geminin is expected to be useful for elucidating the role of Geminin in stem cells and cancer cells, and for manipulating their activity.

  13. Alginate gelation-induced cell death during laser-assisted cell printing

    International Nuclear Information System (INIS)

    Gudapati, Hemanth; Yan, Jingyuan; Huang, Yong; Chrisey, Douglas B

    2014-01-01

    Modified laser-induced forward transfer has emerged as a promising bioprinting technique. Depending on the operating conditions and cell properties, laser cell printing may cause cell injury and even death, which should be carefully elucidated for it to be a viable technology. This study has investigated the effects of alginate gelation, gelation time, alginate concentration, and laser fluence on the post-transfer cell viability of NIH 3T3 fibroblasts. Sodium alginate and calcium chloride are used as the gel precursor and gel reactant solution to form cell-laden alginate microspheres. It is found that the effects of gelation depend on the duration of gelation. Two-minute gelation is observed to increase the cell viability after 24 h incubation, mainly due to the protective cushion effect of the forming gel membrane during droplet landing. Despite the cushion effect from 10 min gelation, it is observed that the cell viability decreases after 24 h incubation because of the forming thick gel membrane that reduces nutrient and oxygen diffusion from the culture medium. In addition, the cell viability after 24 h incubation decreases as the laser fluence or alginate concentration increases. (paper)

  14. Preparation of Single Cells for Imaging Mass Spectrometry

    Energy Technology Data Exchange (ETDEWEB)

    Berman, E S; Fortson, S L; Kulp, K S; Checchi, K D; Wu, L; Felton, J S; Wu, K J

    2007-10-24

    Characterizing chemical changes within single cells is important for determining fundamental mechanisms of biological processes that will lead to new biological insights and improved disease understanding. Imaging biological systems with mass spectrometry (MS) has gained popularity in recent years as a method for creating precise chemical maps of biological samples. In order to obtain high-quality mass spectral images that provide relevant molecular information about individual cells, samples must be prepared so that salts and other cell-culture components are removed from the cell surface and the cell contents are rendered accessible to the desorption beam. We have designed a cellular preparation protocol for imaging MS that preserves the cellular contents for investigation and removes the majority of the interfering species from the extracellular matrix. Using this method, we obtain excellent imaging results and reproducibility in three diverse cell types: MCF7 human breast cancer cells, Madin-Darby canine kidney (MDCK) cells, and NIH/3T3 mouse fibroblasts. This preparation technique allows routine imaging MS analysis of cultured cells, allowing for any number of experiments aimed at furthering scientific understanding of molecular processes within individual cells.

  15. Micropatterned Azopolymer Surfaces Modulate Cell Mechanics and Cytoskeleton Structure.

    Science.gov (United States)

    Rianna, Carmela; Ventre, Maurizio; Cavalli, Silvia; Radmacher, Manfred; Netti, Paolo A

    2015-09-30

    Physical and chemical characteristics of materials are important regulators of cell behavior. In particular, cell elasticity is a fundamental parameter that reflects the state of a cell. Surface topography finely modulates cell fate and function via adhesion mediated signaling and cytoskeleton generated forces. However, how topographies alter cell mechanics is still unclear. In this work we have analyzed the mechanical properties of peripheral and nuclear regions of NIH-3T3 cells on azopolymer substrates with different topographic patterns. Micrometer scale patterns in the form of parallel ridges or square lattices of surface elevations were encoded on light responsive azopolymer films by means of contactless optical methods. Cell mechanics was investigated by atomic force microscopy (AFM). Cells and consequently the cell cytoskeleton were oriented along the linear patterns affecting cytoskeletal structures, e.g., formation of actin stress fibers. Our data demonstrate that topographic substrate patterns are recognized by cells and mechanical information is transferred by the cytoskeleton. Furthermore, cytoskeleton generated forces deform the nucleus, changing its morphology that appears to be related to different mechanical properties in the nuclear region.

  16. Combined cord blood and bone marrow transplantation from the same human leucocyte antigen-identical sibling donor for children with malignant and non-malignant diseases.

    Science.gov (United States)

    Tucunduva, Luciana; Volt, Fernanda; Cunha, Renato; Locatelli, Franco; Zecca, Marco; Yesilipek, Akif; Caniglia, Maurizio; Güngör, Tayfun; Aksoylar, Serap; Fagioli, Franca; Bertrand, Yves; Addari, Maria Carmen; de la Fuente, Josu; Winiarski, Jacek; Biondi, Andrea; Sengeloev, Henrik; Badell, Isabel; Mellgren, Karin; de Heredia, Cristina Díaz; Sedlacek, Petr; Vora, Ajay; Rocha, Vanderson; Ruggeri, Annalisa; Gluckman, Eliane

    2015-04-01

    Umbilical cord blood (UCB) from an human leucocyte antigen (HLA)-identical sibling can be used for transplantation of patients with malignant and non-malignant diseases. However, the low cellular content of most UCB units represents a limitation to this approach. An option to increase cell dose is to harvest bone marrow (BM) cells from the same donor and infuse them along with the UCB. We studied 156 children who received such a combined graft between 1992 and 2011. Median age was 7 years and 78% of patients (n = 122) were transplanted for non-malignant diseases, mainly haemoglobinopathies. Acute leukaemia (n = 26) was the most frequent malignant diagnosis. Most patients (91%) received myeloablative conditioning. Median donor age was 1·7 years, median infused nucleated cell dose was 24·4 × 10(7) /kg and median follow-up was 41 months. Sixty-days neutrophil recovery occurred in 96% of patients at a median of 17 d. The probabilities of grade-II-IV acute and chronic graft-versus-host disease (GVHD) were 19% and 10%, respectively. Four-year overall survival was 90% (68% malignant; 97% non-malignant diseases) with 3% probability of death. In conclusion, combined UCB and BM transplantation from an HLA-identical sibling donor is an effective treatment for children with malignant and non-malignant disorders with high overall survival and low incidence of GVHD. © 2014 John Wiley & Sons Ltd.

  17. The Tol2 transposon system mediates the genetic engineering of T-cells with CD19-specific chimeric antigen receptors for B-cell malignancies.

    Science.gov (United States)

    Tsukahara, T; Iwase, N; Kawakami, K; Iwasaki, M; Yamamoto, C; Ohmine, K; Uchibori, R; Teruya, T; Ido, H; Saga, Y; Urabe, M; Mizukami, H; Kume, A; Nakamura, M; Brentjens, R; Ozawa, K

    2015-02-01

    Engineered T-cell therapy using a CD19-specific chimeric antigen receptor (CD19-CAR) is a promising strategy for the treatment of advanced B-cell malignancies. Gene transfer of CARs to T-cells has widely relied on retroviral vectors, but transposon-based gene transfer has recently emerged as a suitable nonviral method to mediate stable transgene expression. The advantages of transposon vectors compared with viral vectors include their simplicity and cost-effectiveness. We used the Tol2 transposon system to stably transfer CD19-CAR into human T-cells. Normal human peripheral blood lymphocytes were co-nucleofected with the Tol2 transposon donor plasmid carrying CD19-CAR and the transposase expression plasmid and were selectively propagated on NIH3T3 cells expressing human CD19. Expanded CD3(+) T-cells with stable and high-level transgene expression (~95%) produced interferon-γ upon stimulation with CD19 and specifically lysed Raji cells, a CD19(+) human B-cell lymphoma cell line. Adoptive transfer of these T-cells suppressed tumor progression in Raji tumor-bearing Rag2(-/-)γc(-/-) immunodeficient mice compared with control mice. These results demonstrate that the Tol2 transposon system could be used to express CD19-CAR in genetically engineered T-cells for the treatment of refractory B-cell malignancies.

  18. Recurrent, nonmalignant tracheoesophageal fistulas and the need for surgical improvisation.

    Science.gov (United States)

    Altorjay, Aron; Mucs, Mihály; Rüll, Miklós; Tihanyi, Zoltán; Hamvas, Balázs; Madácsy, László; Paál, Balázs

    2010-06-01

    Despite the many recent advances in thoracic surgery, the management of patients with recurrent, nonmalignant tracheoesophageal fistulas remains problematic, controversial, and challenging. Between 1998 and 2008, we treated 8 patients with RTEF. Closure of the original tracheoesophageal fistula had been attempted once in 5 patients, twice in 2 patients, and 4 times in 1 patient, all in different institutions. Four cases necessitated right posterolateral thoracotomy and cervical exposure, 2 cases cervical and abdominal incision, and 1 case right posterolateral thoracotomy, with abdominal and cervical exposure. With the exception of the 2 patients whose excluded esophagus was used to substitute for the trachea membranous wall, the damaged tracheal segment was removed. In general, a pedicled mediastinal pleural flap was pulled into the neck to increase the safety of the tracheal anastomosis formed with the trachea, and (or) to separate the suture lines of the two organs. A single intervention was sufficient for all 8 patients: no reoperation was necessary, and there was no perioperative mortality. Transient reflux, abdominal distention, and dyspnea in response to forced physical exertion occurred in 1 case each. Only 1 patient subsequently takes medication regularly for reflux disease. Separation initiated from the tracheal bifurcation, a pedicled mediastinal pleural flap pulled into the neck, a tracheal anastomosis sewn onto the cricoid cartilage with avoidance of its posterolateral elbow, a shaped Dumon stent (Novatech, Plan de Grasse, France) with an individually fenestrated tracheostomy cannula, and endoscopy-assisted, transhiatal vagal-preserving esophageal exclusion all served as successful elements of our surgical procedures. 2010 The Society of Thoracic Surgeons. Published by Elsevier Inc. All rights reserved.

  19. Cell Matrix Remodeling Ability Shown by Image Spatial Correlation

    Science.gov (United States)

    Chiu, Chi-Li; Digman, Michelle A.; Gratton, Enrico

    2013-01-01

    Extracellular matrix (ECM) remodeling is a critical step of many biological and pathological processes. However, most of the studies to date lack a quantitative method to measure ECM remodeling at a scale comparable to cell size. Here, we applied image spatial correlation to collagen second harmonic generation (SHG) images to quantitatively evaluate the degree of collagen remodeling by cells. We propose a simple statistical method based on spatial correlation functions to determine the size of high collagen density area around cells. We applied our method to measure collagen remodeling by two breast cancer cell lines (MDA-MB-231 and MCF-7), which display different degrees of invasiveness, and a fibroblast cell line (NIH/3T3). We found distinct collagen compaction levels of these three cell lines by applying the spatial correlation method, indicating different collagen remodeling ability. Furthermore, we quantitatively measured the effect of Latrunculin B and Marimastat on MDA-MB-231 cell line collagen remodeling ability and showed that significant collagen compaction level decreases with these treatments. PMID:23935614

  20. High glucose-mediated oxidative stress impairs cell migration.

    Directory of Open Access Journals (Sweden)

    Marcelo L Lamers

    Full Text Available Deficient wound healing in diabetic patients is very frequent, but the cellular and molecular causes are poorly defined. In this study, we evaluate the hypothesis that high glucose concentrations inhibit cell migration. Using CHO.K1 cells, NIH-3T3 fibroblasts, mouse embryonic fibroblasts and primary skin fibroblasts from control and diabetic rats cultured in 5 mM D-glucose (low glucose, LG, 25 mM D-glucose (high glucose, HG or 25 mM L-glucose medium (osmotic control--OC, we analyzed the migration speed, protrusion stability, cell polarity, adhesion maturation and the activity of the small Rho GTPase Rac1. We also analyzed the effects of reactive oxygen species by incubating cells with the antioxidant N-Acetyl-Cysteine (NAC. We observed that HG conditions inhibited cell migration when compared to LG or OC. This inhibition resulted from impaired cell polarity, protrusion destabilization and inhibition of adhesion maturation. Conversely, Rac1 activity, which promotes protrusion and blocks adhesion maturation, was increased in HG conditions, thus providing a mechanistic basis for the HG phenotype. Most of the HG effects were partially or completely rescued by treatment with NAC. These findings demonstrate that HG impairs cell migration due to an increase in oxidative stress that causes polarity loss, deficient adhesion and protrusion. These alterations arise, in large part, from increased Rac1 activity and may contribute to the poor wound healing observed in diabetic patients.

  1. Rat embryo cells immortalized with transfected oncogenes are transformed by gamma irradiation.

    Science.gov (United States)

    Endlich, B; Salavati, R; Sullivan, T; Ling, C C

    1992-12-01

    of myc, raf, Ha-ras, or Ki-ras genes in any REC:myc transformant. DNA from several transformed REC:myc:gamma cell lines induced focus formation in recipient C3H 10T1/2 and NIH 3T3 cells. The NIH 3T3 foci tested positive when hybridized to a probe for rat repetitive DNA. A detailed analysis of the NIH 3T3 transformants generated from REC:myc:gamma 33 and gamma 41 DNA failed to detect Ha-ras, Ki-ras, raf, neu, trk, abl, fms, or src oncogenes of rat origin.(ABSTRACT TRUNCATED AT 400 WORDS)

  2. The Yersinia pseudotuberculosis and Yersinia pestis toxin complex is active against cultured mammalian cells.

    Science.gov (United States)

    Hares, Michelle C; Hinchliffe, Stewart J; Strong, Philippa C R; Eleftherianos, Ioannis; Dowling, Andrea J; ffrench-Constant, Richard H; Waterfield, Nick

    2008-11-01

    The toxin complex (Tc) genes were first identified in the insect pathogen Photorhabdus luminescens and encode approximately 1 MDa protein complexes which are toxic to insect pests. Subsequent genome sequencing projects have revealed the presence of tc orthologues in a range of bacterial pathogens known to be associated with insects. Interestingly, members of the mammalian-pathogenic yersiniae have also been shown to encode Tc orthologues. Studies in Yersinia enterocolitica have shown that divergent tc loci either encode insect-active toxins or play a role in colonization of the gut in gastroenteritis models of rats. So far little is known about the activity of the Tc proteins in the other mammalian-pathogenic yersiniae. Here we present work to suggest that Tc proteins in Yersinia pseudotuberculosis and Yersinia pestis are not insecticidal toxins but have evolved for mammalian pathogenicity. We show that Tc is secreted by Y. pseudotuberculosis strain IP32953 during growth in media at 28 degrees C and 37 degrees C. We also demonstrate that oral toxicity of strain IP32953 to Manduca sexta larvae is not due to Tc expression and that lysates of Escherichia coli BL21 expressing the Yersinia Tc proteins are not toxic to Sf9 insect cells but are toxic to cultured mammalian cell lines. Cell lysates of E. coli BL21 expressing the Y. pseudotuberculosis Tc proteins caused actin ruffles, vacuoles and multi-nucleation in cultured human gut cells (Caco-2); similar morphology was observed after application of a lysate of E. coli BL21 expressing the Y. pestis Tc proteins to mouse fibroblast NIH3T3 cells, but not Caco-2 cells. Finally, transient expression of the individual Tc proteins in Caco-2 and NIH3T3 cell lines reproduced the actin and nuclear rearrangement observed with the topical applications. Together these results add weight to the growing hypothesis that the Tc proteins in Y. pseudotuberculosis and Y. pestis have been adapted for mammalian pathogenicity. We further

  3. Human cell transformation in the study of sunlight-induced cancers in the skin of man

    International Nuclear Information System (INIS)

    Sutherland, B.M.; Bennett, P.V.

    1988-01-01

    Human cell transformation provides a powerful approach to understanding - at the cellular and molecular levels - induction of cancers in the skin of man. A principal approach to this problem is the direct transformation of human skin cells by exposure to ultraviolet and/or near-UV radiation. The frequency of human cells transformed to anchorage independence increases with radiation exposure; the relative transforming efficiencies of different wavelengths implies that direct absorption by nucleic acids is a primary initial event. Partial reversal of potential transforming lesions by photoreactivation suggests that pyrimidine dimers, as well as other lesions, are important in UV transformation of human cells. Human cells can also be transformed by transfection with cloned oncogenes, or with DNAs from tumors or tumor cell lines. Cells treated by the transfection procedure (but without DNA) or cells transfected with DNAs from normal mammalian cells or tissues show only background levels of transformation. Human cells can be transformed to anchorage-independent growth by DNAs ineffective in transformation of NIH 3T3 cells (including most human skin cancers), permitting the analysis of oncogenic molecular changes even in tumor DNAs difficult or impossible to analyze in rodent cell systems. 29 refs.; 4 figs.; 1 table

  4. Effects of drinking desalinated seawater on cell viability and proliferation.

    Science.gov (United States)

    Macarrão, Camila Longhi; Bachi, André Luis Lacerda; Mariano, Mario; Abel, Lucia Jamli

    2017-06-01

    Desalination of seawater is becoming an important means to address the increasing scarcity of freshwater resources in the world. Seawater has been used as drinking water in the health, food, and medical fields and various beneficial effects have been suggested, although not confirmed. Given the presence of 63 minerals and trace elements in drinking desalinated seawater (63 DSW), we evaluated their effects on the behavior of tumorigenic and nontumorigenic cells through the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay and annexin-V-fluorescein isothiocyanate/propidium iodide staining. Our results showed that cell viability and proliferation in the presence of 63 DSW were significantly greater than in mineral water and in the presence of fetal bovine serum in a dose-dependent manner. Furthermore, 63 DSW showed no toxic effect on murine embryonic fibroblast (NIH-3T3) and murine melanoma (B16-F10) cells. In another assay, we also showed that pre-treatment of non-adherent THP-1 cells with 63 DSW reduces apoptosis incidence, suggesting a protective effect against cell death. We conclude that cell viability and proliferation were improved by the mineral components of 63 DSW and this effect can guide further studies on health effects associated with DSW consumption.

  5. Apple derived cellulose scaffolds for 3D mammalian cell culture.

    Directory of Open Access Journals (Sweden)

    Daniel J Modulevsky

    Full Text Available There are numerous approaches for producing natural and synthetic 3D scaffolds that support the proliferation of mammalian cells. 3D scaffolds better represent the natural cellular microenvironment and have many potential applications in vitro and in vivo. Here, we demonstrate that 3D cellulose scaffolds produced by decellularizing apple hypanthium tissue can be employed for in vitro 3D culture of NIH3T3 fibroblasts, mouse C2C12 muscle myoblasts and human HeLa epithelial cells. We show that these cells can adhere, invade and proliferate in the cellulose scaffolds. In addition, biochemical functionalization or chemical cross-linking can be employed to control the surface biochemistry and/or mechanical properties of the scaffold. The cells retain high viability even after 12 continuous weeks of culture and can achieve cell densities comparable with other natural and synthetic scaffold materials. Apple derived cellulose scaffolds are easily produced, inexpensive and originate from a renewable source. Taken together, these results demonstrate that naturally derived cellulose scaffolds offer a complementary approach to existing techniques for the in vitro culture of mammalian cells in a 3D environment.

  6. Induction of Live Cell Phagocytosis by a Specific Combination of Inflammatory Stimuli

    Directory of Open Access Journals (Sweden)

    Takamasa Ishidome

    2017-08-01

    Full Text Available Conditions of severe hyper-inflammation can lead to uncontrolled activation of macrophages, and the ensuing phagocytosis of live cells. However, relationships between inflammatory stimuli and uncontrolled phagocytosis of live cells by macrophages are poorly understood. To identify mediators of this process, we established phagocytosis assays of live cells by stimulating macrophages with CpG DNA, interferon-γ, and anti-interleukin-10 receptor antibody. In this model, various cell surface receptors were upregulated on macrophages, and phagocytosis of live cells was induced in a Rac1-dependent manner. Subsequent inhibition of the ICAM-1, VCAM-1, and both of these receptors abolished in vitro and in vivo phagocytosis of live T cells, myeloid cells, and B cells, respectively. Specifically, the reduction in lymphocyte numbers due to in vivo activation of macrophages was ameliorated in Icam-1-deficient mice. In addition, overexpression of ICAM-1 or VCAM-1 in non-phagocytic NIH3T3 cells led to active phagocytosis of live cells. These data indicate molecular mechanisms underlying live cell phagocytosis induced by hyper-inflammation, and this experimental model will be useful to clarify the pathophysiological mechanisms of hemophagocytosis and to indicate therapeutic targets.

  7. Specitic gene alterations in radiation-induced tumorigenesis

    Energy Technology Data Exchange (ETDEWEB)

    Ahn, Joo Mee; Kang, Chang Mo; Lee, Seung Sook; Cho, Chul Koo; Bae, Sang Woo; Lee, Su Jae; Lee, Yun Sil [Korea Institute of Radiological and Medical Sciences, Seoul (Korea, Republic of)

    2004-07-01

    To identify a set of genes involved in the development of radiation-induced tumorigenesis, we used DNA microarrays consisting of 1,176 mouse genes and compared expression profiles of radioresistant cells, designated NIH3T3-R1 and -R4. These cells were tumorigenic in a nude mouse grafting system, as compared to the parental NIH3T3 cells. Expressions of MDM2, CDK6 and CDC25B were found to increase more than 3-fold. Entactin protein levels were downregulated in NIH3T3-R1 and -R4 cells. Changes in expression genes were confirmed by reverse transcription-PCR or western blotting. When these genes were transfected to NIH3T3 cells, the CDC25B and MDM2 overexpressing NIH3T3 cells showed radioresistance, while 2 CDK6 overexpressing cells did not. In the case of entactin overexpressing NIH3T3-R1 or R-4 cells were still radioresistant. Furthermore, the CDC25B and MDM2 overexpressing cells grafted to nude mice, were tumorigenic. NIH3T3-R1 and R4 cells showed increased radiation-induced apoptosis, accompanied by faster growth rate, rather than and earlier radiation-induced G2/M phase arrest, suggesting that the radioresistance of NIH3T3-R1 and R4 cells was due to faster growth rate, rather than induction of apoptosis. In the case of MDM2 and CDC25B overexpressing cells, similar phenomena, such as increased apoptosis and faster growth rate, were shown. The above results, therefore, demonstrate involvement of CDC25B and MDM2 overexpression in radiation-induced tumorigenesis and provide novel targets for detection of radiation-induced carcinogenesis.

  8. Vitamin E analogue, D-alpha tocopherol succinate, enhances x-ray induced growth delay of human adenocarcinoma cancer cell line

    International Nuclear Information System (INIS)

    Jaworska, A.; Ottesen, T.E.

    2003-01-01

    The purpose of this study was to assess the effects of d-alpha Tocopherol succinate (alpha-TS) in modifying radiation-induced viability reduction and apoptosis occurrence in the model for normal and cancer cells. Our hypothesis was that alpha-TS enhances the growth-inhibitory effect of x-irradiation in cancer cells and that the effect is more pronounced in these cells than in normal cells. Murine NIH 3T3 Swiss albino embryonic cells and HT29 human Caucasian colon adenocarcinoma cells were used in the experiments. Alpha-TS was added to the cultures 1 h prior to irradiation with doses of 2 or 5Gy of x-ray. After irradiation cells were incubated for 73 h. Trypan blue exclusion viability test and estimation of apoptosis and necrosis were made. Apoptotic and necrotic cells were counted in fluorescence microscope using fluorescence dyes: propidium iodide and Hoechst 33342. For experiments with the dose of 5 Gy at least five series of experiments were performed. At lower doses (up to approximately 25μM/ml) treatment with alpha-TS alone enhanced growth of both cell lines. At higher doses treatment with alpha-TS alone delayed the growth of the cell cultures, accompanied by 20-25% necrosis. At the concentrations higher than 25μM/mL alpha-TS alone caused growth delay of both cell cultures, being much more pronounced for the cancer cell line HT29. At the concentrations of 50 μM/mL, responsible for about 30-60% of growth delay, there was observed a synergy effect for x-rays and alpha-TS for both cell lines. The effect was more pronounced for HT29 cells (DMF=0.48 for HT29 versus DMF=0.73 for NIH 3T3). These results may confirm the views of the literature reports suggesting that use of vitamin E together with radiation could be favorable for colon cancer treatment; however, more experiments using more advanced techniques are needed

  9. Magneto-responsive liquid crystalline elastomer nanocomposites as potential candidates for dynamic cell culture substrates

    International Nuclear Information System (INIS)

    Herrera-Posada, Stephany; Mora-Navarro, Camilo; Ortiz-Bermudez, Patricia; Torres-Lugo, Madeline; McElhinny, Kyle M.; Evans, Paul G.; Calcagno, Barbara O.; Acevedo, Aldo

    2016-01-01

    Recently, liquid crystalline elastomers (LCEs) have been proposed as active substrates for cell culture due to their potential to attach and orient cells, and impose dynamic mechanical signals through the application of external stimuli. In this report, the preparation of anisotropic and oriented nematic magnetic-sensitized LCEs with iron oxide nanoparticles, and the evaluation of the effect of particle addition at low concentrations on the resultant structural, thermal, thermo-mechanical, and mechanical properties is presented. Phase transformations produced by heating in alternating magnetic fields were investigated in LCEs in contact with air, water, and a common liquid cell culture medium was also evaluated. The inclusion of nanoparticles into the elastomers displaced the nematic-to-isotropic phase transition, without affecting the nematic structure as evidenced by similar values of the order parameter, while reducing the maximum thermomechanical deformations. Remote and reversible deformations of the magnetic LCEs were achieved through the application of alternating magnetic fields, which induces the nematic–isotropic phase transition through nanoparticle heat generation. Formulation parameters can be modified to allow for remote actuation at values closer to the human physiological temperature range and within the range of deformations that can affect the cellular behavior of fibroblasts. Finally, a collagen surface treatment was performed to improve compatibility with NIH-3T3 fibroblast cultures, which enabled the attachment and proliferation of fibroblasts on substrates with and without magnetic particles under quiescent conditions. The LCEs developed in this work, which are able to deform and experience stress changes by remote contact-less magnetic stimulation, may allow for further studies on the effect of substrate morphology changes and dynamic mechanical properties during in vitro cell culture. - Highlights: • Magnetic LCE nanocomposites were

  10. Magneto-responsive liquid crystalline elastomer nanocomposites as potential candidates for dynamic cell culture substrates

    Energy Technology Data Exchange (ETDEWEB)

    Herrera-Posada, Stephany; Mora-Navarro, Camilo; Ortiz-Bermudez, Patricia; Torres-Lugo, Madeline [Department of Chemical Engineering, Call Box 9000, University of Puerto Rico, Mayagüez PR 00681 (Puerto Rico); McElhinny, Kyle M.; Evans, Paul G. [Department of Materials Science and Engineering, 1509 University Avenue, University of Wisconsin-Madison, WI 53706 (United States); Calcagno, Barbara O. [Department of General Engineering, Call Box 9000, University of Puerto Rico, Mayagüez PR 00681 (Puerto Rico); Acevedo, Aldo, E-mail: aldo.acevedo@upr.edu [Department of Chemical Engineering, Call Box 9000, University of Puerto Rico, Mayagüez PR 00681 (Puerto Rico)

    2016-08-01

    Recently, liquid crystalline elastomers (LCEs) have been proposed as active substrates for cell culture due to their potential to attach and orient cells, and impose dynamic mechanical signals through the application of external stimuli. In this report, the preparation of anisotropic and oriented nematic magnetic-sensitized LCEs with iron oxide nanoparticles, and the evaluation of the effect of particle addition at low concentrations on the resultant structural, thermal, thermo-mechanical, and mechanical properties is presented. Phase transformations produced by heating in alternating magnetic fields were investigated in LCEs in contact with air, water, and a common liquid cell culture medium was also evaluated. The inclusion of nanoparticles into the elastomers displaced the nematic-to-isotropic phase transition, without affecting the nematic structure as evidenced by similar values of the order parameter, while reducing the maximum thermomechanical deformations. Remote and reversible deformations of the magnetic LCEs were achieved through the application of alternating magnetic fields, which induces the nematic–isotropic phase transition through nanoparticle heat generation. Formulation parameters can be modified to allow for remote actuation at values closer to the human physiological temperature range and within the range of deformations that can affect the cellular behavior of fibroblasts. Finally, a collagen surface treatment was performed to improve compatibility with NIH-3T3 fibroblast cultures, which enabled the attachment and proliferation of fibroblasts on substrates with and without magnetic particles under quiescent conditions. The LCEs developed in this work, which are able to deform and experience stress changes by remote contact-less magnetic stimulation, may allow for further studies on the effect of substrate morphology changes and dynamic mechanical properties during in vitro cell culture. - Highlights: • Magnetic LCE nanocomposites were

  11. 3D Printing of Thermo-Responsive Methylcellulose Hydrogels for Cell-Sheet Engineering

    Directory of Open Access Journals (Sweden)

    Andrea Cochis

    2018-04-01

    Full Text Available A possible strategy in regenerative medicine is cell-sheet engineering (CSE, i.e., developing smart cell culture surfaces from which to obtain intact cell sheets (CS. The main goal of this study was to develop 3D printing via extrusion-based bioprinting of methylcellulose (MC-based hydrogels. Hydrogels were prepared by mixing MC powder in saline solutions (Na2SO4 and PBS. MC-based hydrogels were analyzed to investigate the rheological behavior and thus optimize the printing process parameters. Cells were tested in vitro on ring-shaped printed hydrogels; bulk MC hydrogels were used for comparison. In vitro tests used murine embryonic fibroblasts (NIH/3T3 and endothelial murine cells (MS1, and the resulting cell sheets were characterized analyzing cell viability and immunofluorescence. In terms of CS preparation, 3D printing proved to be an optimal approach to obtain ring-shaped CS. Cell orientation was observed for the ring-shaped CS and was confirmed by the degree of circularity of their nuclei: cell nuclei in ring-shaped CS were more elongated than those in sheets detached from bulk hydrogels. The 3D printing process appears adequate for the preparation of cell sheets of different shapes for the regeneration of complex tissues.

  12. 3D Printing of Thermo-Responsive Methylcellulose Hydrogels for Cell-Sheet Engineering.

    Science.gov (United States)

    Cochis, Andrea; Bonetti, Lorenzo; Sorrentino, Rita; Contessi Negrini, Nicola; Grassi, Federico; Leigheb, Massimiliano; Rimondini, Lia; Farè, Silvia

    2018-04-10

    A possible strategy in regenerative medicine is cell-sheet engineering (CSE), i.e., developing smart cell culture surfaces from which to obtain intact cell sheets (CS). The main goal of this study was to develop 3D printing via extrusion-based bioprinting of methylcellulose (MC)-based hydrogels. Hydrogels were prepared by mixing MC powder in saline solutions (Na₂SO₄ and PBS). MC-based hydrogels were analyzed to investigate the rheological behavior and thus optimize the printing process parameters. Cells were tested in vitro on ring-shaped printed hydrogels; bulk MC hydrogels were used for comparison. In vitro tests used murine embryonic fibroblasts (NIH/3T3) and endothelial murine cells (MS1), and the resulting cell sheets were characterized analyzing cell viability and immunofluorescence. In terms of CS preparation, 3D printing proved to be an optimal approach to obtain ring-shaped CS. Cell orientation was observed for the ring-shaped CS and was confirmed by the degree of circularity of their nuclei: cell nuclei in ring-shaped CS were more elongated than those in sheets detached from bulk hydrogels. The 3D printing process appears adequate for the preparation of cell sheets of different shapes for the regeneration of complex tissues.

  13. Highly Efficient Transfer of Chromosomes to a Broad Range of Target Cells Using Chinese Hamster Ovary Cells Expressing Murine Leukemia Virus-Derived Envelope Proteins.

    Directory of Open Access Journals (Sweden)

    Teruhiko Suzuki

    Full Text Available Microcell-mediated chromosome transfer (MMCT is an essential step for introducing chromosomes from donor cells to recipient cells. MMCT allows not only for genetic/epigenetic analysis of specific chromosomes, but also for utilization of human and mouse artificial chromosomes (HACs/MACs as gene delivery vectors. Although the scientific demand for genome scale analyses is increasing, the poor transfer efficiency of the current method has hampered the application of chromosome engineering technology. Here, we developed a highly efficient chromosome transfer method, called retro-MMCT, which is based on Chinese hamster ovary cells expressing envelope proteins derived from ecotropic or amphotropic murine leukemia viruses. Using this method, we transferred MACs to NIH3T3 cells with 26.5 times greater efficiency than that obtained using the conventional MMCT method. Retro-MMCT was applicable to a variety of recipient cells, including embryonic stem cells. Moreover, retro-MMCT enabled efficient transfer of MAC to recipient cells derived from humans, monkeys, mice, rats, and rabbits. These results demonstrate the utility of retro-MMCT for the efficient transfer of chromosomes to various types of target cell.

  14. Smoking history, nicotine dependence and opioid use in patients with chronic non-malignant pain

    DEFF Research Database (Denmark)

    Plesner, K; Jensen, H I; Højsted, J

    2016-01-01

    doses than never smokers and former smokers not using nicotine. CONCLUSIONS: The study supports previous evidence that smoking is associated with chronic pain. Our data suggest that information about use of nicotine substitution in chronic non-malignant patients are relevant both in a clinical setting......BACKGROUND: Previous studies have demonstrated a positive association between smoking and addiction to opioids in patients with chronic non-malignant pain. This could be explained by a susceptibility in some patients to develop addiction. Another explanation could be that nicotine influences both...... pain and the opioid system. The objective of the study was to investigate whether smoking, former smoking ± nicotine use and nicotine dependence in patients with chronic non-malignant pain were associated with opioid use and addiction to opioids. METHODS: The study was a cross-sectional study carried...

  15. An update on the role of opioids in the management of chronic pain of nonmalignant origin.

    Science.gov (United States)

    Højsted, Jette; Sjøgren, Per

    2007-10-01

    To summarize and reflect over primarily recent epidemiological and randomized controlled trials in opioid-treated chronic nonmalignant pain patients, focusing on effects, side effects, risks and long-term consequences of the treatment. In the western world opioids are increasingly being used for long-term treatment of chronic nonmalignant pain. While the long-term benefits of opioids regarding pain relief, functional capacity and health-related quality of life still remain to be proven, studies are emerging that describe serious long-term consequences such as addiction, opioid-induced hyperalgesia, cognitive disorders, and suppression of the immune and reproductive systems. Much more research is needed concerning long-time effects and consequences of opioid therapy in chronic nonmalignant pain patients; however, some clear warning signals have been sent out within recent years.

  16. Multifunctional substrates of thin porous alumina for cell biosensors

    KAUST Repository

    Toccafondi, Chiara; Thorat, Sanjay B.; La Rocca, Rosanna; Scarpellini, Alice; Salerno, Marco; Dante, Silvia; Das, Gobind

    2014-01-01

    We have fabricated anodic porous alumina from thin films (100/500 nm) of aluminium deposited on technological substrates of silicon/glass, and investigated the feasibility of this material as a surface for the development of analytical biosensors aiming to assess the status of living cells. To this goal, porous alumina surfaces with fixed pitch and variable pore size were analyzed for various functionalities. Gold coated (about 25 nm) alumina revealed surface enhanced Raman scattering increasing with the decrease in wall thickness, with factor up to values of approximately 104 with respect to the flat gold surface. Bare porous alumina was employed for micro-patterning and observation via fluorescence images of dye molecules, which demonstrated the surface capability for a drug-loading device. NIH-3T3 fibroblast cells were cultured in vitro and examined after 2 days since seeding, and no significant (P > 0.05) differences in their proliferation were observed on porous and non-porous materials. The effect on cell cultures of pore size in the range of 50–130 nm—with pore pitch of about 250 nm—showed no significant differences in cell viability and similar levels in all cases as on a control substrate. Future work will address combination of all above capabilities into a single device.

  17. Multifunctional substrates of thin porous alumina for cell biosensors

    KAUST Repository

    Toccafondi, Chiara

    2014-02-27

    We have fabricated anodic porous alumina from thin films (100/500 nm) of aluminium deposited on technological substrates of silicon/glass, and investigated the feasibility of this material as a surface for the development of analytical biosensors aiming to assess the status of living cells. To this goal, porous alumina surfaces with fixed pitch and variable pore size were analyzed for various functionalities. Gold coated (about 25 nm) alumina revealed surface enhanced Raman scattering increasing with the decrease in wall thickness, with factor up to values of approximately 104 with respect to the flat gold surface. Bare porous alumina was employed for micro-patterning and observation via fluorescence images of dye molecules, which demonstrated the surface capability for a drug-loading device. NIH-3T3 fibroblast cells were cultured in vitro and examined after 2 days since seeding, and no significant (P > 0.05) differences in their proliferation were observed on porous and non-porous materials. The effect on cell cultures of pore size in the range of 50–130 nm—with pore pitch of about 250 nm—showed no significant differences in cell viability and similar levels in all cases as on a control substrate. Future work will address combination of all above capabilities into a single device.

  18. Cell type-specific suppression of mechanosensitive genes by audible sound stimulation.

    Science.gov (United States)

    Kumeta, Masahiro; Takahashi, Daiji; Takeyasu, Kunio; Yoshimura, Shige H

    2018-01-01

    Audible sound is a ubiquitous environmental factor in nature that transmits oscillatory compressional pressure through the substances. To investigate the property of the sound as a mechanical stimulus for cells, an experimental system was set up using 94.0 dB sound which transmits approximately 10 mPa pressure to the cultured cells. Based on research on mechanotransduction and ultrasound effects on cells, gene responses to the audible sound stimulation were analyzed by varying several sound parameters: frequency, wave form, composition, and exposure time. Real-time quantitative PCR analyses revealed a distinct suppressive effect for several mechanosensitive and ultrasound-sensitive genes that were triggered by sounds. The effect was clearly observed in a wave form- and pressure level-specific manner, rather than the frequency, and persisted for several hours. At least two mechanisms are likely to be involved in this sound response: transcriptional control and RNA degradation. ST2 stromal cells and C2C12 myoblasts exhibited a robust response, whereas NIH3T3 cells were partially and NB2a neuroblastoma cells were completely insensitive, suggesting a cell type-specific response to sound. These findings reveal a cell-level systematic response to audible sound and uncover novel relationships between life and sound.

  19. Znhit1 causes cell cycle arrest and down-regulates CDK6 expression

    International Nuclear Information System (INIS)

    Yang, Zhengmin; Cao, Yonghao; Zhu, Xiaoyan; Huang, Ying; Ding, Yuqiang; Liu, Xiaolong

    2009-01-01

    Cyclin-dependent kinase 6 (CDK6) is the key element of the D-type cyclin holoenzymes which has been found to function in the regulation of G1-phase of the cell cycle and is presumed to play important roles in T cell function. In this study, Znhit1, a member of a new zinc finger protein family defined by a conserved Zf-HIT domain, induced arrest in the G1-phase of the cell cycle in NIH/3T3 cells. Of the G1 cell cycle factors examined, the expression of CDK6 was found to be strongly down-regulated by Znhit1 via transcriptional repression. This effect may have correlations with the decreased acetylation level of histone H4 in the CDK6 promoter region. In addition, considering that CDK6 expression predominates in T cells, the negative regulatory role of Znhit1 in TCR-induced T cell proliferation was validated using transgenic mice. These findings identified Znhit1 as a CDK6 regulator that plays an important role in cell proliferation.

  20. The Impact of MicroRNA-223-3p on IL-17 Receptor D Expression in Synovial Cells.

    Directory of Open Access Journals (Sweden)

    Nozomu Moriya

    Full Text Available Rheumatoid arthritis (RA is an autoimmune inflammatory disease affecting joints. Elevated plasma levels of microRNA-223-3p (miR-223-3p in patients with RA are implicated in the pathogenesis of the disease. This study aimed to analyze the functional role of miR-223-3p in the pathogenesis of RA by overexpressing miR-223-3p in synovial cell lines.Arthritis was induced in the RA model of SKG mice by injection of ß-glucan. The histopathologic features of joints were examined using hematoxylin and eosin and immunohistochemical staining. Plasma levels of miRNA were determined by panel real-time PCR analysis. Target genes of the differentially expressed miRNAs in SKG mice were analyzed using miRNA target prediction algorithms. The dual-luciferase reporter system was used to evaluate the relationship between miR-223-3p and IL-17 receptor D (IL-17RD. The activity of miR-223-3p was analyzed by transfection of plasmid vectors overexpressing miR-223-3p into IL-17RD-expressing NIH3T3 and MH7A cell lines. Il6 and Il17rd mRNA expression was analyzed by quantitative real-time PCR. IL-17RD protein expression was analyzed by western blot analysis.We identified 17 upregulated miRNAs (fold change > 2.0 in plasma of SKG mice injected with ß-glucan relative to untreated SKG mice. Il17rd was identified as the candidate target gene of miR-223-3p using five miRNA target prediction algorithms. The transfection of plasmid vectors overexpressing miR-223-3p into NIH3T3 and MH7A cells resulted in the downregulation of Il17rd expression and upregulation of Il6 expression. Expression of miR-223-3p and Il6 mRNA in MH7A cells was upregulated; however, that of Il17rd mRNA was downregulated following TNF-α stimulation. IL-17RD expression in synovial tissues from SKG mice and RA patients was inversely correlated with the severity of arthritis.This study is the first to demonstrate that miR-223-3p downregulates IL-17RD in both mouse and human cells; miR-223-3p may contribute to

  1. Economic burden of non-malignant blood disorders across Europe: a population-based cost study.

    Science.gov (United States)

    Luengo-Fernandez, Ramon; Burns, Richeal; Leal, Jose

    2016-08-01

    Blood disorders comprise a wide range of diseases including anaemia, malignant blood disorders, and haemorrhagic disorders. Although they are a common cause of disease, no systematic cost-of-illness studies have been done to assess the economic effect of non-malignant blood disorders in Europe. We aimed to assess the economic burden of non-malignant blood disorders across the 28 countries of the European Union (EU), Iceland, Norway, and Switzerland. Non-malignant blood disorder-related costs (WHO International Classification of Diseases, 10th revision [ICD] D50-89) were estimated for 28 EU countries, Iceland, Norway, and Switzerland for 2012. Country-specific costs were estimated with aggregate data on morbidity, mortality, and health-care resource use obtained from international and national sources. Health-care costs were estimated from expenditure on primary care, outpatient care, emergency care, hospital inpatient care, and drugs. Costs of informal care and productivity losses due to morbidity and early death were also included. To these costs we added those due to malignant blood disorders (ICD-10 C81-96 and D47) as estimated in a Burns and colleagues' companion Article to obtain the total costs of blood disorders. Non-malignant disorders of the blood cost the 31 European countries €11 billion in 2012. Health-care costs accounted for €8 billion (75% of total costs), productivity losses for €2 billion (19%), and informal care for less than €1 billion (6%). Averaged across the European population studied, non-malignant disorders of the blood represented an annual health-care cost of €159 per ten citizens. Combining malignant and non-malignant blood disorders, the total cost of blood disorders was €23 billion in 2012. Our study highlights the economic burden that non-malignant blood disorders place on European health-care systems and societies. Our study also shows that blood disorder costs were evenly distributed between malignant and non-malignant

  2. Immunohistochemical and radioimmunological demonstration of alpha/sub 1/-fetoprotein in nonmalignant changes of human gastric mucosa

    Energy Technology Data Exchange (ETDEWEB)

    Falser, N; Lederer, B; Reissigl, H [Innsbruck Univ. (Austria). Pathologisch-Anatomisches Lab.; Innsbruck Univ. (Austria). Gastroenterologisches Lab.)

    1977-07-01

    The occurence of ..cap alpha../sub 1/ fetoprotein in nonmalignant changes of the gastric mucosa was investigated by means of immunohistochemistry and radioimmunonoassay. The investigations were performed in tissue sections, cytological imprint preparations as well as in homogenized tissue samples (obtained by gastroscopy). ..cap alpha../sub 1/ fetoprotein could be demonstrated by immuno-histochemistry in about 90% of the samples originating from the surroundings of gastric ulcer and the region of gastrojejunostomy after B II-resection. The RIA was positive in about 75% of the tissue samples, whereas from gastric juice only 40% of positive results could be obtained. No ..cap alpha../sub 1/ fetoprotein-activity could be demonstrated in serum samples. These investigations indicate that ..cap alpha../sub 1/ fetoprotein is not exclusively synthesized by embryonic or neoplastic tissues and also can be synthesized also by regenerating cell-systems. It may be supposed that this synthesis represents an unspecific answer to growth-stimulation.

  3. Depletion of gamma-glutamylcyclotransferase in cancer cells induces autophagy followed by cellular senescence.

    Science.gov (United States)

    Taniguchi, Keiko; Matsumura, Kengo; Ii, Hiromi; Kageyama, Susumu; Ashihara, Eishi; Chano, Tokuhiro; Kawauchi, Akihiro; Yoshiki, Tatsuhiro; Nakata, Susumu

    2018-01-01

    Gamma-glutamylcyclotransferase (GGCT) was originally identified as a protein highly expressed in bladder cancer tissues by proteomic analysis, and its higher expression in a variety of cancers compared to normal tissues have been shown. Depletion of GGCT in various cancer cells results in antiproliferative effects both in vitro and in vivo ; thus it is considered a promising therapeutic target. Although it has been shown that knockdown of GGCT induces cellular senescence and non-apoptotic cell death, associated with upregulation of cyclin-dependent kinase inhibitors (CDKIs) including p21 WAF1/CIP1 , the cellular events that follow GGCT depletion are not fully understood. Here, we show that GGCT depletion induced autophagy in MCF7 breast and PC3 prostate cancer cells. Conversely, overexpression of GGCT in NIH3T3 fibroblast under conditions of serum deprivation inhibited autophagy and increased proliferation. Simultaneous knockdown of autophagy related-protein 5, a critical effector of autophagy, along with GGCT in MCF7 and PC3 cells led to significant attenuation of the multiple cellular responses, including upregulation of CDKIs, increased numbers of senescence-associated β-galactosidase positive senescent cells, and growth inhibition. Furthermore, we show that autophagy-promoting signaling cascades including activation of the AMPK-ULK1 pathway and/or inactivation of the mTORC2-Akt pathway were triggered in GGCT-depleted cells. These results indicate that autophagy plays an important role in the growth inhibition of cancer cells caused by GGCT depletion.

  4. The non-small cell lung cancer EGFR extracellular domain mutation, M277E, is oncogenic and drug-sensitive

    Directory of Open Access Journals (Sweden)

    Yu S

    2017-09-01

    Full Text Available Su Yu,1,2 Yang Zhang,1 Yunjian Pan,1 Chao Cheng,1,3 Yihua Sun,1,3 Haiquan Chen1–4 1Department of Thoracic Surgery, Fudan University Shanghai Cancer Center, Shanghai, China; 2Cancer Research Center, Fudan University Shanghai Cancer Center, Shanghai, China; 3Department of Oncology, Shanghai Medical College, Fudan University, Shanghai, China; 4Institutes of Biomedical Sciences, Fudan University, Shanghai, China Purpose: To identify novel oncogenic mutations in non-small cell lung cancer patient specimens that lack mutations in known targetable genes (“pan-negative” patients.Methods: Comprehensive mutational analyses were performed on 1,356 lung adenocarcinoma specimens. In this cohort of patients, common lung cancer oncogenic driver mutations were detected in the epidermal growth factor receptor (EGFR kinase domain, the human epidermal growth factor receptor 2 kinase domain, as well as the KRAS, BRAF, ALK, ROS1 and RET genes. A sub-cohort of pan-negative patient specimens was assayed for mutations in the EGFR extracellular domain (ECD. Additionally, EGFR mutant NIH-3T3 stable cell lines were constructed and assessed for protein content, anchorage-independent growth, and tumor formation in xenograft models to identify oncogenic mutations. BaF3 lymphocytes were also used to test sensitivities of the mutations to tyrosine kinase inhibitors.Results: In pan-negative lung adenocarcinoma cases, a novel oncogenic EGFR ECD mutation was identified (M277E. EGFR M277E mutations encoded oncoproteins that transformed NIH-3T3 cells to grow in the absence of exogenous epidermal growth factor. Transformation was further evidenced by anchorage-independent growth and tumor formation in immunocompromised xenograft mouse models. Finally, as seen in the canonical EGFR L858R mutation, the M277E mutation conferred sensitivity to both erlotinib and cetuximab in BaF3 cell lines and to erlotinib in xenograft models.Conclusion: Here, a new EGFR driver mutation, M277E

  5. Primary Care Management of Chronic Nonmalignant Pain in Veterans: A Qualitative Study

    Science.gov (United States)

    Ruiz, Jorge G.; Qadri, S. Sobiya; Nader, Samir; Wang, Jia; Lawler, Timothy; Hagenlocker, Brian; Roos, Bernard A.

    2010-01-01

    Clinicians managing older patients with chronic pain play an important role. This paper explores the attitudes of primary care clinicians (PCPs) toward chronic nonmalignant pain management and their experiences using a clinical decision support system. Our investigation followed a qualitative approach based on grounded theory. Twenty-one PCPs…

  6. Cell type-specific roles of Jak3 in IL-2-induced proliferative signal transduction

    International Nuclear Information System (INIS)

    Fujii, Hodaka

    2007-01-01

    Binding of interleukin-2 (IL-2) to its specific receptor induces activation of two members of Jak family protein tyrosine kinases, Jak1 and Jak3. An IL-2 receptor (IL-2R)-reconstituted NIH 3T3 fibroblast cell line proliferates in response to IL-2 only when hematopoietic lineage-specific Jak3 is ectopically expressed. However, the mechanism of Jak3-dependent proliferation in the fibroblast cell line is not known. Here, I showed that Jak3 expression is dispensable for IL-2-induced activation of Jak1 and Stat proteins and expression of nuclear proto-oncogenes in the IL-2R-reconstituted fibroblast cell line. Jak3 expression markedly enhanced these IL-2-induced signaling events. In contrast, Jak3 expression was essential for induction of cyclin genes involved in the G1-S transition. These data suggest a critical role of Jak3 in IL-2 signaling in the fibroblast cell line and may provide further insight into the cell type-specific mechanism of cytokine signaling

  7. Cell type-specific roles of Jak3 in IL-2-induced proliferative signal transduction

    Science.gov (United States)

    Fujii, Hodaka

    2007-01-01

    Binding of IL-2 to its specific receptor induces activation of two members of Jak family protein tyrosine kinases, Jak1 and Jak3. An IL-2R-reconstituted NIH 3T3 fibroblast cell line proliferates in response to IL-2 only when hematopoietic lineage-specific Jak3 is ectopically expressed. However, the mechanism of Jak3-dependent proliferation in the fibroblast cell line is not known. Here, I showed that Jak3 expression is dispensable for IL-2-induced activation of Jak1 and Stat proteins and expression of nuclear proto-oncogenes in the IL-2R-reconstituted fibroblast cell line. However, Jak3 expression markedly enhanced these IL-2-induced signaling events. In contrast, Jak3 expression was essential for induction of cyclin genes involved in the G1-S transition. These data suggest a critical role of Jak3 in IL-2 signaling in the fibroblast cell line and may provide further insight into the cell type-specific mechanism of cytokine signaling. PMID:17266928

  8. Akt1 intramitochondrial cycling is a crucial step in the redox modulation of cell cycle progression.

    Directory of Open Access Journals (Sweden)

    Valeria Gabriela Antico Arciuch

    2009-10-01

    Full Text Available Akt is a serine/threonine kinase involved in cell proliferation, apoptosis, and glucose metabolism. Akt is differentially activated by growth factors and oxidative stress by sequential phosphorylation of Ser(473 by mTORC2 and Thr(308 by PDK1. On these bases, we investigated the mechanistic connection of H(2O(2 yield, mitochondrial activation of Akt1 and cell cycle progression in NIH/3T3 cell line with confocal microscopy, in vivo imaging, and directed mutagenesis. We demonstrate that modulation by H(2O(2 entails the entrance of cytosolic P-Akt1 Ser(473 to mitochondria, where it is further phosphorylated at Thr(308 by constitutive PDK1. Phosphorylation of Thr(308 in mitochondria determines Akt1 passage to nuclei and triggers genomic post-translational mechanisms for cell proliferation. At high H(2O(2, Akt1-PDK1 association is disrupted and P-Akt1 Ser(473 accumulates in mitochondria in detriment to nuclear translocation; accordingly, Akt1 T308A is retained in mitochondria. Low Akt1 activity increases cytochrome c release to cytosol leading to apoptosis. As assessed by mass spectra, differential H(2O(2 effects on Akt1-PDK interaction depend on the selective oxidation of Cys(310 to sulfenic or cysteic acids. These results indicate that Akt1 intramitochondrial-cycling is central for redox modulation of cell fate.

  9. Metal-Carbon Interactions on Reduced Graphene Oxide under Facile Thermal Treatment: Microbiological and Cell Assay

    Directory of Open Access Journals (Sweden)

    N. L. V. Carreño

    2017-01-01

    Full Text Available Silver-functionalized reduced graphene oxide (Ag-rGO nanosheets were prepared by single chemical and thermal processes, with very low concentration of silver. The resulting carbon framework consists of reduced graphene oxide (rGO sheets or 3D networks, decorated with anchored silver nanoparticles. The Ag-rGO nanosheets were dispersed into a polymer matrix and the composites evaluated for use as biological scaffolds. The rGO material in poly(dimethylsiloxane (PDMS has been tested for antimicrobial activity against Gram-positive Staphylococcus aureus (S. Aureus bacteria, after exposure times of 24 and 120 hours, as well as in the determination of cell viability on cultures of fibroblast cells (NIH/3T3. Using 1 mL of Ag-rGO in PDMS the antibacterial effectiveness against Staphylococcus aureus was limited, showing an increased amount of Colony Forming Units (CFU, after 24 hours of contact. In the cell viability assay, after 48 hours of contact, the group of 1 mL of Ag-rGO with PDMS was the only group that increased cell viability when compared to the control group. In this context, it is believed these behaviors are due to the increase in cell adhesion capacity promoted by the rGO. Thus, the Ag-rGO/PDMS hybrid nanocomposite films can be used as scaffolds for tissue engineering, as they limit antimicrobial activity.

  10. Evaluation of cell behavior on modified polypropylene with swift heavy ion irradiation

    Energy Technology Data Exchange (ETDEWEB)

    Arbeitman, Claudia R., E-mail: arbeitman@tandar.cnea.gov.ar [Gerencia de Investigacion y Aplicaciones, TANDAR-CNEA, Av. Gral. Paz 1499, 1650 San Martin, Bs. As. (Argentina); CONICET, Av. Rivadavia 1917, C1033AAJ, CABA (Argentina); Ibanez, Irene L. [CONICET, Av. Rivadavia 1917, C1033AAJ, CABA (Argentina); Garcia Bermudez, Gerardo [Gerencia de Investigacion y Aplicaciones, TANDAR-CNEA, Av. Gral. Paz 1499, 1650 San Martin, Bs. As. (Argentina); CONICET, Av. Rivadavia 1917, C1033AAJ, CABA (Argentina); Duran, Hebe [CONICET, Av. Rivadavia 1917, C1033AAJ, CABA (Argentina); Gerencia de Desarrollo Tecnologico y Proyectos Especiales, TANDAR-CNEA (Argentina); Grosso, Mariela F. del [Gerencia de Investigacion y Aplicaciones, TANDAR-CNEA, Av. Gral. Paz 1499, 1650 San Martin, Bs. As. (Argentina); CONICET, Av. Rivadavia 1917, C1033AAJ, CABA (Argentina); Salguero, Noelia [Gerencia de Investigacion y Aplicaciones, TANDAR-CNEA, Av. Gral. Paz 1499, 1650 San Martin, Bs. As. (Argentina); Mazzei, Ruben [U.A. Tecnologicas y Agropecuarias, CNEA (Argentina)

    2012-02-15

    Ion beam irradiation is a well known means to change the physico-chemical properties of polymers, and induced bio and citocompatibility in controlled conditions and in selected areas of surface. However, the enhancement of cell adhesion on a modified substrate does not mean that the surface is adequate for functional cells. The purpose of the present work is to study proliferation, changes in cytoskeleton and cell morphology on substrates as a function of irradiation parameters. We irradiated polypropylene with sulfur (S) ion-beam at energies of 110 MeV with fluences between 1 Multiplication-Sign 10{sup 6} and 2 Multiplication-Sign 10{sup 10} ions cm{sup -2}. NIH 3T3 cells were cultured on each sample. Cell morphology was observed using phase contrast microscopy and cytoskeleton proteins with fluorescence microscopy. The analysis show different cellular responses as a functions of irradiation parameter, strongly suggests that different underlying substratum can result in distinct types of cytoskeleton reorganization.

  11. Is there an optimal topographical surface in nano-scale affecting protein adsorption and cell behaviors? Part II

    Energy Technology Data Exchange (ETDEWEB)

    Wang Huajie, E-mail: wanghuajie972001@163.com; Sun Yuanyuan; Cao Ying, E-mail: caoying1130@sina.com; Wang Kui; Yang Lin [Henan Normal University, College of Chemistry and Environmental Science (China); Zhang Yidong; Zheng Zhi [Xuchang University, Institute of Surface Micro and Nano Materials (China)

    2012-05-15

    Although nano-structured surfaces exhibit superior biological activities to the smooth or micro-structured surfaces, whether there is an optimal topographical surface in nano-scale affecting protein adsorption and cell behaviors is still controversial. In this study, porous aluminum oxide membranes with different pore sizes ranging from 25 to 120 nm were prepared by the anodic oxidation technique. The surface morphology, topography and wettability were analyzed by scanning electron microscope, atomic force microscope and water contact angle measurement, respectively. The results indicated that the synergistic action of the nano-topography structure and hydrophilic/hydrophobic properties resulted in a highest protein adsorption on the aluminum oxide membrane with 80 nm pore size. Additionally, the morphological, metabolic and cell counting methods showed that cells had different sensitivity to porous aluminum oxide membranes with different surface features. Furthermore, this sensitivity was cell type dependent. The optimal pore size of aluminum oxide membranes for cell growth was 80 nm for PC12 cells and 50 nm for NIH 3T3 cells.

  12. Long-term opioid treatment of chronic nonmalignant pain: unproven efficacy and neglected safety?

    Directory of Open Access Journals (Sweden)

    Kissin I

    2013-07-01

    Full Text Available Igor Kissin Department of Anesthesiology, Perioperative, and Pain Medicine, Brigham and Women's Hospital, Harvard Medical School, Boston, MA, USA Background: For the past 30 years, opioids have been used to treat chronic nonmalignant pain. This study tests the following hypotheses: (1 there is no strong evidence-based foundation for the conclusion that long-term opioid treatment of chronic nonmalignant pain is effective; and (2 the main problem associated with the safety of such treatment – assessment of the risk of addiction – has been neglected. Methods: Scientometric analysis of the articles representing clinical research in this area was performed to assess (1 the quality of presented evidence (type of study; and (2 the duration of the treatment phase. The sufficiency of representation of addiction was assessed by counting the number of articles that represent (1 editorials; (2 articles in the top specialty journals; and (3 articles with titles clearly indicating that the addiction-related safety is involved (topic-in-title articles. Results: Not a single randomized controlled trial with opioid treatment lasting >3 months was found. All studies with a duration of opioid treatment ≥6 months (n = 16 were conducted without a proper control group. Such studies cannot provide the consistent good-quality evidence necessary for a strong clinical recommendation. There were profound differences in the number of addiction articles related specifically to chronic nonmalignant pain patients and to opioid addiction in general. An inadequate number of chronic pain-related publications were observed with all three types of counted articles: editorials, articles in the top specialty journals, and topic-in-title articles. Conclusion: There is no strong evidence-based foundation for the conclusion that long-term opioid treatment of chronic nonmalignant pain is effective. The above identified signs indicating neglect of addiction associated with the

  13. Improvement of cell infiltration in electrospun polycaprolactone scaffolds for the construction of vascular grafts.

    Science.gov (United States)

    Wang, Kai; Zhu, Meifeng; Li, Ting; Zheng, Wenting; Li, Li; Xu, Mian; Zhao, Qiang; Kong, Deling; Wang, Lianyong

    2014-08-01

    The less-than-ideal cell infiltration resulting from inherently small pore size limits the application of electrospinning scaffold in tissue engineering and regeneration medicine. The present study aims to develop a porogenic method which can significantly increase pore size in electrospinning scaffold and enhance cell migration. With this method, composite scaffolds consisting of poly(epsilon-caprolactone) (PCL) fibers and poly(ethylene oxide) (PEO) microparticles were prepared by simultaneously electrospinning and electrospraying. Removal of the PEO microparticles from the composites generated large pores. In vitro culture of NIH3T3 cells and in vivo subcutaneous implantation both demonstrated that the porogenic scaffolds markedly facilitated cell infiltration. With the same technique, vascular grafts with alternative dense and loose layers were prepared by turning on or off electrospraying PEO. SEM showed that there was no a clear delamination between the loose and dense layers. The mechanical strength and burst pressure of these vascular grafts could meet the requirements of vascular implantation. In conclusion, electrospinning PCL fibers with electrospraying PEO microparticles may be an effective and controllable method to increase pore size in electrospinning scaffold and provides a useful tool for the fabrication of vascular grafts that meets the need of blood vessel replacement.

  14. Porous Silicon Nanoparticle Photosensitizers for Singlet Oxygen and Their Phototoxicity Against Cancer Cells

    Science.gov (United States)

    Xiao, Ling; Gu, Luo; Howell, Stephen B.; Sailor, Michael J.

    2011-01-01

    Porous Si nanoparticles, prepared from electrochemically etched single crystal Si wafers, function as photosensitizers to generate 1O2 in ethanol and in aqueous media. The preparation conditions for the porous Si nanoparticles were optimized to maximize (1) the yield of material; (2) its quantum yield of 1O2 production; and (3) its in vitro degradation properties. The optimal formulation was determined to consist of nanoparticles 146 ± 7 nm in diameter, with nominal pore sizes of 12 ± 4 nm. The quantum yield for 1O2 production is 0.10 ± 0.02 in ethanol and 0.17 ± 0.01 in H2O. HeLa or NIH-3T3 cells treated with 100 µg/mL porous Si nanoparticles and exposed to 60 J/cm2 white light (infrared filtered, 100 mW/cm2 for 10 min) exhibit ~ 45% cell death, while controls containing no nanoparticles show 10% or 25% cell death, respectively. The dark control experiment yields < 10% cytotoxicity for either cell type. PMID:21452822

  15. Tumorigenesis of K-ras mutation in human endometrial carcinoma via upregulation of estrogen receptor.

    Science.gov (United States)

    Tu, Zheng; Gui, Liming; Wang, Jianliu; Li, Xiaoping; Sun, Pengming; Wei, Lihui

    2006-05-01

    To investigate the tumorigenesis of mutant [12Asp]-K-ras in endometrial carcinoma and its relationship with ER. We constructed pcDI-[12Asp]K-ras4B by inserting full-length [12Asp]K-ras4B from human endometrial carcinoma Hec-1A cells, into pcDI vector. Cell proliferation of NIH3T3 after transfection with pcDI-[12Asp]K-ras4B was measured by MTT assay. The cell transformation was determined by colony formation and tumor nodule development. [12Asp]-K-ras4B-NIH3T3 cells were transfected with constitutively active pCMV-RafCAAX and dominant-negative pCMV-RafS621A. Cell growth was measured by MTT assay and [3H]thymidine incorporation. After transfected with pcDI-[12Asp]K-ras4B or pCMV-RafS621A, the cells were harvested for Western blot and reporter assay to determine the expression and transcriptional activity of ERalpha and ERbeta, respectively. [12Asp]-K-ras4B enhanced NIH3T3 cells proliferation after 48 h post-transfection (P ras4B-NIH3T3 cells (13.48%) than pcDI-NIH3T3 (4.26%) or untreated NIH3T3 (2.33%). The pcDI-[12Asp]-K-ras4B-NIH3T3 cells injected to the nude mice Balb/C developed tumor nodules with poor-differentiated cells after 12 days. An increase of ERalpha and ERbeta was observed in pcDI-[12Asp]-K-ras4B-NIH3T3 cells. RafS621A downregulated ERalpha and ERbeta expression. Estrogen induced the ER transcriptional activity by 5-fold in pcDI-NIH3T3 cells, 13-fold in pcDI-[12Asp]K-ras4B-NIH3T3 and 19-fold in HEC-1A. RafS621A suppressed the ER transcriptional activity. K-ras mutation induces tumorigenesis in endometrium, and this malignant transformation involves Raf signaling pathway and ER.

  16. Positive autoregulation of the transcription factor Pax6 in response to increased levels of either of its major isoforms, Pax6 or Pax6(5a, in cultured cells

    Directory of Open Access Journals (Sweden)

    Mason John O

    2006-05-01

    Full Text Available Abstract Background Pax6 is a transcription factor essential for normal development of the eyes and nervous system. It has two major isoforms, Pax6 and Pax6(5a, and the ratios between their expression levels vary within narrow limits. We tested the effects of overexpressing either one or other isoform on endogenous Pax6 expression levels in Neuro2A and NIH3T3 cells. Results We found that both isoforms caused an up-regulation of endogenous Pax6 expression in cells with (Neuro2A or without (NIH3T3 constitutive Pax6 expression. Western blots showed that cells stably transfected with constructs expressing either Pax6 or Pax6(5a contained raised levels of both Pax6 and Pax6(5a. Quantitative RT-PCR confirmed an increase in levels of Pax6(5a mRNA in cells containing Pax6-expressing constructs and an increase in levels of Pax6 mRNA in cells containing Pax6(5a-expressing constructs. The fact that the introduction of constructs expressing only one isoform increased the cellular levels of not only that isoform but also the other indicates that activation of the endogenous Pax6 locus occurred. The ratio between the levels of the two isoforms was maintained close to physiological values. The overexpression of either isoform in neuroblastoma (Neuro2A cell lines also promoted morphological change and an increase in β-III-tubulin expression, indicating an increase in neurogenesis. Conclusion Our results demonstrate that Pax6 can up-regulate production of Pax6 protein from an entire intact endogenous Pax6 locus in its genomic environment. This adds to previous studies showing that Pax6 can up-regulate reporter expression driven by isolated Pax6 regulatory elements. Furthermore, our results suggest that an important function of positive feedback might be to stabilise the relative levels of Pax6 and Pax6(5a.

  17. A minimal murine Msx-1 gene promoter. Organization of its cis-regulatory motifs and their role in transcriptional activation in cells in culture and in transgenic mice.

    Science.gov (United States)

    Takahashi, T; Guron, C; Shetty, S; Matsui, H; Raghow, R

    1997-09-05

    To dissect the cis-regulatory elements of the murine Msx-1 promoter, which lacks a conventional TATA element, a putative Msx-1 promoter DNA fragment (from -1282 to +106 base pairs (bp)) or its congeners containing site-specific alterations were fused to luciferase reporter and introduced into NIH3T3 and C2C12 cells, and the expression of luciferase was assessed in transient expression assays. The functional consequences of the sequential 5' deletions of the promotor revealed that multiple positive and negative regulatory elements participate in regulating transcription of the Msx-1 gene. Surprisingly, however, the optimal expression of Msx-1 promoter in either NIH3T3 or C2C12 cells required only 165 bp of the upstream sequence to warrant detailed examination of its structure. Therefore, the functional consequences of site-specific deletions and point mutations of the cis-acting elements of the minimal Msx-1 promoter were systematically examined. Concomitantly, potential transcriptional factor(s) interacting with the cis-acting elements of the minimal promoter were also studied by gel electrophoretic mobility shift assays and DNase I footprinting. Combined analyses of the minimal promoter by DNase I footprinting, electrophoretic mobility shift assays, and super shift assays with specific antibodies revealed that 5'-flanking regions from -161 to -154 and from -26 to -13 of the Msx-1 promoter contains an authentic E box (proximal E box), capable of binding a protein immunologically related to the upstream stimulating factor 1 (USF-1) and a GC-rich sequence motif which can bind to Sp1 (proximal Sp1), respectively. Additionally, we observed that the promoter activation was seriously hampered if the proximal E box was removed or mutated, and the promoter activity was eliminated completely if the proximal Sp1 site was similarly altered. Absolute dependence of the Msx-1 minimal promoter on Sp1 could be demonstrated by transient expression assays in the Sp1-deficient

  18. Fragrance chemicals lyral and lilial decrease viability of HaCat cells' by increasing free radical production and lowering intracellular ATP level: protection by antioxidants.

    Science.gov (United States)

    Usta, Julnar; Hachem, Yassmine; El-Rifai, Omar; Bou-Moughlabey, Yolla; Echtay, Karim; Griffiths, David; Nakkash-Chmaisse, Hania; Makki, Rajaa Fakhoury

    2013-02-01

    We investigate in this study the biochemical effects on cells in culture of two commonly used fragrance chemicals: lyral and lilial. Whereas both chemicals exerted a significant effect on primary keratinocyte(s), HaCat cells, no effect was obtained with any of HepG2, Hek293, Caco2, NIH3T3, and MCF7 cells. Lyral and lilial: (a) decreased the viability of HaCat cells with a 50% cell death at 100 and 60 nM respectively; (b) decreased significantly in a dose dependant manner the intracellular ATP level following 12-h of treatment; (c) inhibited complexes I and II of electron transport chain in liver sub-mitochondrial particles; and (d) increased reactive oxygen species generation that was reversed by N-acetyl cysteine and trolox and the natural antioxidant lipoic acid, without influencing the level of free and/or oxidized glutathione. Lipoic acid protected HaCat cells against the decrease in viability induced by either compound. Dehydrogenation of lyral and lilial produce α,β-unsaturated aldehydes, that reacts with lipoic acid requiring proteins resulting in their inhibition. We propose lyral and lilial as toxic to mitochondria that have a direct effect on electron transport chain, increase ROS production, derange mitochondrial membrane potential, and decrease cellular ATP level, leading thus to cell death. Copyright © 2012 Elsevier Ltd. All rights reserved.

  19. Cell Adhesion on Surface-Functionalized Magnesium.

    Science.gov (United States)

    Wagener, Victoria; Schilling, Achim; Mainka, Astrid; Hennig, Diana; Gerum, Richard; Kelch, Marie-Luise; Keim, Simon; Fabry, Ben; Virtanen, Sannakaisa

    2016-05-18

    The biocompatibility of commercially pure magnesium-based (cp Mg) biodegradable implants is compromised of strong hydrogen evolution and surface alkalization due to high initial corrosion rates of cp Mg in the physiological environment. To mitigate this problem, the addition of corrosion-retarding alloying elements or coating of implant surfaces has been suggested. In the following work, we explored the effect of organic coatings on long-term cell growth. cp Mg was coated with aminopropyltriehtoxysilane + vitamin C (AV), carbonyldiimidazole (CDI), or stearic acid (SA). All three coatings have been previously suggested to reduce initial corrosion and to enhance protein adsorption and hence cell adhesion on magnesium surfaces. Endothelial cells (DH1+/+) and osteosarcoma cells (MG63) were cultured on coated samples for up to 20 days. To quantify Mg corrosion, electrochemical impedance spectroscopy (EIS) was measured after 1, 3, and 5 days of cell culture. We also investigated the speed of initial cell spreading after seeding using fluorescently labeled fibroblasts (NIH/3T3). Hydrogen evolution after contact with cell culture medium was markedly decreased on AV- and SA-coated Mg compared to uncoated Mg. These coatings also showed improved cell adhesion and spreading after 24 h of culture comparable to tissue-treated plastic surfaces. On AV-coated cp Mg, a confluent layer of endothelial cells formed after 5 days and remained intact for up to 20 days. Together, these data demonstrate that surface coating with AV is a viable strategy for improving long-term biocompatibility of cp Mg-based implants. EIS measurements confirmed that the presence of a confluent cell layer increased the corrosion resistance.

  20. Lithium treatment elongates primary cilia in the mouse brain and in cultured cells

    Energy Technology Data Exchange (ETDEWEB)

    Miyoshi, Ko, E-mail: miyoshi@cc.okayama-u.ac.jp [Department of Brain Science, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University, 2-5-1 Shikatacho, Okayama 700-8558 (Japan); Kasahara, Kyosuke; Miyazaki, Ikuko; Asanuma, Masato [Department of Brain Science, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University, 2-5-1 Shikatacho, Okayama 700-8558 (Japan)

    2009-10-30

    The molecular mechanisms underlying the therapeutic effects of lithium, a first-line antimanic mood stabilizer, have not yet been fully elucidated. Treatment of the algae Chlamydomonas reinhardtii with lithium has been shown to induce elongation of their flagella, which are analogous structures to vertebrate cilia. In the mouse brain, adenylyl cyclase 3 (AC3) and certain neuropeptide receptors colocalize to the primary cilium of neuronal cells, suggesting a chemosensory function for the primary cilium in the nervous system. Here we show that lithium treatment elongates primary cilia in the mouse brain and in cultured cells. Brain sections from mice chronically fed with Li{sub 2}CO{sub 3} were subjected to immunofluorescence study. Primary cilia carrying both AC3 and the receptor for melanin-concentrating hormone (MCH) were elongated in the dorsal striatum and nucleus accumbens of lithium-fed mice, as compared to those of control animals. Moreover, lithium-treated NIH3T3 cells and cultured striatal neurons exhibited elongation of the primary cilia. The present results provide initial evidence that a psychotropic agent can affect ciliary length in the central nervous system, and furthermore suggest that lithium exerts its therapeutic effects via the upregulation of cilia-mediated MCH sensing. These findings thus contribute novel insights into the pathophysiology of bipolar mood disorder and other psychiatric diseases.

  1. Lithium treatment elongates primary cilia in the mouse brain and in cultured cells

    International Nuclear Information System (INIS)

    Miyoshi, Ko; Kasahara, Kyosuke; Miyazaki, Ikuko; Asanuma, Masato

    2009-01-01

    The molecular mechanisms underlying the therapeutic effects of lithium, a first-line antimanic mood stabilizer, have not yet been fully elucidated. Treatment of the algae Chlamydomonas reinhardtii with lithium has been shown to induce elongation of their flagella, which are analogous structures to vertebrate cilia. In the mouse brain, adenylyl cyclase 3 (AC3) and certain neuropeptide receptors colocalize to the primary cilium of neuronal cells, suggesting a chemosensory function for the primary cilium in the nervous system. Here we show that lithium treatment elongates primary cilia in the mouse brain and in cultured cells. Brain sections from mice chronically fed with Li 2 CO 3 were subjected to immunofluorescence study. Primary cilia carrying both AC3 and the receptor for melanin-concentrating hormone (MCH) were elongated in the dorsal striatum and nucleus accumbens of lithium-fed mice, as compared to those of control animals. Moreover, lithium-treated NIH3T3 cells and cultured striatal neurons exhibited elongation of the primary cilia. The present results provide initial evidence that a psychotropic agent can affect ciliary length in the central nervous system, and furthermore suggest that lithium exerts its therapeutic effects via the upregulation of cilia-mediated MCH sensing. These findings thus contribute novel insights into the pathophysiology of bipolar mood disorder and other psychiatric diseases.

  2. [Comparison of fibroblastic cell compatibility of type I collagen-immobilized titanium between electrodeposition and immersion].

    Science.gov (United States)

    Kyuragi, Takeru

    2014-03-01

    Titanium is widely used for medical implants. While many techniques for surface modification have been studied for optimizing its biocompatibility with hard tissues, little work has been undertaken to explore ways of maximizing its biocompatibility with soft tissues. We investigated cell attachment to titanium surfaces modified with bovine Type I collagen immobilized by either electrodeposition or a conventional immersion technique. The apparent thickness and durability of the immobilized collagen layer were evaluated prior to incubation of the collagen-immobilized titanium surfaces with NIH/3T3 mouse embryonic fibroblasts. The initial cell attachment and expression of actin and vinculin were evaluated. We determined that the immobilized collagen layer was much thicker and more durable when placed using the electrodeposition technique than the immersion technique. Both protocols produced materials that promoted better cell attachment, growth and structural protein expression than titanium alone. However, electrodeposition was ultimately superior to immersion because it is quicker to perform and produces a more durable collagen coating. We conclude that electrodeposition is an effective technique for immobilizing type I collagen on titanium surfaces, thus improving their cytocompatibility with fibroblasts.

  3. Model-based investigation of the circadian clock and cell cycle coupling in mouse embryonic fibroblasts: Prediction of RevErb-α up-regulation during mitosis.

    Science.gov (United States)

    Traynard, Pauline; Feillet, Céline; Soliman, Sylvain; Delaunay, Franck; Fages, François

    2016-11-01

    Experimental observations have put in evidence autonomous self-sustained circadian oscillators in most mammalian cells, and proved the existence of molecular links between the circadian clock and the cell cycle. Some mathematical models have also been built to assess conditions of control of the cell cycle by the circadian clock. However, recent studies in individual NIH3T3 fibroblasts have shown an unexpected acceleration of the circadian clock together with the cell cycle when the culture medium is enriched with growth factors, and the absence of such acceleration in confluent cells. In order to explain these observations, we study a possible entrainment of the circadian clock by the cell cycle through a regulation of clock genes around the mitosis phase. We develop a computational model and a formal specification of the observed behavior to investigate the conditions of entrainment in period and phase. We show that either the selective activation of RevErb-α or the selective inhibition of Bmal1 transcription during the mitosis phase, allow us to fit the experimental data on both period and phase, while a uniform inhibition of transcription during mitosis seems incompatible with the phase data. We conclude on the arguments favoring the RevErb-α up-regulation hypothesis and on some further predictions of the model. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

  4. A bioaccumulative cyclometalated platinum(II) complex with two-photon-induced emission for live cell imaging.

    Science.gov (United States)

    Koo, Chi-Kin; Wong, Ka-Leung; Man, Cornelia Wing-Yin; Lam, Yun-Wah; So, Leo King-Yan; Tam, Hoi-Lam; Tsao, Sai-Wah; Cheah, Kok-Wai; Lau, Kai-Chung; Yang, Yang-Yi; Chen, Jin-Can; Lam, Michael Hon-Wah

    2009-02-02

    The cyclometalated platinum(II) complex [Pt(L)Cl], where HL is a new cyclometalating ligand 2-phenyl-6-(1H-pyrazol-3-yl)pyridine containing C(phenyl), N(pyridyl), and N(pyrazolyl) donor moieties, was found to possess two-photon-induced luminescent properties. The two-photon-absorption cross section of the complex in N,N-dimethylformamide at room temperature was measured to be 20.8 GM. Upon two-photon excitation at 730 nm from a Ti:sapphire laser, bright-green emission was observed. Besides its two-photon-induced luminescent properties, [Pt(L)Cl] was able to be rapidly accumulated in live HeLa and NIH3T3 cells. The two-photon-induced luminescence of the complex was retained after live cell internalization and can be observed by two-photon confocal microscopy. Its bioaccumulation properties enabled time-lapse imaging of the internalization process of the dye into living cells. Cytotoxicity of [Pt(L)Cl] to both tested cell lines was low, according to MTT assays, even at loadings as high as 20 times the dose concentration for imaging for 6 h.

  5. Improved cellular response of ion modified poly(lactic acid-co-glycolic acid) substrates for mouse fibroblast cells

    Energy Technology Data Exchange (ETDEWEB)

    Adhikari, Ananta Raj, E-mail: aa8381@gmail.com [Department of Sciences, Wentworth Institute of Technology, Boston MA 02115 (United States); Geranpayeh, Tanya [Department of Biology and Biochemistry, University of Houston, Houston, TX 77204 (United States); Chu, Wei Kan [Texas Center for Superconductivity, University of Houston, Houston, TX 77204 (United States); Department of Physics, University of Houston, Houston, TX 77204 (United States); Otteson, Deborah C. [Department of Biology and Biochemistry, University of Houston, Houston, TX 77204 (United States); Department of Basic and Vision Sciences, College of Optometry, University of Houston, Houston, TX 77204 (United States)

    2016-03-01

    In this report, the effects of argon (Ar) ion irradiation on poly(lactic acid-co-glycolic acid) (PLGA) substrates on biocompatibility were studied. PLGA scaffold substrates were prepared by spin coating glass surfaces with PLGA dissolved in anhydrous chloroform. Previously, we showed that surface modifications of PLGA films using ion irradiation modulate the inherent hydrophobicity of PLGA surface. Here we show that with increasing ion dose (1 × 10{sup 12} to 1 × 10{sup 14} ions/cm{sup 2}), hydrophobicity and surface roughness decreased. Biocompatibility for NIH3T3 mouse fibroblast cells was increased by argon irradiation of PLGA substrates. On unirradiated PLGA films, fibroblasts had a longer doubling time and cell densities were 52% lower than controls after 48 h in vitro. Argon irradiated PLGA substrates supported growth rates similar to control. Despite differences in cell cycle kinetics, there was no detectible cytotoxicity observed on any substrate. This demonstrates that argon ion irradiation can be used to tune the surface microstructure and generate substrates that are more compatible for the cell growth and proliferation. - Highlights: • Argon irradiation modifies surface chemistry and increases hydrophilicity of poly(lactic-glycolic) acid (PLGA) films. • Both native and irradiated PLGA films were not cytotoxic for mouse fibroblasts. • Fibroblast proliferation increased on PLGA substrates modified with higher doses of Argon irradiation. • Surface modification with Argon irradiation increases biocompatibility of PLGA films.

  6. Improved cellular response of ion modified poly(lactic acid-co-glycolic acid) substrates for mouse fibroblast cells

    International Nuclear Information System (INIS)

    Adhikari, Ananta Raj; Geranpayeh, Tanya; Chu, Wei Kan; Otteson, Deborah C.

    2016-01-01

    In this report, the effects of argon (Ar) ion irradiation on poly(lactic acid-co-glycolic acid) (PLGA) substrates on biocompatibility were studied. PLGA scaffold substrates were prepared by spin coating glass surfaces with PLGA dissolved in anhydrous chloroform. Previously, we showed that surface modifications of PLGA films using ion irradiation modulate the inherent hydrophobicity of PLGA surface. Here we show that with increasing ion dose (1 × 10 12 to 1 × 10 14 ions/cm 2 ), hydrophobicity and surface roughness decreased. Biocompatibility for NIH3T3 mouse fibroblast cells was increased by argon irradiation of PLGA substrates. On unirradiated PLGA films, fibroblasts had a longer doubling time and cell densities were 52% lower than controls after 48 h in vitro. Argon irradiated PLGA substrates supported growth rates similar to control. Despite differences in cell cycle kinetics, there was no detectible cytotoxicity observed on any substrate. This demonstrates that argon ion irradiation can be used to tune the surface microstructure and generate substrates that are more compatible for the cell growth and proliferation. - Highlights: • Argon irradiation modifies surface chemistry and increases hydrophilicity of poly(lactic-glycolic) acid (PLGA) films. • Both native and irradiated PLGA films were not cytotoxic for mouse fibroblasts. • Fibroblast proliferation increased on PLGA substrates modified with higher doses of Argon irradiation. • Surface modification with Argon irradiation increases biocompatibility of PLGA films.

  7. Health care costs, work productivity and activity impairment in non-malignant chronic pain patients

    DEFF Research Database (Denmark)

    Kronborg, Christian; Handberg, Gitte; Axelsen, Flemming

    2009-01-01

    This study explores the costs of non-malignant chronic pain in patients awaiting treatment in a multidisciplinary pain clinic in a hospital setting. Health care costs due to chronic pain are particular high during the first year after pain onset, and remain high compared with health care costs be...... before pain onset. The majority of chronic pain patients incur the costs of alternative treatments. Chronic pain causes production losses at work, as well as impairment of non-work activities.......This study explores the costs of non-malignant chronic pain in patients awaiting treatment in a multidisciplinary pain clinic in a hospital setting. Health care costs due to chronic pain are particular high during the first year after pain onset, and remain high compared with health care costs...

  8. Air Pollution and Nonmalignant Respiratory Mortality in 16 Cohorts within the ESCAPE Project

    DEFF Research Database (Denmark)

    Dimakopoulou, Konstantina; Samoli, Evangelia; Beelen, Rob

    2014-01-01

    Rationale: Prospective cohort studies have shown that chronic exposure to particulate matter and traffic related air pollution is associated with reduced survival. However, the effects on non-malignant respiratory mortality are less studied and those reported are less consistent. Objectives: We...... have investigated the relationship of long-term exposure to air pollution and non-malignant respiratory mortality in 16 cohorts with individual level data within the multi center European Study of Cohorts for Air Pollution Effects (ESCAPE). Methods: Data from 16 ongoing cohort studies from Europe were...... used. The total number of subjects was 307,553. There were 1,559 respiratory deaths during follow-up. Measurements: Air pollution exposure was estimated by land use regression models at the baseline residential addresses of study participants and traffic-proximity variables were derived from...

  9. Outcomes of Follow-Up Visits to Chronic Nonmalignant Pain Patients

    DEFF Research Database (Denmark)

    Sørensen, Jan

    2010-01-01

    Follow-up visits by clinical nurse specialists are beneficial for patients with various chronic conditions. It is unknown whether patients with chronic nonmalignant pain can achieve similar benefit. The aim of this study was to assess outcomes of follow-up visits by clinical nurse specialists...... to chronic nonmalignant pain patients regarding health-related quality of life (HRQoL), pain, opioid treatment, quality of sleep, and depression. A total of 102 patients were enrolled in a prospective randomized controlled trial during a 2-year period after discharge from multidisciplinary pain treatment...... and randomized to intervention or control group. Intervention group patients (n = 52) received home visits every fourth month for 2 years. The findings showed that HRQoL improved generally more in the intervention group. Statistically significant improvements were observed for physical function and bodily pain...

  10. Distinctive transforming genes in x-ray-transformed mammalian cells

    International Nuclear Information System (INIS)

    Borek, C.; Ong, A.; Mason, H.

    1987-01-01

    DNAs from hamster embryo cells and mouse C3H/10T1/2 cells transformed in vitro by x-irradiation into malignant cells transmit the radiation transformation phenotype by producing transformed colonies (transfectants) in two mouse recipient lines, the NIH 3T3 and C3H/101/2 cells, and in a rat cell line, the Rat-2 cells. DNAs from unirradiated cells or irradiated and visibly untransformed cells do not produce transformed colonies. The transfectant grow in agar and form tumors in nude mice. Treatment of the DNAs with restriction endonucleases prior to transfection indicates that the same transforming gene (oncogene) is present in each of the transformed mouse cells and is the same in each of the transformed hamster cells. Southern blot analysis of 3T3 or Rat-2 transfectants carrying oncogenes from radiation-transformed C3H/10T1/2 or hamster cells indicates that the oncogenes responsible for the transformation of 3T3 cells are not the Ki-ras, Ha-ras, N-ras genes, nor are they neu, trk, raf, abl, or fms. The work demonstrates that DNAs from mammalian cells transformed into malignancy by direct exposure in vitro to radiation contain genetic sequences with detectable transforming activity in three recipient cell lines. The results provide evidence that DNA is the target of radiation carcinogenesis induced at a cellular level in vitro. The experiments indicate that malignant radiogenic transformation in vitro of hamster embryo and mouse C3H/10T1/2 cells involves the activation of unique non-ras transforming genes, which heretofore have not been described

  11. C-reactive protein and serum amyloid A levels in discriminating malignant from non-malignant pleural effusion

    Directory of Open Access Journals (Sweden)

    Hala Mohamed Shalaby Samaha

    2015-10-01

    Conclusion: Measurement of SAA and CRP levels in pleural fluid has good diagnostic utility in differentiation between malignant and non-malignant pleural effusion and pleural SAA has a better diagnostic performance than CRP.

  12. Identification and characterization of an alternative splice variant of Mpl with a high affinity for TPO and its activation of ERK1/2 signaling.

    Science.gov (United States)

    Wang, Qiong; Sun, Rui; Wu, Leyan; Huang, Junfeng; Wang, Ping; Yuan, Hailong; Qiu, Feifei; Xu, Xiaohong; Wu, Di; Yu, Ying; Liu, Xin; Zhang, Qing

    2013-12-01

    The thrombopoietin receptor is a crucial element in thrombopoietin-initiated signaling pathways, which stimulates the differentiation of normal hematopoietic progenitor cells, the maturation of megakaryocytes, and the generation of platelets. In this study, we identified a novel activating variant of thrombopoietin receptor, termed Mpl-D, in human megakaryoblastic leukemia Dami cells and demonstrated that the binding affinity of the Mpl-D receptor for thrombopoietin is enhanced. Cell cycle analysis revealed that in the presence of thrombopoietin, most Mpl-D expressing NIH3T3 (NIH3T3/Mpl-D) cells were prevalent in G1 phase while the S and G2/M populations were less frequently observed. Unexpectedly, thrombopoietin induced strong and prolonged ERK1/2 signaling in NIH3T3/Mpl-D cells compared with its receptor wild-type expressing NIH3T3 (NIH3T3/Mpl-F) cells. Further analysis of the mRNA levels of cyclin D1/D2 in NIH3T3/Mpl-D cells demonstrated markedly down-regulated expression compared to NIH3T3/Mpl-F cells in the presence of thrombopoietin. Thus, the prolonged activation of ERK1/2 by Mpl-D might lead to G1 cell cycle arrest through a profound reduction of cyclin D1/D2 in order to support cell survival without proliferation. We also provided tertiary structural basis for the Mpl-D and thrombopoietin interaction, which might provide insights into how Mpl-D effectively increases binding to thrombopoietin and significantly contributes to its specific signaling pathway. These results suggest a new paradigm for the regulation of cytokine receptor expression and function through the alternative splicing variant of Mpl in Dami cells, which may play a role in the pathogenesis of megakaryoblastic leukemia. Copyright © 2013 Elsevier Ltd. All rights reserved.

  13. Structural requirements for cub domain containing protein 1 (CDCP1 and Src dependent cell transformation.

    Directory of Open Access Journals (Sweden)

    Gwendlyn Kollmorgen

    Full Text Available Cub domain containing protein 1 (CDCP1 is strongly expressed in tumors derived from lung, colon, ovary, or kidney. It is a membrane protein that is phosphorylated and then bound by Src family kinases. Although expression and phosphorylation of CDCP1 have been investigated in many tumor cell lines, the CDCP1 features responsible for transformation have not been fully evaluated. This is in part due to the lack of an experimental system in which cellular transformation depends on expression of exogenous CDCP1 and Src. Here we use retrovirus mediated co-overexpression of c-Src and CDCP1 to induce focus formation of NIH3T3 cells. Employing different mutants of CDCP1 we show that for a full transformation capacity, the intact amino- and carboxy-termini of CDCP1 are essential. Mutation of any of the core intracellular tyrosine residues (Y734, Y743, or Y762 abolished transformation, and mutation of a palmitoylation motif (C689,690G strongly reduced it. Src kinase binding to CDCP1 was not required since Src with a defective SH2 domain generated even more CDCP1 dependent foci whereas Src myristoylation was necessary. Taken together, the focus formation assay allowed us to define structural requirements of CDCP1/Src dependent transformation and to characterize the interaction of CDCP1 and Src.

  14. wnt3a but not wnt11 supports self-renewal of embryonic stem cells

    International Nuclear Information System (INIS)

    Singla, Dinender K.; Schneider, David J.; LeWinter, Martin M.; Sobel, Burton E.

    2006-01-01

    wnt proteins (wnts) promote both differentiation of midbrain dopaminergic cells and self-renewal of haematopoietic stem cells. Mouse embryonic stem (ES) cells can be maintained and self-renew on mouse feeder cell layers or in media containing leukemia inhibitory factor (LIF). However, the effects of wnts on ES cells self-renewal and differentiation are not clearly understood. In the present study, we found that conditioned medium prepared from L cells expressing wnt3a can replace feeder cell layers and medium containing LIF in maintaining ES cells in the proliferation without differentiation (self-renewal) state. By contrast, conditioned medium from NIH3T3 cells expressing wnt11 did not. Alkaline phosphatase staining and compact colony formation were used as criteria of cells being in the undifferentiated state. ES cells maintained in medium conditioned by Wnt3a expressing cells underwent freezing and thawing while maintaining properties seen with LIF maintained ES cells. Purified wnt3a did not maintain self-renewal of ES cells for prolonged intervals. Thus, other factors in the medium conditioned by wnt3a expressing cells may have contributed to maintenance of ES cells in a self-renewal state. Pluripotency of ES cells was determined with the use of embryoid bodies in vitro. PD98059, a MEK specific inhibitor, promoted the growth of undifferentiated ES cells maintained in conditioned medium from wnt3a expressing cells. By contrast, the P38 MAPK inhibitor SB230580 did not, suggesting a role for the MEK pathway in self-renewal and differentiation of ES cells maintained in the wnt3a cell conditioned medium. Thus, our results show that conditioned medium from wnt3a but not wnt11 expressing cells can maintain ES cells in self-renewal and in a pluripotent state

  15. Decoration of a Poly(methyl vinyl ether-co-maleic anhydride)-Shelled Selol Nanocapsule with Folic Acid Increases Its Activity Against Different Cancer Cell Lines In Vitro.

    Science.gov (United States)

    Ganassin, Rayane; Souza, Ludmilla Regina de; Py-Daniel, Karen Rapp; Longo, João Paulo Figueiró; Coelho, Janaína Moreira; Rodrigues, Mosar Correa; Jiang, Cheng-Shi; Gu, Jinsong; Morais, Paulo César de; Mosiniewicz-Szablewska, Ewa; Suchocki, Piotr; Báo, Sônia Nair; Azevedo, Ricardo Bentes; Muehlmann, Luis Alexandre

    2018-01-01

    Due to the low therapeutic index of different chemotherapeutic drugs used for cancer treatment, the development of new anticancer drugs remains an intense field of research. A recently developed mixture of selenitetriacylglycerides, selol, was shown to be active against different cancer cells in vitro. As this compound is highly hydrophobic, it was encapsulated, in a previous study, into poly(methyl vinyl ether-co-maleic anhydride)-shelled nanocapsules in order to improve its dispersibility in aqueous media. Following this line of research, the present report aimed at enhancing the In Vitro activity of the selol nanocapsules against cancerous cells by decorating their surface with folic acid. It is known that several cancer cells overexpress folate receptors. Stable folic acid-decorated selol nanocapsules (SNP-FA) were obtained, which showed to be spherical, with a hydro-dynamic diameter of 364 nm, and zeta potential of -24 mV. In comparison to non-decorated selol nanocapsules, SNP-FA presented higher activity against 4T1, MCF-7 and HeLa cells. Moreover, the decoration of the nanocapsules did not alter their toxicity towards fibroblasts, NIH-3T3 cells. These results show that the decoration with folic acid increased the toxicity of selol nanocapsules to cancer cells. These nanocapsules, besides enabling to disperse selol in an aqueous medium, increased the toxicity of this drug In Vitro, and may be useful to treat cancer in vivo, potentially increasing the specificity of selol towards cancer cells.

  16. Transgene expression, but not gene delivery, is improved by adhesion-assisted lipofection of hematopoietic cells.

    Science.gov (United States)

    Keller, H; Yunxu, C; Marit, G; Pla, M; Reiffers, J; Thèze, J; Froussard, P

    1999-05-01

    In contrast to adherent cells, cells growing in suspension and particularly hematopoietic cells, are notoriously difficult to transfect in vitro using nonviral approaches. In the present study, the effect of cell adhesion on gene transfer efficacy was investigated by allowing hematopoietic cells to bind to an adherent cell monolayer (ACM) before being subjected to cationic liposome-mediated DNA transfer. Human CD34 and T CD4 cell lines were cultivated on an ACM constituted of murine fibroblast NIH3T3 cells and transfected with a plasmid carrying the beta-galactosidase gene. X-gal staining showed that up to 27% of the cells expressed the transgene. In contrast, less than 0.1% of these cells were positively transfected in suspension. This adhesion-assisted lipofection (AAL) procedure was also successfully tested on blood lymphocytes, since it resulted in up to 30% of transfected human primary T lymphocytes. Flow cytometry analysis performed on T lymphocyte subsets revealed that 8 and 9%, respectively, of CD4 and CD8 cells could be transfected with a plasmid carrying the green fluorescent protein gene. Other adherent cells, such as MS5 murine stromal cells or HeLa epithelial cells, were also a compatible matrix for AAL. Moreover, the pCMV beta plasmid was present in similar amounts in the nuclei of TF1 cells transfected in suspension or with the AAL procedure. These data raise the possibility that cell matrix/hematopoietic cell interactions might govern expression of the transgene in hematopoietic cells growing usually in suspension, but not endocytosis of liposome/DNA particles and plasmid migration ot the cell nucleus.

  17. Radiation therapy for nonmalignant diseases in Germany. Current concepts and future perspectives

    International Nuclear Information System (INIS)

    Heinrich Seegenschmiedt, M.; Micke, O.; Willich, N.

    2004-01-01

    Background: radiotherapy (RT) of nonmalignant diseases has a long-standing tradition in Germany. Over the past decade significant theoretical and clinical progress has been made in this field to be internationally recognized as an important segment of clinical RT. This development is reflected in a national patterns-of-care study (PCS) conducted during the years 2001-2002. Material and methods: in 2001 and 2002, a questionnaire was mailed to all RT facilities in Germany to assess equipment, patient accrual, RT indications, and treatment concepts. 146 of 180 institutions (81%) returned all requested data: 23 university hospitals (UNI), 95 community hospitals (COM), and 28 private institutions (PRIV). The specific diseases treated at each institution and the RT concepts were analyzed for frequencies and ratios between the different institution types. All data were compared to the first PCS in 1994-1996. Results: in 137 institutions (94%) 415 megavoltage units (mean 1.7; range 1-4), and in 78 institutions (53%) 112 orthovoltage units (mean 1.1; range 0-2) were available. A mean of 37,410 patients were treated per year in all institutions: 503 (1.3%) for inflammatory disorders, 23,752 (63.5%) for degenerative, 1,252 (3.3%) for hypertrophic, and 11,051 (29.5%) for functional, other and unspecified disorders. In comparison to the first PCS there was a significant increase of patients per year (from 20,082 to 37,410; +86.3%) in most nonmalignant diseases during the past 7-8 years. Most disorders were treated in accordance with the national consensus guidelines: the prescribed dose concepts (single and total doses) varied much less during the period 2001-2002 in comparison with the previous PCS in 1994-1996. Only five institutions (3.4%) received recommendations to change single or total doses and/or treatment delivery. Univariate analysis detected significant institutional differences in the use of RT for various disorders. Conclusion: RT is increasingly accepted in

  18. A new fluorescent pyrene–pyridine dithiocarbamate probe: A chemodosimeter to detect Hg2+ in pure aqueous medium and in live cells

    International Nuclear Information System (INIS)

    Singh, Vikram; Srivastava, Priyanka; PrakashVerma, Shiv; Misra, Arvind; Das, Parimal; Singh, Nanhai

    2014-01-01

    A new pyrene–pyridine dithiocarbamate based fluorescent chemodosimeter, potassium (pyren-1-ylmethyl)(pyridin-2-ylmethyl)dithiocarbamate (L1) has been designed and synthesized. The chemodosimeter shows high selectivity and sensitivity (5.2 ppb) for Hg 2+ in pure aqueous medium in which emission intensity was quenched by ≈80% due to the formation of new cyclized species, 1. The probe behaves as a chemodosimeter for Hg 2+ ions and forms Hg 2+ triggered cyclised imidazoline species with approximate detection time of 50 s and exhibits both colorimetric and fluorometric changes on detection of Hg 2+ ion. Color of the probe (L1) changed from green to colorless visible to the naked eye and from green to dark blue upon the addition of Hg 2+ ions under UV light. The Hg 2+ triggered cyclization reaction was confirmed by spectral data analysis and a single crystal structure determination of the cyclised entity 2 obtained from the model compound potassium benzyl(pyridin-2-ylmethyl) dithiocarbamate (L2). L1 finds its application for detection of Hg 2+ ions on paper strips, and in BSA (bovine serum albumin) medium. L1 is also applicable for the monitoring of Hg 2+ ion in NIH3T3 live cells. - Highlights: • Efficient chemodosimeter to detect Hg 2+ ions in pure aqueous medium. • Hg 2+ triggered cyclisation and formation of imidazoline species. • Probe exhibit both colorimetric and fluorometric changes • Probe is applicable to detect Hg 2+ in live cells and on cellulose paper strips

  19. Regulation of cell proliferation and apoptosis in neuroblastoma cells by ccp1, a FGF2 downstream gene

    Directory of Open Access Journals (Sweden)

    Inman Gareth J

    2010-11-01

    Full Text Available Abstract Background Coiled-coil domain containing 115 (Ccdc115 or coiled coil protein-1 (ccp1 was previously identified as a downstream gene of Fibroblast Growth Factor 2 (FGF2 highly expressed in embryonic and adult brain. However, its function has not been characterised to date. Here we hypothesized that ccp1 may be a downstream effecter of FGF2, promoting cell proliferation and protecting from apoptosis. Methods Forced ccp1 expression in mouse embryonic fibroblast (MEF and neuroblastoma SK-N-SH cell line, as well as down-regulation of ccp1 expression by siRNA in NIH3T3, was used to characterize the role of ccp1. Results Ccp1 over-expression increased cell proliferation, whereas down-regulation of ccp1 expression reduced it. Ccp1 was able to increase cell proliferation in the absence of serum. Furthermore, ccp1 reduced apoptosis upon withdrawal of serum in SK-N-SH. The mitogen-activated protein kinase (MAPK or ERK Kinase (MEK inhibitor, U0126, only partially inhibited the ccp1-dependent BrdU incorporation, indicating that other signaling pathway may be involved in ccp1-induced cell proliferation. Induction of Sprouty (SPRY upon FGF2 treatment was accelerated in ccp1 over-expressing cells. Conclusions All together, the results showed that ccp1 regulates cell number by promoting proliferation and suppressing cell death. FGF2 was shown to enhance the effects of ccp1, however, it is likely that other mitogenic factors present in the serum can also enhance the effects. Whether these effects are mediated by FGF2 influencing the ccp1 function or by increasing the ccp1 expression level is still unclear. At least some of the proliferative regulation by ccp1 is mediated by MAPK, however other signaling pathways are likely to be involved.

  20. Plasma polymer-coated contact lenses for the culture and transfer of corneal epithelial cells in the treatment of limbal stem cell deficiency.

    Science.gov (United States)

    Brown, Karl David; Low, Suet; Mariappan, Indumathi; Abberton, Keren Maree; Short, Robert; Zhang, Hong; Maddileti, Savitri; Sangwan, Virender; Steele, David; Daniell, Mark

    2014-02-01

    Extensive damage to the limbal region of the cornea leads to a severe form of corneal blindness termed as limbal stem cell deficiency (LSCD). Whereas most cases of corneal opacity can be treated with full thickness corneal transplants, LSCD requires stem cell transplantation for successful ocular surface reconstruction. Current treatments for LSCD using limbal stem cell transplantation involve the use of murine NIH 3T3 cells and human amniotic membranes as culture substrates, which pose the threat of transmission of animal-derived pathogens and donor tissue-derived cryptic infections. In this study, we aimed to produce surface modified therapeutic contact lenses for the culture and delivery of corneal epithelial cells for the treatment of LSCD. This approach avoids the possibility of suture-related complications and is completely synthetic. We used plasma polymerization to deposit acid functional groups onto the lenses at various concentrations. Each surface was tested for its suitability to promote corneal epithelial cell adhesion, proliferation, retention of stem cells, and differentiation and found that acid-based chemistries promoted better cell adhesion and proliferation. We also found that the lenses coated with a higher percentage of acid functional groups resulted in a higher number of cells transferred onto the corneal wound bed in rabbit models of LSCD. Immunohistochemistry of the recipient cornea confirmed the presence of autologous, transplanted 5-bromo-2'-deoxyuridine (BrdU)-labeled cells. Hematoxylin staining has also revealed the presence of a stratified epithelium at 26 days post-transplantation. This study provides the first evidence for in vivo transfer and survival of cells transplanted from a contact lens to the wounded corneal surface. It also proposes the possibility of using plasma polymer-coated contact lenses with high acid functional groups as substrates for the culture and transfer of limbal cells in the treatment of LSCD.

  1. Raf oncogene is associated with a radiation-resistant human laryngeal cancer

    International Nuclear Information System (INIS)

    Kasid, U.; Pfeifer, A.; Weichselbaum, R.R.; Dritschilo, A.; Mark, G.E.

    1987-01-01

    In order to identify the genetic factors associated with the radiation-resistant human laryngeal carcinoma cell line (SQ-20B), tumor cell DNA was transfected into NIH/3T3 cells. A high incidence (six out of six) of raf sequences was found in transfected NIH/3T3 clones and the tumorigenic potential of SQ-20B DNA could be linked to genomic fragments that represent most of the kinase domain of human c-raf-1. An apparently unaltered 3.5-kilobase pair (kb) human c-raf transcript was identified in SQ-20B cells but was not observed in the transfected NIH/3T3 cell clones. Two new transcripts (4.2 kb and 2.6 kb) were found in tumorigenic clones; the large transcript was missing in a very poorly tumorigenic clone. Cytogenetic analysis indicated that the normal autosomes of chromosome 3 were absent in SQ-20B karyotypes and had formed apparently stable marker chromosomes. Unlike the recipient NIH/3T3 cell line, 30% of the transformed clone-1 metaphases had minute and double-minute chromosomes representative of amplified DNA sequences. The frequency of the c-raf-1 identification by NIH/3T3 transfection of SQ-20B DNA suggests the presence of some genetic abnormality within this locus

  2. Ca2+ signals, cell membrane disintegration, and activation of TMEM16F during necroptosis.

    Science.gov (United States)

    Ousingsawat, Jiraporn; Cabrita, Inês; Wanitchakool, Podchanart; Sirianant, Lalida; Krautwald, Stefan; Linkermann, Andreas; Schreiber, Rainer; Kunzelmann, Karl

    2017-01-01

    Activated receptor-interacting protein kinase 3 (RIPK3) and mixed lineage kinase domain like (MLKL) are essential components of the necroptotic pathway. Phosphorylated MLKL (pMLKL) is thought to induce membrane leakage, leading to cell swelling and disintegration of the cell membrane. However, the molecular identity of the necroptotic membrane pore remains unclear, and the role of pMLKL for membrane permeabilization is currently disputed. We observed earlier that the phospholipid scramblase and ion channel TMEM16F/anoctamin 6 cause large membrane currents, cell swelling, and cell death when activated by a strong increase in intracellular Ca 2+ . We, therefore, asked whether TMEM16F is also central to necroptotic cell death and other cellular events during necroptosis. Necroptosis was induced by TNFα, smac mimetic, and Z-VAD (TSZ) in NIH3T3 fibroblasts and the four additional cell lines HT 29 , 16HBE, H441, and L929. Time-dependent changes in intracellular Ca 2+ , cell morphology, and membrane currents were recorded. TSZ induced a small and only transient oscillatory rise in intracellular Ca 2+ , which was paralleled by the activation of outwardly rectifying Cl - currents, which were typical for TMEM16F/ANO6. Ca 2+ oscillations were due to Ca 2+ release from endoplasmic reticulum, and were independent of extracellular Ca 2+ . The initial TSZ-induced cell swelling was followed by cell shrinkage. Using typical channel blockers and siRNA-knockdown, the Cl - currents were shown to be due to the activation of ANO6. However, the knockdown of ANO6 or inhibitors of ANO6 did not inhibit necroptotic cell death. The present data demonstrate the activation of ANO6 during necroptosis, which, however, is not essential for cell death.

  3. Photoprotective efficiency of PLGA-curcumin nanoparticles versus curcumin through the involvement of ERK/AKT pathway under ambient UV-R exposure in HaCaT cell line.

    Science.gov (United States)

    Chopra, Deepti; Ray, Lipika; Dwivedi, Ashish; Tiwari, Shashi Kant; Singh, Jyoti; Singh, Krishna P; Kushwaha, Hari Narayan; Jahan, Sadaf; Pandey, Ankita; Gupta, Shailendra K; Chaturvedi, Rajnish Kumar; Pant, Aditya Bhushan; Ray, Ratan Singh; Gupta, Kailash Chand

    2016-04-01

    Curcumin (Cur) has been demonstrated to have wide pharmacological window including anti-oxidant and anti-inflammatory properties. However, phototoxicity under sunlight exposure and poor biological availability limits its applicability. We have synthesized biodegradable and non-toxic polymer-poly (lactic-co-glycolic) acid (PLGA) encapsulated formulation of curcumin (PLGA-Cur-NPs) of 150 nm size range. Photochemically free curcumin generates ROS, lipid peroxidation and induces significant UVA and UVB mediated impaired mitochondrial functions leading to apoptosis/necrosis and cell injury in two different origin cell lines viz., mouse fibroblasts-NIH-3T3 and human keratinocytes-HaCaT as compared to PLGA-Cur-NPs. Molecular docking studies suggested that intact curcumin from nanoparticles, bind with BAX in BIM SAHB site and attenuate it to undergo apoptosis while upregulating anti-apoptotic genes like BCL2. Real time studies and western blot analysis with specific phosphorylation inhibitor of ERK1 and AKT1/2/3 confirm the involvement of ERK/AKT signaling molecules to trigger the survival cascade in case of PLGA-Cur-NPs. Our finding demonstrates that low level sustained release of curcumin from PLGA-Cur-NPs could be a promising way to protect the adverse biological interactions of photo-degradation products of curcumin upon the exposure of UVA and UVB. Hence, the applicability of PLGA-Cur-NPs could be suggested as prolonged radical scavenging ingredient in curcumin containing products. Copyright © 2016 Elsevier Ltd. All rights reserved.

  4. Non-malignant disease mortality in meat workers: a model for studying the role of zoonotic transmissible agents in non-malignant chronic diseases in humans.

    Science.gov (United States)

    Johnson, E S; Zhou, Y; Sall, M; Faramawi, M El; Shah, N; Christopher, A; Lewis, N

    2007-12-01

    Current research efforts have mainly concentrated on evaluating the role of substances present in animal food in the aetiology of chronic diseases in humans, with relatively little attention given to evaluating the role of transmissible agents that are also present. Meat workers are exposed to a variety of transmissible agents present in food animals and their products. This study investigates mortality from non-malignant diseases in workers with these exposures. A cohort mortality study was conducted between 1949 and 1989, of 8520 meat workers in a union in Baltimore, Maryland, who worked in manufacturing plants where animals were killed or processed, and who had high exposures to transmissible agents. Mortality in meat workers was compared with that in a control group of 6081 workers in the same union, and also with the US general population. Risk was estimated by proportional mortality and standardised mortality ratios (SMRs) and relative SMR. A clear excess of mortality from septicaemia, subarachnoid haemorrhage, chronic nephritis, acute and subacute endocarditis, functional diseases of the heart, and decreased risk of mortality from pre-cerebral, cerebral artery stenosis were observed in meat workers when compared to the control group or to the US general population. The authors hypothesise that zoonotic transmissible agents present in food animals and their products may be responsible for the occurrence of some cases of circulatory, neurological and other diseases in meat workers, and possibly in the general population exposed to these agents.

  5. Early growth response 4 is involved in cell proliferation of small cell lung cancer through transcriptional activation of its downstream genes.

    Directory of Open Access Journals (Sweden)

    Taisuke Matsuo

    Full Text Available Small cell lung cancer (SCLC is aggressive, with rapid growth and frequent bone metastasis; however, its detailed molecular mechanism remains poorly understood. Here, we report the critical role of early growth factor 4 (EGR4, a DNA-binding, zinc-finger transcription factor, in cell proliferation of SCLC. EGR4 overexpression in HEK293T cells conferred significant upregulation of specific splice variants of the parathyroid hormone-related protein (PTHrP gene, resulting in enhancement of the secretion of PTHrP protein, a known mediator of osteolytic bone metastasis. More importantly, depletion of EGR4 expression by siRNA significantly suppressed growth of the SCLC cell lines, SBC-5, SBC-3 and NCI-H1048. On the other hand, introduction of EGR4 into NIH3T3 cells significantly enhanced cell growth. We identified four EGR4 target genes, SAMD5, RAB15, SYNPO and DLX5, which were the most significantly downregulated genes upon depletion of EGR4 expression in all of the SCLC cells examined, and demonstrated the direct recruitment of EGR4 to their promoters by ChIP and luciferase reporter analysis. Notably, knockdown of the expression of these genes by siRNA remarkably suppressed the growth of all the SCLC cells. Taken together, our findings suggest that EGR4 likely regulates the bone metastasis and proliferation of SCLC cells via transcriptional regulation of several target genes, and may therefore be a promising target for the development of anticancer drugs for SCLC patients.

  6. Cell-patterned glass spray for direct drug assay using mass spectrometry

    International Nuclear Information System (INIS)

    Wu, Jing; Wang, Shiqi; Chen, Qiushui; Jiang, Hao; Liang, Shuping; Lin, Jin-Ming

    2015-01-01

    In this work, the establishment of a glass spray mass spectrometry (GS-MS) platform for direct cell-based drug assay was described. Cell co-culture, drug-induced cell apoptosis, proliferation analysis and intracellular drug absorption measurement were performed simultaneously on this specifically designed platform. Two groups of co-cultured cells (NIH-3T3/HepG2 and HepG2/MCF-7) were cultivated and they showed high viability within 3 days. The biocompatibility of the platform facilitated the subsequent bioassays, in which, cyclophosphamide (CPA) and genistein were used as the model drugs. The distinctions of cell apoptosis and proliferation between the mono-cultured and co-cultured cells were clearly observed and well explained by in situ GS-MS measurements. A satisfactory linearity of the calibration curve between the relative MS intensity and CPA concentrations was obtained using stable isotope labeling method (y = 0.16545 + 0.0985x, R"2 = 0.9937). The variations in the quantity of absorbed drug were detected and the results were consistent with the concentration-dependence of cell apoptosis. All the results demonstrated that direct cell-based drug assay could be performed on the stable isotope labeling assisted GS-MS platform in a facile and quantitative manner. - Highlights: • A versatile glass spray mass spectrometry (GS-MS) platform for direct cell-based drug assay was developed in this paper. • It has characteristics of the atmospheric pressure ionization method. • It is multifunctional for cell co-culture, bioassays, qualitative and quantitative intracellular drug absorption measurement. • GS-MS has the potential to increase the use of mass spectrometry in biological analysis.

  7. Real-time monitoring of cisplatin cytotoxicity on three-dimensional spheroid tumor cells

    Directory of Open Access Journals (Sweden)

    Baek NH

    2016-07-01

    Full Text Available NamHuk Baek,1,* Ok Won Seo,1,* Jaehwa Lee,1 John Hulme,2 Seong Soo A An2 1Department of Research and Development, NanoEntek Inc., Seoul, 2Department of BioNano Technology, Gachon University, Gyeonggi-do, Korea *These authors contributed equally to this work Abstract: Three-dimensional (3D cell cultivation is a powerful technique for monitoring and understanding diverse cellular mechanisms in developmental cancer and neuronal biology, tissue engineering, and drug development. 3D systems could relate better to in vivo models than two-dimensional (2D cultures. Several factors, such as cell type, survival rate, proliferation rate, and gene and protein expression patterns, determine whether a particular cell line can be adapted to a 3D system. The 3D system may overcome some of the limitations of 2D cultures in terms of cell–cell communication and cell networks, which are essential for understanding differentiation, structural organization, shape, and extended connections with other cells or organs. Here, the effect of the anticancer drug cisplatin, also known as cis-diamminedichloroplatinum (II or CDDP, on adenosine triphosphate (ATP generation was investigated using 3D spheroid-forming cells and real-time monitoring for 7 days. First, 12 cell lines were screened for their ability to form 3D spheroids: prostate (DU145, testis (F9, embryonic fibroblast (NIH-3T3, muscle (C2C12, embryonic kidney (293T, neuroblastoma (SH-SY5Y, adenocarcinomic alveolar basal epithelial cell (A549, cervical cancer (HeLa, HeLa contaminant (HEp2, pituitary epithelial-like cell (GH3, embryonic cell (PA317, and osteosarcoma (U-2OS cells. Of these, eight cell lines were selected: NIH-3T3, C2C12, 293T, SH-SY5Y, A549, HeLa, PA317, and U-2OS; and five underwent real-time monitoring of CDDP cytotoxicity: HeLa, A549, 293T, SH-SY5Y, and U-2OS. ATP generation was blocked 1 day after addition of 50 µM CDDP, but cytotoxicity in HeLa, A549, SH-SY5Y, and U-2OS cells could be

  8. Use of electroporation for high-molecular-weight DNA-mediated gene transfer.

    Science.gov (United States)

    Jastreboff, M M; Ito, E; Bertino, J R; Narayanan, R

    1987-08-01

    Electroporation was used to introduce high-molecular-weight DNA into murine hematopoietic cells and NIH3T3 cells. CCRF-CEM cells were stably transfected with SV2NEO plasmid and the genomic DNA from G-418-resistant clones (greater than 65 kb) was introduced into mouse bone marrow and NIH3T3 cells by electroporation. NEO sequences and expression were detected in the hematopoietic tissues of lethally irradiated mice, with 24% of individual spleen colonies expressing NEO. The frequency of genomic DNA transfer into NIH3T3 cells was 0.25 X 10(-3). Electroporation thus offers a powerful mode of gene transfer not only of cloned genes but also of high-molecular-weight DNA into cells.

  9. Cell motility regulation on a stepped micro pillar array device (SMPAD) with a discrete stiffness gradient.

    Science.gov (United States)

    Lee, Sujin; Hong, Juhee; Lee, Junghoon

    2016-02-28

    Our tissues consist of individual cells that respond to the elasticity of their environment, which varies between and within tissues. To better understand mechanically driven cell migration, it is necessary to manipulate the stiffness gradient across a substrate. Here, we have demonstrated a new variant of the microfabricated polymeric pillar array platform that can decouple the stiffness gradient from the ECM protein area. This goal is achieved via a "stepped" micro pillar array device (SMPAD) in which the contact area with the cell was kept constant while the diameter of the pillar bodies was altered to attain the proper mechanical stiffness. Using double-step SU-8 mold fabrication, the diameter of the top of every pillar was kept uniform, whereas that of the bottom was changed, to achieve the desired substrate rigidity. Fibronectin was immobilized on the pillar tops, providing a focal adhesion site for cells. C2C12, HeLa and NIH3T3 cells were cultured on the SMPAD, and the motion of the cells was observed by time-lapse microscopy. Using this simple platform, which produces a purely physical stimulus, we observed that various types of cell behavior are affected by the mechanical stimulus of the environment. We also demonstrated directed cell migration guided by a discrete rigidity gradient by varying stiffness. Interestingly, cell velocity was highest at the highest stiffness. Our approach enables the regulation of the mechanical properties of the polymeric pillar array device and eliminates the effects of the size of the contact area. This technique is a unique tool for studying cellular motion and behavior relative to various stiffness gradients in the environment.

  10. Modulation of intracellular calcium and proliferative activity of invertebrate and vertebrate cells by ethylene

    Directory of Open Access Journals (Sweden)

    Müller Werner EG

    2001-05-01

    Full Text Available Abstract Background Ethylene is a widely distributed alkene product which is formed enzymatically (e.g., in plants or by photochemical reactions (e.g., in the upper oceanic layers from dissolved organic carbon. This gaseous compound was recently found to induce in cells from the marine sponge Suberites domuncula, an increase in intracellular Ca2+ level ([Ca2+]i and an upregulation of the expression of two genes, the potential ethylene-responsive gene, SDERR, and a Ca2+/calmodulin-dependent protein kinase. Results Here we describe for the first time, that besides sponge cells, mammalian cell lines (mouse NIH-3T3 and human HeLa and SaOS-2 cells respond to ethylene, generated by ethephon, with an immediate and strong, transient increase in [Ca2+]i level, as demonstrated using Fura-2 imaging method. A rise of [Ca2+]i level was also found following exposure to ethylene gas of cells kept under pressure (SaOS-2 cells. The upregulation of [Ca2+]i was associated with an increase in the level of the cell cycle-associated Ki-67 antigen. In addition, we show that the effect of ethephon addition to S. domuncula cells depends on the presence of calcium in the extracellular milieu. Conclusion The results presented in this paper indicate that ethylene, previously known to act as a mediator (hormone in plants only, deserves also attention as a potential signaling molecule in higher vertebrates. Further studies are necessary to clarify the specificity and physiological significance of the effects induced by ethylene in mammalian cells.

  11. Differential expression of the ufo/axl oncogene in human leukemia-lymphoma cell lines.

    Science.gov (United States)

    Challier, C; Uphoff, C C; Janssen, J W; Drexler, H G

    1996-05-01

    The ufo protein (also termed axl) is a member of a new family of receptor tyrosine kinases and is encoded by a transforming gene that was initially isolated from primary human myeloid leukemia cells by DNA-mediated transformation of NIH/3T3 cells. The ligand, Gas6, a protein S-related molecule lacking any known function yet, has recently been identified. We report the expression pattern of ufo mRNA in a panel of 76 human continuous leukemia-lymphoma cell lines. The gene was not expressed in cell lines derived from lymphoid malignancies (n=28), but transcription was seen in 3/11 myeloid, 0/6 monocytic, 9/13 erythroid and 11/18 megakaryocytic cell lines. Several cell lines were treated with phorbol ester leading to significant upregulation of the ufo message in constitutively positive cells. An apparent ufo mRNA overexpression was not found in any of the positive leukemia cell lines, but was identified in the drug-resistant subclones of the cervix carcinoma cell line HeLa. Southern blot analysis of restriction enzyme-digested genomic DNA did not provide evidence for gene amplification, but the HeLa subclones showed banding patterns suggestive of gene rearrangement. Two main ufo mRNA bands of 3.2 and 5.0 kb were identified; no differences in the half-lives (t1/2 = 2.5 h) of these two mRNA species could be identified. In summary, ufo, representing a novel type of receptor tyrosine kinase, is expressed solely in myeloid and erythro-megakaryocytic leukemias but not in lymphoid malignancies. These and previous data suggest an involvement of the ufo receptor tyrosine kinase in normal and malignant myelopoiesis; however, its exact role, if any, and mode of operation in leukemogenesis remains to be determined.

  12. Cytosolic NADP(+)-dependent isocitrate dehydrogenase status modulates oxidative damage to cells.

    Science.gov (United States)

    Lee, Su Min; Koh, Ho-Jin; Park, Dong-Chan; Song, Byoung J; Huh, Tae-Lin; Park, Jeen-Woo

    2002-06-01

    NADPH is an important cofactor in many biosynthesis pathways and the regeneration of reduced glutathione, critically important in cellular defense against oxidative damage. It is mainly produced by glucose 6-phosphate dehydrogenase (G6PD), malic enzyme, and the cytosolic form of NADP(+)-dependent isocitrate dehydrogenase (IDPc). Little information is available about the role of IDPc in antioxidant defense. In this study we investigated the role of IDPc against cytotoxicity induced by oxidative stress by comparing the relative degree of cellular responses in three different NIH3T3 cells with stable transfection with the cDNA for mouse IDPc in sense and antisense orientations, where IDPc activities were 3-4-fold higher and 35% lower, respectively, than that in the parental cells carrying the vector alone. Although the activities of other antioxidant enzymes, such as superoxide dismutase, catalase, glutathione reductase, glutathione peroxidase, and G6PD, were comparable in all transformed cells, the ratio of GSSG to total glutathione was significantly higher in the cells expressing the lower level of IDPc. This finding indicates that IDPc is essential for the efficient glutathione recycling. Upon transient exposure to increasing concentrations of H(2)O(2) or menadione, an intracellular source of free radicals and reactive oxygen species, the cells with low levels of IDPc became more sensitive to oxidative damage by H(2)O(2) or menadione. Lipid peroxidation, oxidative DNA damage, and intracellular peroxide generation were higher in the cell-line expressing the lower level of IDPc. However, the cells with the highly over-expressed IDPc exhibited enhanced resistance against oxidative stress, compared to the control cells. This study provides direct evidence correlating the activities of IDPc and the maintenance of the cellular redox state, suggesting that IDPc plays an important role in cellular defense against oxidative stress.

  13. Probing cellular mechanoadaptation using cell-substrate de-adhesion dynamics: experiments and model.

    Science.gov (United States)

    S S, Soumya; Sthanam, Lakshmi Kavitha; Padinhateeri, Ranjith; Inamdar, Mandar M; Sen, Shamik

    2014-01-01

    Physical properties of the extracellular matrix (ECM) are known to regulate cellular processes ranging from spreading to differentiation, with alterations in cell phenotype closely associated with changes in physical properties of cells themselves. When plated on substrates of varying stiffness, fibroblasts have been shown to exhibit stiffness matching property, wherein cell cortical stiffness increases in proportion to substrate stiffness up to 5 kPa, and subsequently saturates. Similar mechanoadaptation responses have also been observed in other cell types. Trypsin de-adhesion represents a simple experimental framework for probing the contractile mechanics of adherent cells, with de-adhesion timescales shown to scale inversely with cortical stiffness values. In this study, we combine experiments and computation in deciphering the influence of substrate properties in regulating de-adhesion dynamics of adherent cells. We first show that NIH 3T3 fibroblasts cultured on collagen-coated polyacrylamide hydrogels de-adhere faster on stiffer substrates. Using a simple computational model, we qualitatively show how substrate stiffness and cell-substrate bond breakage rate collectively influence de-adhesion timescales, and also obtain analytical expressions of de-adhesion timescales in certain regimes of the parameter space. Finally, by comparing stiffness-dependent experimental and computational de-adhesion responses, we show that faster de-adhesion on stiffer substrates arises due to force-dependent breakage of cell-matrix adhesions. In addition to illustrating the utility of employing trypsin de-adhesion as a biophysical tool for probing mechanoadaptation, our computational results highlight the collective interplay of substrate properties and bond breakage rate in setting de-adhesion timescales.

  14. Intracellular lysyl oxidase: Effect of a specific inhibitor on nuclear mass in proliferating cells

    Energy Technology Data Exchange (ETDEWEB)

    Saad, Fawzy A. [Laboratory for the Study of Skeletal Disorders and Rehabilitation, Department of Orthopedics, Children' s Hospital Boston, 300 Longwood Avenue EN926, Boston, MA 02115 (United States); Harvard Medical School, Boston, MA 02115 (United States); Torres, Marie [Laboratory for the Study of Skeletal Disorders and Rehabilitation, Department of Orthopedics, Children' s Hospital Boston, 300 Longwood Avenue EN926, Boston, MA 02115 (United States); Wang, Hao [Laboratory for the Study of Skeletal Disorders and Rehabilitation, Department of Orthopedics, Children' s Hospital Boston, 300 Longwood Avenue EN926, Boston, MA 02115 (United States); Harvard Medical School, Boston, MA 02115 (United States); Graham, Lila, E-mail: lilagraham@cs.com [Laboratory for the Study of Skeletal Disorders and Rehabilitation, Department of Orthopedics, Children' s Hospital Boston, 300 Longwood Avenue EN926, Boston, MA 02115 (United States); Harvard Medical School, Boston, MA 02115 (United States)

    2010-06-11

    LOX, the principal enzyme involved in crosslinking of collagen, was the first of several lysyl oxidase isotypes to be characterized. Its active form was believed to be exclusively extracellular. Active LOX was later reported to be present in cell nuclei; its function there is unknown. LOX expression opposes the effect of mutationally activated Ras, which is present in about 30% of human cancers. The mechanism of LOX in countering the action of Ras is also unknown. In the present work, assessment of nuclear protein for possible effects of lysyl oxidase activity led to the discovery that proliferating cells dramatically increase their nuclear protein content when exposed to BAPN ({beta}-aminopropionitrile), a highly specific lysyl oxidase inhibitor that reportedly blocks LOX inhibition of Ras-induced oocyte maturation. In three cell types (PC12 cells, A7r5 smooth muscle cells, and NIH 3T3 fibroblasts), BAPN caused a 1.8-, 1.7-, and 2.1-fold increase in total nuclear protein per cell, respectively, affecting all major components in both nuclear matrix and chromatin fractions. Since nuclear size is correlated with proliferative status, enzyme activity restricting nuclear growth may be involved in the lysyl oxidase tumor suppressive effect. Evidence is also presented for the presence of apparent lysyl oxidase isotype(s) containing a highly conserved LOX active site sequence in the nuclei of PC12 cells, which do not manufacture extracellular lysyl oxidase substrates. Results reported here support the hypothesis that nuclear lysyl oxidase regulates nuclear growth, and thereby modulates cell proliferation.

  15. Fabrication of Nanostructured Poly-ε-caprolactone 3D Scaffolds for 3D Cell Culture Technology

    KAUST Repository

    Schipani, Rossana

    2015-04-21

    Tissue engineering is receiving tremendous attention due to the necessity to overcome the limitations related to injured or diseased tissues or organs. It is the perfect combination of cells and biomimetic-engineered materials. With the appropriate biochemical factors, it is possible to develop new effective bio-devices that are capable to improve or replace biological functions. Latest developments in microfabrication methods, employing mostly synthetic biomaterials, allow the production of three-dimensional (3D) scaffolds that are able to direct cell-to-cell interactions and specific cellular functions in order to drive tissue regeneration or cell transplantation. The presented work offers a rapid and efficient method of 3D scaffolds fabrication by using optical lithography and micro-molding techniques. Bioresorbable polymer poly-ε-caprolactone (PCL) was the material used thanks to its high biocompatibility and ability to naturally degrade in tissues. 3D PCL substrates show a particular combination in the designed length scale: cylindrical shaped pillars with 10μm diameter, 10μm height, arranged in a hexagonal lattice with spacing of 20μm were obtained. The sidewalls of the pillars were nanostructured by attributing a 3D architecture to the scaffold. The suitability of these devices as cell culture technology supports was evaluated by plating NIH/3T3 mouse embryonic fibroblasts and human Neural Stem Cells (hNSC) on them. Scanning Electron Microscopy (SEM) analysis was carried out in order to examine the micro- and nano-patterns on the surface of the supports. In addition, after seeding of cells, SEM and immunofluorescence characterization of the fabricated systems were performed to check adhesion, growth and proliferation. It was observed that cells grow and develop healthy on the bio-polymeric devices by giving rise to well-interconnected networks. 3D PCL nano-patterned pillared scaffold therefore may have considerable potential as effective tool for

  16. Predictors of long-term opioid use among chronic nonmalignant pain patients

    DEFF Research Database (Denmark)

    Hansen, Carrinna; Abrahamsen, Bo

    2016-01-01

    , socioeconomic and demographic characteristics. Data analysis is ongoing. Conclusions: It is expected that this study will serve as a significant supplement of existing knowledge in the area of opioid consumption among CNP patients in Denmark. In a future perspective of prevention and health promotion......Aims: 1) To determine the distribution and determinants of opioid use among chronic nonmalignant pain(CNP) patients. 2) To identify the patient, treatment and socioeconomic characteristics as determinants for potential risk groups. We hypothesized that CNP patient who use opioids for more than 1...... product using the ATC codes N02AA01-N02AX06. Patients were then classified as either opioid use for more than 1 year(group A), as opioid use for more than 6 months but less than 1 year(group B) and opioid use equal to or less than 6 months(group C). Results: The quantity of sold opioids has been...

  17. Colorectal cancer and non-malignant respiratory disease in asbestos cement and cement workers

    International Nuclear Information System (INIS)

    Jacobsson, K.

    1993-09-01

    Radiologically visible parenchymal changes (small opacities >= 1/0;ILO 1980 classification) were present in 20% of a sample of workers (N=174), employed for 20 years (median) in an asbestos cement plant. Exposure-response relationships were found, after controlling for age and smoking habits. In a sample of asbestos cement workers with symptoms and signs suggestive of pulmonary disease (N=33), increased lung density measured by x-ray computed tomography, and reduced static lung volumes and lung compliance was found. In a cohort of asbestos cement workers (N=1.929) with an estimated median exposure of 1.2 fibres/ml, the mortality from non-malignant respiratory disease was increased in comparison to a regional reference cohort (N=1.233). A two-to three-fold increase of non-malignant respiratory mortality was noted among workers employed for more than a decade in the asbestos cement plant, compared to cement workers (N=1.526), who in their turn did not experience and increased risk compared to the general population. In the cohorts of asbestos cement and cement workers, there was a tow-to three-fold increased incidence of cancer in the right part of the colon, compared to the general population as well as to external reference cohorts of other industrial workers (N=3.965) and fishermen (N=8.092). A causal relation with the exposure to mineral dust and fibres was supported by the findings of higher risk estimated in subgroups with high cumulated asbestos doses or longer duration of cement work. The incidence of cancer in the left part of the colon was not increased. Morbidity data, but not mortality data, disclosed the subsite-specific risk pattern. Both asbestos cement workers and cement workers has an increased incidence of rectal cancer, compared with the general population, and with the fishermen. The risk was, however, of the same magnitude among the other industrial workers. 181 refs

  18. Non-malignant consequences of decreasing asbestos exposure in the Brazil chrysotile mines and mills.

    Science.gov (United States)

    Bagatin, E; Neder, J A; Nery, L E; Terra-Filho, M; Kavakama, J; Castelo, A; Capelozzi, V; Sette, A; Kitamura, S; Favero, M; Moreira-Filho, D C; Tavares, R; Peres, C; Becklake, M R

    2005-06-01

    To investigate the consequences of improvement in the workplace environment over six decades (1940-96) in asbestos miners and millers from a developing country (Brazil). A total of 3634 Brazilian workers with at least one year of exposure completed a respiratory symptoms questionnaire, chest radiography, and a spirometric evaluation. The study population was separated into three groups whose working conditions improved over time: group I (1940-66, n = 180), group II (1967-76, n = 1317), and group III (1977-96, n = 2137). Respiratory symptoms were significantly related to spirometric abnormalities, smoking, and latency time. Breathlessness, in particular, was also associated with age, pleural abnormality and increased cumulative exposure to asbestos fibres. The odds ratios (OR) for parenchymal and/or non-malignant pleural disease were significantly lower in groups II and III compared to group I subjects (0.29 (0.12-0.69) and 0.19 (0.08-0.45), respectively), independent of age and smoking status. Similar results were found when groups were compared at equivalent latency times (groups I v II: 30-45 years; groups II v III: 20-25 years). Ageing, dyspnoea, past and current smoking, and radiographic abnormalities were associated with ventilatory impairment. Lower spirometric values were found in groups I and II compared to group III: lung function values were also lower in higher quartiles of latency and of cumulative exposure in these subjects. Progressive improvement in occupational hygiene in a developing country is likely to reduce the risk of non-malignant consequences of dust inhalation in asbestos miners and millers.

  19. Reduced-intensity conditioning for the treatment of malignant and life-threatening non-malignant disorders.

    Science.gov (United States)

    Slavin, Shimon; Aker, Mehmet; Shapira, Michael Y; Resnick, Igor; Bitan, Menachem; Or, Reuven

    2003-01-01

    Allogeneic bone marrow or blood stem cell transplantation (BMT) represents an important therapeutic tool for the treatment of an otherwise incurable broad spectrum of malignant and non-malignant diseases. Until recently, BMT was used primarily to replace a malignant, genetically abnormal or deficient immunohematopoietic compartment and therefore, highly toxic myeloablative regimens were considered mandatory for more effective eradication of all undesirable host-derived hematopoietic cells, including stem cells and their progeny. Our preclinical and ongoing clinical studies indicated that much more effective eradication of host immunohematopoietic system cells can be mediated by donor lymphocytes in the process of adoptive allogeneic cell therapy following BMT. Thus, eradication of all malignant cells, especially in patients with CML and, to a lesser extent, in patients with other hematologic malignancies can be accomplished despite complete resistance of puch tumor cells to maximally tolerated doses of chemoradiotherapy. Our cumulative experience suggested that graft-versus-malignancy effects might be used as a tool for eradication of otherwise resistant tumor cells of host origin. We speculated that the therapeutic benefit of BMT may be improved by using safer conditioning for engraftment of donor stem cells induce host-versus-graft unresponsiveness to enable engraftment of donor lymphocytes for subsequent induction of graft-versus-malignancy effects, or even graft-versus-autoimmunity and graft-versus-genetically abnormal cells. In other words, focusing on more selective and smarter rather than stronger modalities. Effective BMT procedures may be accomplished without lethal conditioning of the host, using a new, well-tolerated and user-friendly non-myeloablative regimen, thus eliminating or minimizing immediate and late procedure-related toxicity and mortality. It appears that initial induction of graft tolerance, mediated by engraftment of donor stem cells, leads

  20. Monitoring the effects of doxorubicin on 3D-spheroid tumor cells in real-time

    Directory of Open Access Journals (Sweden)

    Baek N

    2016-11-01

    Full Text Available NamHuk Baek,1,* Ok Won Seo,1,* MinSung Kim,1 John Hulme,2 Seong Soo A An2 1Department of R & D, NanoEntek Inc., Seoul, Republic of Korea; 2Department of BioNano Technology Gachon University, Gyeonggi-do, Republic of Korea *These authors contributed equally to this work Abstract: Recently, increasing numbers of cell culture experiments with 3D spheroids presented better correlating results in vivo than traditional 2D cell culture systems. 3D spheroids could offer a simple and highly reproducible model that would exhibit many characteristics of natural tissue, such as the production of extracellular matrix. In this paper numerous cell lines were screened and selected depending on their ability to form and maintain a spherical shape. The effects of increasing concentrations of doxorubicin (DXR on the integrity and viability of the selected spheroids were then measured at regular intervals and in real-time. In total 12 cell lines, adenocarcinomic alveolar basal epithelial (A549, muscle (C2C12, prostate (DU145, testis (F9, pituitary epithelial-like (GH3, cervical cancer (HeLa, HeLa contaminant (HEp2, embryo (NIH3T3, embryo (PA317, neuroblastoma (SH-SY5Y, osteosarcoma U2OS, and embryonic kidney cells (293T, were screened. Out of the 12, 8 cell lines, NIH3T3, C2C12, 293T, SH-SY5Y, A549, HeLa, PA317, and U2OS formed regular spheroids and the effects of DXR on these structures were measured at regular intervals. Finally, 5 cell lines, A549, HeLa, SH-SY5Y, U2OS, and 293T, were selected for real-time monitoring and the effects of DXR treatment on their behavior were continuously recorded for 5 days. A potential correlation regarding the effects of DXR on spheroid viability and ATP production was measured on days 1, 3, and 5. Cytotoxicity of DXR seemed to occur after endocytosis, since the cellular activities and ATP productions were still viable after 1 day of the treatment in all spheroids, except SH-SY5Y. Both cellular activity and ATP production were

  1. Graphene as a nanocarrier for tamoxifen induces apoptosis in transformed cancer cell lines of different origins.

    Science.gov (United States)

    Misra, Santosh K; Kondaiah, Paturu; Bhattacharya, Santanu; Rao, C N R

    2012-01-09

    A cationic amphiphile, cholest-5en-3β-oxyethyl pyridinium bromide (PY(+) -Chol), is able to efficiently disperse exfoliated graphene (GR) in water by the physical adsorption of PY(+) -Chol on the surface of GR to form stable, dark aqueous suspensions at room temperature. The GR-PY(+) -Chol suspension can then be used to solubilize Tamoxifen Citrate (TmC), a breast cancer drug, in water. The resulting TmC-GR-PY(+) -Chol is stable for a long time without any precipitation. Fluorescence emission and UV absorption spectra indicate the existence of noncovalent interactions between TmC, GR, and PY(+) -Chol in these suspensions. Electron microscopy shows the existence of segregated GR sheets and TmC 'ribbons' in the composite suspensions. Atomic force microscopy indicates the presence of 'extended' structures of GR-PY(+) -Chol, which grows wider in the presence of TmC. The slow time-dependent release of TmC is noticed in a reconstituted cell culture medium, a property useful as a drug carrier. TmC-GR-PY(+) -Chol selectively enhanced the cell death (apoptosis) of the transformed cancer cells compared to normal cells. This potency is found to be true for a wide range of transformed cancer cells viz. HeLa, A549, ras oncogene-transformed NIH3T3, HepG2, MDA-MB231, MCF-7, and HEK293T compared to the normal cell HEK293 in vitro. Confocal microscopy confirmed the high efficiency of TmC-GR-PY(+) -Chol in delivering the drug to the cells, compared to the suspensions devoid of GR. Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  2. E3B1, a human homologue of the mouse gene product Abi-1, sensitizes activation of Rap1 in response to epidermal growth factor

    International Nuclear Information System (INIS)

    Jenei, Veronika; Andersson, Tommy; Jakus, Judit; Dib, Karim

    2005-01-01

    E3B1, a human homologue of the mouse gene product Abi-1, has been implicated in growth-factor-mediated regulation of the small GTPases p21 Ras and Rac. E3b1 is a regulator of Rac because it can form a complex with Sos-1 and eps8, and such a Sos-1-e3B1-eps8 complex serves as a guanine nucleotide exchange factor for Rac. In the present study, we found that overexpression of e3B1 in NIH3T3/EGFR cells sensitized EGF-induced activation of Rac1, whereas it had no impact on EGF-induced activation of p21 Ras . Remarkably, we found that EGF-induced activation of the p21 Ras -related GTPase Rap1 was also sensitized in NIH3T3/EGFR-e3B1 cells. Thus, in NIH3T3/EGFR-e3B1 cells, maximal EGF-induced activation of Rap1 occurs with a dose of EGF much lower than in NIH3T3/EGFR cells. We also report that overexpression of e3B1 in NIH3T3/EGFR cells renders EGF-induced activation of Rap1 completely dependent on Src tyrosine kinases but not on c-Abl. However, EGF-induced tyrosine phosphorylation of the Rap GEF C3G occurred regardless of whether e3B1 was overexpressed or not, and this did not involve Src tyrosine kinases. Accordingly, we propose that overexpression of e3B1 in NIH3T3/EGFR cells leads to mobilization of Src tyrosine kinases that participate in EGF-induced activation of Rap1 and inhibition of cell proliferation

  3. BRAFV600E negatively regulates the AKT pathway in melanoma cell lines.

    Science.gov (United States)

    Chen, Brenden; Tardell, Christine; Higgins, Brian; Packman, Kathryn; Boylan, John F; Niu, Huifeng

    2012-01-01

    Cross-feedback activation of MAPK and AKT pathways is implicated as a resistance mechanism for cancer therapeutic agents targeting either RAF/MEK or PI3K/AKT/mTOR. It is thus important to have a better understanding of the molecular resistance mechanisms to improve patient survival benefit from these agents. Here we show that BRAFV600E is a negative regulator of the AKT pathway. Expression of BRAFV600E in NIH3T3 cells significantly suppresses MEK inhibitor (RG7167) or mTORC1 inhibitor (rapamycin) induced AKT phosphorylation (pAKT) and downstream signal activation. Treatment-induced pAKT elevation is found in BRAF wild type melanoma cells but not in a subset of melanoma cell lines harboring BRAFV600E. Knock-down of BRAFV600E in these melanoma cells elevates basal pAKT and downstream signals, whereas knock-down of CRAF, MEK1/2 or ERK1/2 or treatment with a BRAF inhibitor have no impact on pAKT. Mechanistically, we show that BRAFV600E interacts with rictor complex (mTORC2) and regulates pAKT through mTORC2. BRAFV600E is identified in mTORC2 after immunoprecipitation of rictor. Knock-down of rictor abrogates BRAFV600E depletion induced pAKT. Knock-down of BRAFV600E enhances cellular enzyme activity of mTORC2. Aberrant activation of AKT pathway by PTEN loss appears to override the negative impact of BRAFV600E on pAKT. Taken together, our findings suggest that in a subset of BRAFV600E melanoma cells, BRAFV600E negatively regulates AKT pathway in a rictor-dependent, MEK/ERK and BRAF kinase-independent manner. Our study reveals a novel molecular mechanism underlying the regulation of feedback loops between the MAPK and AKT pathways.

  4. BRAFV600E negatively regulates the AKT pathway in melanoma cell lines.

    Directory of Open Access Journals (Sweden)

    Brenden Chen

    Full Text Available Cross-feedback activation of MAPK and AKT pathways is implicated as a resistance mechanism for cancer therapeutic agents targeting either RAF/MEK or PI3K/AKT/mTOR. It is thus important to have a better understanding of the molecular resistance mechanisms to improve patient survival benefit from these agents. Here we show that BRAFV600E is a negative regulator of the AKT pathway. Expression of BRAFV600E in NIH3T3 cells significantly suppresses MEK inhibitor (RG7167 or mTORC1 inhibitor (rapamycin induced AKT phosphorylation (pAKT and downstream signal activation. Treatment-induced pAKT elevation is found in BRAF wild type melanoma cells but not in a subset of melanoma cell lines harboring BRAFV600E. Knock-down of BRAFV600E in these melanoma cells elevates basal pAKT and downstream signals, whereas knock-down of CRAF, MEK1/2 or ERK1/2 or treatment with a BRAF inhibitor have no impact on pAKT. Mechanistically, we show that BRAFV600E interacts with rictor complex (mTORC2 and regulates pAKT through mTORC2. BRAFV600E is identified in mTORC2 after immunoprecipitation of rictor. Knock-down of rictor abrogates BRAFV600E depletion induced pAKT. Knock-down of BRAFV600E enhances cellular enzyme activity of mTORC2. Aberrant activation of AKT pathway by PTEN loss appears to override the negative impact of BRAFV600E on pAKT. Taken together, our findings suggest that in a subset of BRAFV600E melanoma cells, BRAFV600E negatively regulates AKT pathway in a rictor-dependent, MEK/ERK and BRAF kinase-independent manner. Our study reveals a novel molecular mechanism underlying the regulation of feedback loops between the MAPK and AKT pathways.

  5. Reactive oxygen species-dependent HSP90 protein cleavage participates in arsenical As+3- and MMA+3-induced apoptosis through inhibition of telomerase activity via JNK activation

    International Nuclear Information System (INIS)

    Shen, S.-C.; Yang, L.-Y.; Lin, H.-Y.; Wu, C.-Y.; Su, T.-H.; Chen, Y.-C.

    2008-01-01

    The effects of six arsenic compounds including As +3 , MMA +3 , DMA +3 , As +5 , MMA +5 , and DMA +5 on the viability of NIH3T3 cells were examined. As +3 and MMA +3 , but not the others, exhibited significant cytotoxic effects in NIH3T3 cells through apoptosis induction. The apoptotic events such as DNA fragmentation and chromosome condensation induced by As +3 and MMA +3 were prevented by the addition of NAC and CAT, and induction of HO-1 gene expression in accordance with cleavage of the HSP90 protein, and suppression of telomerase activity were observed in NIH3T3 cells under As +3 and MMA +3 treatments. An increase in the intracellular peroxide level was examined in As +3 - and MMA +3 -treated NIH3T3 cells, and As +3 - and MMA +3 -induced apoptotic events were blocked by NAC, CAT, and DPI addition. HSP90 inhibitors, GA and RD, significantly attenuated the telomerase activity in NIH3T3 cells with an enhancement of As +3 - and MMA +3 -induced cytotoxicity. Suppression of JNKs significantly inhibited As +3 - and MMA +3 -induced apoptosis by blocking HSP90 protein cleavage and telomerase reduction in NIH3T3 cells. Furthermore, Hb, SnPP, and dexferosamine showed no effect against As +3 - and MMA +3 -induced apoptosis, and overexpression of HO-1 protein or inhibition of HO-1 protein expression did not affect the apoptosis induced by As +3 or MMA +3 . These data provide the first evidence to indicate that apoptosis induced by As +3 and MMA +3 is mediated by an ROS-dependent degradation of HSP90 protein and reduction of telomerase via JNK activation, and HO-1 induction might not be involved

  6. Interaction of Prevotella intermedia strain 17 leucine-rich repeat domain protein AdpF with eukaryotic cells promotes bacterial internalization.

    Science.gov (United States)

    Sengupta, Dipanwita; Kang, Dae-Joong; Anaya-Bergman, Cecilia; Wyant, Tiana; Ghosh, Arnab K; Miyazaki, Hiroshi; Lewis, Janina P

    2014-06-01

    Prevotella intermedia is an oral bacterium implicated in a variety of oral diseases. Although internalization of this bacterium by nonphagocytic host cells is well established, the molecular players mediating the process are not well known. Here, the properties of a leucine-rich repeat (LRR) domain protein, designated AdpF, are described. This protein contains a leucine-rich region composed of 663 amino acid residues, and molecular modeling shows that it folds into a classical curved solenoid structure. The cell surface localization of recombinant AdpF (rAdpF) was confirmed by electron and confocal microscopy analyses. The recombinant form of this protein bound fibronectin in a dose-dependent manner. Furthermore, the protein was internalized by host cells, with the majority of the process accomplished within 30 min. The internalization of rAdpF was inhibited by nystatin, cytochalasin, latrunculin, nocodazole, and wortmannin, indicating that microtubules, microfilaments, and signal transduction are required for the invasion. It is noteworthy that preincubation of eukaryotic cells with AdpF increased P. intermedia 17 internalization by 5- and 10-fold for HeLa and NIH 3T3 fibroblast cell lines, respectively. The addition of the rAdpF protein was also very effective in inducing bacterial internalization into the oral epithelial cell line HN4, as well as into primary cells, including human oral keratinocytes (HOKs) and human umbilical vein endothelial cells (HUVECs). Finally, cells exposed to P. intermedia 17 internalized the bacteria more readily upon reinfection. Taken together, our data demonstrate that rAdpF plays a role in the internalization of P. intermedia 17 by a variety of host cells.

  7. Live-cell visualization of intracellular interaction between a nuclear migration protein (hNUDC) and the thrombopoietin receptor (Mpl).

    Science.gov (United States)

    Zheng, Yuan-Bin; Xiao, Ying-Ying; Tan, Peng; Zhang, Qing; Xu, Peilin

    2012-01-01

    We previously demonstrated that endogenous hNUDC and Mpl co-localized in the perinuclear and cytoplasmic regions of megakaryocyte cells by indirect immunofluorescence. We further reported that hNUDC accumulated in the Golgi when NIH 3T3 cells were transfected with an hNUDC expression vector alone. However, co-transfection with hNUDC and Mpl expression vectors caused both proteins to co-localize predominantly in the cytosol. These observations led us to hypothesize that a complex containing hNUDC and Mpl may alter hNUDC subcellular location and induce its secretion. In the present study, we test this hypothesis by employing bimolecular fluorescence complementation (BiFC) to detect and visualize the complex formation of hNUDC/Mpl in living cells. We further examined in detail the subcellular locations of the hNUDC/Mpl complex by co-transfection of BiFC chimeras with known subcellular markers. The distribution of hNUDC/Mpl in the endoplasmic reticulum (ER), Golgi and cell surface was determined. Furthermore, the N-terminal 159 amino acids of hNUDC, but not C-terminal half, bound to Mpl in vivo and exhibited a similar localization pattern to that of full-length hNUDC in Cos-1 cells. Adenovirus-mediated overexpression of hNUDC or its N-terminal 159 residues in a human megakaryocyte cell line (Dami) resulted in increased levels of hNUDC or hNUDC(1-159) secretion. In contrast, depletion of Mpl by transfecting Dami cells with adenovirus bearing Mpl-targeting siRNA significantly blocked hNUDC secretion. Thus, we provide the first evidence that the N-terminal region of hNUDC contains all of the necessary information to complex with Mpl and traffic through the secretory pathway.

  8. Live-cell visualization of intracellular interaction between a nuclear migration protein (hNUDC and the thrombopoietin receptor (Mpl.

    Directory of Open Access Journals (Sweden)

    Yuan-Bin Zheng

    Full Text Available We previously demonstrated that endogenous hNUDC and Mpl co-localized in the perinuclear and cytoplasmic regions of megakaryocyte cells by indirect immunofluorescence. We further reported that hNUDC accumulated in the Golgi when NIH 3T3 cells were transfected with an hNUDC expression vector alone. However, co-transfection with hNUDC and Mpl expression vectors caused both proteins to co-localize predominantly in the cytosol. These observations led us to hypothesize that a complex containing hNUDC and Mpl may alter hNUDC subcellular location and induce its secretion. In the present study, we test this hypothesis by employing bimolecular fluorescence complementation (BiFC to detect and visualize the complex formation of hNUDC/Mpl in living cells. We further examined in detail the subcellular locations of the hNUDC/Mpl complex by co-transfection of BiFC chimeras with known subcellular markers. The distribution of hNUDC/Mpl in the endoplasmic reticulum (ER, Golgi and cell surface was determined. Furthermore, the N-terminal 159 amino acids of hNUDC, but not C-terminal half, bound to Mpl in vivo and exhibited a similar localization pattern to that of full-length hNUDC in Cos-1 cells. Adenovirus-mediated overexpression of hNUDC or its N-terminal 159 residues in a human megakaryocyte cell line (Dami resulted in increased levels of hNUDC or hNUDC(1-159 secretion. In contrast, depletion of Mpl by transfecting Dami cells with adenovirus bearing Mpl-targeting siRNA significantly blocked hNUDC secretion. Thus, we provide the first evidence that the N-terminal region of hNUDC contains all of the necessary information to complex with Mpl and traffic through the secretory pathway.

  9. Subanesthetic, Subcutaneous Ketamine Infusion Therapy in the Treatment of Chronic Nonmalignant Pain.

    Science.gov (United States)

    Zekry, Olfat; Gibson, Stephen B; Aggarwal, Arun

    2016-06-01

    This study was designed to describe the efficacy and toxicity of subcutaneous ketamine infusions and sublingual ketamine lozenges for the treatment of chronic nonmalignant pain. Data were collected prospectively on 70 subjects managed in an academic, tertiary care hospital between 2007 and 2012 who received between 3 and 7 days of subanesthetic, subcutaneous ketamine infusion. Data were analyzed for efficacy, adverse effects, and reduction in use of opioid medication. We also analyzed whether subsequent treatment with sublingual ketamine lozenges resulted in longer-term efficacy of the beneficial effects of the initial ketamine infusion. There was a significant reduction in pain intensity measured by numerical rating scale (NRS) from mean of 6.38 before ketamine to 4.60 after ketamine (P ketamine infusion from a mean morphine equivalent dose (MMED) of 216 mg/day before ketamine to 89 mg/day after ketamine (P ketamine infusion was 59%. No subjects increased their use of opioids during their hospitalization for the ketamine infusion. A small proportion of subjects who responded to the infusion were continued on ketamine lozenges. This group was followed for between 3 months and 2 years. The use of ketamine lozenges after the infusion resulted in 31% of these subjects being able to cease their use of opioids compared with only 6% who did not receive ketamine lozenges. Eleven percent of subjects who received lozenges subsequently increased their opioid usage. Adverse effects were fairly common, but only mild, with 46% of patients experiencing light-headedness and dizziness, 25% tiredness and sedation, 12% headaches, 12% hallucinations, and 8% vivid dreams. Adverse effects were easily managed by reducing the rate of the ketamine infusion. The administration of subanesthetic, subcutaneous ketamine infusion was well tolerated, with mostly mild adverse effects and no serious adverse effects. The infusion provided significant pain relief in subjects who had failed a wide

  10. Non-malignant causes of hypercalcemia in cancer patients: a frequent and neglected occurrence.

    Science.gov (United States)

    Soyfoo, M S; Brenner, K; Paesmans, M; Body, J J

    2013-05-01

    Hypercalcemia is a frequent finding in cancer patients and can be observed in any type of cancer. The physician in charge of cancer patients often ignores non-malignant causes of hypercalcemia. Our objective was to review the causes of hypercalcemia in a large series of cancer patients. We have retrospectively studied in a Cancer Centre all consecutive hypercalcemic (Ca> 10.5 mg/dl) patients over an 8-year period. Of 699 evaluated patients, 642 were analyzed after exclusion of patients whose hypercalcemia resolved after rehydration or who had a normal Ca level after correction for protein concentrations. Clinical information was gathered on the type of cancer, its histology, whether the disease was active or in complete remission, and on the presence of bone metastases. Biochemical data included serum Ca, P(i), proteins in all patients, PTH in most patients, and PTHrP, 25OH-Vitamin D, 1,25(OH)(2)-Vitamin D, TSH, and T4 in selected cases. By order of decreasing frequency, the main causes of hypercalcemia were cancer (69.0 %), primary hyperparathyroidism (24.6 %), hyperthyroidism (2.2 %), milk alkali syndrome (0.9 %), and sarcoidosis (0.45 %). In cancer-related causes, bone metastases accounted for 53.0 % of the cases, humoral hypercalcemia of malignancy (HHM) for 35.3 % while there were 11.7 % of cases apparently due to both HHM and bone metastases. Hypercalcemia was not due to cancer in 97 % (84/87) of the patients who were in complete remission. Even in patients with active neoplastic disease, the number of patients whose hypercalcemia was not due to cancer remained clinically relevant (115/555 = 20.5 %). In the 158 patients with primary hyperparathyroidism, 92 patients were in complete remission and 66 patients had active neoplastic disease. In this large series of hypercalcemia in cancer patients, the cause was not due to cancer in almost one third of the cases. Most patients considered to be in complete remission had hypercalcemia due to a benign

  11. Therapeutic effects of diaphragmatic plication for acquired unilateral non-malignant diaphragm paralysis in twenty patients

    Directory of Open Access Journals (Sweden)

    Reza Bagheri

    2013-12-01

    Full Text Available Background: Acquired paralysis of the diaphragm is a condition caused by trauma, surgical injuries, (lung cancer surgery, esophageal surgery, cardiac surgery, thoracic surgery, and is sometimes of an unknown etiology. It can lead to dyspnea and can affect ventilatory function and patients activity. Diaphragmatic plication is a treatment method which decreases inconsistent function of diaphragm. The aim of this study is to evaluate the outcome of diaphragmatic plication in patients with acquired unilateral non-malignant diaphragmatic paralysis. Methods: From 1991 to 2011, 20 patients with acquired unilateral diaphragmatic paralysis who underwent surgery enrolled in our study in Ghaem Hospital Mashhad University of Medical Science. Patients were evaluated in terms of age, sex, BMI, clinical symptoms, dyspnea score (DS, etiology of paralysis, diagnostic methods, respiratory function tests and complication of surgery. Some tests including dyspnea score were carried out again six months after surgery. We evaluated patients with SPSS version 11.5 and Paired t-test or nonparametric equivalent. Results: Twenty patients enrolled in our study. 14 were male and 6 were female. The mean age was 58 years and the average time interval between diagnosis to surgical treatment was 38.3 months. Acquired diaphragmatic paralysis was mostly caused by trauma (in 11 patients and almost occurred on the left side (in 15 patients. Diagnostic methods included chest x-ray, CT scan, ultrasonography and sniff. Test prior to surgery the average FVC was 41.4±7 percent and the average FEV1 was 52.4±6 percent and after surgery they were 80.1±8.6 percent and 74.4±1 percent respectively. The average increase in FEV1 and FVC 63.4±4, 61.1±7.8. Performing surgery also leads to a noticeable improvement in dyspnea score in our study. Conclusion: In patients with acquired unilateral non-malignant diaphragm paralysis diaphragmatic plication is highly recommended due to the

  12. Human Umbilical Cord Blood Serum Has Higher Potential in Inducing Proliferation of Fibroblast than Fetal Bovine Serum

    Directory of Open Access Journals (Sweden)

    Ferry Sandra

    2017-09-01

    Full Text Available Background: Cytokines and growth factors were reported to play an important role in stimulating fibroblast proliferation. In vitro culture, fibroblast is mostly culture in medium containing fetal bovine serum (FBS.  Human umbilical cord blood (hUCB has been reported to have low immunogenic property and potential in wound healing, so therefore hUCB serum (hUCBS could be potential and were investigated in current study. Materials and Methods: Five hUCBs were collected from healthy volunteers with normal delivering procedure. hUCB was ex utero immediately collected from umbilical vein in vacutainers and processed. NIH3T3 cells were cultured in DMEM with 10% FBS or 5-20% hUCBS for 48 hours. Cells were then quantified using MTT assay. Protein concentration of FBS and hUCBS were quantified using Bradford assay. Results: NIH3T3 cells density grown in DMEM with 10% FBS was the lowest. NIH3T3 cells densities were increased along with the increment of hUCBS concentrations. MTT results showed that average number of NIH3T3 cells grown in DMEM with 10% FBS was 6,185±1,243. Meanwhile average numbers of NIH3T3 cells grown in DMEM with 5%, 10% and 20% hUCBS were 8,126±628, 9,685±313 and 12,200±304, respectively. Average numbers of NIH3T3 cells grown in DMEM with 5% hUCBS were significantly higher than the ones with 10% FBS (p=0.000. Bradford results showed that concentration of hUCBS was significantly higher than the one of FBS (p=0.000. Conclusion: hUCBS could induce higher proliferation rate of NIH3T3 cells than FBS. Hence hUCBS could be suggested as an alternate of FBS in inducing fibroblast. Keywords: NIH3T3, fibroblast, UCB, serum, FBS, proliferation

  13. Nonmalignant respiratory mortality and long-term exposure to PM10 and SO2: A 12-year cohort study in northern China

    International Nuclear Information System (INIS)

    Chen, Xi; Wang, Xue; Huang, Jia-ju; Zhang, Li-wen; Song, Feng-ju; Mao, Hong-jun; Chen, Ke-xin; Chen, Jie; Liu, Ya-min; Jiang, Guo-hong; Dong, Guang-hui; Bai, Zhi-peng

    2017-01-01

    Highlights: • The relationship between air pollution and respiratory disease is proposed. • Nonmalignant respiratory disease mortality was associated with PM 10 and SO 2 . • Passive smokers are susceptible to the harmful effects of air pollution.

  14. Opposing effects of low versus high concentrations of water soluble vitamins/dietary ingredients Vitamin C and niacin on colon cancer stem cells (CSCs).

    Science.gov (United States)

    Sen, Utsav; Shenoy P, Sudheer; Bose, Bipasha

    2017-10-01

    Colorectal cancer is one of the global causes of cancer deaths. Cancer stem cells (CSCs) inside the tumour niche responsible for metastasis and relapses, and hence need to be targeted for cancer therapeutics. Although dietary fibre and lifestyle changes have been recommended as measures for colorectal cancer prevention, no such recommendations are available for using water soluble vitamins as prophylaxis measure for colorectal cancers. High dose of Vitamin C has been proven to selectively kill colon cancer cells having BRAF and KRAS mutations by inducing oxidative stress. In this study, we show for the first time the opposing effects of the low and high dose of Vitamin C and vitamin B3 on colon CSCs isolated from HT-29 and HCT-15 colorectal carcinoma cell lines. At small doses, both of these vitamins exerted a cell proliferative effect only on CSCs, while there was no change in the proliferation status of non-stem cancer cells and wild-type (WT) populations. On the other hand, the death effects induced by high doses of Vitamin C and B3 were of the order of 50-60% and ∼30% on CSCs from HT-29 and HCT15, respectively. Interestingly, the control fibroblast cell line (NIH3T3) was highly refractory all the tested concentrations of Vitamin C and B3, except for the highest dose - 10,000 μg of Vitamin C that induced only 15% of cell death. Hence, these results indicate the future scope of use of therapeutic doses of Vitamin C and B3 especially in patients with advanced colorectal cancer. © 2017 International Federation for Cell Biology.

  15. Faculty Communication Knowledge, Attitudes, and Skills Around Chronic Non-Malignant Pain Improve with Online Training.

    Science.gov (United States)

    Donovan, Anna K; Wood, Gordon J; Rubio, Doris M; Day, Hollis D; Spagnoletti, Carla L

    2016-11-01

    Many physicians struggle to communicate with patients with chronic, non-malignant pain (CNMP). Through the use of a Web module, the authors aimed to improve faculty participants' communication skills knowledge and confidence, use of skills in clinical practice, and actual communication skills. The module was implemented for faculty development among clinician-educators with university faculty appointments, outpatient clinical practices, and teaching roles. Participants completed the Collaborative Opioid Prescribing Education Risk Evaluation and Mitigation Strategy (COPE-REMS®) module, a free Web module designed to improve provider communication around opioid prescribing. Main study outcomes were improvements in CNMP communication knowledge, attitudes, and skills. Skills were assessed by comparing a subset of participants' Observed Structured Clinical Exam (OSCE) performance before and after the curriculum. Sixty-two percent of eligible participants completed the curriculum in 2013. Knowledge-based test scores improved with curriculum completion (75% vs. 90%; P communication skills on the OSCE improved after the curriculum (mean 67% vs. 79%, P = 0.03). Experienced clinician-educators improved their communication knowledge, attitudes, and skills in managing patients with CNMP after implementation of this curriculum. The improvements in attitudes were sustained at six months. A Web-based curriculum such as COPE-REMS® may be useful for other programs seeking improvement in faculty communication with patients who have CNMP. © 2016 American Academy of Pain Medicine. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  16. Polysomnographic characteristics in nonmalignant chronic pain populations: A review of controlled studies.

    Science.gov (United States)

    Bjurstrom, Martin F; Irwin, Michael R

    2016-04-01

    Sleep and pain are critical homeostatic systems that interact in a bidirectional manner. Complaints of sleep disturbance are ubiquitous among patients with chronic pain disorders, and conversely, patients with persistent insomnia symptoms commonly report suffering from chronic pain. Sleep deprivation paradigms demonstrate that partial or complete sleep loss induce hyperalgesia, possibly due to shared mechanistic pathways including neuroanatomic and molecular substrates. Further, chronic pain conditions and sleep disturbances are intertwined through comorbidities, which together cause detrimental psychological and physical consequences. This critical review examines 29 polysomnography studies to evaluate whether nonmalignant chronic pain patients, as compared to controls, show differences in objective measures of sleep continuity and sleep architecture. Whereas these controlled studies did not reveal a consistent pattern of objective sleep disturbances, alterations of sleep continuity were commonly reported. Alterations of sleep architecture such as increases in light sleep or decreases in slow-wave sleep were less commonly reported and findings were mixed and also inconsistent. Methodological flaws were identified, which complicated interpretation and limited conclusions; hence, recommendations for future research are suggested. Knowledge of abnormalities in the sleep process has implications for understanding the pathophysiology of chronic pain conditions, which might also direct the development of novel intervention strategies. Copyright © 2015 Elsevier Ltd. All rights reserved.

  17. Radiotherapy for non-malignant disorders: state of the art and update of the evidence-based practice guidelines

    Science.gov (United States)

    Micke, O; Muecke, R

    2015-01-01

    Every year in Germany about 50,000 patients are referred and treated by radiotherapy (RT) for “non-malignant disorders”. This highly successful treatment is applied only for specific indications such as preservation or recovery of the quality of life by means of pain reduction or resolution and/or an improvement of formerly impaired physical body function owing to specific disease-related symptoms. Since 1995, German radiation oncologists have treated non-malignant disorders according to national consensus guidelines; these guidelines were updated and further developed over 3 years by implementation of a systematic consensus process to achieve national upgraded and accepted S2e clinical practice guidelines. Throughout this process, international standards of evaluation were implemented. This review summarizes most of the generally accepted indications for the application of RT for non-malignant diseases and presents the special treatment concepts. The following disease groups are addressed: painful degenerative skeletal disorders, hyperproliferative disorders and symptomatic functional disorders. These state of the art guidelines may serve as a platform for daily clinical work; they provide a new starting point for quality assessment, future clinical research, including the design of prospective clinical trials, and outcome research in the underrepresented and less appreciated field of RT for non-malignant disorders. PMID:25955230

  18. Multifunctional fluorescent iron quantum clusters for non-invasive radiofrequency ablationof cancer cells.

    Science.gov (United States)

    Jose, Akhila; Surendran, Mrudula; Fazal, Sajid; Prasanth, Bindhu-Paul; Menon, Deepthy

    2018-05-01

    This work reports the potential of iron quantum clusters (FeQCs) as a hyperthermia agent for cancer, by testing its in-vitro response to shortwave (MHz range), radiofrequency (RF) waves non-invasively. Stable, fluorescent FeQCs of size ∼1 nm prepared by facile aqueous chemistry from endogenous protein haemoglobin were found to give a high thermal response, with a ΔT ∼50 °C at concentrationsas low as165 μg/mL. The as-prepared nanoclusters purified by lyophilization as well as dialysis showed a concentration, power and time-dependent RF response, with the lyophilized FeQCs exhibiting pronounced heating effects. FeQCs were found to be cytocompatible to NIH-3T3 fibroblast and 4T1 cancer cells treated at concentrations upto 1000 μg/mL for 24 h. Upon incubation with FeQCs and exposure to RF waves, significant cancer cell death was observed which proves its therapeutic ability. The fluorescent ability of the clusters could additionally be utilized for imaging cancer cells upon excitation at ∼450 nm. Further, to demonstrate the feasibility of imparting additional functionality such as drug/biomolecule/dye loading to FeQCs, they were self assembled with cationic polymers to form nanoparticles. Self assembly did not alter the RF heating potential of FeQCs and additionally enhanced its fluorescence. The multifunctional fluorescent FeQCs therefore show good promise as a novel therapeutic agent for RF hyperthermia and drug loading. Copyright © 2018 Elsevier B.V. All rights reserved.

  19. Hematopoietic Stem Cell Transplant for High Risk Hemoglobinopathies

    Science.gov (United States)

    2017-12-03

    Sickle Cell Disease; Transfusion Dependent Alpha- or Beta- Thalassemia; Diamond Blackfan Anemia; Paroxysmal Nocturnal Hemoglobinuria; Glanzmann Thrombasthenia; Severe Congenital Neutropenia; Shwachman-Diamond Syndrome; Non-Malignant Hematologic Disorders

  20. Hydrophobic and electrostatic interactions between cell penetrating peptides and plasmid DNA are important for stable non-covalent complexation and intracellular delivery.

    Science.gov (United States)

    Upadhya, Archana; Sangave, Preeti C

    2016-10-01

    Cell penetrating peptides are useful tools for intracellular delivery of nucleic acids. Delivery of plasmid DNA, a large nucleic acid, poses a challenge for peptide mediated transport. The paper investigates and compares efficacy of five novel peptide designs for complexation of plasmid DNA and subsequent delivery into cells. The peptides were designed to contain reported DNA condensing agents and basic cell penetrating sequences, octa-arginine (R 8 ) and CHK 6 HC coupled to cell penetration accelerating peptides such as Bax inhibitory mutant peptide (KLPVM) and a peptide derived from the Kaposi fibroblast growth factor (kFGF) membrane translocating sequence. A tryptophan rich peptide, an analogue of Pep-3, flanked with CH 3 on either ends was also a part of the study. The peptides were analysed for plasmid DNA complexation, protection of peptide-plasmid DNA complexes against DNase I, serum components and competitive ligands by simple agarose gel electrophoresis techniques. Hemolysis of rat red blood corpuscles (RBCs) in the presence of the peptides was used as a measure of peptide cytotoxicity. Plasmid DNA delivery through the designed peptides was evaluated in two cell lines, human cervical cancer cell line (HeLa) and (NIH/3 T3) mouse embryonic fibroblasts via expression of the secreted alkaline phosphatase (SEAP) reporter gene. The importance of hydrophobic sequences in addition to cationic sequences in peptides for non-covalent plasmid DNA complexation and delivery has been illustrated. An alternative to the employment of fatty acid moieties for enhanced gene transfer has been proposed. Comparison of peptides for plasmid DNA complexation and delivery of peptide-plasmid DNA complexes to cells estimated by expression of a reporter gene, SEAP. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.

  1. Murine hematopoietic stem cell dormancy controlled by induction of a novel short form of PSF1 by histone deacetylase inhibitors

    International Nuclear Information System (INIS)

    Han, Yinglu; Gong, Zhi-Yuan; Takakura, Nobuyuki

    2015-01-01

    Hematopoietic stem cells (HSCs) can survive long-term in a state of dormancy. Little is known about how histone deacetylase inhibitors (HDACi) affect HSC kinetics. Here, we use trichostatin A (TSA), a histone deacetylase inhibitor, to enforce histone acetylation and show that this suppresses cell cycle entry by dormant HSCs. Previously, we found that haploinsufficiency of PSF1, a DNA replication factor, led to attenuation of the bone marrow (BM) HSC pool size and lack of acute proliferation after 5-FU ablation. Because PSF1 protein is present in CD34 + transiently amplifying HSCs but not in CD34 − long-term reconstituting-HSCs which are resting in a dormant state, we analyzed the relationship between dormancy and PSF1 expression, and how a histone deacetylase inhibitor affects this. We found that CD34 + HSCs produce long functional PSF1 (PSF1a) but CD34 − HSCs produce a shorter possibly non-functional PSF1 (PSF1b, c, dominantly PSF1c). Using PSF1a-overexpressing NIH-3T3 cells in which the endogenous PSF1 promoter is suppressed, we found that TSA treatment promotes production of the shorter form of PSF1 possibly by inducing recruitment of E2F family factors upstream of the PSF1 transcription start site. Our data document one mechanism by which histone deacetylase inhibitors affect the dormancy of HSCs by regulating the DNA replication factor PSF1. - Highlights: • Hematopoetic stem cell dormancy is controlled by histone deacetylation inhibitors. • Dormancy of HSCs is associated with a shorter form of non-functional PSF1. • Histone deacetylase inhibitors suppress PSF1 promoter activity

  2. Murine hematopoietic stem cell dormancy controlled by induction of a novel short form of PSF1 by histone deacetylase inhibitors

    Energy Technology Data Exchange (ETDEWEB)

    Han, Yinglu; Gong, Zhi-Yuan [Department of Signal Transduction, Research Institute for Microbial Diseases, Osaka University, 3-1 Yamada-oka, Suita, Osaka 565-0871 (Japan); Takakura, Nobuyuki, E-mail: ntakaku@biken.osaka-u.ac.jp [Department of Signal Transduction, Research Institute for Microbial Diseases, Osaka University, 3-1 Yamada-oka, Suita, Osaka 565-0871 (Japan); Japan Science Technology Agency, CREST, K' s Gobancho, 7, Gobancho, Chiyoda-ku, Tokyo 102-0076 (Japan)

    2015-06-10

    Hematopoietic stem cells (HSCs) can survive long-term in a state of dormancy. Little is known about how histone deacetylase inhibitors (HDACi) affect HSC kinetics. Here, we use trichostatin A (TSA), a histone deacetylase inhibitor, to enforce histone acetylation and show that this suppresses cell cycle entry by dormant HSCs. Previously, we found that haploinsufficiency of PSF1, a DNA replication factor, led to attenuation of the bone marrow (BM) HSC pool size and lack of acute proliferation after 5-FU ablation. Because PSF1 protein is present in CD34{sup +} transiently amplifying HSCs but not in CD34{sup −} long-term reconstituting-HSCs which are resting in a dormant state, we analyzed the relationship between dormancy and PSF1 expression, and how a histone deacetylase inhibitor affects this. We found that CD34{sup +} HSCs produce long functional PSF1 (PSF1a) but CD34{sup −} HSCs produce a shorter possibly non-functional PSF1 (PSF1b, c, dominantly PSF1c). Using PSF1a-overexpressing NIH-3T3 cells in which the endogenous PSF1 promoter is suppressed, we found that TSA treatment promotes production of the shorter form of PSF1 possibly by inducing recruitment of E2F family factors upstream of the PSF1 transcription start site. Our data document one mechanism by which histone deacetylase inhibitors affect the dormancy of HSCs by regulating the DNA replication factor PSF1. - Highlights: • Hematopoetic stem cell dormancy is controlled by histone deacetylation inhibitors. • Dormancy of HSCs is associated with a shorter form of non-functional PSF1. • Histone deacetylase inhibitors suppress PSF1 promoter activity.

  3. Supercritical CO2 Foaming of Thermoplastic Materials Derived from Maize: Proof-of-Concept Use in Mammalian Cell Culture Applications

    Science.gov (United States)

    Trujillo-de Santiago, Grissel; Portales-Cabrera, Cynthia Guadalupe; Portillo-Lara, Roberto; Araiz-Hernández, Diana; Del Barone, Maria Cristina; García-López, Erika; Rojas-de Gante, Cecilia; de los Angeles De Santiago-Miramontes, María; Segoviano-Ramírez, Juan Carlos; García-Lara, Silverio; Rodríguez-González, Ciro Ángel; Alvarez, Mario Moisés; Di Maio, Ernesto; Iannace, Salvatore

    2015-01-01

    Background Foams are high porosity and low density materials. In nature, they are a common architecture. Some of their relevant technological applications include heat and sound insulation, lightweight materials, and tissue engineering scaffolds. Foams derived from natural polymers are particularly attractive for tissue culture due to their biodegradability and bio-compatibility. Here, the foaming potential of an extensive list of materials was assayed, including slabs elaborated from whole flour, the starch component only, or the protein fraction only of maize seeds. Methodology/Principal Findings We used supercritical CO2 to produce foams from thermoplasticized maize derived materials. Polyethylene-glycol, sorbitol/glycerol, or urea/formamide were used as plasticizers. We report expansion ratios, porosities, average pore sizes, pore morphologies, and pore size distributions for these materials. High porosity foams were obtained from zein thermoplasticized with polyethylene glycol, and from starch thermoplasticized with urea/formamide. Zein foams had a higher porosity than starch foams (88% and 85%, respectively) and a narrower and more evenly distributed pore size. Starch foams exhibited a wider span of pore sizes and a larger average pore size than zein (208.84 vs. 55.43 μm2, respectively). Proof-of-concept cell culture experiments confirmed that mouse fibroblasts (NIH 3T3) and two different prostate cancer cell lines (22RV1, DU145) attached to and proliferated on zein foams. Conclusions/Significance We conducted screening and proof-of-concept experiments on the fabrication of foams from cereal-based bioplastics. We propose that a key indicator of foamability is the strain at break of the materials to be foamed (as calculated from stress vs. strain rate curves). Zein foams exhibit attractive properties (average pore size, pore size distribution, and porosity) for cell culture applications; we were able to establish and sustain mammalian cell cultures on zein

  4. Supercritical CO2 foaming of thermoplastic materials derived from maize: proof-of-concept use in mammalian cell culture applications.

    Science.gov (United States)

    Trujillo-de Santiago, Grissel; Portales-Cabrera, Cynthia Guadalupe; Portillo-Lara, Roberto; Araiz-Hernández, Diana; Del Barone, Maria Cristina; García-López, Erika; Rojas-de Gante, Cecilia; de Los Angeles De Santiago-Miramontes, María; Segoviano-Ramírez, Juan Carlos; García-Lara, Silverio; Rodríguez-González, Ciro Ángel; Alvarez, Mario Moisés; Di Maio, Ernesto; Iannace, Salvatore

    2015-01-01

    Foams are high porosity and low density materials. In nature, they are a common architecture. Some of their relevant technological applications include heat and sound insulation, lightweight materials, and tissue engineering scaffolds. Foams derived from natural polymers are particularly attractive for tissue culture due to their biodegradability and bio-compatibility. Here, the foaming potential of an extensive list of materials was assayed, including slabs elaborated from whole flour, the starch component only, or the protein fraction only of maize seeds. We used supercritical CO2 to produce foams from thermoplasticized maize derived materials. Polyethylene-glycol, sorbitol/glycerol, or urea/formamide were used as plasticizers. We report expansion ratios, porosities, average pore sizes, pore morphologies, and pore size distributions for these materials. High porosity foams were obtained from zein thermoplasticized with polyethylene glycol, and from starch thermoplasticized with urea/formamide. Zein foams had a higher porosity than starch foams (88% and 85%, respectively) and a narrower and more evenly distributed pore size. Starch foams exhibited a wider span of pore sizes and a larger average pore size than zein (208.84 vs. 55.43 μm2, respectively). Proof-of-concept cell culture experiments confirmed that mouse fibroblasts (NIH 3T3) and two different prostate cancer cell lines (22RV1, DU145) attached to and proliferated on zein foams. We conducted screening and proof-of-concept experiments on the fabrication of foams from cereal-based bioplastics. We propose that a key indicator of foamability is the strain at break of the materials to be foamed (as calculated from stress vs. strain rate curves). Zein foams exhibit attractive properties (average pore size, pore size distribution, and porosity) for cell culture applications; we were able to establish and sustain mammalian cell cultures on zein foams for extended time periods.

  5. Supercritical CO2 foaming of thermoplastic materials derived from maize: proof-of-concept use in mammalian cell culture applications.

    Directory of Open Access Journals (Sweden)

    Grissel Trujillo-de Santiago

    Full Text Available Foams are high porosity and low density materials. In nature, they are a common architecture. Some of their relevant technological applications include heat and sound insulation, lightweight materials, and tissue engineering scaffolds. Foams derived from natural polymers are particularly attractive for tissue culture due to their biodegradability and bio-compatibility. Here, the foaming potential of an extensive list of materials was assayed, including slabs elaborated from whole flour, the starch component only, or the protein fraction only of maize seeds.We used supercritical CO2 to produce foams from thermoplasticized maize derived materials. Polyethylene-glycol, sorbitol/glycerol, or urea/formamide were used as plasticizers. We report expansion ratios, porosities, average pore sizes, pore morphologies, and pore size distributions for these materials. High porosity foams were obtained from zein thermoplasticized with polyethylene glycol, and from starch thermoplasticized with urea/formamide. Zein foams had a higher porosity than starch foams (88% and 85%, respectively and a narrower and more evenly distributed pore size. Starch foams exhibited a wider span of pore sizes and a larger average pore size than zein (208.84 vs. 55.43 μm2, respectively. Proof-of-concept cell culture experiments confirmed that mouse fibroblasts (NIH 3T3 and two different prostate cancer cell lines (22RV1, DU145 attached to and proliferated on zein foams.We conducted screening and proof-of-concept experiments on the fabrication of foams from cereal-based bioplastics. We propose that a key indicator of foamability is the strain at break of the materials to be foamed (as calculated from stress vs. strain rate curves. Zein foams exhibit attractive properties (average pore size, pore size distribution, and porosity for cell culture applications; we were able to establish and sustain mammalian cell cultures on zein foams for extended time periods.

  6. Aurora-A overexpression enhances cell-aggregation of Ha-ras transformants through the MEK/ERK signaling pathway

    International Nuclear Information System (INIS)

    Tseng, Ya-Shih; Lee, Jenq-Chang; Huang, Chi-Ying F; Liu, Hsiao-Sheng

    2009-01-01

    Overexpression of Aurora-A and mutant Ras (Ras V12 ) together has been detected in human bladder cancer tissue. However, it is not clear whether this phenomenon is a general event or not. Although crosstalk between Aurora-A and Ras signaling pathways has been reported, the role of these two genes acting together in tumorigenesis remains unclear. Real-time PCR and sequence analysis were utilized to identify Ha- and Ki-ras mutation (Gly -> Val). Immunohistochemistry staining was used to measure the level of Aurora-A expression in bladder and colon cancer specimens. To reveal the effect of overexpression of the above two genes on cellular responses, mouse NIH3T3 fibroblast derived cell lines over-expressing either Ras V12 and wild-type Aurora-A (designated WT) or Ras V12 and kinase-inactivated Aurora-A (KD) were established. MTT and focus formation assays were conducted to measure proliferation rate and focus formation capability of the cells. Small interfering RNA, pharmacological inhibitors and dominant negative genes were used to dissect the signaling pathways involved. Overexpression of wild-type Aurora-A and mutation of Ras V12 were detected in human bladder and colon cancer tissues. Wild-type Aurora-A induces focus formation and aggregation of the Ras V12 transformants. Aurora-A activates Ral A and the phosphorylation of AKT as well as enhances the phosphorylation of MEK, ERK of WT cells. Finally, the Ras/MEK/ERK signaling pathway is responsible for Aurora-A induced aggregation of the Ras V12 transformants. Wild-type-Aurora-A enhances focus formation and aggregation of the Ras V12 transformants and the latter occurs through modulating the Ras/MEK/ERK signaling pathway

  7. Coatings of Different Carbon Nanotubes on Platinum Electrodes for Neuronal Devices: Preparation, Cytocompatibility and Interaction with Spiral Ganglion Cells.

    Science.gov (United States)

    Burblies, Niklas; Schulze, Jennifer; Schwarz, Hans-Christoph; Kranz, Katharina; Motz, Damian; Vogt, Carla; Lenarz, Thomas; Warnecke, Athanasia; Behrens, Peter

    2016-01-01

    Cochlear and deep brain implants are prominent examples for neuronal prostheses with clinical relevance. Current research focuses on the improvement of the long-term functionality and the size reduction of neural interface electrodes. A promising approach is the application of carbon nanotubes (CNTs), either as pure electrodes but especially as coating material for electrodes. The interaction of CNTs with neuronal cells has shown promising results in various studies, but these appear to depend on the specific type of neurons as well as on the kind of nanotubes. To evaluate a potential application of carbon nanotube coatings for cochlear electrodes, it is necessary to investigate the cytocompatibility of carbon nanotube coatings on platinum for the specific type of neuron in the inner ear, namely spiral ganglion neurons. In this study we have combined the chemical processing of as-delivered CNTs, the fabrication of coatings on platinum, and the characterization of the electrical properties of the coatings as well as a general cytocompatibility testing and the first cell culture investigations of CNTs with spiral ganglion neurons. By applying a modification process to three different as-received CNTs via a reflux treatment with nitric acid, long-term stable aqueous CNT dispersions free of dispersing agents were obtained. These were used to coat platinum substrates by an automated spray-coating process. These coatings enhance the electrical properties of platinum electrodes, decreasing the impedance values and raising the capacitances. Cell culture investigations of the different CNT coatings on platinum with NIH3T3 fibroblasts attest an overall good cytocompatibility of these coatings. For spiral ganglion neurons, this can also be observed but a desired positive effect of the CNTs on the neurons is absent. Furthermore, we found that the well-established DAPI staining assay does not function on the coatings prepared from single-wall nanotubes.

  8. Development, Characterization and Cell Cultural Response of 3D Biocompatible Micro-Patterned Poly-ε-Caprolactone Scaffolds Designed and Fabricated Integrating Lithography and Micromolding Fabrication Techniques

    KAUST Repository

    Limongi, Tania; Miele, Ermanno; Shalabaeva, Victoria; Rocca, Rosanna La; Schipani, Rossana; Malara, Natalia; Angelis, Francesco de; Giugni, Andrea; Di Fabrizio, Enzo M.

    2014-01-01

    Scaffold design and fabrication are very important subjects for biomaterial, tissue engineering and regenerative medicine research playing a unique role in tissue regeneration and repair. Among synthetic biomaterials Poly-ε- Caprolactone (PCL) is very attractive bioresorbable polyester due to its high permeability, biodegradability and capacity to be blended with other biopolymers. Thanks to its ability to naturally degrade in tissues, PCL has a great potential as a new material for implantable biomedical micro devices. This work focuses on the establishment of a micro fabrication process, by integrating lithography and micromolding fabrication techniques, for the realization of 3D microstructure PCL devices. Scaffold surface exhibits a combination in the patterned length scale; cylindrical pillars of 10 μm height and 10 μm diameter are arranged in a hexagonal lattice with periodicity of 30 μm and their sidewalls are nano-sculptured, with a regular pattern of grooves leading to a spatial modulation in the z direction. In order to demonstrate that these biocompatible pillared PCL substrates are suitable for a proper cell growth, NIH/3T3 mouse embryonic fibroblasts were seeded on them and cells key adhesion parameters were evaluated. Scanning Electron Microscopy and immunofluorescence analysis were carried out to check cell survival, proliferation and adhesion; cells growing on the PCL substrates appeared healthy and formed a well-developed network in close contact with the micro and nano features of the pillared surface. Those 3D scaffolds could be a promising solution for a wide range of applications within tissue engineering and regenerative medicine applications.

  9. Development, Characterization and Cell Cultural Response of 3D Biocompatible Micro-Patterned Poly-ε-Caprolactone Scaffolds Designed and Fabricated Integrating Lithography and Micromolding Fabrication Techniques

    KAUST Repository

    Limongi, Tania

    2014-12-12

    Scaffold design and fabrication are very important subjects for biomaterial, tissue engineering and regenerative medicine research playing a unique role in tissue regeneration and repair. Among synthetic biomaterials Poly-ε- Caprolactone (PCL) is very attractive bioresorbable polyester due to its high permeability, biodegradability and capacity to be blended with other biopolymers. Thanks to its ability to naturally degrade in tissues, PCL has a great potential as a new material for implantable biomedical micro devices. This work focuses on the establishment of a micro fabrication process, by integrating lithography and micromolding fabrication techniques, for the realization of 3D microstructure PCL devices. Scaffold surface exhibits a combination in the patterned length scale; cylindrical pillars of 10 μm height and 10 μm diameter are arranged in a hexagonal lattice with periodicity of 30 μm and their sidewalls are nano-sculptured, with a regular pattern of grooves leading to a spatial modulation in the z direction. In order to demonstrate that these biocompatible pillared PCL substrates are suitable for a proper cell growth, NIH/3T3 mouse embryonic fibroblasts were seeded on them and cells key adhesion parameters were evaluated. Scanning Electron Microscopy and immunofluorescence analysis were carried out to check cell survival, proliferation and adhesion; cells growing on the PCL substrates appeared healthy and formed a well-developed network in close contact with the micro and nano features of the pillared surface. Those 3D scaffolds could be a promising solution for a wide range of applications within tissue engineering and regenerative medicine applications.

  10. Meta-ethnography to understand healthcare professionals’ experience of treating adults with chronic non-malignant pain

    Science.gov (United States)

    Seers, Kate; Barker, Karen L

    2017-01-01

    Objectives We aimed to explore healthcare professionals’ experience of treating chronic non-malignant pain by conducting a qualitative evidence synthesis. Understanding this experience from the perspective of healthcare professionals will contribute to improvements in the provision of care. Design Qualitative evidence synthesis using meta-ethnography. We searched five electronic bibliographic databases from inception to November 2016. We included studies that explore healthcare professionals’ experience of treating adults with chronic non-malignant pain. We used the GRADE-CERQual framework to rate confidence in review findings. Results We screened the 954 abstracts and 184 full texts and included 77 published studies reporting the experiences of over 1551 international healthcare professionals including doctors, nurses and other health professionals. We abstracted six themes: (1) a sceptical cultural lens, (2) navigating juxtaposed models of medicine, (3) navigating the geography between patient and clinician, (4) challenge of dual advocacy, (5) personal costs and (6) the craft of pain management. We rated confidence in review findings as moderate to high. Conclusions This is the first qualitative evidence synthesis of healthcare professionals’ experiences of treating people with chronic non-malignant pain. We have presented a model that we developed to help healthcare professionals to understand, think about and modify their experiences of treating patients with chronic pain. Our findings highlight scepticism about chronic pain that might explain why patients feel they are not believed. Findings also indicate a dualism in the biopsychosocial model and the complexity of navigating therapeutic relationships. Our model may be transferable to other patient groups or situations. PMID:29273663

  11. A mega-ethnography of eleven qualitative evidence syntheses exploring the experience of living with chronic non-malignant pain.

    Science.gov (United States)

    Toye, Fran; Seers, Kate; Hannink, Erin; Barker, Karen

    2017-08-01

    Each year over five million people develop chronic non-malignant pain and can experience healthcare as an adversarial struggle. The aims of this study were: (1) to bring together qualitative evidence syntheses that explore patients' experience of living with chronic non-malignant pain and develop conceptual understanding of what it is like to live with chronic non-malignant pain for improved healthcare; (2) to undertake the first mega-ethnography of qualitative evidence syntheses using the methods of meta-ethnography. We used the seven stages of meta-ethnography refined for large studies. The innovation of mega-ethnography is to use conceptual findings from qualitative evidence syntheses as primary data. We searched 7 bibliographic databases from inception until February 2016 to identify qualitative evidence syntheses that explored patients' experience of living with chronic non-malignant pain. We identified 82 potential studies from 556 titles, screened 34 full text articles and included 11 qualitative evidence syntheses synthesising a total of 187 qualitative studies reporting more than 5000 international participants living with chronic pain. We abstracted concepts into 7 conceptual categories: (1) my life is impoverished and confined; (2) struggling against my body to be me; (3) the quest for the diagnostic 'holy grail'; (4) lost personal credibility; (5) trying to keep up appearances; (6) need to be treated with dignity; and (7) deciding to end the quest for the grail is not easy. Each conceptual category was supported by at least 7 of the 11 qualitative evidence syntheses. This is the first mega-ethnography, or synthesis of qualitative evidence syntheses using the methods of meta-ethnography. Findings help us to understand that the decision to end the quest for a diagnosis can leave patients feeling vulnerable and this may contribute to the adversarial nature of the clinical encounter. This knowledge demonstrates that treating a patient with a sense that they

  12. Pharmacological consequences of long-term morphine treatment in patients with cancer and chronic non-malignant pain

    DEFF Research Database (Denmark)

    Andersen, Gertrud; Sjøgren, Per; Hansen, Steen Honoré

    2004-01-01

    In patients with pain of malignant origin morphine may be administered in high and often increasing doses during extended periods of time. In patients with chronic pain of non-malignant origin morphine may be an important remedy, and in these cases the goal is to keep the morphine dose stable. Th....... The pharmacokinetic as well as the pharmacodynamic consequences of long-term morphine treatment with special reference to the two most important metabolites of morphine morphine-6-glucuronide (M-6-G) and morphine-3-glucuronide (M-3-G) remain to be settled....

  13. Oral opioid administration and hyperalgesia in patients with cancer or chronic nonmalignant pain.

    Science.gov (United States)

    Reznikov, Igor; Pud, Dorit; Eisenberg, Elon

    2005-09-01

    Previous research has reported on reduced paw withdrawal latencies to heat and mechanical stimuli after parenteral administration of opioids in animals and on increased pain sensitivity in humans subsequent to postoperative infusions of short-acting opioids or in drug addicts. The aim of the present study was to explore the possibility that oral opioid treated patients with cancer-related or chronic nonmalignant pain differ in their pain sensitivity from patients treated with non-opioid analgesics. The study population consisted of 224 patients, including 142 in the opioid-treated group and 82 in the non-opioid-treated group. Pain thresholds for punctuate measured by von Frey filaments (g), mechanical pressure measured by pressure algometer (mmHg), heat stimuli measured by quantitative sensory testing (degrees C), as well as suprathreshold tonic heat pain intensity (46.5 degrees C for 1 min) measured by 0-10 numerical pain scale (NPS) were obtained at a nonpainful site (thenar eminence) in all patients. No differences between the groups were found for gender, age, duration of pain, or duration of treatment (independent variables). No significant differences between the groups were found in punctuate (difference = 17.0 g (95% CI -8.8, 42.8), P = 0.19), pressure (2.2 mmHg (-28.7, 33.2), P = 0.89) and heat (-0.3 degrees C (-1.5, 0.9), P = 0.70) pain thresholds, or in suprathreshold heat pain intensity (difference between maximal pain intensities -0.4 NPS units (95% CI -1.2, 0.4), P = 0.31). Pearson correlations within the opioid-treated group failed to show significant relationships between any of the independent variables and the outcome measures. A further comparison of the outcomes between the 'weak' opioid-treated subgroup and the 'strong' opioid-treated subgroup again revealed insignificant results. These results suggest that the administration of 'commonly used' dosages of oral opioids does not result in abnormal pain sensitivity beyond that of patients

  14. Enantioselective cellular localisation of europium(iii) coordination complexes.

    Science.gov (United States)

    Frawley, Andrew T; Linford, Holly V; Starck, Matthieu; Pal, Robert; Parker, David

    2018-01-28

    The selective mitochondrial localisation of the Λ enantiomer of three different emissive europium(iii) complexes in NIH 3T3 and MCF7 cells contrasts with the behaviour of the Δ enantiomer, for which a predominant lysosomal localisation was observed by confocal microscopy. In each case, cell uptake occurs via macropinocytosis.

  15. Journal of Chemical Sciences | Indian Academy of Sciences

    Indian Academy of Sciences (India)

    Hence, an anticancer agent conjugated to them may render more toxicity in cancer cells due to higher uptake. ... It acts by inducing apoptosis through G2/M phase arrest and encouragingly, is much less toxic to nontumorigenic human embryonic kidney (HEK-293T) and mouse embryonic fibroblast (NIH 3T3) cell lines in vitro ...

  16. In vitro Protective Effect of Ganoderol A Isolated from ...

    African Journals Online (AJOL)

    Apoptosis and cell-cycle arrest of NIH/3T3 fibroblast cells were assayed by ... by chronic exposure to solar UV radiation, is the most critical factor in the ..... This work was supported by Key Projects in the. National ... Mechanisms of photoaging.

  17. HIV-1 Vif's Capacity To Manipulate the Cell Cycle Is Species Specific.

    Science.gov (United States)

    Evans, Edward L; Becker, Jordan T; Fricke, Stephanie L; Patel, Kishan; Sherer, Nathan M

    2018-04-01

    Cells derived from mice and other rodents exhibit profound blocks to HIV-1 virion production, reflecting species-specific incompatibilities between viral Tat and Rev proteins and essential host factors cyclin T1 (CCNT1) and exportin-1 (XPO1, also known as CRM1), respectively. To determine if mouse cell blocks other than CCNT1 and XPO1 affect HIV's postintegration stages, we studied HIV-1 NL4-3 gene expression in mouse NIH 3T3 cells modified to constitutively express HIV-1-compatible versions of CCNT1 and XPO1 (3T3.CX cells). 3T3.CX cells supported both Rev-independent and Rev-dependent viral gene expression and produced relatively robust levels of virus particles, confirming that CCNT1 and XPO1 represent the predominant blocks to these stages. Unexpectedly, however, 3T3.CX cells were remarkably resistant to virus-induced cytopathic effects observed in human cell lines, which we mapped to the viral protein Vif and its apparent species-specific capacity to induce G 2 /M cell cycle arrest. Vif was able to mediate rapid degradation of human APOBEC3G and the PPP2R5D regulatory B56 subunit of the PP2A phosphatase holoenzyme in mouse cells, thus demonstrating that Vif NL4-3 's modulation of the cell cycle can be functionally uncoupled from some of its other defined roles in CUL5-dependent protein degradation. Vif was also unable to induce G 2 /M cell cycle arrest in other nonhuman cell types, including cells derived from nonhuman primates, leading us to propose that one or more human-specific cofactors underpin Vif's ability to modulate the cell cycle. IMPORTANCE Cells derived from mice and other rodents exhibit profound blocks to HIV-1 replication, thus hindering the development of a low-cost small-animal model for studying HIV/AIDS. Here, we engineered otherwise-nonpermissive mouse cells to express HIV-1-compatible versions of two species-specific host dependency factors, cyclin T1 (CCNT1) and exportin-1 (XPO1) (3T3.CX cells). We show that 3T3.CX cells rescue HIV-1

  18. Stimulation of MMP-11 (stromelysin-3) expression in mouse fibroblasts by cytokines, collagen and co-culture with human breast cancer cell lines

    International Nuclear Information System (INIS)

    Selvey, Saxon; Haupt, Larisa M; Thompson, Erik W; Matthaei, Klaus I; Irving, Michael G; Griffiths, Lyn R

    2004-01-01

    Matrix metalloproteinases (MMPs) are central to degradation of the extracellular matrix and basement membrane during both normal and carcinogenic tissue remodeling. MT1-MMP (MMP-14) and stromelysin-3 (MMP-11) are two members of the MMP family of proteolytic enzymes that have been specifically implicated in breast cancer progression. Expressed in stromal fibroblasts adjacent to epithelial tumour cells, the mechanism of MT1-MMP and MMP-11 induction remains unknown. To investigate possible mechanisms of induction, we examined the effects of a number of plausible regulatory agents and treatments that may physiologically influence MMP expression during tumour progression. Thus NIH3T3 and primary mouse embryonic fibroblasts (MEFs) were: a) treated with the cytokines IL-1β, IL-2, IL-6, IL-8 and TGF-β for 3, 6, 12, 24, and 48 hours; b) grown on collagens I, IV and V; c) treated with fibronectin, con-A and matrigel; and d) co-cultured with a range of HBC (human breast cancer) cell lines of varied invasive and metastatic potential. Competitive quantitative RT-PCR indicated that MMP-11 expression was stimulated to a level greater than 100%, by 48 hour treatments of IL-1β, IL-2, TGF-β, fibronectin and collagen V. No other substantial changes in expression of MMP-11 or MT1-MMP in either tested fibroblast culture, under any treatment conditions, were observed. We have demonstrated significant MMP-11 stimulation in mouse fibroblasts using cytokines, matrix constituents and HBC cell lines, and also some inhibition of MT1-MMP. Our data suggest that the regulation of these genes in the complex stromal-epithelial interactions that occur in human breast carcinoma, is influenced by several mechanisms

  19. Stimulation of MMP-11 (stromelysin-3 expression in mouse fibroblasts by cytokines, collagen and co-culture with human breast cancer cell lines

    Directory of Open Access Journals (Sweden)

    Matthaei Klaus I

    2004-07-01

    Full Text Available Abstract Background Matrix metalloproteinases (MMPs are central to degradation of the extracellular matrix and basement membrane during both normal and carcinogenic tissue remodeling. MT1-MMP (MMP-14 and stromelysin-3 (MMP-11 are two members of the MMP family of proteolytic enzymes that have been specifically implicated in breast cancer progression. Expressed in stromal fibroblasts adjacent to epithelial tumour cells, the mechanism of MT1-MMP and MMP-11 induction remains unknown. Methods To investigate possible mechanisms of induction, we examined the effects of a number of plausible regulatory agents and treatments that may physiologically influence MMP expression during tumour progression. Thus NIH3T3 and primary mouse embryonic fibroblasts (MEFs were: a treated with the cytokines IL-1β, IL-2, IL-6, IL-8 and TGF-β for 3, 6, 12, 24, and 48 hours; b grown on collagens I, IV and V; c treated with fibronectin, con-A and matrigel; and d co-cultured with a range of HBC (human breast cancer cell lines of varied invasive and metastatic potential. Results Competitive quantitative RT-PCR indicated that MMP-11 expression was stimulated to a level greater than 100%, by 48 hour treatments of IL-1β, IL-2, TGF-β, fibronectin and collagen V. No other substantial changes in expression of MMP-11 or MT1-MMP in either tested fibroblast culture, under any treatment conditions, were observed. Conclusion We have demonstrated significant MMP-11 stimulation in mouse fibroblasts using cytokines, matrix constituents and HBC cell lines, and also some inhibition of MT1-MMP. Our data suggest that the regulation of these genes in the complex stromal-epithelial interactions that occur in human breast carcinoma, is influenced by several mechanisms.

  20. Will Post-Transplantation Cell Therapies for Pediatric Patients Become Standard of Care?

    NARCIS (Netherlands)

    Lankester, Arjan C.; Locatelli, Franco; Bader, Peter; Rettinger, Eva; Egeler, Maarten; Katewa, Satyendra; Pulsipher, Michael A.; Nierkens, Stefan; Schultz, Kirk; Handgretinger, Rupert; Grupp, Stephan A.; Boelens, Jaap Jan; Bollard, Catherine M.

    Although allogeneic hematopoietic stem cell transplantation (HSCT) is a curative approach for many pediatric patients with hematologic malignancies and some nonmalignant disorders, some critical obstacles remain to be overcome, including relapse, engraftment failure, graft-versus-host disease

  1. Functional analysis of protein N-myristoylation: Metabolic labeling studies using three oxygen-substituted analogs of myristic acid and cultured mammalian cells provide evidence for protein-sequence-specific incorporation and analog-specific redistribution

    International Nuclear Information System (INIS)

    Johnson, D.R.; Heuckeroth, R.O.; Gordon, J.I.; Cox, A.D.; Solski, P.A.; Buss, J.E.; Devadas, B.; Adams, S.P.; Leimgruber, R.M.

    1990-01-01

    Covalent attachment of myristic acid (C14:0) to the NH 2 -terminal glycine residue of a number of cellular, viral, and oncogene-encoded proteins is essential for full expression of their biological function. Substitution of oxygen for methylene groups in this fatty acid does not produce a significant change in chain length or stereochemistry but does result in a reduction in hydrophobicity. These heteroatom-containing analogs serve as alternative substrates for mammalian myristoyl-CoA: protein N-myristoyltransferase and offer the opportunity to explore structure/function relationships of myristate in N-myristoyltransferase proteins. The authors have synthesized three tritiated analogs of myristate with oxygen substituted for methylene groups at C6, C11, and C13. Metabolic labeling studies were performed with these compounds and (i) a murine myocyte cell line (BC 3 H1), (ii) a rat fibroblast cell that produces p60 v-src (3Xsrc), or (iii) NIH 3T3 cells that have been engineered to express a fusion protein consisting of an 11-residue myristoylation signal from the Rasheed sarcoma virus (RaSV) gag protein linked to c-Ha-ras with a Cys → Ser-186 mutation. Two-dimensional gel electrophoresis of membrane and soluble fractions prepared from cell lysates revealed different patterns of incorporation of the analogs into cellular N-myristoyl proteins. The demonstration that these analogs differ in the extent to which they are incorporated and in their ability to cause redistribution of any single protein suggests that they may also have sufficient selectivity to be of potential therapeutic value

  2. Peptide array-based interaction assay of solid-bound peptides and anchorage-dependant cells and its effectiveness in cell-adhesive peptide design.

    Science.gov (United States)

    Kato, Ryuji; Kaga, Chiaki; Kunimatsu, Mitoshi; Kobayashi, Takeshi; Honda, Hiroyuki

    2006-06-01

    Peptide array, the designable peptide library covalently synthesized on cellulose support, was applied to assay peptide-cell interaction, between solid-bound peptides and anchorage-dependant cells, to study objective peptide design. As a model case, cell-adhesive peptides that could enhance cell growth as tissue engineering scaffold material, was studied. On the peptide array, the relative cell-adhesion ratio of NIH/3T3 cells was 2.5-fold higher on the RGDS (Arg-Gly-Asp-Ser) peptide spot as compared to the spot with no peptide, thus indicating integrin-mediated peptide-cell interaction. Such strong cell adhesion mediated by the RGDS peptide was easily disrupted by single residue substitution on the peptide array, thus indicating that the sequence recognition accuracy of cells was strictly conserved in our optimized scheme. The observed cellular morphological extension with active actin stress-fiber on the RGD motif-containing peptide supported our strategy that peptide array-based interaction assay of solid-bound peptide and anchorage-dependant cells (PIASPAC) could provide quantitative data on biological peptide-cell interaction. The analysis of 180 peptides obtained from fibronectin type III domain (no. 1447-1629) yielded 18 novel cell-adhesive peptides without the RGD motif. Taken together with the novel candidates, representative rules of ineffective amino acid usage were obtained from non-effective candidate sequences for the effective designing of cell-adhesive peptides. On comparing the amino acid usage of the top 20 and last 20 peptides from the 180 peptides, the following four brief design rules were indicated: (i) Arg or Lys of positively charged amino acids (except His) could enhance cell adhesion, (ii) small hydrophilic amino acids are favored in cell-adhesion peptides, (iii) negatively charged amino acids and small amino acids (except Gly) could reduce cell adhesion, and (iv) Cys and Met could be excluded from the sequence combination since they have

  3. Hypoxic conditions induce a cancer-like phenotype in human breast epithelial cells

    DEFF Research Database (Denmark)

    Vaapil, Marica; Helczynska, Karolina; Villadsen, René

    2012-01-01

    Solid tumors are less oxygenated than their tissue of origin. Low intra-tumor oxygen levels are associated with worse outcome, increased metastatic potential and immature phenotype in breast cancer. We have reported that tumor hypoxia correlates to low differentiation status in breast cancer. Less...... is known about effects of hypoxia on non-malignant cells. Here we address whether hypoxia influences the differentiation stage of non-malignant breast epithelial cells and potentially have bearing on early stages of tumorigenesis....

  4. Transjugular local thrombolysis with/without TIPS in patients with acute non-cirrhotic, non-malignant portal vein thrombosis.

    Science.gov (United States)

    Klinger, Christoph; Riecken, Bettina; Schmidt, Arthur; De Gottardi, Andrea; Meier, Benjamin; Bosch, Jaime; Caca, Karel

    2017-12-01

    Therapeutic anticoagulation is the standard treatment in patients with acute non-cirrhotic portal vein thrombosis (PVT). In critically ill patients, anticoagulation only may not suffice to achive rapid and stable recanalization. This study evaluates efficacy and safety of transjugular interventional therapy in acute non-cirrhotic PVT. This retrospective study includes 17 consecutive patients with acute noncirrhotic, non-malignant PVT. Main indication for interventional therapy was imminent intestinal infarction (n=10). Treatment consisted of a combination of transjugular thrombectomy, local fibrinolysis and - depending on thrombus resolution - transjugular intrahepatic portosystemic shunt. Recanalization was successful in 94.1%. One- and two-year secondary PV patency rates were 88.2%. Major complications (n=3) resolved spontaneously in all but one patient (heparin induced thrombocytopenia type 2 with intestinal infarction). Symptoms improved in all patients. However, segmental bowel resection had to be performed in two (11.8%). During a median follow-up of 28.6 months, no patient experienced portal hypertensive complications. Presence of JAK2 V617F mutation predicted both short-term and long-term technical success. Transjugular recanalization is safe and effective in patients with acute non-cirrhotic, non-malignant PVT. It should be considered especially in patients with imminent bowel infarction and low likelihood of recanalization following therapeutic anticoagulation. Patients with JAK2 mutation ought to be followed meticulously. Copyright © 2017 Editrice Gastroenterologica Italiana S.r.l. Published by Elsevier Ltd. All rights reserved.

  5. A new fluorescent pyrene–pyridine dithiocarbamate probe: A chemodosimeter to detect Hg{sup 2+} in pure aqueous medium and in live cells

    Energy Technology Data Exchange (ETDEWEB)

    Singh, Vikram; Srivastava, Priyanka [Department of Chemistry, Faculty of Science, Banaras Hindu University, Varanasi-221005 (India); PrakashVerma, Shiv [Centre for Genetic Disorders, Faculty of Science, Banaras Hindu University, Varanasi-221005 (India); Misra, Arvind [Department of Chemistry, Faculty of Science, Banaras Hindu University, Varanasi-221005 (India); Das, Parimal [Centre for Genetic Disorders, Faculty of Science, Banaras Hindu University, Varanasi-221005 (India); Singh, Nanhai, E-mail: nsinghbhu@gmail.com [Department of Chemistry, Faculty of Science, Banaras Hindu University, Varanasi-221005 (India)

    2014-10-15

    A new pyrene–pyridine dithiocarbamate based fluorescent chemodosimeter, potassium (pyren-1-ylmethyl)(pyridin-2-ylmethyl)dithiocarbamate (L1) has been designed and synthesized. The chemodosimeter shows high selectivity and sensitivity (5.2 ppb) for Hg{sup 2+} in pure aqueous medium in which emission intensity was quenched by ≈80% due to the formation of new cyclized species, 1. The probe behaves as a chemodosimeter for Hg{sup 2+} ions and forms Hg{sup 2+} triggered cyclised imidazoline species with approximate detection time of 50 s and exhibits both colorimetric and fluorometric changes on detection of Hg{sup 2+} ion. Color of the probe (L1) changed from green to colorless visible to the naked eye and from green to dark blue upon the addition of Hg{sup 2+} ions under UV light. The Hg{sup 2+} triggered cyclization reaction was confirmed by spectral data analysis and a single crystal structure determination of the cyclised entity 2 obtained from the model compound potassium benzyl(pyridin-2-ylmethyl) dithiocarbamate (L2). L1 finds its application for detection of Hg{sup 2+} ions on paper strips, and in BSA (bovine serum albumin) medium. L1 is also applicable for the monitoring of Hg{sup 2+} ion in NIH3T3 live cells. - Highlights: • Efficient chemodosimeter to detect Hg{sup 2+} ions in pure aqueous medium. • Hg{sup 2+} triggered cyclisation and formation of imidazoline species. • Probe exhibit both colorimetric and fluorometric changes • Probe is applicable to detect Hg{sup 2+} in live cells and on cellulose paper strips.

  6. p21-ras effector domain mutants constructed by "cassette" mutagenesis

    DEFF Research Database (Denmark)

    Stone, J C; Vass, W C; Willumsen, B M

    1988-01-01

    A series of mutations encoding single-amino-acid substitutions within the v-rasH effector domain were constructed, and the ability of the mutants to induce focal transformation of NIH 3T3 cells was studied. The mutations, which spanned codons 32 to 40, were made by a "cassette" mutagenesis...

  7. Experiment list: SRX262797 [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available 3T3_SAP1_03 || cell line=NIH3T3 fibroblasts || genotype=normal || chip antibody=SAP-1a || chip antibody vendor=Santa Cruz Biotechnolo...gy http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/eachDa

  8. Experiment list: SRX262799 [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available H3T3_SAP1_LAT || cell line=NIH3T3 fibroblasts || genotype=normal || chip antibody=SAP-1a || chip antibody vendor=Santa Cruz Biotechno...logy http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/each

  9. Experiment list: SRX262786 [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available H3T3_MRTFA_15 || cell line=NIH3T3 fibroblasts || genotype=normal || chip antibody=MRTF-A || chip antibody vendor=Santa Cruz Biotechno...logy http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/each

  10. Experiment list: SRX262791 [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available IH3T3_MRTFB_LAT || cell line=NIH3T3 fibroblasts || genotype=normal || chip antibody=MRTF-B || chip antibody vendor=Santa Cruz Biotech...nology http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/ea

  11. Experiment list: SRX262788 [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available IH3T3_MRTFA_UO || cell line=NIH3T3 fibroblasts || genotype=normal || chip antibody=MRTF-A || chip antibody vendor=Santa Cruz Biotechn...ology http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/eac

  12. Experiment list: SRX262787 [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available IH3T3_MRTFA_LAT || cell line=NIH3T3 fibroblasts || genotype=normal || chip antibody=MRTF-A || chip antibody vendor=Santa Cruz Biotech...nology http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/ea

  13. AFRRI Reports Third Quarter, July - September 1991

    Science.gov (United States)

    1991-10-01

    electrical activity induced by 11202 in isolated canine myocytes. In OAygen Radicals in Biology and Medicine (eds Simic M. G., Taylor K A., Ward J. 1...radioresistance in human osteosarcoma cells (manuscript in preparation). Since others have suggested that response to -radiation ’ c-myc among NIH 3T3

  14. Screening and Mechanism of Trapping Ligand Antagonist Peptide ...

    African Journals Online (AJOL)

    Purpose: The aim of the present study was to develop peptide H9 as an efficient antagonist of human cytomegalovirus (HCMV) chemokine receptor US28. Methods: US28 gene was amplified from HCMV, and a stable expression system was constructed using NIH/3T3 cells. Interaction between peptide H9 and receptor ...

  15. The anti-apoptotic activity associated with phosphatidylinositol transfer protein alpha activates the MAPK and Akt/PKB pathway.

    Science.gov (United States)

    Schenning, Martijn; Goedhart, Joachim; Gadella, Theodorus W J; Avram, Diana; Wirtz, Karel W A; Snoek, Gerry T

    2008-10-01

    The conditioned medium (CM) from mouse NIH3T3 fibroblast cells overexpressing phosphatidylinositol transfer protein alpha (PI-TPalpha; SPIalpha cells) demonstrates an increased anti-apoptotic activity compared with CM from wild type NIH3T3 (wtNIH3T3) cells. As previously shown, the anti-apoptotic activity acts by activating a G protein-coupled receptor, most probably a cannabinoid 1 (CB1)-like receptor as the activity was blocked by both pertussis toxin and rimonabant [M. Schenning, C.M. van Tiel, D. Van Manen, J.C. Stam, B.M. Gadella, K.W. Wirtz and G.T. Snoek, Phosphatidylinositol transfer protein alpha regulates growth and apoptosis of NIH3T3 cells: involvement of a cannabinoid 1-like receptor, J. Lipid Res. 45 (2004) 1555-1564]. The CB1 receptor appears to be expressed in mouse fibroblast cells, at levels in the order SPIalpha>wtNIH3T3>SPIbeta cells (i.e. wild type cells overexpressing PI-TPbeta). Upon incubation of SPIbeta cells with the PI-TPalpha-dependent anti-apoptotic factors, both the ERK/MAP kinase and the Akt/PKB pathway are activated in a CB1 receptor dependent manner as shown by Western blotting. In addition, activation of ERK2 was also shown by EYFP-ERK2 translocation to the nucleus, as visualized by confocal laser scanning microscopy. The subsequent activation of the anti-apoptotic transcription factor NF-kappaB is in line with the increased resistance towards UV-induced apoptosis. On the other hand, receptor activation by CM from SPIalpha cells was not linked to phospholipase C activation as the YFP-labelled C2-domain of protein kinase C was not translocated to the plasma membrane of SPIbeta cells as visualized by confocal laser scanning microscopy.

  16. Feline leukemia virus infection requires a post-receptor binding envelope-dependent cellular component.

    Science.gov (United States)

    Hussain, Naveen; Thickett, Kelly R; Na, Hong; Leung, Cherry; Tailor, Chetankumar S

    2011-12-01

    Gammaretrovirus receptors have been suggested to contain the necessary determinants to mediate virus binding and entry. Here, we show that murine NIH 3T3 and baby hamster kidney (BHK) cells overexpressing receptors for subgroup A, B, and C feline leukemia viruses (FeLVs) are weakly susceptible (10(1) to 10(2) CFU/ml) to FeLV pseudotype viruses containing murine leukemia virus (MLV) core (Gag-Pol) proteins, whereas FeLV receptor-expressing murine Mus dunni tail fibroblast (MDTF) cells are highly susceptible (10(4) to 10(6) CFU/ml). However, NIH 3T3 cells expressing the FeLV subgroup B receptor PiT1 are highly susceptible to gibbon ape leukemia virus pseudotype virus, which differs from the FeLV pseudotype viruses only in the envelope protein. FeLV resistance is not caused by a defect in envelope binding, low receptor expression levels, or N-linked glycosylation. Resistance is not alleviated by substitution of the MLV core in the FeLV pseudotype virus with FeLV core proteins. Interestingly, FeLV resistance is alleviated by fusion of receptor-expressing NIH 3T3 and BHK cells with MDTF or human TE671 cells, suggesting the absence of an additional cellular component in NIH 3T3 and BHK cells that is required for FeLV infection. The putative FeLV-specific cellular component is not a secreted factor, as MDTF conditioned medium does not alleviate the block to FeLV infection. Together, our findings suggest that FeLV infection requires an additional envelope-dependent cellular component that is absent in NIH 3T3 and BHK cells but that is present in MDTF and TE671 cells.

  17. Ectopic expression of PTTG1/securin promotes tumorigenesis in human embryonic kidney cells

    Directory of Open Access Journals (Sweden)

    Malik Mohammed T

    2005-01-01

    Full Text Available Abstract Background Pituitary tumor transforming gene1 (PTTG1 is a novel oncogene that is expressed in most tumors. It encodes a protein that is primarily involved in the regulation of sister chromatid separation during cell division. The oncogenic potential of PTTG1 has been well characterized in the mouse, particularly mouse fibroblast (NIH3T3 cells, in which it induces cell proliferation, promotes tumor formation and angiogenesis. Human tumorigenesis is a complex and a multistep process often requiring concordant expression of a number of genes. Also due to differences between rodent and human cell biology it is difficult to extrapolate results from mouse models to humans. To determine if PTTG1 functions similarly as an oncogene in humans, we have characterized its effects on human embryonic kidney (HEK293 cells. Results We report that introduction of human PTTG1 into HEK293 cells through transfection with PTTG1 cDNA resulted in increased cell proliferation, anchorage-independent growth in soft agar, and formation of tumors after subcutaneous injection of nu/nu mice. Pathologic analysis revealed that these tumors were poorly differentiated. Both analysis of HEK293 cells transiently transfected with PTTG1 cDNA and analysis of tumors developed on injection of HEK293 cells that had been stably transfected with PTTG1 cDNA indicated significantly higher levels of secretion and expression of bFGF, VEGF and IL-8 compared to HEK293 cells transfected with pcDNA3.1 vector or uninvolved tissues collected from the mice. Mutation of the proline-rich motifs at the C-terminal of PTTG1 abolished its oncogenic properties. Mice injected with this mutated PTTG1 either did not form tumors or formed very small tumors. Taken together our results suggest that PTTG1 is a human oncogene that possesses the ability to promote tumorigenesis in human cells at least in part through the regulation of expression or secretion of bFGF, VEGF and IL-8. Conclusions Our results

  18. A prototypical non-malignant epithelial model to study genome dynamics and concurrently monitor micro-RNAs and proteins in situ during oncogene-induced senescence

    DEFF Research Database (Denmark)

    Komseli, Eirini Stavroula; Pateras, Ioannis S.; Krejsgaard, Thorbjørn

    2018-01-01

    limitations achieving for the first time simultaneous detection of both a micro-RNA and a protein in the biological context of cellular senescence, utilizing the new commercially available SenTraGorTM compound. The method was applied in a prototypical human non-malignant epithelial model of oncogene...

  19. A software solution for recording circadian oscillator features in time-lapse live cell microscopy

    Directory of Open Access Journals (Sweden)

    Salmon Patrick

    2010-07-01

    Full Text Available Abstract Background Fluorescent and bioluminescent time-lapse microscopy approaches have been successfully used to investigate molecular mechanisms underlying the mammalian circadian oscillator at the single cell level. However, most of the available software and common methods based on intensity-threshold segmentation and frame-to-frame tracking are not applicable in these experiments. This is due to cell movement and dramatic changes in the fluorescent/bioluminescent reporter protein during the circadian cycle, with the lowest expression level very close to the background intensity. At present, the standard approach to analyze data sets obtained from time lapse microscopy is either manual tracking or application of generic image-processing software/dedicated tracking software. To our knowledge, these existing software solutions for manual and automatic tracking have strong limitations in tracking individual cells if their plane shifts. Results In an attempt to improve existing methodology of time-lapse tracking of a large number of moving cells, we have developed a semi-automatic software package. It extracts the trajectory of the cells by tracking theirs displacements, makes the delineation of cell nucleus or whole cell, and finally yields measurements of various features, like reporter protein expression level or cell displacement. As an example, we present here single cell circadian pattern and motility analysis of NIH3T3 mouse fibroblasts expressing a fluorescent circadian reporter protein. Using Circadian Gene Express plugin, we performed fast and nonbiased analysis of large fluorescent time lapse microscopy datasets. Conclusions Our software solution, Circadian Gene Express (CGE, is easy to use and allows precise and semi-automatic tracking of moving cells over longer period of time. In spite of significant circadian variations in protein expression with extremely low expression levels at the valley phase, CGE allows accurate and

  20. A software solution for recording circadian oscillator features in time-lapse live cell microscopy.

    Science.gov (United States)

    Sage, Daniel; Unser, Michael; Salmon, Patrick; Dibner, Charna

    2010-07-06

    Fluorescent and bioluminescent time-lapse microscopy approaches have been successfully used to investigate molecular mechanisms underlying the mammalian circadian oscillator at the single cell level. However, most of the available software and common methods based on intensity-threshold segmentation and frame-to-frame tracking are not applicable in these experiments. This is due to cell movement and dramatic changes in the fluorescent/bioluminescent reporter protein during the circadian cycle, with the lowest expression level very close to the background intensity. At present, the standard approach to analyze data sets obtained from time lapse microscopy is either manual tracking or application of generic image-processing software/dedicated tracking software. To our knowledge, these existing software solutions for manual and automatic tracking have strong limitations in tracking individual cells if their plane shifts. In an attempt to improve existing methodology of time-lapse tracking of a large number of moving cells, we have developed a semi-automatic software package. It extracts the trajectory of the cells by tracking theirs displacements, makes the delineation of cell nucleus or whole cell, and finally yields measurements of various features, like reporter protein expression level or cell displacement. As an example, we present here single cell circadian pattern and motility analysis of NIH3T3 mouse fibroblasts expressing a fluorescent circadian reporter protein. Using Circadian Gene Express plugin, we performed fast and nonbiased analysis of large fluorescent time lapse microscopy datasets. Our software solution, Circadian Gene Express (CGE), is easy to use and allows precise and semi-automatic tracking of moving cells over longer period of time. In spite of significant circadian variations in protein expression with extremely low expression levels at the valley phase, CGE allows accurate and efficient recording of large number of cell parameters, including

  1. Tracking of [{sup 18}F]FDG-labeled natural killer cells to HER2/neu-positive tumors

    Energy Technology Data Exchange (ETDEWEB)

    Meier, Reinhard [Department of Radiology, University of California San Francisco (United States)], E-mail: reinhardt.meier@gmail.com; Piert, Morand [Department of Radiology, Division of Nuclear Medicine, University of Michigan (United States); Piontek, Guido; Rudelius, Martina [Institute of Pathology, Klinikum rechts der Isar, Technische Universitaet Muenchen (Germany); Oostendorp, Robert A. [3rd Department of Internal Medicine, Klinikum rechts der Isar, Technische Universitaet Muenchen (Germany); Senekowitsch-Schmidtke, Reingard [Department of Nuclear Medicine, Klinikum rechts der Isar, Technische Universitaet Muenchen (Germany); Henning, Tobias D. [Department of Radiology, University of California San Francisco (United States); Wels, Winfried S.; Uherek, Christoph [Chemotherapeutisches Forschungsinstitut, Georg-Speyer-Haus, Frankfurt am Main (Germany); Rummeny, Ernst J. [Department of Radiology, Klinikum rechts der Isar, Technische Universitaet Muenchen (Germany); Daldrup-Link, Heike E. [Department of Radiology, University of California San Francisco (United States)

    2008-07-15

    Introduction: The objective of this study was to label the human natural killer (NK) cell line NK-92 with [{sup 18}F]fluoro-deoxy-glucose (FDG) for subsequent in vivo tracking to HER2/neu-positive tumors. Methods: NK-92 cells were genetically modified to NK-92-scFv(FRP5)-zeta cells, which express a chimeric antigen receptor that is specific to the tumor-associated ErbB2 (HER2/neu) antigen. NK-92 and NK-92-scFv(FRP5)-zeta cells were labeled with [{sup 18}F]FDG by simple incubation at different settings. Labeling efficiency was evaluated by a gamma counter. Subsequently, [{sup 18}F]FDG-labeled parental NK-92 or NK-92-scFv(FRP5)-zeta cells were intravenously injected into mice with implanted HER2/neu-positive NIH/3T3 tumors. Radioactivity in tumors was quantified by digital autoradiography and correlated with histopathology. Results: The NK-92 and NK-92-scFv(FRP5)-zeta cells could be efficiently labeled with [{sup 18}F]FDG by simple incubation. Optimal labeling efficiencies (80%) were achieved using an incubation period of 60 min and additional insulin (10 IU/ml). After injection of 5x10{sup 6} [{sup 18}F]FDG-labeled NK-92-scFv(FRP5)-zeta cells into tumor-bearing mice, digital autoradiography showed an increased uptake of radioactivity in HER2/neu-positive tumors at 60 min postinjection. Conversely, injection of 5x10{sup 6} NK-92 cells not directed against HER2/neu receptors did not result in increased uptake of radioactivity in the tumors. Histopathology confirmed an accumulation of the NK-92-scFv(FRP5)-zeta cells, but not the parental NK cells, in tumor tissues. Conclusion: The human NK cell line NK-92 can be directed against HER2/neu antigens by genetic modification. The genetically modified NK cells can be efficiently labeled with [{sup 18}F]FDG, and the accumulation of these labeled NK cells in HER2/neu-positive tumors can be monitored with autoradiography.

  2. Tracking of [18F]FDG-labeled natural killer cells to HER2/neu-positive tumors

    International Nuclear Information System (INIS)

    Meier, Reinhard; Piert, Morand; Piontek, Guido; Rudelius, Martina; Oostendorp, Robert A.; Senekowitsch-Schmidtke, Reingard; Henning, Tobias D.; Wels, Winfried S.; Uherek, Christoph; Rummeny, Ernst J.; Daldrup-Link, Heike E.

    2008-01-01

    Introduction: The objective of this study was to label the human natural killer (NK) cell line NK-92 with [ 18 F]fluoro-deoxy-glucose (FDG) for subsequent in vivo tracking to HER2/neu-positive tumors. Methods: NK-92 cells were genetically modified to NK-92-scFv(FRP5)-zeta cells, which express a chimeric antigen receptor that is specific to the tumor-associated ErbB2 (HER2/neu) antigen. NK-92 and NK-92-scFv(FRP5)-zeta cells were labeled with [ 18 F]FDG by simple incubation at different settings. Labeling efficiency was evaluated by a gamma counter. Subsequently, [ 18 F]FDG-labeled parental NK-92 or NK-92-scFv(FRP5)-zeta cells were intravenously injected into mice with implanted HER2/neu-positive NIH/3T3 tumors. Radioactivity in tumors was quantified by digital autoradiography and correlated with histopathology. Results: The NK-92 and NK-92-scFv(FRP5)-zeta cells could be efficiently labeled with [ 18 F]FDG by simple incubation. Optimal labeling efficiencies (80%) were achieved using an incubation period of 60 min and additional insulin (10 IU/ml). After injection of 5x10 6 [ 18 F]FDG-labeled NK-92-scFv(FRP5)-zeta cells into tumor-bearing mice, digital autoradiography showed an increased uptake of radioactivity in HER2/neu-positive tumors at 60 min postinjection. Conversely, injection of 5x10 6 NK-92 cells not directed against HER2/neu receptors did not result in increased uptake of radioactivity in the tumors. Histopathology confirmed an accumulation of the NK-92-scFv(FRP5)-zeta cells, but not the parental NK cells, in tumor tissues. Conclusion: The human NK cell line NK-92 can be directed against HER2/neu antigens by genetic modification. The genetically modified NK cells can be efficiently labeled with [ 18 F]FDG, and the accumulation of these labeled NK cells in HER2/neu-positive tumors can be monitored with autoradiography

  3. In vivo tracking of genetically engineered, anti-HER2/neu directed natural killer cells to HER2/neu positive mammary tumors with magnetic resonance imaging

    International Nuclear Information System (INIS)

    Daldrup-Link, Heike E.; Meier, Reinhardt; Metz, Stephan; Settles, Marcus; Rummeny, Ernst J.; Rudelius, Martina; Piontek, Guido; Schlegel, Juergen; Piert, Morand; Uherek, Christoph; Wels, Winfried

    2005-01-01

    The purpose of this study is to optimize labeling of the human natural killer (NK) cell line NK-92 with iron-oxide-based contrast agents and to monitor the in vivo distribution of genetically engineered NK-92 cells, which are directed against HER2/neu receptors, to HER2/neu positive mammary tumors with magnetic resonance (MR) imaging. Parental NK-92 cells and genetically modified HER2/neu specific NK-92-scFv(FRP5)-zeta cells, expressing a chimeric antigen receptor specific to the tumor-associated ErbB2 (HER2/neu) antigen, were labeled with ferumoxides and ferucarbotran using simple incubation, lipofection and electroporation techniques. Labeling efficiency was evaluated by MR imaging, Prussian blue stains and spectrometry. Subsequently, ferucarbotran-labeled NK-92-scFv(FRP5)-zeta (n=3) or parental NK-92 cells were intravenously injected into the tail vein of six mice with HER2/neu-positive NIH 3T3 mammary tumors, implanted in the mammary fat pad. The accumulation of the cells in the tumors was monitored by MR imaging before and 12 and 24 h after cell injection (p.i.). MR data were correlated with histopathology. Both the parental NK-92 and the genetically modified NK-92-scFv(FRP5)-zeta cells could be labeled with ferucarbotran and ferumoxides by lipofection and electroporation, but not by simple incubation. The intracellular cytoplasmatic iron-oxide uptake was significantly higher after labeling with ferucarbotran than ferumoxides (P 6 NK-92-scFv(FRP5)-zeta cells into tumor-bearing mice, MR showed a progressive signal decline in HER2/neu-positive mammary tumors at 12 and 24 h (p.i.). Conversely, injection of 5 x 10 6 parental NK-92 control cells, not directed against HER2/neu receptors, did not cause significant signal intensity changes of the tumors. Histopathology confirmed an accumulation of the former, but not the latter cells in tumor tissue. The human natural killer cell line NK-92 can be efficiently labeled with clinically applicable iron-oxide contrast

  4. In vivo tracking of genetically engineered, anti-HER2/neu directed natural killer cells to HER2/neu positive mammary tumors with magnetic resonance imaging

    Energy Technology Data Exchange (ETDEWEB)

    Daldrup-Link, Heike E. [UCSF Medical Center, Department of Radiology, San Francisco, CA (United States); Meier, Reinhardt; Metz, Stephan; Settles, Marcus; Rummeny, Ernst J. [Technical University Munich, Department of Radiology, Munich (Germany); Rudelius, Martina; Piontek, Guido; Schlegel, Juergen [Technical University Munich, Institute of Pathology, Division of Neuropathology, Munich (Germany); Piert, Morand [Technical University Munich, Department of Nuclear Medicine, Munich (Germany); Uherek, Christoph; Wels, Winfried [University of Frankfurt, Georg Speyer House, Frankfurt (Germany)

    2005-01-01

    The purpose of this study is to optimize labeling of the human natural killer (NK) cell line NK-92 with iron-oxide-based contrast agents and to monitor the in vivo distribution of genetically engineered NK-92 cells, which are directed against HER2/neu receptors, to HER2/neu positive mammary tumors with magnetic resonance (MR) imaging. Parental NK-92 cells and genetically modified HER2/neu specific NK-92-scFv(FRP5)-zeta cells, expressing a chimeric antigen receptor specific to the tumor-associated ErbB2 (HER2/neu) antigen, were labeled with ferumoxides and ferucarbotran using simple incubation, lipofection and electroporation techniques. Labeling efficiency was evaluated by MR imaging, Prussian blue stains and spectrometry. Subsequently, ferucarbotran-labeled NK-92-scFv(FRP5)-zeta (n=3) or parental NK-92 cells were intravenously injected into the tail vein of six mice with HER2/neu-positive NIH 3T3 mammary tumors, implanted in the mammary fat pad. The accumulation of the cells in the tumors was monitored by MR imaging before and 12 and 24 h after cell injection (p.i.). MR data were correlated with histopathology. Both the parental NK-92 and the genetically modified NK-92-scFv(FRP5)-zeta cells could be labeled with ferucarbotran and ferumoxides by lipofection and electroporation, but not by simple incubation. The intracellular cytoplasmatic iron-oxide uptake was significantly higher after labeling with ferucarbotran than ferumoxides (P<0.05). After intravenous injection of 5 x 10{sup 6} NK-92-scFv(FRP5)-zeta cells into tumor-bearing mice, MR showed a progressive signal decline in HER2/neu-positive mammary tumors at 12 and 24 h (p.i.). Conversely, injection of 5 x 10{sup 6} parental NK-92 control cells, not directed against HER2/neu receptors, did not cause significant signal intensity changes of the tumors. Histopathology confirmed an accumulation of the former, but not the latter cells in tumor tissue. The human natural killer cell line NK-92 can be efficiently

  5. Anemia and Long-Term Renal Prognosis in Patients with Post-Renal Acute Kidney Injury of Nonmalignant Cause.

    Science.gov (United States)

    Sasaki, Sho; Kawarazaki, Hiroo; Hasegawa, Takeshi; Shima, Hideaki; Naganuma, Toshihide; Shibagaki, Yugo

    2017-01-01

    The renal prognosis of post-renal acute kidney injury (PoR-AKI) has not been verified so far. The objective of this study was to assess the association of baseline anemia with long-term renal prognosis in patients with PoR-AKI. We performed a multicenter retrospective cohort study. Consecutive adult patients from December 2006 to February 2010, who met the requirements as mentioned in the definition of PoR-AKI, were included. Patients without data on baseline renal function and at 6 months after PoR-AKI were excluded. We set baseline hemoglobin (Hb) level (g/dl) as the main exposure to be tested. The main outcome measure was long-term renal prognosis as determined by the difference between proximate estimated glomerular filtration rate (eGFR) at 6 months after diagnosis of PoR-AKI and baseline eGFR prior to the occurrence of the present PoR-AKI (ΔeGFR after 6 months) using the general linear model. We included 136 patients with PoR-AKI. The most frequent cause of PoR-AKI was malignancy, accounting for 39.0% (n = 53) of cases. Multivariate analysis adjusted for possible confounders showed that ΔeGFR after 6 months significantly changed by -4.28 ml/min/1.73 m2 for every 1 g/dl lower Hb at diagnosis (95% CI 1.86-6.69, p < 0.01). An additional multivariate analysis that was stratified by the presence or absence of malignancy as the cause of PoR-AKI yielded the same significant result only in the stratum of the nonmalignant cause of PoR-AKI. Patients with a nonmalignant cause of PoR-AKI who have baseline anemia may have poor long-term renal prognosis. In these cases, close observation of renal function after renal recovery may be required. © 2016 S. Karger AG, Basel.

  6. Green synthesis of selenium nanoparticles using Acinetobacter sp. SW30: optimization, characterization and its anticancer activity in breast cancer cells

    Directory of Open Access Journals (Sweden)

    Wadhwani SA

    2017-09-01

    Full Text Available Sweety A Wadhwani,1 Mahadeo Gorain,2 Pinaki Banerjee,2 Utkarsha U Shedbalkar,3 Richa Singh,1 Gopal C Kundu,2 Balu A Chopade1,4 1Department of Microbiology, Savitribai Phule Pune University, 2Laboratory of Tumor Biology, Angiogenesis and Nanomedicine Research, National Center for Cell Science, Savitribai Phule Pune University Campus, Pune, 3Department of Biochemistry, The Institute of Science, Mumbai, 4Dr Babasaheb Ambedkar Marathwada University, Aurangabad, Maharashtra, India Abstract: The aim of this study was to synthesize selenium nanoparticles (SeNPs using cell suspension and total cell protein of Acinetobacter sp. SW30 and optimize its synthesis by studying the influence of physiological and physicochemical parameters. Also, we aimed to compare its anticancer activity with that of chemically synthesized SeNPs in breast cancer cells. Cell suspension of Acinetobacter sp. SW30 was exposed to various physiological and physicochemical conditions in the presence of sodium selenite to study their effects on the synthesis and morphology of SeNPs. Breast cancer cells (4T1, MCF-7 and noncancer cells (NIH/3T3, HEK293 were exposed to different concentrations of SeNPs. The 18 h grown culture with 2.7×109 cfu/mL could synthesize amorphous nanospheres of size 78 nm at 1.5 mM and crystalline nanorods at above 2.0 mM Na2SeO3 concentration. Polygonal-shaped SeNPs of average size 79 nm were obtained in the supernatant of 4 mg/mL of total cell protein of Acinetobacter sp. SW30. Chemical SeNPs showed more anticancer activity than SeNPs synthesized by Acinetobacter sp. SW30 (BSeNPs, but they were found to be toxic to noncancer cells also. However, BSeNPs were selective against breast cancer cells than chemical ones. Results suggest that BSeNPs are a good choice of selection as anticancer agents. Keywords: comparison, selective, 4T1, MCF7

  7. Safety, efficacy, and response predictors of anticoagulation for the treatment of nonmalignant portal-vein thrombosis in patients with cirrhosis: a propensity score matching analysis

    Directory of Open Access Journals (Sweden)

    Jung Wha Chung

    2014-12-01

    Full Text Available Background/AimsPortal-vein thrombosis (PVT develops in 10-25% of cirrhotic patients and may aggravate portal hypertension. There are few data regarding the effects of anticoagulation on nonmalignant PVT in liver cirrhosis. The aim of this study was to elucidate the safety, efficacy, and predictors of response to anticoagulation therapy in cirrhotic patients.MethodsPatients with liver cirrhosis and nonmalignant PVT were identified by a hospital electronic medical record system (called BESTCARE. Patients with malignant PVT, Budd-Chiari syndrome, underlying primary hematologic disorders, or preexisting extrahepatic thrombosis were excluded from the analysis. Patients were divided into two groups (treatment and nontreatment, and propensity score matching analysis was performed to identify control patients. The sizes of the thrombus and spleen were evaluated using multidetector computed tomography.ResultsTwenty-eight patients were enrolled in this study between 2003 and 2014: 14 patients who received warfarin for nonmalignant PVT and 14 patients who received no anticoagulation. After 112 days of treatment, 11 patients exhibited significantly higher response rates (complete in 6 and partial in 5 compared to the control patients, with decreases in thrombus size of >30%. Compared to nonresponders, the 11 responders were older, and had a thinner spleen and fewer episodes of previous endoscopic variceal ligations, whereas pretreatment liver function and changes in prothrombin time after anticoagulation did not differ significantly between the two groups. Two patients died after warfarin therapy, but the causes of death were not related to anticoagulation.ConclusionsWarfarin can be safely administered to cirrhotic patients with nonmalignant PVT. The presence of preexisting portal hypertension is a predictor of nonresponse to anticoagulation.

  8. Utility of FDG-PETCT and magnetic resonance spectroscopy in differentiating between cerebral lymphoma and non-malignant CNS lesions in HIV-infected patients

    Energy Technology Data Exchange (ETDEWEB)

    Westwood, Thomas D., E-mail: tdwestwood@googlemail.com [Department of Radiology, The Christie NHS Foundation Trust, Wilmslow Road, Manchester (United Kingdom); Hogan, Celia, E-mail: celiahogan@hotmail.com [Monsall Unit, Department of Infectious Diseases and Tropical Medicine, North Manchester General Hospital, Pennine Acute Hospitals NHS Trust (United Kingdom); Julyan, Peter J., E-mail: Peter.Julyan@christie.nhs.uk [Christie Medical Physics and Engineering, The Christie NHS Foundation Trust, Wilmslow Road, Manchester (United Kingdom); Coutts, Glyn, E-mail: Glyn.Coutts@christie.nhs.uk [Christie Medical Physics and Engineering, The Christie NHS Foundation Trust, Wilmslow Road, Manchester (United Kingdom); Bonington, Suzie, E-mail: suzi.bonington@christie.nhs.uk [Department of Radiology, The Christie NHS Foundation Trust, Wilmslow Road, Manchester (United Kingdom); Carrington, Bernadette, E-mail: Bernadette.Carrington@christie.nhs.uk [Department of Radiology, The Christie NHS Foundation Trust, Wilmslow Road, Manchester (United Kingdom); Taylor, Ben, E-mail: Ben.taylor@christie.nhs.uk [Department of Radiology, The Christie NHS Foundation Trust, Wilmslow Road, Manchester (United Kingdom); Khoo, Saye, E-mail: S.H.Khoo@liverpool.ac.uk [Department of Infectious Diseases and Tropical Medicine, Royal Liverpool Hospital, Liverpool (United Kingdom); Bonington, Alec, E-mail: Alec.Bonington@pat.nhs.uk [Monsall Unit, Department of Infectious Diseases and Tropical Medicine, North Manchester General Hospital, Pennine Acute Hospitals NHS Trust (United Kingdom)

    2013-08-15

    Background and purpose: In HIV infected patients, MRI cannot reliably differentiate between central nervous system (CNS) lymphoma and non-malignant CNS lesions, particularly cerebral toxoplasmosis (CTOX). This study prospectively investigates the utility of FDG PET-CT and magnetic resonance spectroscopy (MRS) in discriminating CNS lymphoma from non-malignant CNS lesions in HIV infected patients, and assesses the ability of FDG PET-CT to guide the use of early brain biopsy. Methods: 10 HIV patients with neurological symptoms and contrast enhancing lesions on MRI were commenced on anti-toxoplasmosis therapy before undergoing FDG PET-CT and MRS. Brain biopsies were sought in those with FDG PET-CT suggestive of CNS lymphoma, and in those with a negative FDG PET-CT scan who failed to respond to therapy. Final diagnosis was based on histology or treatment response. Results: Two patients were confirmed to have CNS lymphoma and FDG PET-CT was consistent with this diagnosis in both. Six patients had cerebral toxoplasmosis in all of whom FDG PET-CT was consistent with non-malignant disease. One patient had progressive multifocal leukoencephalopathy (PML), FDG PET-CT was equivocal. One patient had a haemorrhagic brain metastasis and FDG PET-CT wrongly suggested non-malignant disease. MRS was performed successfully in eight subjects: three results were suggestive of CNS lymphoma (one true positive, two false positive), four suggested CTOX (two false negative, two true negative), one scan was equivocal. Conclusion: FDG PET-CT correctly identified all cases of CNS lymphoma and CTOX, supporting its use in this situation. MRS was unhelpful in our cohort.

  9. Utility of FDG-PETCT and magnetic resonance spectroscopy in differentiating between cerebral lymphoma and non-malignant CNS lesions in HIV-infected patients

    International Nuclear Information System (INIS)

    Westwood, Thomas D.; Hogan, Celia; Julyan, Peter J.; Coutts, Glyn; Bonington, Suzie; Carrington, Bernadette; Taylor, Ben; Khoo, Saye; Bonington, Alec

    2013-01-01

    Background and purpose: In HIV infected patients, MRI cannot reliably differentiate between central nervous system (CNS) lymphoma and non-malignant CNS lesions, particularly cerebral toxoplasmosis (CTOX). This study prospectively investigates the utility of FDG PET-CT and magnetic resonance spectroscopy (MRS) in discriminating CNS lymphoma from non-malignant CNS lesions in HIV infected patients, and assesses the ability of FDG PET-CT to guide the use of early brain biopsy. Methods: 10 HIV patients with neurological symptoms and contrast enhancing lesions on MRI were commenced on anti-toxoplasmosis therapy before undergoing FDG PET-CT and MRS. Brain biopsies were sought in those with FDG PET-CT suggestive of CNS lymphoma, and in those with a negative FDG PET-CT scan who failed to respond to therapy. Final diagnosis was based on histology or treatment response. Results: Two patients were confirmed to have CNS lymphoma and FDG PET-CT was consistent with this diagnosis in both. Six patients had cerebral toxoplasmosis in all of whom FDG PET-CT was consistent with non-malignant disease. One patient had progressive multifocal leukoencephalopathy (PML), FDG PET-CT was equivocal. One patient had a haemorrhagic brain metastasis and FDG PET-CT wrongly suggested non-malignant disease. MRS was performed successfully in eight subjects: three results were suggestive of CNS lymphoma (one true positive, two false positive), four suggested CTOX (two false negative, two true negative), one scan was equivocal. Conclusion: FDG PET-CT correctly identified all cases of CNS lymphoma and CTOX, supporting its use in this situation. MRS was unhelpful in our cohort

  10. Cost-Saving Early Diagnosis of Functional Pain in Nonmalignant Pain: A Noninferiority Study of Diagnostic Accuracy.

    Science.gov (United States)

    Cámara, Rafael J A; Merz, Christian; Wegmann, Barbara; Stauber, Stefanie; von Känel, Roland; Egloff, Niklaus

    2016-01-01

    Objectives. We compared two index screening tests for early diagnosis of functional pain: pressure pain measurement by electronic diagnostic equipment, which is accurate but too specialized for primary health care, versus peg testing, which is cost-saving and more easily manageable but of unknown sensitivity and specificity. Early distinction of functional (altered pain perception; nervous sensitization) from neuropathic or nociceptive pain improves pain management. Methods. Clinicians blinded for the index screening tests assessed the reference standard of this noninferiority diagnostic accuracy study, namely, comprehensive medical history taking with all previous findings and treatment outcomes. All consenting patients referred to a university hospital for nonmalignant musculoskeletal pain participated. The main analysis compared the receiver operating characteristic (ROC) curves of both index screening tests. Results. The area under the ROC curve for peg testing was not inferior to that of electronic equipment: it was at least 95% as large for finger measures (two-sided p = 0.038) and at least equally as large for ear measures (two-sided p = 0.003). Conclusions. Routine diagnostic testing by peg, which is accessible for general practitioners, is at least as accurate as specialized equipment. This may shorten time-to-treatment in general practices, thereby improving the prognosis and quality of life.

  11. Occupational exposure to respirable crystalline silica and chronic non-malignant renal disease: systematic review and meta-analysis.

    Science.gov (United States)

    Möhner, Matthias; Pohrt, Anne; Gellissen, Johannes

    2017-10-01

    While occupational exposure to respirable silica is known to lead to lung disease, most notably silicosis, its association with chronic kidney disease is unclear. This review explores the association between occupational exposure to respirable silica and chronic non-malignant renal disease such as glomerulonephritis. The evidence has been collected and compiled. Possible sources of bias are thoroughly discussed. Cohort studies with silica exposure and case-control studies of renal disease were searched in PubMed until January 2015. Two authors independently abstracted data; any disagreement was resolved by consulting a third reviewer. A meta-analysis was performed to evaluate the association to silica exposure. A total of 23 cohort and four case-control studies were included in the analysis. The meta-analysis of cohort studies yielded elevated overall SMRs for renal disease. Some studies, however, included dose-response analyses, most of which did not show a positive trend. The approaches and results of the case-control studies were very heterogeneous. While the studies of cohorts exposed to silica found elevated SMRs for renal disease, no clear evidence of a dose-response relationship emerged. The elevated risk may be attributed to diagnostic and methodological issues. In order to permit a reliable estimation of a possible causal link, exposed cohorts should be monitored for renal disease, as the information from mortality studies is hardly reliable in this field.

  12. Bone marrow-derived CD13+ cells sustain tumor progression

    Science.gov (United States)

    Dondossola, Eleonora; Corti, Angelo; Sidman, Richard L; Arap, Wadih; Pasqualini, Renata

    2014-01-01

    Non-malignant cells found within neoplastic lesions express alanyl (membrane) aminopeptidase (ANPEP, best known as CD13), and CD13-null mice exhibit limited tumor growth and angiogenesis. We have recently demonstrated that a subset of bone marrow-derived CD11b+CD13+ myeloid cells accumulate within neoplastic lesions in several murine models of transplantable cancer to promote angiogenesis. If these findings were confirmed in clinical settings, CD11b+CD13+ myeloid cells could become a non-malignant target for the development of novel anticancer regimens. PMID:25339996

  13. A prototypical non-malignant epithelial model to study genome dynamics and concurrently monitor micro-RNAs and proteins in situ during oncogene-induced senescence.

    Science.gov (United States)

    Komseli, Eirini-Stavroula; Pateras, Ioannis S; Krejsgaard, Thorbjørn; Stawiski, Konrad; Rizou, Sophia V; Polyzos, Alexander; Roumelioti, Fani-Marlen; Chiourea, Maria; Mourkioti, Ioanna; Paparouna, Eleni; Zampetidis, Christos P; Gumeni, Sentiljana; Trougakos, Ioannis P; Pefani, Dafni-Eleftheria; O'Neill, Eric; Gagos, Sarantis; Eliopoulos, Aristides G; Fendler, Wojciech; Chowdhury, Dipanjan; Bartek, Jiri; Gorgoulis, Vassilis G

    2018-01-10

    Senescence is a fundamental biological process implicated in various pathologies, including cancer. Regarding carcinogenesis, senescence signifies, at least in its initial phases, an anti-tumor response that needs to be circumvented for cancer to progress. Micro-RNAs, a subclass of regulatory, non-coding RNAs, participate in senescence regulation. At the subcellular level micro-RNAs, similar to proteins, have been shown to traffic between organelles influencing cellular behavior. The differential function of micro-RNAs relative to their subcellular localization and their role in senescence biology raises concurrent in situ analysis of coding and non-coding gene products in senescent cells as a necessity. However, technical challenges have rendered in situ co-detection unfeasible until now. In the present report we describe a methodology that bypasses these technical limitations achieving for the first time simultaneous detection of both a micro-RNA and a protein in the biological context of cellular senescence, utilizing the new commercially available SenTraGor TM compound. The method was applied in a prototypical human non-malignant epithelial model of oncogene-induced senescence that we generated for the purposes of the study. For the characterization of this novel system, we applied a wide range of cellular and molecular techniques, as well as high-throughput analysis of the transcriptome and micro-RNAs. This experimental setting has three advantages that are presented and discussed: i) it covers a "gap" in the molecular carcinogenesis field, as almost all corresponding in vitro models are fibroblast-based, even though the majority of neoplasms have epithelial origin, ii) it recapitulates the precancerous and cancerous phases of epithelial tumorigenesis within a short time frame under the light of natural selection and iii) it uses as an oncogenic signal, the replication licensing factor CDC6, implicated in both DNA replication and transcription when over

  14. FS laser processing of bio-polymer thin films for studying cell-to-substrate specific response

    Energy Technology Data Exchange (ETDEWEB)

    Daskalova, A., E-mail: a_daskalova@code.bg [Institute of Electronics, Bulgarian Academy of Sciences, 72, Tsarigradsko Chaussee Blvd., 1784 Sofia (Bulgaria); Nathala, Chandra S.R. [Institute of General Physics, Vienna University of Technology, Wiedner Hauptstr. 8-10/134, A-1040 Wien (Austria); Spectra-Physics Vienna, Fernkorngasse 10, 1100 Wien (Austria); Kavatzikidou, P.; Ranella, A. [Institute for Electronic Structure and Lasers-FORTH, P.O. Box 1385, Vassilika Vouton, 711 10 Heraklion, Crete (Greece); Szoszkiewicz, R. [Faculty of Materials Science and Engineering, Warsaw University of Technology, 141 Woloska Str., 02-507 Warsaw, Poland (Poland); Husinsky, W. [Institute of General Physics, Vienna University of Technology, Wiedner Hauptstr. 8-10/134, A-1040 Wien (Austria); Fotakis, C. [Institute for Electronic Structure and Lasers-FORTH, P.O. Box 1385, Vassilika Vouton, 711 10 Heraklion, Crete (Greece)

    2016-09-30

    Highlights: • Systematic research in the field of fs laser interaction with biopolymers for application in tissue engineering. • Utilizing a new biopolymer blend of collagen/elastin material for studying the interaction process in the fs domain. • Obtaining of improved, circularly shaped, interconnected nanopores, with high reproducibility from collagen/elastin layer. • Observation of randomly arranged pattern outside modification zone due to formation of an impact wave over biofilm surface. • NIH/3T3 cell-interface interaction reveal a preferable cell migration on fs laser-modified surface array. - Abstract: The use of ultra-short pulses for nanoengineering of biomaterials opens up possibilities for biological, medical and tissue engineering applications. Structuring the surface of a biomaterial into arrays with micro- and nanoscale features and architectures, defines new roadmaps to innovative engineering of materials. Thin films of novel collagen/elastin composite and gelatin were irradiated by Ti:sapphire fs laser in air at central wavelength 800 nm, with pulse durations in the range of 30 fs. The size and shape as well as morphological forms occurring in the resulted areas of interaction were analyzed as a function of irradiation fluence and number of pulses by atomic force microscopy (AFM). The fs interaction regime allows generation of well defined micro porous surface arrays. In this study we examined a novel composite consisting of collagen and elastin in order to create a biodegradable matrix to serve as a biomimetic surface for cell attachment. Confocal microscopy images of modified zones reveal formation of surface fringe patterns with orientation direction alongside the area of interaction. Outside the crater rim a wave-like topography pattern is observed. Structured, on a nanometer scale, surface array is employed for cell-culture experiments for testing cell’s responses to substrate morphology. Mice fibroblasts migration was monitored

  15. FS laser processing of bio-polymer thin films for studying cell-to-substrate specific response

    International Nuclear Information System (INIS)

    Daskalova, A.; Nathala, Chandra S.R.; Kavatzikidou, P.; Ranella, A.; Szoszkiewicz, R.; Husinsky, W.; Fotakis, C.

    2016-01-01

    Highlights: • Systematic research in the field of fs laser interaction with biopolymers for application in tissue engineering. • Utilizing a new biopolymer blend of collagen/elastin material for studying the interaction process in the fs domain. • Obtaining of improved, circularly shaped, interconnected nanopores, with high reproducibility from collagen/elastin layer. • Observation of randomly arranged pattern outside modification zone due to formation of an impact wave over biofilm surface. • NIH/3T3 cell-interface interaction reveal a preferable cell migration on fs laser-modified surface array. - Abstract: The use of ultra-short pulses for nanoengineering of biomaterials opens up possibilities for biological, medical and tissue engineering applications. Structuring the surface of a biomaterial into arrays with micro- and nanoscale features and architectures, defines new roadmaps to innovative engineering of materials. Thin films of novel collagen/elastin composite and gelatin were irradiated by Ti:sapphire fs laser in air at central wavelength 800 nm, with pulse durations in the range of 30 fs. The size and shape as well as morphological forms occurring in the resulted areas of interaction were analyzed as a function of irradiation fluence and number of pulses by atomic force microscopy (AFM). The fs interaction regime allows generation of well defined micro porous surface arrays. In this study we examined a novel composite consisting of collagen and elastin in order to create a biodegradable matrix to serve as a biomimetic surface for cell attachment. Confocal microscopy images of modified zones reveal formation of surface fringe patterns with orientation direction alongside the area of interaction. Outside the crater rim a wave-like topography pattern is observed. Structured, on a nanometer scale, surface array is employed for cell-culture experiments for testing cell’s responses to substrate morphology. Mice fibroblasts migration was monitored

  16. Factors predicting the duration of adrenal insufficiency in patients successfully treated for Cushing disease and nonmalignant primary adrenal Cushing syndrome.

    Science.gov (United States)

    Prete, Alessandro; Paragliola, Rosa Maria; Bottiglieri, Filomena; Rota, Carlo Antonio; Pontecorvi, Alfredo; Salvatori, Roberto; Corsello, Salvatore Maria

    2017-03-01

    Successful treatment of Cushing syndrome causes transient or permanent adrenal insufficiency deriving from endogenous hypercortisolism-induced hypothalamus-pituitary-adrenal-axis suppression. We analyzed pre-treatment factors potentially affecting the duration of adrenal insufficiency. We conducted a retrospective analysis on patients successfully treated for Cushing disease (15 patients) who underwent transsphenoidal surgery, and nonmalignant primary adrenal Cushing syndrome (31 patients) who underwent unilateral adrenalectomy, divided into patients with overt primary adrenal Cushing syndrome (14 patients) and subclinical primary adrenal Cushing syndrome (17 patients). Epidemiological data, medical history, and hormonal parameters depending on the etiology of hypercortisolism were collected and compared to the duration of adrenal insufficiency. The median duration of follow-up after surgery for Cushing disease and primary adrenal Cushing syndrome was 70 and 48 months, respectively. In the Cushing disease group, the median duration of adrenal insufficiency after transsphenoidal surgery was 15 months: younger age at diagnosis and longer duration of signs and symptoms of hypercortisolism before diagnosis and surgery were associated with longer duration of adrenal insufficiency. The median duration of adrenal insufficiency was 6 months for subclinical primary adrenal Cushing syndrome and 18.5 months for overt primary adrenal Cushing syndrome. The biochemical severity of hypercortisolism, the grade of hypothalamus-pituitary-adrenal-axis suppression, and treatment with ketoconazole before surgery accounted for longer duration of adrenal insufficiency. In patients with Cushing disease, younger age and delayed diagnosis and treatment predict longer need for glucocorticoid replacement therapy after successful transsphenoidal surgery. In patients with primary adrenal Cushing syndrome, the severity of hypercortisolism plays a primary role in influencing the duration of

  17. Synergistic apoptosis of CML cells by buthionine sulfoximine and hydroxychavicol correlates with activation of AIF and GSH-ROS-JNK-ERK-iNOS pathway.

    Directory of Open Access Journals (Sweden)

    Avik Acharya Chowdhury

    Full Text Available BACKGROUND: Hydroxychavicol (HCH, a constituent of Piper betle leaf has been reported to exert anti-leukemic activity through induction of reactive oxygen species (ROS. The aim of the study is to optimize the oxidative stress -induced chronic myeloid leukemic (CML cell death by combining glutathione synthesis inhibitor, buthionine sulfoximine (BSO with HCH and studying the underlying mechanism. MATERIALS AND METHODS: Anti-proliferative activity of BSO and HCH alone or in combination against a number of leukemic (K562, KCL22, KU812, U937, Molt4, non-leukemic (A549, MIA-PaCa2, PC-3, HepG2 cancer cell lines and normal cell lines (NIH3T3, Vero was measured by MTT assay. Apoptotic activity in CML cell line K562 was detected by flow cytometry (FCM after staining with annexin V-FITC/propidium iodide (PI, detection of reduced mitochondrial membrane potential after staining with JC-1, cleavage of caspase- 3 and poly (ADP-ribose polymerase proteins by western blot analysis and translocation of apoptosis inducing factor (AIF by confocal microscopy. Intracellular reduced glutathione (GSH was measured by colorimetric assay using GSH assay kit. 2',7'-dichlorodihydrofluorescein diacetate (DCF-DA and 4-amino-5-methylamino-2',7'-difluorofluorescein (DAF-FM were used as probes to measure intracellular increase in ROS and nitric oxide (NO levels respectively. Multiple techniques like siRNA transfection and pharmacological inhibition were used to understand the mechanisms of action. RESULTS: Non-apoptotic concentrations of BSO significantly potentiated HCH-induced apoptosis in K562 cells. BSO potentiated apoptosis-inducing activity of HCH in CML cells by caspase-dependent as well as caspase-independent but apoptosis inducing factor (AIF-dependent manner. Enhanced depletion of intracellular GSH induced by combined treatment correlated with induction of ROS. Activation of ROS- dependent JNK played a crucial role in ERK1/2 activation which subsequently induced the

  18. Kidney dysfunction after allogeneic stem cell transplantation

    NARCIS (Netherlands)

    Kersting, S.

    2008-01-01

    Allogeneic stem cell transplantation (SCT) is a widely accepted approach for malignant and nonmalignant hematopoietic diseases. Unfortunately complications can occur because of the treatment, leading to treatment-related mortality. We studied kidney dysfunction after allogeneic SCT in 2 cohorts of

  19. Dose and dose rate extrapolation factors for malignant and non-malignant health endpoints after exposure to gamma and neutron radiation

    Energy Technology Data Exchange (ETDEWEB)

    Tran, Van; Little, Mark P. [National Cancer Institute, Radiation Epidemiology Branch, Rockville, MD (United States)

    2017-11-15

    Murine experiments were conducted at the JANUS reactor in Argonne National Laboratory from 1970 to 1992 to study the effect of acute and protracted radiation dose from gamma rays and fission neutron whole body exposure. The present study reports the reanalysis of the JANUS data on 36,718 mice, of which 16,973 mice were irradiated with neutrons, 13,638 were irradiated with gamma rays, and 6107 were controls. Mice were mostly Mus musculus, but one experiment used Peromyscus leucopus. For both types of radiation exposure, a Cox proportional hazards model was used, using age as timescale, and stratifying on sex and experiment. The optimal model was one with linear and quadratic terms in cumulative lagged dose, with adjustments to both linear and quadratic dose terms for low-dose rate irradiation (<5 mGy/h) and with adjustments to the dose for age at exposure and sex. After gamma ray exposure there is significant non-linearity (generally with upward curvature) for all tumours, lymphoreticular, respiratory, connective tissue and gastrointestinal tumours, also for all non-tumour, other non-tumour, non-malignant pulmonary and non-malignant renal diseases (p < 0.001). Associated with this the low-dose extrapolation factor, measuring the overestimation in low-dose risk resulting from linear extrapolation is significantly elevated for lymphoreticular tumours 1.16 (95% CI 1.06, 1.31), elevated also for a number of non-malignant endpoints, specifically all non-tumour diseases, 1.63 (95% CI 1.43, 2.00), non-malignant pulmonary disease, 1.70 (95% CI 1.17, 2.76) and other non-tumour diseases, 1.47 (95% CI 1.29, 1.82). However, for a rather larger group of malignant endpoints the low-dose extrapolation factor is significantly less than 1 (implying downward curvature), with central estimates generally ranging from 0.2 to 0.8, in particular for tumours of the respiratory system, vasculature, ovary, kidney/urinary bladder and testis. For neutron exposure most endpoints, malignant and

  20. Leukemic Cells "Gas Up" Leaky Bone Marrow Blood Vessels.

    Science.gov (United States)

    Itkin, Tomer; Rafii, Shahin

    2017-09-11

    In this issue of Cancer Cell, Passaro et al. demonstrate how leukemia through aberrant induction of reactive oxygen species and nitric oxide production trigger marrow vessel leakiness, instigating pro-leukemic function. Disrupted tumor blood vessels promote exhaustion of non-malignant stem and progenitor cells and may facilitate leukemia relapse following chemotherapeutic treatment. Copyright © 2017. Published by Elsevier Inc.

  1. IND-2, a pyrimido[1″,2″:1,5]pyrazolo[3,4-b]quinoline derivative, circumvents multi-drug resistance and causes apoptosis in colon cancer cells.

    Science.gov (United States)

    Karthikeyan, Chandrabose; Lee, Crystal; Moore, Joshua; Mittal, Roopali; Suswam, Esther A; Abbott, Kodye L; Pondugula, Satyanarayana R; Manne, Upender; Narayanan, Narayanan K; Trivedi, Piyush; Tiwari, Amit K

    2015-02-01

    Naturally occurring condensed quinolines have anticancer properties. In efforts to find active analogues, we designed and synthesized eight polycyclic heterocycles with a pyrimido[1″,2″:1,5]pyrazolo[3,4-b]quinoline framework (IND series). The compounds were evaluated for activity against colon (HCT-116 and S1-MI-80), prostate (PC3 and DU-145), breast (MCF-7 and MDAMB-231), ovarian (ov2008 and A2780), and hepatocellular (HepG2) cancer cells and against non-cancerous Madin Darby canine kidney (MDCK), mouse embryonic fibroblast (NIH/3T3), and human embryonic kidney cells (HEK293). IND-2, a 4-chloro-2-methyl pyrimido[1″,2″:1,5]pyrazolo[3,4-b]quinoline, exhibited more than ten-fold selectivity and potent cytotoxic activity against colon cancer cells relative to the other cancer and non-cancer cells. With five additional colon cancer cell lines (HT-29, HCT-15, LS-180, LS-174, and LoVo), IND-2 had similar cytotoxicity and selectivity, and sub-micromolar concentrations caused changes in the morphology of HCT-116 and HCT-15 cells. IND-2 did not activate the transactivating function of the pregnane X receptor (PXR), indicating that it does not induce PXR-regulated ABCB1 or ABCG2 transporters. Indeed, IND-2 was not a substrate of ABCB1 or ABCG2, and it induced cytotoxicity in HEK293 cells overexpressing ABCB1 or ABCG2 to the same extent as in normal HEK293 cells. IND-2 was cytotoxic to resistant colon carcinoma S1-MI-80 cells, approximately three- and five-fold more than SN-38 and topotecan, respectively. In HCT-116 colon cancer cells, IND-2 produced concentration-dependent changes in mitochondrial membrane potential, leading to apoptosis, and sub-micromolar concentrations caused chromosomal DNA fragmentation. These findings suggest that, by increasing apoptosis, IND-2 has potential therapeutic efficacy for colorectal cancer. Copyright © 2014 Elsevier Ltd. All rights reserved.

  2. The Role of the Y-Located TSPY Gene in Prostatic Oncogenesis

    Science.gov (United States)

    2007-02-01

    Coordination of growth with cell division in the yeast Saccharomyces cerevisiae . Exp Cell Res 1977;105:79-98. 50. Sonenberg N. Translation factors as...cyclins in budding yeast and frogs [17]. In Sac- charomyces cerevisiae , cells that lack NAP-1, the Clb2 (B- type cyclin) was unable to efficiently induce... origenic , the development of small tumors in nude mice inoculated with TSPY expressing NIH3T3 cells suggests that TSPY could potentially play the role

  3. Macropinocytosis is the Entry Mechanism of Amphotropic Murine Leukemia Virus

    DEFF Research Database (Denmark)

    Rasmussen, Izabela; Vilhardt, Frederik

    2015-01-01

    of infection. Understanding how pathogens and toxins exploit or divert endocytosis pathways has advanced our understanding of membrane trafficking pathways, which benefits development of new therapeutical schemes and methods of drug delivery. We show here that Murine Leukemia Virus (A-MLV) pseudotyped......, or NIH-3T3 cells knocked-down for caveolin expression, was unaffected. Conversely, A-MLV infection of NIH-3T3 and HeLa cells was sensitive to amiloride analogues and actin-depolymerizing drugs that interfere with macropinocytosis. Further manipulation of the actin cytoskeleton through conditional...... with the amphotropic (expands the host range to many mammalian cells) envelope protein gains entry into host cells by macropinocytosis. Macropinosomes form as large, fluid-filled vacuoles (up to 10 μm) following collapse of cell surface protrusions and membrane scission. We use drugs or introduction of mutant proteins...

  4. Intact and cleaved forms of the urokinase receptor enhance discrimination of cancer from non-malignant conditions in patients presenting with symptoms related to colorectal cancer

    DEFF Research Database (Denmark)

    Lomholt, A F; Høyer-Hansen, G; Nielsen, H J

    2009-01-01

    plasminogen activator receptor (suPAR) was proposed as a marker in CRC patients. This study was undertaken to evaluate the individual molecular forms of suPAR as discriminators in a group of patients undergoing endoscopical examination following symptoms related to colorectal cancer. METHODS: In a case......-control study comprising 308 patients undergoing endoscopical examination following CRC-related symptoms, 77 CRC patients with adenocarcinoma were age and gender matched to: 77 patients with adenomas; 77 with other non-malignant findings, and 77 with no findings. The different uPAR forms were measured...

  5. Determining pathogenetic connection between disorders of lipid and carbohydrate metabolism and non-malignant pathology of thyroid gland in children , born from parents, Chernobyl accident survivors

    International Nuclear Information System (INIS)

    Kopilova, O.V.; Stepanenko, O.A.; Belyingyio, T.O.

    2014-01-01

    The 92 children aged 12-17 years were examined with the purpose to study the links between carbohydrate and lipid metabolic abnormalities and non-malignant thyroid disorders in descendants of the Chernobyl accident survivors. Clinical, anthropometrical studies and hormonal assays were applied. Carbohydrate and lipid metabolic abnormalities were revealed in every third case of thyroid disease. It confirms our supposition of such a possibility being due to the fact that radiation impact even in low doses can result in pronounced metabolic disorders lading to entire endocrine disregulation. It is relevant in children of the puberty age

  6. Effect of ionizing radiation on the biological activity of activated oncogenes and dormant proto-oncogenes

    International Nuclear Information System (INIS)

    Angenent, G.C.; Berg, K.J. van den.

    1984-01-01

    The authors have studied the effect of ionizing radiation on the cloned human activated Ha-ras oncogene, on the Ha-ras gene in integrated form and on the dormant proto-oncogene murine c-mos using the NIH/3T3 transfection system. NIH/3T3 cells were transfected with DNA from the plasmid pT24 carrying the cloned Ha-ras oncogene of the T24 bladder carcinoma cell line. Various individual foci which developed were injected into nude mice. DNA was isolated from tumours, digested with the restriction enzyme Bam HI, electrophoresed on agarose and blotted onto nitrocellulose filter according to Southern. Hybridization with a pT24 probe showed that all the primary foci of transformed cells contained various fragments of the pT24 plasmid indicating that fibroblast transformation had been induced by introduction of the Ha-ras oncogene. (Auth.)

  7. Anti-proliferative and mutagenic activities of aqueous and methanol extracts of leaves from Pereskia bleo (Kunth) DC (Cactaceae).

    Science.gov (United States)

    Er, Hui Meng; Cheng, En-Hsiang; Radhakrishnan, Ammu Kutty

    2007-09-25

    The anti-proliferative effects of the aqueous and methanol extracts of leaves of Pereskia bleo (Kunth) DC (Cactaceae) against a mouse mammary cancer cell line (4T1) and a normal mouse fibroblast cell line (NIH/3T3) were evaluated under an optimal (in culture medium containing 10% foetal bovine serum (FBS)) and a sub-optimal (in culture medium containing 0.5% FBS) conditions. Under the optimal condition, the aqueous extract showed a significant (pCactaceae) do not have appreciable anti-proliferative effect on the 4T1 and NIH/3T3 cells as the EC(50) values obtained are greater than 50 microg/mL when tested under optimal culture condition. Moreover, the aqueous extract may form mutagenic compound(s) upon the metabolisation by liver enzymes.

  8. Global mapping of cell type-specific open chromatin by FAIRE-seq reveals the regulatory role of the NFI family in adipocyte differentiation.

    Directory of Open Access Journals (Sweden)

    Hironori Waki

    2011-10-01

    Full Text Available Identification of regulatory elements within the genome is crucial for understanding the mechanisms that govern cell type-specific gene expression. We generated genome-wide maps of open chromatin sites in 3T3-L1 adipocytes (on day 0 and day 8 of differentiation and NIH-3T3 fibroblasts using formaldehyde-assisted isolation of regulatory elements coupled with high-throughput sequencing (FAIRE-seq. FAIRE peaks at the promoter were associated with active transcription and histone modifications of H3K4me3 and H3K27ac. Non-promoter FAIRE peaks were characterized by H3K4me1+/me3-, the signature of enhancers, and were largely located in distal regions. The non-promoter FAIRE peaks showed dynamic change during differentiation, while the promoter FAIRE peaks were relatively constant. Functionally, the adipocyte- and preadipocyte-specific non-promoter FAIRE peaks were, respectively, associated with genes up-regulated and down-regulated by differentiation. Genes highly up-regulated during differentiation were associated with multiple clustered adipocyte-specific FAIRE peaks. Among the adipocyte-specific FAIRE peaks, 45.3% and 11.7% overlapped binding sites for, respectively, PPARγ and C/EBPα, the master regulators of adipocyte differentiation. Computational motif analyses of the adipocyte-specific FAIRE peaks revealed enrichment of a binding motif for nuclear family I (NFI transcription factors. Indeed, ChIP assay showed that NFI occupy the adipocyte-specific FAIRE peaks and/or the PPARγ binding sites near PPARγ, C/EBPα, and aP2 genes. Overexpression of NFIA in 3T3-L1 cells resulted in robust induction of these genes and lipid droplet formation without differentiation stimulus. Overexpression of dominant-negative NFIA or siRNA-mediated knockdown of NFIA or NFIB significantly suppressed both induction of genes and lipid accumulation during differentiation, suggesting a physiological function of these factors in the adipogenic program. Together, our

  9. cDNA sequence of human transforming gene hst and identification of the coding sequence required for transforming activity

    International Nuclear Information System (INIS)

    Taira, M.; Yoshida, T.; Miyagawa, K.; Sakamoto, H.; Terada, M.; Sugimura, T.

    1987-01-01

    The hst gene was originally identified as a transforming gene in DNAs from human stomach cancers and from a noncancerous portion of stomach mucosa by DNA-mediated transfection assay using NIH3T3 cells. cDNA clones of hst were isolated from the cDNA library constructed from poly(A) + RNA of a secondary transformant induced by the DNA from a stomach cancer. The sequence analysis of the hst cDNA revealed the presence of two open reading frames. When this cDNA was inserted into an expression vector containing the simian virus 40 promoter, it efficiently induced the transformation of NIH3T3 cells upon transfection. It was found that one of the reading frames, which coded for 206 amino acids, was responsible for the transforming activity

  10. The carboxyl terminal tyrosine 417 residue of NOK has an autoinhibitory effect on NOK-mediated signaling transductions

    International Nuclear Information System (INIS)

    Li Yinghua; Zhong Shan; Rong Zhili; Ren Yongming; Li Zhiyong; Zhang Shuping; Chang Zhijie; Liu Li

    2007-01-01

    Receptor protein tyrosine kinases (RPTKs) are essential mediators of cell growth, differentiation, migration, and metabolism. Recently, a novel RPTK named NOK has been cloned and characterized. In current study, we investigated the role of the carboxyl terminal tyrosine 417 residue of NOK in the activations of different signaling pathways. A single tyrosine to phenylalanine point mutation at Y417 site (Y417 F) not only dramatically enhanced the NOK-induced activation of extracellular signal-regulated kinase (ERK), but also markedly promoted the NOK-mediated activation of both signal transducer and activator of transcription 1 and 3 (STAT1 and 3). Moreover, the proliferation potential of NIH3T3-NOK (Y417F) stable cells were significantly elevated as compared with that of NIH3T3-NOK. Overall, our results demonstrate that the tyrosine Y417 residue at the carboxyl tail of NOK exhibits an autoinhibitory role in NOK-mediated signaling transductions

  11. Role of NADP+-dependent isocitrate dehydrogenase (NADP+-ICDH) on cellular defence against oxidative injury by gamma-rays.

    Science.gov (United States)

    Lee, S H; Jo, S H; Lee, S M; Koh, H J; Song, H; Park, J W; Lee, W H; Huh, T L

    2004-09-01

    To investigate the regulation of NADPH-producing isocitrate dehydrogenase (ICDH) in cytosol (IDPc) and mitochondria (IDPm) upon gamma-ray irradiation, and the roles of IDPc and IDPm in the protection against cellular damage induced by gamma-ray irradiation. Changes of IDPc and IDPm proteins upon gamma-ray irradiation to NIH3T3 cells were analysed by immunoblotting. To increase or decrease the expression of IDPc or IDPm, NIH3T3 cells were stably transfected with mouse IDPc or IDPm cDNA in either the sense or the antisense direction. The transfected cells with either increased or decreased IDPc or IDPm were exposed to gamma-rays, and the levels of reactive oxygen species generation, protein oxidation and lipid peroxidation were measured. Both IDPc and IDPm activities were induced by gamma-ray in NIH3T3 cells. Cells with decreased expression of IDPc or IDPm had elevated reactive oxygen species generation, lipid peroxidation and protein oxidation. Conversely, overproduction of IDPc or IDPm protein partially protected the cells from oxidative damage induced by gamma-ray irradiation. The protective role of IDPc and IDPm against gamma-ray-induced cellular damage can be attributed to elevated NADPH, reducing equivalents needed for recycling reduced glutathione in the cytosol and mitochondria. Thus, a primary biological function of the ICDHs may be production of NADPH, which is a prerequisite for some cellular defence systems against oxidative damage.

  12. Stem cell comparison : What can we learn clinically from unrelated cord blood transplantation as an alternative stem cell source?

    NARCIS (Netherlands)

    Milano, Filippo; Boelens, Jaap Jan

    2015-01-01

    Allogeneic hematopoietic cell transplantation (HCT) is an important therapeutic option for a variety of malignant and non-malignant disorders (NMD). The use of umbilical cord blood transplantation (UCBT) has made HCT available to many more patients. The increased level of human leukocyte antigen

  13. Correlates and management of nonmalignant pain in the nursing home. SAGE Study Group. Systematic Assessment of Geriatric drug use via Epidemiology.

    Science.gov (United States)

    Won, A; Lapane, K; Gambassi, G; Bernabei, R; Mor, V; Lipsitz, L A

    1999-08-01

    Nonmalignant pain is a common problem among older people. The prevalence of pain in the nursing home is not well studied. We looked at the association between nonmalignant pain, psychological and functional health, and the practice patterns for pain management in the nursing home. A cross-sectional study. Nursing Home in four US states. A total of 49,971 nursing home residents from 1992 to 1995. We used the SAGE database (Systematic Assessment of Geriatric drug use via Epidemiology), which linked information from the Minimum Data Set and nursing home drug utilization data. The MDS items measured included pain, activities of daily living (ADL) function, mood, and time involved in activities. The use of analgesics, anxiolytics, and antidepressants was also documented. Daily pain was reported in 26% of nursing home residents. The prevalence was lower among men, persons more than age 85, and racial minorities. Persons suffering pain daily were more likely to have severe ADL impairment, odds ratio (OR) (2.47 (95% CI, 2.34-2.60)), more depressive signs and symptoms (OR 1.66 (95% CI, 1.57-1.75)), and less frequent involvement in activities (OR 1.35 (95% CI, 1.29-1.40)). Approximately 25% of persons with daily pain received no analgesics. Residents who were more than 85 years old (OR 1.15 (95% CI 1.02-1.28)), cognitively impaired, (OR 1.44 (95% CI, 1.29-1.61)), of male gender (OR 1.17 (95% CI, 1.06-1.29)), or a racial minority (OR 1.69 (95% CI, 1.40-2.05) and OR 1.56 (95% CI, 0.70-1.04) for blacks and Hispanics, respectively) were at greater risk of not receiving analgesics. Approximately 50% of those in pain used physical and occupational therapies, which was more than two times higher (OR 2.44 (95% CI, 2.34-2.54)), than use for those not in pain. Daily nonmalignant pain is prevalent among nursing home residents and is often associated with impairments in ADL, mood, and decreased activity involvement. Even when pain was recognized, men, racial minorities, and cognitively

  14. Mechanisms of Resistance to Chemotherapies Targeting BRCA-Mutant Breast Cancer

    Science.gov (United States)

    2016-12-01

    Amsterdam, The Netherlands. 4Laboratory of Endocrinology and Receptor Biology, National Institute of Diabetes and Digestive and Kidney Diseases, National...at www.nature.com/reprints. The authors declare no competing financial interests. Readers are welcome to comment on the online version of the paper...SURVEYOR nuclease assay was performed by transfecting sgRNA into NIH3T3 cells and PCR amplifying the edited genomic region. Nuclease S was used to digest

  15. Size- and dose-dependent toxicity of cellulose nanocrystals (CNC) on human fibroblasts and colon adenocarcinoma.

    Science.gov (United States)

    Hanif, Zahid; Ahmed, Farrukh R; Shin, Seung Won; Kim, Young-Kee; Um, Soong Ho

    2014-07-01

    A controlled preparation of cellulose nanocrystals of different sizes and shapes has been carried out by acid hydrolysis of microcrystalline cellulose. The size- and concentration-dependent toxicity effects of the resulting cellulose nanocrystals were evaluated against two different cell lines, NIH3T3 murine embryo fibroblasts and HCT116 colon adenocarcinoma. It could serve as a therapeutic platform for cancer treatment. Copyright © 2014 Elsevier B.V. All rights reserved.

  16. Genomic response to Wnt signalling is highly context-dependent - Evidence from DNA microarray and chromatin immunoprecipitation screens of Wnt/TCF targets

    International Nuclear Information System (INIS)

    Railo, Antti; Pajunen, Antti; Itaeranta, Petri; Naillat, Florence; Vuoristo, Jussi; Kilpelaeinen, Pekka; Vainio, Seppo

    2009-01-01

    Wnt proteins are important regulators of embryonic development, and dysregulated Wnt signalling is involved in the oncogenesis of several human cancers. Our knowledge of the downstream target genes is limited, however. We used a chromatin immunoprecipitation-based assay to isolate and characterize the actual gene segments through which Wnt-activatable transcription factors, TCFs, regulate transcription and an Affymetrix microarray analysis to study the global transcriptional response to the Wnt3a ligand. The anti-β-catenin immunoprecipitation of DNA-protein complexes from mouse NIH3T3 fibroblasts expressing a fusion protein of β-catenin and TCF7 resulted in the identification of 92 genes as putative TCF targets. GeneChip assays of gene expression performed on NIH3T3 cells and the rat pheochromocytoma cell line PC12 revealed 355 genes in NIH3T3 and 129 genes in the PC12 cells with marked changes in expression after Wnt3a stimulus. Only 2 Wnt-regulated genes were shared by both cell lines. Surprisingly, Disabled-2 was the only gene identified by the chromatin immunoprecipitation approach that displayed a marked change in expression in the GeneChip assay. Taken together, our approaches give an insight into the complex context-dependent nature of Wnt pathway transcriptional responses and identify Disabled-2 as a potential new direct target for Wnt signalling.

  17. High-density growth arrest in Ras-transformed cells: low Cdk kinase activities in spite of absence of p27Kip Cdk-complexes

    DEFF Research Database (Denmark)

    Groth, Anja; Willumsen, Berthe Marie

    2005-01-01

    The ras oncogene transforms immortalized, contact-inhibited non-malignant murine fibroblasts into cells that are focus forming, exhibit increased saturation density, and are malignant in suitable hosts. Here, we examined changes in cell cycle control complexes as normal and Ras-transformed cells...

  18. Inhibition of glucose turnover by 3-bromopyruvate counteracts pancreatic cancer stem cell features and sensitizes cells to gemcitabine

    OpenAIRE

    Isayev, Orkhan; Rausch, Vanessa; Bauer, Nathalie; Liu, Li; Fan, Pei; Zhang, Yiyao; Gladkich, Jury; Nwaeburu, Clifford C.; Mattern, Jürgen; Mollenhauer, Martin; Rückert, Felix; Zach, Sebastian; Haberkorn, Uwe; Gross, Wolfgang; Schönsiegel, Frank

    2014-01-01

    According to the cancer stem cell (CSC) hypothesis, the aggressive growth and early metastasis of pancreatic ductal adenocarcinoma (PDA) is due to the activity of CSCs, which are not targeted by current therapies. Otto Warburg suggested that the growth of cancer cells is driven by a high glucose metabolism. Here, we investigated whether glycolysis inhibition targets CSCs and thus may enhance therapeutic efficacy. Four established and 3 primary PDA cell lines, non-malignant cells, and 3 patien...

  19. Inverse dose-rate-effects on the expressions of extra-cellular matrix-related genes in low-dose-rate γ-ray irradiated murine cells

    International Nuclear Information System (INIS)

    Sugihara, Takashi; Tanaka, Kimio; Oghiso, Yoichi; Murano, Hayato

    2008-01-01

    Based on the results of previous microarray analyses of murine NIH3T3/PG13Luc cells irradiated with continuous low-dose-rate (LDR) γ-ray or end-high-dose-rate-irradiations (end-HDR) at the end of the LDR-irradiation period, the inverse dose-rate-effects on gene expression levels were observed. To compare differences of the effects between LDR-irradiation and HDR-irradiation, HDR-irradiations at 2 different times, one (ini-HDR) at the same time at the start of LDR-irradiation and the other (end-HDR), were performed. The up-regulated genes were classified into two types, in which one was up-regulated in LDR-, ini-HDR-, and end-HDR irradiation such as Cdkn1a and Ccng1, which were reported as p53-dependent genes, and the other was up-regulated in LDR- and ini-HDR irradiations such as pro-collagen TypeIa2/Colla2, TenascinC/Tnc, and Fibulin5/Fbln5, which were reported as extra-cellular matrix-related (ECM) genes. The time dependent gene expression patterns in LDR-irradiation were also classified into two types, in which one was an early response such as in Cdkn1a and Ccng1 and the other was a delayed response such as the ECM genes which have no linearity to total dose. The protein expression pattern of Cdkn1a increased dose dependently in LDR- and end-HDR-irradiations, but those of p53Ser15/18 and MDM2 in LDR-irradiations were different from end-HDR-irradiations. Furthermore, the gene expression levels of the ECM genes in embryonic fibroblasts from p53-deficient mice were not increased by LDR- and end-HDR-irradiation, so the delayed expressions of the ECM genes seem to be regulated by p53. Consequently, the inverse dose-rate-effects on the expression levels of the ECM genes in LDR- and end-HDR-irradiations may be explained from different time responses by p53 status. (author)

  20. Cancer Specific Proliferating Cell Nuclear Antigen as a Novel Diagnostic Marker for the Detection of Breast Cancer

    National Research Council Canada - National Science Library

    Hoelz, Derek

    2003-01-01

    ... (which is now referred to as the cancer-specific form of PCNA or csPCNA). Additionally, a basic form of PCNA was also observed in the malignant cells, but this isoform was the only isoform found in non-malignant cells and tissues...

  1. Integrin-mediated signal transduction linked to Ras pathway by GRB2 binding to focal adhesion kinase.

    Science.gov (United States)

    Schlaepfer, D D; Hanks, S K; Hunter, T; van der Geer, P

    The cytoplasmic focal adhesion protein-tyrosine kinase (FAK) localizes with surface integrin receptors at sites where cells attach to the extracellular matrix. Increased FAK tyrosine phosphorylation occurs upon integrin engagement with fibronectin. Here we show that adhesion of murine NIH3T3 fibroblasts to fibronectin promotes SH2-domain-mediated association of the GRB2 adaptor protein and the c-Src protein-tyrosine kinase (PTK) with FAK in vivo, and also results in activation of mitogen-activated protein kinase (MAPK). In v-Src-transformed NIH3T3, the association of v-Src, GRB2 and Sos with FAK is independent of cell adhesion to fibronectin. The GRB2 SH2 domain binds directly to tyrosine-phosphorylated FAK. Mutation of tyrosine residue 925 of FAK (YENV motif) to phenylalanine blocks GRB2 SH2-domain binding to FAK in vitro. Our results show that fibronectin binding to integrins on NIH3T3 fibroblasts promotes c-Src and FAK association and formation of an integrin-activated signalling complex. Phosphorylation of FAK at Tyr 925 upon fibronectin stimulation creates an SH2-binding site for GRB2 which may link integrin engagement to the activation of the Ras/MAPK signal transduction pathway.

  2. The role of autophagy in cytotoxicity induced by new oncogenic B-Raf inhibitor UI-152 in v-Ha-ras transformed fibroblasts

    International Nuclear Information System (INIS)

    Ahn, Jun-Ho; Ahn, Soon Kil; Lee, Michael

    2012-01-01

    Highlights: ► We recently discovered a potent and selective B-Raf inhibitor, UI-152. ► UI-152 displayed a selective cytotoxicity toward v-Ha-ras transformed cells. ► UI-152-induced growth inhibition was largely meditated by autophagy. ► UI-152 induced paradoxical activation of Raf-1. -- Abstract: In human cancers, B-Raf is the most frequently mutated protein kinase in the MAPK signaling cascade, making it an important therapeutic target. We recently discovered a potent and selective B-Raf inhibitor, UI-152, by using a structure-based drug design strategy. In this study, we examined whether B-Raf inhibition by UI-152 may be an effective therapeutic strategy for eliminating cancer cells transformed with v-Ha-ras (Ras-NIH 3T3). UI-152 displayed selective cytotoxicity toward Ras-NIH 3T3 cells while having little to no effect on non-transformed NIH 3T3 cells. We found that treatment with UI-152 markedly increased autophagy and, to a lesser extent, apoptosis. However, inhibition of autophagy by addition of 3-MA failed to reverse the cytotoxic effects of UI-152 on Ras-NIH 3T3 cells, demonstrating that apoptosis and autophagy can act as cooperative partners to induce growth inhibition in Ras-NIH 3T3 cells treated with UI-152. Most interestingly, cell responses to UI-152 appear to be paradoxical. Here, we showed that although UI-152 inhibited ERK, it induced B-Raf binding to Raf-1 as well as Raf-1 activation. This paradoxical activation of Raf-1 by UI-152 is likely to be coupled with the inhibition of the mTOR pathway, an intracellular signaling pathway involved in autophagy. We also showed for the first time that, in multi-drug resistant cells, the combination of UI-152 with verapamil significantly decreased cell proliferation and increased autophagy. Thus, our findings suggest that the inhibition of autophagy, in combination with UI-152, offers a more effective therapeutic strategy for v-Ha-ras-transformed cells harboring wild-type B-Raf.

  3. State-of-the-art fertility preservation in children and adolescents undergoing haematopoietic stem cell transplantation

    DEFF Research Database (Denmark)

    Dalle, J-H; Lucchini, G; Balduzzi, A

    2017-01-01

    Nowadays, allogeneic haematopoietic stem cell transplantation (allo-HSCT) is a well-established treatment procedure and often the only cure for many patients with malignant and non-malignant diseases. Decrease in short-term complications has substantially contributed to increased survival. Theref...

  4. Positive Response to Thermobalancing Therapy Enabled by Therapeutic Device in Men with Non-Malignant Prostate Diseases: BPH and Chronic Prostatitis

    Directory of Open Access Journals (Sweden)

    Ivan Gerasimovich Aghajanyan

    2016-04-01

    Full Text Available Background: The most common types of non-malignant prostate diseases are benign prostatic hyperplasia (BPH and chronic prostatitis (CP. The aim of this study was to find out whether thermobalancing therapy with a physiotherapeutic device is effective for BPH and CP. Methods: During a 2.5-year period, 124 men with BPH over the age of 55 were investigated. Clinical parameters were tested twice: via the International Prostate Symptom Score (IPSS and via ultrasound measurement of prostate volume (PV and uroflowmetry maximum flow rate (Qmax, before and after six months of therapy. In 45 men with CP under the age of 55, the dynamics of the National Institute of Health Chronic Prostatitis Symptom Index (NIH-CPSI were studied. Results: The results of the investigated index tests in men with BPH confirmed a decrease in IPSS (p < 0.001, a reduction in PV (p < 0.001, an increase in Qmax (p < 0.001, and an improvement of quality of life (QoL (p < 0.001. NIH-CPSI scores in men with CP indicated positive dynamics. Conclusions: The observed positive changes in IPSS, PV, and Qmax in men with BPH and the improvement in NIH-CPSI-QoL in patients with CP after using a physiotherapeutic device for six months as mono-therapy, support the view that thermobalancing therapy with the device can be recommended for these patients. Furthermore, the therapeutic device is free of side effects.

  5. Positive Response to Thermobalancing Therapy Enabled by Therapeutic Device in Men with Non-Malignant Prostate Diseases: BPH and Chronic Prostatitis.

    Science.gov (United States)

    Aghajanyan, Ivan Gerasimovich; Allen, Simon

    2016-04-18

    The most common types of non-malignant prostate diseases are benign prostatic hyperplasia (BPH) and chronic prostatitis (CP). The aim of this study was to find out whether thermobalancing therapy with a physiotherapeutic device is effective for BPH and CP. During a 2.5-year period, 124 men with BPH over the age of 55 were investigated. Clinical parameters were tested twice: via the International Prostate Symptom Score (IPSS) and via ultrasound measurement of prostate volume (PV) and uroflowmetry maximum flow rate (Q max ), before and after six months of therapy. In 45 men with CP under the age of 55, the dynamics of the National Institute of Health Chronic Prostatitis Symptom Index (NIH-CPSI) were studied. The results of the investigated index tests in men with BPH confirmed a decrease in IPSS ( p BPH and the improvement in NIH-CPSI-QoL in patients with CP after using a physiotherapeutic device for six months as mono-therapy, support the view that thermobalancing therapy with the device can be recommended for these patients. Furthermore, the therapeutic device is free of side effects.

  6. Early CD3+/CD15+ peripheral blood leukocyte chimerism patterns correlate with long-term engraftment in non-malignant hematopoietic SCT.

    Science.gov (United States)

    Ketterl, T G; Flesher, M; Shanley, R; Miller, W

    2014-04-01

    Following hematopoietic SCT (HSCT) for non-malignant disorders (NMDs) variable donor chimerism among lympho-hematopoietic lines may be observed. We retrospectively evaluated early post-HSCT, lineage-sorted (CD3+ and CD15+) peripheral blood leukocyte chimerism data to characterize patterns and assess for association with long-term CD15+ engraftment. 'Early' was defined as the first value obtained between days +14 and +42, 'late' as the last recorded value after day +90. 'High' donor chimerism was defined as 80% on either fraction at all time-points. Patients were classified into four subgroups with respect to early CD3+/CD15+ chimerism patterns (high/low) then analyzed for long-term CD15+ chimerism status. A total of 135 transplants were evaluable, with all three time-points available in 97. Underlying disease, graft source, patient age and conditioning intensity varied. 'Split' early chimerism (discordant high/low CD3+/CD15+ status) was common. Multivariable analysis revealed strong association between conditioning regimen and primary disease on early CD3+/CD15+ chimerism patterns and a dominant predictive effect of early CD15+ chimerism on long-term CD15+ donor engraftment (observed at median day +365). These data may guide real-time clinician decisions (restraint vs intervention, when available) when faced with unfavorable or unusual early lympho-hematopoietic chimerism patterns following HSCT for NMD.

  7. Transjugular portal vein recanalization with creation of intrahepatic portosystemic shunt (PVR-TIPS) in patients with chronic non-cirrhotic, non-malignant portal vein thrombosis.

    Science.gov (United States)

    Klinger, Christoph; Riecken, Bettina; Schmidt, Arthur; De Gottardi, Andrea; Meier, Benjamin; Bosch, Jaime; Caca, Karel

    2018-03-01

    To determine safety and efficacy of transjugular portal vein recanalization with creation of intrahepatic portosystemic shunt (PVR-TIPS) in patients with chronic non-cirrhotic, non-malignant portal vein thrombosis (PVT). This retrospective study includes 17 consecutive patients with chronic non-cirrhotic PVT (cavernous transformation n = 15). PVR-TIPS was indicated because of variceal bleeding (n = 13), refractory ascites (n = 2), portal biliopathy with recurrent cholangitis (n = 1), or abdominal pain (n = 1). Treatment consisted of a combination of transjugular balloon angioplasty, mechanical thrombectomy, and-depending on extent of residual thrombosis-transjugular intrahepatic portosystemic shunt and additional stenting of the portal venous system. Recanalization was successful in 76.5 % of patients despite cavernous transformation in 88.2 %. Both 1- and 2-year secondary PV and TIPS patency rates were 69.5 %. Procedure-related bleeding complications occurred in 2 patients (intraperitoneal bleeding due to capsule perforation, n = 1; liver hematoma, n = 1) and resolved spontaneously. However, 1 patient died due to subsequent nosocomial pneumonia. During follow-up, 3 patients with TIPS occlusion and PVT recurrence experienced portal hypertensive complications. PVR-TIPS is safe and effective in selected patients with chronic non-cirrhotic PVT. Due to technical complexity and possible complications, it should be performed only in specialized centers with high experience in TIPS procedures. © Georg Thieme Verlag KG Stuttgart · New York.

  8. Establishment of a normal-derived estrogen receptor-positive cell line comparable to the prevailing human breast cancer subtype

    DEFF Research Database (Denmark)

    Hopkinson, Branden Michael; Klitgaard, Marie Christine; Petersen, Ole William

    2017-01-01

    Understanding human cancer increasingly relies on insight gained from subtype specific comparisons between malignant and non-malignant cells. The most frequent subtype in breast cancer is the luminal. By far the most frequently used model for luminal breast cancer is the iconic estrogen receptor-...

  9. Decoupling Internalization, Acidification and Phagosmal-Endosomal/Iysosomal Phagocytosis of Internalin A coated Beads in epithelial cells

    Energy Technology Data Exchange (ETDEWEB)

    Blanchette, C D; Woo, Y; Thomas, C; Shen, N; Sulchek, T A; Hiddessen, A L

    2008-12-22

    process to demonstrate an additional, real31 time method for tracking bead binding, internalization and phagosomal acidification in both MDCK and Caco-2 cells, as well as 1 NIH 3T3 fibroblast cells, using FITC conjugated to InlA-beads or fibronectin-coated beads. Using this method, we found that the time scales for internalization, phagosomal acidification and phagosomal-endosomal/lysosomal fusion were 23-32 min, 3-4 min and 74-120 min, respectively, for epithelial cells, MDCK and Caco-2, which are slower than the kinetics observed in professional phagocytes such as macrophages. Both the static and real-time methods developed here are expected to be readily and broadly applicable, as they simply require conjugation of a fluorophore to a pathogen or mimetic of interest in combination with common cell labeling dyes, and are not limited to the InlA ligand or cell types used here. As such, these methods hold promise for future measurements of receptor-mediated internalization in other cell systems, e.g. other pathogen-host systems.

  10. Decreased expression of cytochrome P450 protein in non-malignant colonic tissue of patients with colonic adenoma

    DEFF Research Database (Denmark)

    Bergheim, I.; Bode, C.; Parlesak, Alexandr

    2005-01-01

    BACKGROUND: Cytochrome P450 (CYP) enzymes in epithelial cells lining the alimentary tract play an important role in both the elimination and activation of (pro-)carcinogens. To estimate the role of cytochrome P450 in carcinogenesis of the colon, expression patterns and protein levels of four...... representative CYPs (CYP2C, CYP2E1, CYP3A4 and CYP3A5) were determined in colon mucosa of normal and adenomatous colonic tissue of patients with adenomas and disease-free controls. METHODS: Expression of CYP2C, CYP2E1, CYP3A4, and CYP3A5 in colon mucosa of normal and adenomatous colonic tissue of patients...... with adenoma and disease-free controls was determined by RT-PCR. Protein concentration of CYPs was determined using Western blot. RESULTS: With the exception of CYP3A5, expression of CYP mRNA was similar among groups and tissues (e.g. normal colon mucosa and adenoma). CYP3A5 mRNA expression was significantly...

  11. Correlation between histopathological and endoscopic findings of non-malignant gastrointestinal lesions: an experience of a tertiary care teaching hospital from Northern India

    Directory of Open Access Journals (Sweden)

    Manpreet Kaur

    2018-04-01

    Full Text Available Background: Gastrointestinal tract endoscopy along with biopsy is an established procedure for investigating a wide range of gastrointestinal conditions especially inflammatory and malignant diseases. The aim was to study and categorizing the morphological lesions of non-malignant origin at various sites of gastrointestinal tract and to compare with its endoscopic findings.Materials and Methods: This study was conducted on 280 benign GI biopsies received in the Department of Pathology of Sri Guru Ram Das Institute of Medical Sciences and Research, Amritsar, Punjab, India.Results: Maximum cases (39% were observed in the age group of 41-60 years. Male patients outnumbered the females (male to female ratio was 1.4:1. There were total 33 esophageal biopsies amongst which the most common lesion was non-specific esophagitis with least common being Barrett’s esophagus. Correlating the results of endoscopic and histopathological features of acute and chronic gastritis a positive predictive value of 80% with sensitivity of 44.4% was seen. Total 83 duodenal biopsies were analyzed with non-specific duodenitis being the most commonly diagnosed lesion followed by celiac disease. Correlation of endoscopic and histopathological findings in celiac disease revealed a sensitivity of 50% and positive predictive value of 42.86%. In both sigmoid colon and rectum, non-specific colitis was the commonest diagnosis followed by ulcerative colitis. Endoscopic findings were correlated with the histopathological features in ulcerative colitis, revealing a sensitivity of 57.14% along with the positive predictive value of 80%.  Conclusion: Histopathology remains the gold standard for diagnosing a case along with endoscopic findings and endoscopic findings alone cannot make the final diagnosis.

  12. Characterization of variants in the promoter of BZLF1 gene of EBV in nonmalignant EBV-associated diseases in Chinese children

    Directory of Open Access Journals (Sweden)

    Yang Shuang

    2010-05-01

    Full Text Available Abstract Background Diseases associated with Epstein-Barr virus (EBV infections, such as infectious mononucleosis (IM, EBV-associated hemophagocytic lymphohistiocytosis (EBV-HLH and chronic active EBV infection (CAEBV are not rare in Chinese children. The association of type 1 or type 2 EBV and variants of the EBV BZLF1 promoter zone (Zp with these diseases is unclear. Results The objective of this study was to investigate the relationship between EBV genotypes (Zp variants and EBV type 1 and 2 and the clinical phenotypes of EBV-associated diseases in Chinese children. The Zp region was directly sequenced in 206 EBV-positive DNA samples from the blood of patients with IM, EBV-HLH, CAEBV, and healthy controls. Type 1 or type 2 EBV was examined by PCR for EBNA2 and EBNA3C subtypes. Four polymorphic Zp variants were identified: Zp-P, Zp-V3, Zp-P4 and Zp-V1, a new variant. The Zp-V3 variant was significantly associated with CAEBV (P ≤ 0.01. The frequency of co-infection with Zp variants was higher in patients with CAEBV and EBV-HLH, compared with IM and healthy controls, mostly as Zp-P+V3 co-infection. Type 1 EBV was predominant in all categories (81.3-95% and there was no significant difference in the frequency of the EBV types 1 and 2 in different categories (P > 0.05. Conclusions Type 1 EBV and BZLF1 Zp-P of EBV were the predominant genotypes in nonmalignant EBV associated diseases in Chinese children and Zp-V3 variant may correlates with the developing of severe EBV infection diseases, such as CAEBV and EBV-HLH.

  13. Aspects and Intensity of Pediatric Palliative Case Management Provided by a Hospital-Based Case Management Team: A Comparative Study Between Children With Malignant and Nonmalignant Disease.

    Science.gov (United States)

    Jagt-van Kampen, Charissa T; Colenbrander, Derk A; Bosman, Diederik K; Grootenhuis, Martha A; Kars, Marijke C; Schouten-van Meeteren, Antoinette Yn

    2018-01-01

    Anticipating case management is considered crucial in pediatric palliative care. In 2012, our children's university hospital initiated a specialized pediatric palliative care team (PPCT) to deliver inbound and outbound case management for children with life-shortening disease. The aim of this report is to gain insight in the first 9 months of this PPCT. Aspects of care during the first 9 months of the PPCT are presented, and comparison is made between patients with malignant disease (MD) and nonmalignant disease (NMD) in a retrospective study design. Insight in the aspects of care of all patients with a life-shortening disease was retrieved from web-based files and the hour registrations from the PPCT. Forty-three children were supported by the PPCT during the first 9 months: 22 with MD with a median of 50 (1-267) days and 29 minutes (4-615) of case management per patient per day and 21 patients with NMD with a median of 79.5 (5-211) days and 16 minutes of case management per day (6-64). Our data show significantly more interprofessional contacts for patients with MD and more in-hospital contacts for patients with NMD. The median number of admission days per patient was 11 (0-22) for MD (44% for anticancer therapy) and 44 (0-303) for NMD (36% for infectious diseases). This overview of aspects of pediatric palliative case management shows shorter but more intensive case management for MD in comparison with NMD. This insight in palliative case management guides the design of a PPCT.

  14. Expression of estrogen receptors in non-malignant mammary tissue modifies the association between insulin-like growth factor 1 and breast cancer risk.

    Science.gov (United States)

    Samoli, E; Lagiou, A; Zourna, P; Barbouni, A; Georgila, C; Tsikkinis, A; Vassilarou, D; Minaki, P; Sfikas, C; Spanos, E; Trichopoulos, D; Lagiou, P

    2015-04-01

    Several studies have reported that the insulin-like growth factor 1 (IGF-1) is positively associated with estrogen receptor-positive [ER(+)] breast cancer risk, whereas there is little or no association with respect to ER(-) breast cancer. All comparisons of ER(+) breast cancer cases, however, have been made versus healthy controls, for whom there is no information about the ER expression in their mammary gland. In the context of a case-control investigation conducted in Athens, Greece, we studied 102 women with incident ERα(+) breast cancer and compared their IGF-1 blood levels with those of 178 ERα(+) and 83 ERα(-) women with benign breast disease (BBD) who underwent biopsies in the context of their standard medical care. Data were analysed using multiple logistic regression and controlling for potential confounding variables. ERα(+) breast cancer patients had higher IGF-1 levels compared with women with BBD [odds ratio (OR) 1.36, 95% confidence interval (CI): 0.95-1.94, per 1 standard deviation (SD) increase in IGF-1 levels]. When ERα status of women with BBD was taken into account, the difference in IGF-1 levels between ERα(+) breast cancer patients and women with BBD was clearly driven by the comparison with BBD women who were ERα(+) (OR = 1.95, 95% CI: 1.31-2.89 per 1 SD increase in IGF-1 levels), whereas there was essentially no association with IGF-1 levels when ERα(+) breast cancer patients were compared with ERα(-) BBD women. These contrasts were particularly evident among post/peri-menopausal women. We found evidence in support of an interaction of IGF-1 with the expression of ERα in the non-malignant mammary tissue in the context of breast cancer pathogenesis. This is in line with previous evidence suggesting that IGF-1 increases the risk of ER(+) breast cancer. Published by Oxford University Press on behalf of the European Society for Medical Oncology 2014.

  15. Mortality from non-malignant respiratory diseases among people with silicosis in Hong Kong: exposure-response analyses for exposure to silica dust.

    Science.gov (United States)

    Tse, L A; Yu, I T S; Leung, C C; Tam, W; Wong, T W

    2007-02-01

    To examine the exposure-response relationships between various indices of exposure to silica dust and the mortality from non-malignant respiratory diseases (NMRDs) or chronic obstructive pulmonary diseases (COPDs) among a cohort of workers with silicosis in Hong Kong. The concentrations of respirable silica dust were assigned to each industry and job task according to historical industrial hygiene measurements documented previously in Hong Kong. Exposure indices included cumulative dust exposure (CDE) and mean dust concentration (MDC). Penalised smoothing spline models were used as a preliminary step to detect outliers and guide further analyses. Multiple Cox's proportional hazard models were used to estimate the dust effects on the risk of mortality from NMRDs or COPDs after truncating the highest exposures. 371 of the 853 (43.49%) deaths occurring among 2789 workers with silicosis during 1981-99 were from NMRDs, and 101 (27.22%) NMRDs were COPDs. Multiple Cox's proportional hazard models showed that CDE (p = 0.009) and MDC (pcaisson workers and among those ever employed in other occupations with high exposure to silica dust. No exposure-response relationship was observed for surface construction workers with low exposures. A clear upward trend for both NMRDs and COPDs mortality was found with increasing severity of radiological silicosis. This study documented an exposure-response relationship between exposure to silica dust and the risk of death from NMRDs or COPDs among workers with silicosis, except for surface construction workers with low exposures. The risk of mortality from NMRDs increased significantly with the progression of International Labor Organization categories, independent of dust effects.

  16. Quantification of acetaldehyde and carbon dioxide in the headspace of malignant and non-malignant lung cells in vitro by SIFT-MS

    Czech Academy of Sciences Publication Activity Database

    Sule-Suso, J.; Pysanenko, Andriy; Španěl, Patrik; Smith, D.

    2009-01-01

    Roč. 134, č. 12 (2009), s. 2419-2425 ISSN 0003-2654 R&D Projects: GA ČR GA202/09/0800; GA ČR GA203/09/0256 Institutional research plan: CEZ:AV0Z40400503 Keywords : acetaldehyde * SIFT-MS * carbon dioxide Subject RIV: CF - Physical ; Theoretical Chemistry Impact factor: 3.272, year: 2009

  17. Helicobacter pylori and nonmalignant diseases.

    LENUS (Irish Health Repository)

    Alakkari, Alaa

    2012-02-01

    Research published over the past year has documented the continued decline of Helicobacter pylori-related peptic ulcer disease and increased recognition of non-H. pylori, non-steroidal anti-inflammatory drugs ulcer disease--idiopathic ulcers. Despite reduced prevalence of uncomplicated PUD, rates of ulcer complications and associated mortality remain stubbornly high. The role of H. pylori in functional dyspepsia is unclear, with some authors considering H. pylori-associated nonulcer dyspepsia a distinct organic entity. There is increasing acceptance of an inverse relationship between H. pylori and gastroesophageal reflux disease (GERD), but little understanding of how GERD might be more common\\/severe in H. pylori-negative subjects. Research has focused on factors such as different H. pylori phenotypes, weight gain after H. pylori eradication, and effects on hormones such as ghrelin that control appetite.

  18. Molecular mechanisms of anti-angiogenic effect of curcumin.

    Science.gov (United States)

    Gururaj, Anupama E; Belakavadi, Madesh; Venkatesh, Deepak A; Marmé, Dieter; Salimath, Bharathi P

    2002-10-04

    Modulation of pathological angiogenesis by curcumin (diferuloylmethane), the active principle of turmeric, seems to be an important possibility meriting mechanistic investigations. In this report, we have studied the effect of curcumin on the growth of Ehrlich ascites tumor cells and endothelial cells in vitro. Further, regulation of tumor angiogenesis by modulation of angiogenic ligands and their receptor gene expression in tumor and endothelial cells, respectively, by curcumin was investigated. Curcumin, when injected intraperitoneally (i.p) into mice, effectively decreased the formation of ascites fluid by 66% in EAT bearing mice in vivo. Reduction in the number of EAT cells and human umbelical vein endothelial cells (HUVECs) in vitro by curcumin, without being cytotoxic to these cells, is attributed to induction of apoptosis by curcumin, as is evident by an increase in cells with fractional DNA content seen in our results on FACS analysis. However, curcumin had no effect on the growth of NIH3T3 cells. Curcumin proved to be a potent angioinhibitory compound, as demonstrated by inhibition of angiogenesis in two in vivo angiogenesis assay systems, viz. peritoneal angiogenesis and chorioallantoic membrane assay. The angioinhibitory effect of curcumin in vivo was corroborated by the results on down-regulation of the expression of proangiogenic genes, in EAT, NIH3T3, and endothelial cells by curcumin. Our results on Northern blot analysis clearly indicated a time-dependent (0-24h) inhibition by curcumin of VEGF, angiopoietin 1 and 2 gene expression in EAT cells, VEGF and angiopoietin 1 gene expression in NIH3T3 cells, and KDR gene expression in HUVECs. Further, decreased VEGF levels in conditioned media from cells treated with various doses of curcumin (1 microM-1mM) for various time periods (0-24h) confirm its angioinhibitory action at the level of gene expression. Because of its non-toxic nature, curcumin could be further developed to treat chronic diseases that

  19. A Novel Therapeutic Vaccine for Metastatic Mammary Carcinoma: Focusing MHC/Peptide Complexes to Lipid Rafts

    Science.gov (United States)

    2006-11-01

    reportable outcomes). Briefly, the T cell lymphoma EL4 and the immortalized fibroblast cell line DAP (both expressing ova) were used to measure...and Use Committee. Cells , transfections, and antibodies B16.BL6 8.2, A20, EL4 and EL4 /ova were cultured as described (20-22). NIH3T3 cells were...types can donate MHC class I molecules to DC. To determine if the levels of MHC class I on the donor cell affected the efficiency of transfer, EL4 /ova

  20. Human corneal epithelial subpopulations

    DEFF Research Database (Denmark)

    Søndergaard, Chris Bath

    2013-01-01

    Corneal epithelium is being regenerated throughout life by limbal epithelial stem cells (LESCs) believed to be located in histologically defined stem cell niches in corneal limbus. Defective or dysfunctional LESCs result in limbal stem cell deficiency (LSCD) causing pain and decreased visual acuity...... subpopulations in human corneal epithelium using a combination of laser capture microdissection and RNA sequencing for global transcriptomic profiling. We compared dissociation cultures, using either expansion on γ-irradiated NIH/3T3 feeder cells in serum-rich medium or expansion directly on plastic in serum...

  1. [Carcinogenesis and its mechanism of mutant-type[12Asp]K-ras4B gene].

    Science.gov (United States)

    Gui, Li-ming; Wei, Li-hui; Zhang, Ying-mei; Wang, Jian-liu; Wang, Ying; Chen, Ying; Ma, Da-long

    2002-01-01

    Ras gene plays an important role in the extra- and intra-cellular signal transduction pathway. It mediates series cascade reactions, and eventually actives transcriptional factors in nucleus. It is unknown on the mechanism of carcinogenesis of Ras gene in endometrial carcinoma, though K-ras mutant is very common in endometrial atypical hyperplasia and carcinoma. On basis of discovering the mutation in 12th codon of K-ras in endometrial carcinoma cell line, HEC-1A, we explored the carcinogenesis and molecular mechanism of mutant-type [12Asp] K-ras4B gene. (1) Full-length [12Asp]K-ras4B cDNA was amplified with RT-PCR, then inserted into pcDI eukaryotic expressive vector. (2) Morphological change, growth kinetics in vitro and tumorigencity in nude mice in vivo after-before transfection were observed. (3) To test the cell growth kinetics by methyl thiazolium tetrazolium (MTT) and [3H]thymidine incorporation method. (1) The authors have successfully constructed eukaryotic expression plasmid pcDI-[12Asp] K-ras4B; (2) To confirm that [12Asp] K-ras4B mutant can trigger the neoplastic transformation of NIH3T3 cells by test in vitro and in vivo. (3) After pMCV-RasN17 plasmid, a Ras mutant were transfected into pcDI-[12Asp] K-ras4B cells, the growth of this cell were restrained significantly in comparison with control group. (4) These findings indicate the expression of RafS621A resulted in remarkable inhibition in proliferation of pcDI-[12Asp]K-ras4B cell (P ras4B cell growth (P ras4B gene alone is able to cause neoplastic transformation in NIH3T3 cells in vitro and in vivo. (2) [12Asp]K-ras4B-induced NIH3T3 cells neoplastic transformation required Raf signaling pathway.

  2. Entrapping quercetin in silica/polyethylene glycol hybrid materials: Chemical characterization and biocompatibility

    Energy Technology Data Exchange (ETDEWEB)

    Catauro, Michelina, E-mail: michelina.catauro@unina2.it [Department of Industrial and Information Engineering, Second University of Naples, Via Roma 29, 81031 Aversa (Italy); Bollino, Flavia [Department of Industrial and Information Engineering, Second University of Naples, Via Roma 29, 81031 Aversa (Italy); Nocera, Paola; Piccolella, Simona; Pacifico, Severina [Department Environmental, Biological and Pharmaceutical Sciences and Technologies, Second University of Naples, Via Vivaldi 43, 81100 Caserta (Italy)

    2016-11-01

    Sol-gel synthesis was exploited to entrap quercetin, a natural occurring antioxidant polyphenol, in silica-based hybrid materials, which differed in their polyethylene glycol (PEG) content (6, 12, 24 and 50 wt%). The materials obtained, whose nano-composite nature was ascertained by Scanning Electron Microscopy (SEM), were chemically characterized by Fourier Transform InfraRed (FT-IR) and UV-Vis spectroscopies. The results prove that a reaction between the polymer and the drug occurred. Bioactivity tests showed their ability to induce hydroxyapatite nucleation on the sample surfaces. The direct contact method was applied to screen the cytotoxicity of the synthetized materials towards fibroblast NIH 3T3 cells, commonly used for in vitro biocompatibility studies, and three nervous system cell lines (neuroblastoma SH-SY5Y, glioma U251, and pheochromocytoma PC12 cell lines), adopted as models in oxidative stress related studies. Using the MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) assay NIH 3T3 proliferation was assessed and the morphology was not compromised by direct exposure to the materials. Analogously, PC-12, and U-251 cell lines were not affected by new materials. SH-SY5Y appeared to be the most sensitive cell line with cytotoxic effects of 20–35%. - Highlights: • SiO{sub 2}/PEG quercetin organic-inorganic hybrids were synthesized via sol-gel. • The formation of apatite on materials surface after SBF proved their bioactivity. • Viability of NIH-3T3 cells was significantly increased by exposure to the hybrids. • Viability of PC-12 and U-251 cell lines was not affected by new materials. • SH-SY5Y cell proliferation was inhibited and their morphology was changed by hybrids.

  3. Investigation on the role of IGF-1 signal transduction in the biological radiation responses

    Energy Technology Data Exchange (ETDEWEB)

    Jung, U Hee; Jo, Sung Kee; Park, Hae Ran; Oh, Soo Jin; Cho, Eun Hee; Eom, Hyun Soo; Ju, Eun Jin

    2009-05-15

    Effects of {gamma}-irradiation on the IGF-1 related gene expressions and activations in various cell lines - Various expression patterns of IGF-1 and IGF-1R following {gamma}-irradiation were observed according to the cell lines - The increased expressions of IGF-1 and IGF-1R were observed in Balb/3T3 and NIH/3T3 cells - Among the IGF-1 downstream signaling molecules, the phosphorylated ERK5 were not changed by {gamma}-irradiation in all three examined cell lines, whereas the phosphorylated p65 were increased by {gamma} -irradiation in all cell lines. The role of IGF-1 and p38 signaling in {gamma}-irradiated mouse embryonic fibroblast (MEF) cells - In MEF cells, IGF-1 signaling molecules were decreased and p21/phosphorylated p38 were increased by {gamma}-irradiation - The experiments with IGF-1R inhibitor (AG1024) and p38 inhibitor (SB203580) revealed that IGF-1 signaling is involved but not essential in radiation-induced cell growth arrest and senescence and that p38 MAP kinase play a important role in this cellular radiation response. The role of IGF-1 and p38 signaling in {gamma}-irradiated mouse fibroblast (NIH/3T3) cell - In NIH/3T3 cells, IGF-1 signaling molecules and p21/phosphorylated p38 were increased by {gamma} -irradiation. - However, the experiments with IGF-1R inhibitor (AG1024) and p38 inhibitor (SB203580) revealed that IGF-1 and p38 signaling do not play a crucial role in radiation-induced cell growth arrest and senescence in NIH/3T3 cells. Effects of {gamma}-irradiation on the expressions and activations on the genes related to the IGF-1 signaling in mouse tissues - In {gamma}-irradiated mice, the increased expressions of IGF-1 and IGF-1R were observed in the lung and kidney at 2 months after irradiation, and in all the tissues examined (lung, liver and kidney) at 6 months after irradiation. - In the lung of {gamma}-irradiated mice at 6 months after irradiation, the increases of IGF-1R, phosphorylated FOXO3a, p65, p38, p21 were observed. - The

  4. Investigation on the role of IGF-1 signal transduction in the biological radiation responses

    International Nuclear Information System (INIS)

    Jung, U Hee; Jo, Sung Kee; Park, Hae Ran; Oh, Soo Jin; Cho, Eun Hee; Eom, Hyun Soo; Ju, Eun Jin

    2009-05-01

    Effects of γ-irradiation on the IGF-1 related gene expressions and activations in various cell lines - Various expression patterns of IGF-1 and IGF-1R following γ-irradiation were observed according to the cell lines - The increased expressions of IGF-1 and IGF-1R were observed in Balb/3T3 and NIH/3T3 cells - Among the IGF-1 downstream signaling molecules, the phosphorylated ERK5 were not changed by γ-irradiation in all three examined cell lines, whereas the phosphorylated p65 were increased by γ -irradiation in all cell lines. The role of IGF-1 and p38 signaling in γ-irradiated mouse embryonic fibroblast (MEF) cells - In MEF cells, IGF-1 signaling molecules were decreased and p21/phosphorylated p38 were increased by γ-irradiation - The experiments with IGF-1R inhibitor (AG1024) and p38 inhibitor (SB203580) revealed that IGF-1 signaling is involved but not essential in radiation-induced cell growth arrest and senescence and that p38 MAP kinase play a important role in this cellular radiation response. The role of IGF-1 and p38 signaling in γ-irradiated mouse fibroblast (NIH/3T3) cell - In NIH/3T3 cells, IGF-1 signaling molecules and p21/phosphorylated p38 were increased by γ -irradiation. - However, the experiments with IGF-1R inhibitor (AG1024) and p38 inhibitor (SB203580) revealed that IGF-1 and p38 signaling do not play a crucial role in radiation-induced cell growth arrest and senescence in NIH/3T3 cells. Effects of γ-irradiation on the expressions and activations on the genes related to the IGF-1 signaling in mouse tissues - In γ-irradiated mice, the increased expressions of IGF-1 and IGF-1R were observed in the lung and kidney at 2 months after irradiation, and in all the tissues examined (lung, liver and kidney) at 6 months after irradiation. - In the lung of γ-irradiated mice at 6 months after irradiation, the increases of IGF-1R, phosphorylated FOXO3a, p65, p38, p21 were observed. - The patterns of altered expressions showed significant

  5. The proto-oncogene product c-Crk associates with insulin receptor substrate-1 and 4PS. Modulation by insulin growth factor-I (IGF) and enhanced IGF-I signaling.

    Science.gov (United States)

    Beitner-Johnson, D; Blakesley, V A; Shen-Orr, Z; Jimenez, M; Stannard, B; Wang, L M; Pierce, J; LeRoith, D

    1996-04-19

    The Crk proto-oncogene product is an SH2 and SH3 domain-containing adaptor protein which we have previously shown to become rapidly tyrosine phosphorylated in response to stimulation with insulin-like growth factor I (IGF-I) in NIH-3T3 cells. In order to further characterize the role of Crk in the IGF-I signaling pathway, NIH-3T3 and 293 cells were stably transfected with an expression vector containing the Crk cDNA. The various resultant 3T3-Crk clones expressed Crk at approximately 2-15-fold higher levels than parental 3T3 cells. In 3T3-Crk cells, Crk immunoreactivity was detected in insulin receptor substrate-1 (IRS-1) immunoprecipitates. Stimulation with IGF-I resulted in a dissociation of Crk protein from IRS-1. In contrast, the association of the related adaptor protein Grb2 with IRS-1 was enhanced by IGF-I stimulation. Similar results were obtained in stably transfected 293-Crk cells, which express both IRS-1 and the IRS-1-related signaling protein 4PS. In these cells, IRS-1 and 4PS both associated with Crk, and this association was also decreased by IGF-I treatment, whereas the association of Grb2 with IRS-1 and 4PS was enhanced by IGF-I. Overexpression of Crk also enhanced IGF-I-induced mitogenesis of NIH-3T3 cells, as measured by [3H]thymidine incorporation. The levels of IGF-I-induced mitogenesis were proportional to the level of Crk expression. These results suggest that Crk is a positive effector of IGF-I signaling, and may mediate its effects via interaction with IRS-1 and/or 4PS.

  6. Radiobiological basis of total body irradiation with different dose rate and fractionation: repair capacity of hemopoietic cells

    International Nuclear Information System (INIS)

    Song, C.W.; Kim, T.H.; Khan, F.M.; Kersey, J.H.; Levitt, S.H.

    1981-01-01

    Total body irradiation (TBI) followed by bone marrow transplantation is being used in the treatment of malignant or non-malignant hemopoietic disorders. It has been believed that the ability of hemopoietic cells to repair sublethal radiation damage is negligible. Therefore, several schools of investigators suggested that TBI in a single exposure at extremely low dose rate (5 rad/min) over several hours, or in several fractions in 2-3 days, should yield a higher therapeutic gain, as compared with a single exposure at a high dose rate (26 rad/min). We reviewed the existing data in the literature, in particular, the response of hemopoietic cells to fractionated doses of irradiation and found that the repair capacity of both malignant and non-malignant hemopoietic cells might be greater than has been thought. It is concluded that we should not underestimate the ability of hemopoietic cells to repair sublethal radiation damage in using TBI

  7. The role of volume-sensitive ion transport systems in regulation of epithelial transport

    DEFF Research Database (Denmark)

    Hoffmann, Else Kay; Schettino, T; Marshall, W S

    2007-01-01

    This review focuses on using the knowledge on volume-sensitive transport systems in Ehrlich ascites tumour cells and NIH-3T3 cells to elucidate osmotic regulation of salt transport in epithelia. Using the intestine of the European eel (Anguilla anguilla) (an absorptive epithelium of the type...... on the apical side and the Na+/K+ ATPase, NKCC1 and a K+ channel on the basolateral side. Osmotic control of Cl- secretion across the operculum epithelium includes: (i) hyperosmotic shrinkage activation of NKCC1 via PKC, MLCK, p38, OSR1 and SPAK; (ii) deactivation of NKCC by hypotonic cell swelling...

  8. Characterization of a human MSX-2 cDNA and its fragment isolated as a transformation suppressor gene against v-Ki-ras oncogene.

    Science.gov (United States)

    Takahashi, C; Akiyama, N; Matsuzaki, T; Takai, S; Kitayama, H; Noda, M

    1996-05-16

    A cDNA (termed CT124) encoding a carboxyl-terminal fragment of the human homeobox protein MSX-2 was found to induce flat reversion when expressed in v-Ki-ras-transformed NIH3T3 cells. Although the expression of endogenous MSX-2 gene is low in most of the normal adult tissues examined, it is frequently activated in carcinoma-derived cell lines. Likewise, the gene is inactive in NIH3T3 cells but is transcriptionally activated after transformation by v-Ki-ras oncogene, suggesting that the intact MSX-2 may play a positive, rather than suppressive, role in cell transformation. To test this possibility, we isolated a near full-length human MSX-2 cDNA and tested its activities in two cell systems, i.e. fibroblast and myoblast. In NIH3T3 fibroblasts, although the gene by itself failed to confer a transformed phenotype, antisense MSX-2 cDNA as well as truncated CT124 cDNA interfered with the transforming activities of v-Ki-ras oncogene. In C2C12 myoblasts, MSX-2 was found to suppress MyoD gene expression, as do activated ras oncogenes, under certain culture conditions, and CT124 was found to inhibit the activities of both MSX-2 and ras in this system as well. Our findings not only suggest that CT124 may act as a dominant suppressor of MSX-2 but also raise the possibility that MSX-2 gene may be an important downstream target for the Ras signaling pathways.

  9. In vitro cytotoxicity and phototoxicity of surface-modified gold nanoparticles associated with neutral red as a potential drug delivery system in phototherapy

    Energy Technology Data Exchange (ETDEWEB)

    Verissimo, Tanira V. [School of Health Sciences, University of Brasilia, Brasilia (Brazil); Laboratory of Photochemistry and Nanobiotechnology, Faculty of Ceilandia, University of Brasilia, Brasilia (Brazil); Santos, Naiara T. [School of Health Sciences, University of Brasilia, Brasilia (Brazil); Silva, Jaqueline R.; Azevedo, Ricardo B. [Institute of Biological Sciences, University of Brasilia, Brasilia (Brazil); Gomes, Anderson J., E-mail: ajgomes@unb.br [School of Health Sciences, University of Brasilia, Brasilia (Brazil); Laboratory of Photochemistry and Nanobiotechnology, Faculty of Ceilandia, University of Brasilia, Brasilia (Brazil); Lunardi, Claure N., E-mail: clunardi@unb.br [School of Health Sciences, University of Brasilia, Brasilia (Brazil); Laboratory of Photochemistry and Nanobiotechnology, Faculty of Ceilandia, University of Brasilia, Brasilia (Brazil)

    2016-08-01

    The surface of gold nanoparticles (AuNP) was modified, improving their interaction with neutral red (NR), by using sodium thioglycolate (TGA) as a covering agent. The resulting NR-AuNPTGA system was evaluated as a potential drug delivery system for photodynamic therapy (PDT). The associations of NR with the gold nanoparticles were evaluated using UV-vis spectrometry and measurement of their zeta potential and size distribution. The toxicity and phototoxicity of NR, AuNPTGA and NR-AuNPTGA were evaluated in NIH-3T3 fibroblast and 4T1 tumor cell lines. The compounds NR and NR-AuNPTGA induced toxicity in 4T1 tumor cells and NIH-3T3 fibroblasts under visible light irradiation. Modification of the surface of AuNP with TGA prevented nanoparticle aggregation and allowed greater association with NR molecules than for naked AuNP. The photosensitizer (PS) characteristics were not affected by its association with the modified surface of the gold nanoparticles, leading to a reduction of cell viability in both cell lines assayed. This NR-AuNPTGA system is a promising drug delivery system for photodynamic cancer therapy. - Highlights: • Modified gold nanoparticle (AuNP) by sodium thioglicolate (TGA) prevents aggregation. • Neutral red (NR) adsorbed on the surface of modified gold nanoparticles (AuNPTGA). • AuNPTGA is suitable as a platform to deliver the NR under irradiation process. • Photodamage of 90% was achieved by NR added to AuNPTGA in 4T1 and NIH-3T3 cells.

  10. B cell lymphomas express CX3CR1 a non-B cell lineage adhesion molecule

    DEFF Research Database (Denmark)

    Andreasson, U.; Ek, S.; Merz, H.

    2008-01-01

    normally is not expressed on B cells, is expressed both at the mRNA and protein level in several subtypes of lymphoma. CX3CR1 has also shown to be involved in the homing to specific tissues that express the ligand, CX3CL1, in breast and prostate cancer and may thus be involved in dissemination of lymphoma......To study the differential expression of cell membrane-bound receptors and their potential role in growth and/or survival of the tumor cells, highly purified follicular lymphoma cells were analyzed, using gene expression analysis, and compared to non-malignant B cell populations. Filtering...... the genome for overexpressed genes coding for cell membrane-bound proteins/receptors resulted in a hit list of 27 identified genes. Among these, we have focused on the aberrant over expression of CX3CR1, in different types of B cell lymphoma, as compared to non-malignant B cells. We show that CX3CR1, which...

  11. Synthesis and in vitro bioactivity of mesoporous bioactive glass scaffolds

    Energy Technology Data Exchange (ETDEWEB)

    Shih, C.J., E-mail: cjshih@kmu.edu.tw [Department of Fragrance and Cosmetic Science, College of Pharmacy, Kaohsiung Medical University, Kaohsiung 807, Taiwan (China); Chen, H.T. [Department of Fragrance and Cosmetic Science, College of Pharmacy, Kaohsiung Medical University, Kaohsiung 807, Taiwan (China); Huang, L.F. [School of Pharmacy, College of Pharmacy, Kaohsiung Medical University, Kaohsiung 807, Taiwan (China); Lu, P.S.; Chang, H.F. [Department of Fragrance and Cosmetic Science, College of Pharmacy, Kaohsiung Medical University, Kaohsiung 807, Taiwan (China); Chang, I.L., E-mail: 84004@cch.org.tw [Department of Orthopaedic Surgery, Chang-Hua Christian Hospital, Changhua 500, Taiwan (China)

    2010-06-15

    The main objective of the present study was to determine the effect of thermal treatment procedures (calcination temperature, heating rate and duration time) on the synthesis of SiO{sub 2}-CaO-P{sub 2}O{sub 5} mesoporous bioactive glass scaffolds. This is accomplished by thermogravimetric analyses, Fourier transform infrared (FTIR) absorption spectra, X-ray diffraction (XRD) and by analysis of nitrogen adsorption/desorption isotherms. In vitro bioactivity can also be assessed by the cytotoxic effect of the glasses on the NIH-3T3 cell line, and by characterization of MC-3T3-E1 cell attachment.

  12. Synthesis and in vitro bioactivity of mesoporous bioactive glass scaffolds

    International Nuclear Information System (INIS)

    Shih, C.J.; Chen, H.T.; Huang, L.F.; Lu, P.S.; Chang, H.F.; Chang, I.L.

    2010-01-01

    The main objective of the present study was to determine the effect of thermal treatment procedures (calcination temperature, heating rate and duration time) on the synthesis of SiO 2 -CaO-P 2 O 5 mesoporous bioactive glass scaffolds. This is accomplished by thermogravimetric analyses, Fourier transform infrared (FTIR) absorption spectra, X-ray diffraction (XRD) and by analysis of nitrogen adsorption/desorption isotherms. In vitro bioactivity can also be assessed by the cytotoxic effect of the glasses on the NIH-3T3 cell line, and by characterization of MC-3T3-E1 cell attachment.

  13. Titanium tungsten coatings for bioelectrochemical applications

    DEFF Research Database (Denmark)

    Wierzbicki, Rafal; Amato, Letizia; Łopacińska, J.

    2011-01-01

    This paper presents an assessment of titanium tungsten (TiW) coatings and their applicability as components of biosensing systems. The focus is put on using TiW as an electromechanical interface layer between carbon nanotube (CNT) forests and silicon nanograss (SiNG) cell scaffolds. Cytotoxicity......, applicability to plasma-enhanced chemical vapor deposition (PECVD) of aligned CNT forests, and electrochemical performance are investigated. Experiments include culturing of NIH3T3 mouse embryonic fibroblast cells on TiW coated silicon scaffolds, CNT growth on TiW substrates with nickel catalyst, and cyclic...

  14. Eps15R is a tyrosine kinase substrate with characteristics of a docking protein possibly involved in coated pits-mediated internalization

    DEFF Research Database (Denmark)

    Coda, L; Salcini, A E; Confalonieri, S

    1998-01-01

    in NIH-3T3 cells overexpressing the receptor, even at low levels of receptor occupancy, thus behaving as physiological substrates. A role for eps15R in clathrin-mediated endocytosis is suggested by its localization in plasma membrane-coated pits and in vivo association to the coated pits' adapter protein...... AP-2. Finally, we demonstrate that a sizable fraction of eps15R exists in the cell as a complex with eps15 and that its EH domains exhibit binding specificities that are partially distinct from those of eps15. We propose that eps15 and eps15R are multifunctional binding proteins that serve...

  15. Endothelial Cell Migration and Vascular Endothelial Growth Factor Expression Are the Result of Loss of Breast Tissue Polarity

    Energy Technology Data Exchange (ETDEWEB)

    Chen, Amy; Cuevas, Ileana; Kenny, Paraic A; Miyake, Hiroshi; Mace, Kimberley; Ghajar, Cyrus; Boudreau, Aaron; Bissell, Mina; Boudreau, Nancy

    2009-05-26

    Recruiting a new blood supply is a rate-limiting step in tumor progression. In a three-dimensional model of breast carcinogenesis, disorganized, proliferative transformed breast epithelial cells express significantly higher expression of angiogenic genes compared with their polarized, growth-arrested nonmalignant counterparts. Elevated vascular endothelial growth factor (VEGF) secretion by malignant cells enhanced recruitment of endothelial cells (EC) in heterotypic cocultures. Significantly, phenotypic reversion of malignant cells via reexpression of HoxD10, which is lost in malignant progression, significantly attenuated VEGF expression in a hypoxia-inducible factor 1{alpha}-independent fashion and reduced EC migration. This was due primarily to restoring polarity: forced proliferation of polarized, nonmalignant cells did not induce VEGF expression and EC recruitment, whereas disrupting the architecture of growth-arrested, reverted cells did. These data show that disrupting cytostructure activates the angiogenic switch even in the absence of proliferation and/or hypoxia and restoring organization of malignant clusters reduces VEGF expression and EC activation to levels found in quiescent nonmalignant epithelium. These data confirm the importance of tissue architecture and polarity in malignant progression.

  16. In vitro toxicological nanoparticle studies under flow exposure

    Energy Technology Data Exchange (ETDEWEB)

    Sambale, Franziska, E-mail: sambale@iftc.uni-hannover.de; Stahl, Frank; Bahnemann, Detlef; Scheper, Thomas [Gottfried Wilhelm Leibniz University Hanover, Institute for Technical Chemistry (Germany)

    2015-07-15

    The use of nanoparticles is becoming increasingly common in industry and everyday objects. Thus, extensive risk management concerning the potential health risk of nanoparticles is important. Currently, in vitro nanoparticle testing is mainly performed under static culture conditions without any shear stress. However, shear stress is an important biomechanical parameter. Therefore, in this study, a defined physiological flow to different mammalian cell lines such as A549 cells and NIH-3T3 cells has been applied. The effects of zinc oxide and titanium dioxide nanoparticles (TiO{sub 2}-NP), respectively, were investigated under both static and dynamic conditions. Cell viability, cell morphology, and adhesion were proven and compared to the static cell culture. Flow exposure had an impact on the cellular morphology of the cells. NIH-3T3 cells were elongated in the direction of flow and A549 cells exhibited vesicles inside the cells. Zinc oxide nanoparticles reduced the cell viability in the static and in the dynamic culture; however, the dynamic cultures were more sensitive. In the static culture and in the dynamic culture, TiO{sub 2}-NP did not affect cell viability. In conclusion, dynamic culture conditions are important for further in vitro investigations and provide more relevant results than static culture conditions.

  17. In vitro toxicological nanoparticle studies under flow exposure

    International Nuclear Information System (INIS)

    Sambale, Franziska; Stahl, Frank; Bahnemann, Detlef; Scheper, Thomas

    2015-01-01

    The use of nanoparticles is becoming increasingly common in industry and everyday objects. Thus, extensive risk management concerning the potential health risk of nanoparticles is important. Currently, in vitro nanoparticle testing is mainly performed under static culture conditions without any shear stress. However, shear stress is an important biomechanical parameter. Therefore, in this study, a defined physiological flow to different mammalian cell lines such as A549 cells and NIH-3T3 cells has been applied. The effects of zinc oxide and titanium dioxide nanoparticles (TiO 2 -NP), respectively, were investigated under both static and dynamic conditions. Cell viability, cell morphology, and adhesion were proven and compared to the static cell culture. Flow exposure had an impact on the cellular morphology of the cells. NIH-3T3 cells were elongated in the direction of flow and A549 cells exhibited vesicles inside the cells. Zinc oxide nanoparticles reduced the cell viability in the static and in the dynamic culture; however, the dynamic cultures were more sensitive. In the static culture and in the dynamic culture, TiO 2 -NP did not affect cell viability. In conclusion, dynamic culture conditions are important for further in vitro investigations and provide more relevant results than static culture conditions

  18. Reference: 453 [Arabidopsis Phenome Database[Archive

    Lifescience Database Archive (English)

    Full Text Available ctor. We demonstrate that this protein functions as a transcriptional repressor in vivo. The express...ion of all members of the CYCLINA2 (CYCA2) family was reduced in an ILP1 overexpressing l...ine, and the mouse (Mus musculus) homolog of ILP1 repressed cyclin A2 expression in mouse NIH3T3 cells. T-DN...A insertion mutants of ILP1 showed reduced polyploidy and upregulated all CYCA2 express...ion. Furthermore, loss of CYCA2;1 expression induces an increase in polyploidy in Arabidopsis. We demo

  19. Lipid droplets interact with mitochondria using SNAP23

    DEFF Research Database (Denmark)

    Jägerström, Sara; Polesie, Sam; Wickström, Ylva

    2009-01-01

    peroxisomes and the endoplasmic reticulum. We have used electron and confocal microscopy to demonstrate that LD form complexes with mitochondria in NIH 3T3 fibroblasts. Using an in vitro system of purified LD and mitochondria, we also show the formation of the LD-mitochondria complex, in which cytosolic...... factors are involved. Moreover, the presence of LD markers in mitochondria isolated by subcellular fractionations is demonstrated. Finally, ablation of SNAP23 using siRNA reduced complex formation and beta oxidation, which suggests that the LD-mitochondria complex is functional in the cell....

  20. Imaging in haematopoietic stem cell transplantation

    International Nuclear Information System (INIS)

    Evans, A.; Steward, C.G.; Lyburn, I.D.; Grier, D.J.

    2003-01-01

    Haematopoietic stem cell transplantation (SCT) is used to treat a wide range of malignant and non-malignant haematological conditions, solid malignancies, and metabolic and autoimmune diseases. Although imaging has a limited role before SCT, it is important after transplantation when it may support the clinical diagnosis of a variety of complications. It may also be used to monitor the effect of therapy and to detect recurrence of the underlying disease if the transplant is unsuccessful. We present a pictorial review of the imaging of patients who have undergone SCT, based upon 15 years experience in a large unit performing both adult and paediatric transplants

  1. Imaging in haematopoietic stem cell transplantation

    Energy Technology Data Exchange (ETDEWEB)

    Evans, A.; Steward, C.G.; Lyburn, I.D.; Grier, D.J

    2003-03-01

    Haematopoietic stem cell transplantation (SCT) is used to treat a wide range of malignant and non-malignant haematological conditions, solid malignancies, and metabolic and autoimmune diseases. Although imaging has a limited role before SCT, it is important after transplantation when it may support the clinical diagnosis of a variety of complications. It may also be used to monitor the effect of therapy and to detect recurrence of the underlying disease if the transplant is unsuccessful. We present a pictorial review of the imaging of patients who have undergone SCT, based upon 15 years experience in a large unit performing both adult and paediatric transplants.

  2. Is the carbohydrate sialosyl-Tn a marker for altered, non-malignant activity in squamous epithelium in the head and neck region?

    DEFF Research Database (Denmark)

    Bryne, M.; Gravdahl, C.; Koppang, H.S.

    1995-01-01

    on basal cells in some lesions with epithelial hyperplasia and dysplasia. The aim of this study was to carry out a systematic investigation of STn expression on epithelial basal cells in hyperplastic, 'borderline' malignant, and malignant head and neck lesions, to see if the expression of STn is associated...... with basal cells in other locations. The most highly differentiated lesions, such as focal epithelial hyperplasia and verrucous hyperplasia, revealed a high percentage (86 per cent in both cases) of STn reactivity. The least-differentiated verrucous carcinomas (VCs) and keratoacanthomas (KAs) did not express...

  3. Tumour-initiating cells vs. cancer 'stem' cells and CD133: What's in the name?

    International Nuclear Information System (INIS)

    Neuzil, Jiri; Stantic, Marina; Zobalova, Renata; Chladova, Jaromira; Wang, Xiufang; Prochazka, Lubomir; Dong, Lanfeng; Andera, Ladislav; Ralph, Stephen J.

    2007-01-01

    Recent evidence suggests that a subset of cells within a tumour have 'stem-like' characteristics. These tumour-initiating cells, distinct from non-malignant stem cells, show low proliferative rates, high self-renewing capacity, propensity to differentiate into actively proliferating tumour cells, resistance to chemotherapy or radiation, and they are often characterised by elevated expression of the stem cell surface marker CD133. Understanding the molecular biology of the CD133 + cancer cells is now essential for developing more effective cancer treatments. These may include drugs targeting organelles, such as mitochondria or lysosomes, using highly efficient and selective inducers of apoptosis. Alternatively, agents or treatment regimens that enhance sensitivity of these therapy-resistant 'tumour stem cells' to the current or emerging anti-tumour drugs would be of interest as well

  4. A comparison of cell proliferation in normal and neoplastic intestinal epithelia following either biogenic amine depletion or monoamine oxidase inhibition.

    Science.gov (United States)

    Tutton, P J; Barkla, D H

    1976-08-11

    Epithelial cell proliferation was studied in the jejunum and in the colon of normal rats, in the colon of dimethylhydrazine-treated rats and in dimethylhydrazine-induced adenocarcinoma of the colon using a stathmokinetic technique. Estimates of cell proliferation rates in these four tissues were then repeated in animals which had been depleted of biogenic animes by treatment with reserpine and in animals whose monoamine oxidase was inhibited by treatment with nialamide. In amine-depleted animals cell proliferation essentially ceased in all four tissues examined. Inhibition of monoamine oxidase did not significantly influence cell proliferation in nonmalignant tissues but accelerated cell division in colonic tumours.

  5. Rplp1 bypasses replicative senescence and contributes to transformation

    Energy Technology Data Exchange (ETDEWEB)

    Artero-Castro, A. [Pathology Department, Fundacio Institut de Recerca Hospital Vall d' Hebron, Passeig Vall d' Hebron 119-129, 08035 Barcelona (Spain); Kondoh, H. [Department of Geriatric Medicine, Graduate School of Medicine, Kyoto University, 54 Kawahara-cho, Shogoin, Sakyo-ku, Kyoto 606-8507 (Japan); Fernandez-Marcos, P.J.; Serrano, M. [Spanish National Cancer Research Center (CNIO), 3 Melchor Fernandez Almagro St, Madrid 28029 (Spain); Ramon y Cajal, S. [Pathology Department, Fundacio Institut de Recerca Hospital Vall d' Hebron, Passeig Vall d' Hebron 119-129, 08035 Barcelona (Spain); LLeonart, M.E., E-mail: melleona@ir.vhebron.net [Pathology Department, Fundacio Institut de Recerca Hospital Vall d' Hebron, Passeig Vall d' Hebron 119-129, 08035 Barcelona (Spain)

    2009-05-01

    To determine whether genes expressed by embryonic stem cells have a proliferative effect in primary cells, primary mouse embryonic fibroblasts were infected with an ES cell cDNA library. This led to identification of the ribosomal protein, Rplp1, a member of the P group of ribosomal proteins, whose putative role for bypassing replicative senescence in MEFs was investigated. Our results show that Rplp1 produces a two-fold increase in the expression of an E2F1 promoter and upregulation of cyclin E in MEFs. Therefore, this study is the first to show that overexpression of a single ribosomal protein, Rplp1, is a cause and not a consequence of cell proliferation. In addition, co-expression of Rplp1 with mutant ras{sup Val12} contributed to transformation in NIH3T3 cells, as was evidenced by colony production in soft-agar assays. Moreover, the Rplp1 protein was upregulated in MEFs and NIH3T3 cells upon expression of a p53 dominant negative mutant gene designated p53R175H. Hence, mutation of p53 may facilitate immortalization in vitro by upregulating Rplp1. Lastly, Rplp1 mRNA was found to be upregulated in 16 of 26 human colon cancer biopsy specimens, a finding that may be of relevance to cancer research.

  6. Rplp1 bypasses replicative senescence and contributes to transformation

    International Nuclear Information System (INIS)

    Artero-Castro, A.; Kondoh, H.; Fernandez-Marcos, P.J.; Serrano, M.; Ramon y Cajal, S.; LLeonart, M.E.

    2009-01-01

    To determine whether genes expressed by embryonic stem cells have a proliferative effect in primary cells, primary mouse embryonic fibroblasts were infected with an ES cell cDNA library. This led to identification of the ribosomal protein, Rplp1, a member of the P group of ribosomal proteins, whose putative role for bypassing replicative senescence in MEFs was investigated. Our results show that Rplp1 produces a two-fold increase in the expression of an E2F1 promoter and upregulation of cyclin E in MEFs. Therefore, this study is the first to show that overexpression of a single ribosomal protein, Rplp1, is a cause and not a consequence of cell proliferation. In addition, co-expression of Rplp1 with mutant ras Val12 contributed to transformation in NIH3T3 cells, as was evidenced by colony production in soft-agar assays. Moreover, the Rplp1 protein was upregulated in MEFs and NIH3T3 cells upon expression of a p53 dominant negative mutant gene designated p53R175H. Hence, mutation of p53 may facilitate immortalization in vitro by upregulating Rplp1. Lastly, Rplp1 mRNA was found to be upregulated in 16 of 26 human colon cancer biopsy specimens, a finding that may be of relevance to cancer research.

  7. Hyaluronic acid oligosaccharide modified redox-responsive mesoporous silica nanoparticles for targeted drug delivery.

    Science.gov (United States)

    Zhao, Qinfu; Geng, Hongjian; Wang, Ying; Gao, Yikun; Huang, Jiahao; Wang, Yan; Zhang, Jinghai; Wang, Siling

    2014-11-26

    A redox-responsive delivery system based on colloidal mesoporous silica (CMS) has been developed, in which 6-mercaptopurine (6-MP) was conjugated to vehicles by cleavable disulfide bonds. The oligosaccharide of hyaluronic acid (oHA) was modified on the surface of CMS by disulfide bonds as a targeting ligand and was able to increase the stability and biocompatibility of CMS under physiological conditions. In vitro release studies indicated that the cumulative release of 6-MP was less than 3% in the absence of glutathione (GSH), and reached nearly 80% within 2 h in the presence of 3 mM GSH. Confocal microscopy and fluorescence-activated cell sorter (FACS) methods were used to evaluate the cellular uptake performance of fluorescein isothiocyanate (FITC) labeled CMS, with and without oHA modification. The CMS-SS-oHA exhibited a higher cellular uptake performance via CD44 receptor-mediated endocytosis in HCT-116 (CD44 receptor-positive) cells than in NIH-3T3 (CD44 receptor-negative) cells. 6-MP loaded CMS-SS-oHA exhibited greater cytotoxicity against HCT-116 cells than NIH-3T3 cells due to the enhanced cell uptake behavior of CMS-SS-oHA. This study provides a novel strategy to covalently link bioactive drug and targeting ligand to the interiors and exteriors of mesoporous silica to construct a stimulus-responsive targeted drug delivery system.

  8. Resveratrol Sensitizes Selectively Thyroid Cancer Cell to 131-Iodine Toxicity

    Directory of Open Access Journals (Sweden)

    Seyed Jalal Hosseinimehr

    2014-01-01

    Full Text Available Background. In this study, the radiosensitizing effect of resveratrol as a natural product was investigated on cell toxicity induced by 131I in thyroid cancer cell. Methods. Human thyroid cancer cell and human nonmalignant fibroblast cell (HFFF2 were treated with 131I and/or resveratrol at different concentrations for 48 h. The cell proliferation was measured by determination of the percent of the survival cells using 3-(4,5-dimethylthiazol-2-yl-2,5-diphenyltetrazolium bromide (MTT assay. Results. Findings of this study show that resveratrol enhanced the cell death induced by 131I on thyroid cancer cell. Also, resveratrol exhibited a protective effect on normal cells against 131I toxicity. Conclusion. This result indicates a promising effect of resveratrol on improvement of cellular toxicity during iodine therapy.

  9. Endothelial cell subpopulations in vitro: cell volume, cell cycle, and radiosensitivity

    International Nuclear Information System (INIS)

    Rubin, D.B.; Drab, E.A.; Bauer, K.D.

    1989-01-01

    Vascular endothelial cells (EC) are important clinical targets of radiation and other forms of free radical/oxidant stresses. In this study, we found that the extent of endothelial damage may be determined by the different cytotoxic responses of EC subpopulations. The following characteristics of EC subpopulations were examined: (1) cell volume; (2) cell cycle position; and (3) cytotoxic indexes for both acute cell survival and proliferative capacity after irradiation (137Cs, gamma, 0-10 Gy). EC cultured from bovine aortas were separated by centrifugal elutriation into subpopulations of different cell volumes. Through flow cytometry, we found that cell volume was related to the cell cycle phase distribution. The smallest EC were distributed in G1 phase and the larger cells were distributed in either early S, middle S, or late S + G2M phases. Cell cycle phase at the time of irradiation was not associated with acute cell loss. However, distribution in the cell cycle did relate to cell survival based on proliferative capacity (P less than 0.01). The order of increasing radioresistance was cells in G1 (D0 = 110 cGy), early S (135 cGy), middle S (145 cGy), and late S + G2M phases (180 cGy). These findings (1) suggest an age-related response to radiation in a nonmalignant differentiated cell type and (2) demonstrate EC subpopulations in culture

  10. AFM indentation study of breast cancer cells

    International Nuclear Information System (INIS)

    Li, Q.S.; Lee, G.Y.H.; Ong, C.N.; Lim, C.T.

    2008-01-01

    Mechanical properties of individual living cells are known to be closely related to the health and function of the human body. Here, atomic force microscopy (AFM) indentation using a micro-sized spherical probe was carried out to characterize the elasticity of benign (MCF-10A) and cancerous (MCF-7) human breast epithelial cells. AFM imaging and confocal fluorescence imaging were also used to investigate their corresponding sub-membrane cytoskeletal structures. Malignant (MCF-7) breast cells were found to have an apparent Young's modulus significantly lower (1.4-1.8 times) than that of their non-malignant (MCF-10A) counterparts at physiological temperature (37 deg. C), and their apparent Young's modulus increase with loading rate. Both confocal and AFM images showed a significant difference in the organization of their sub-membrane actin structures which directly contribute to their difference in cell elasticity. This change may have facilitated easy migration and invasion of malignant cells during metastasis

  11. Possible involvement of MSX-2 homeoprotein in v-ras-induced transformation.

    Science.gov (United States)

    Takahashi, C; Akiyama, N; Kitayama, H; Takai, S; Noda, M

    1997-04-01

    A truncated MSX-2 homeoprotein was found to induce flat reversion when expressed in v-Ki-ras-transformed NIH3T3 cells. Although the expression of endogenous MSX-2 gene is low in most of the normal adult tissues examined, it is frequently activated in carcinoma-derived cell lines. Likewise, the gene is inactive in untransformed cells but is transcriptionally activated after transformation by v-Ki-ras oncogene, suggesting that the intact MSX-2 may play a positive, rather than suppressive, role in cell transformation. To test this possibility, we isolated a full-length human MSX-2 cDNA and tested its activities in two cell systems: fibroblast and myoblast. In NIH3T3 fibroblasts, although the gene by itself failed to confer a transformed phenotype, antisense MSX-2 cDNA as well as truncated MSX-2 cDNA interfered with the transforming activities of both v-Ki-ras and v-raf oncogene. In C2C12 myoblasts, MSX-2 was found to suppress MyoD gene expression, as do activated ras oncogenes, under certain culture conditions, and truncated MSX-2 cDNA was found to inhibit the activities of both MSX-2 and ras in this system as well. Our findings not only suggest that the truncated version MSX-2 may act as a dominant suppressor of intact MSX-2 but also raise the possibility that MSX-2 gene may be an important downstream target for the Ras signaling pathways.

  12. Characterization of KRAS Rearrangements in Metastatic Prostate Cancer

    Science.gov (United States)

    Wang, Xiao-Song; Shankar, Sunita; Dhanasekaran, Saravana M.; Ateeq, Bushra; Sasaki, Atsuo T.; Jing, Xiaojun; Robinson, Daniel; Cao, Qi; Prensner, John R.; Yocum, Anastasia K.; Wang, Rui; Fries, Daniel F.; Han, Bo; Asangani, Irfan A.; Cao, Xuhong; Li, Yong; Omenn, Gilbert S.; Pflueger, Dorothee; Gopalan, Anuradha; Reuter, Victor E.; Kahoud, Emily Rose; Cantley, Lewis C.; Rubin, Mark A.; Palanisamy, Nallasivam; Varambally, Sooryanarayana; Chinnaiyan, Arul M.

    2011-01-01

    Using an integrative genomics approach called Amplification Breakpoint Ranking and Assembly (ABRA) analysis, we nominated KRAS as a gene fusion with the ubiquitin-conjugating enzyme UBE2L3 in the DU145 cell line, originally derived from prostate cancer metastasis to the brain. Interestingly, analysis of tissues revealed that 2 of 62 metastatic prostate cancers harbored aberrations at the KRAS locus. In DU145 cells, UBE2L3-KRAS produces a fusion protein, specific knock-down of which, attenuates cell invasion and xenograft growth. Ectopic expression of the UBE2L3-KRAS fusion protein exhibits transforming activity in NIH 3T3 fibroblasts and RWPE prostate epithelial cells in vitro and in vivo. In NIH 3T3 cells, UBE2L3-KRAS attenuates MEK/ERK signaling, commonly engaged by oncogenic mutant KRAS, and instead signals via AKT and p38 MAPK pathways. This is the first report of a gene fusion involving Ras family suggesting that this aberration may drive metastatic progression in a rare subset of prostate cancers. PMID:22140652

  13. Synthesis and Evaluation of New Potential Benzo[a]phenoxazinium Photosensitizers for Anticancer Photodynamic Therapy

    Directory of Open Access Journals (Sweden)

    Juan Zhang

    2018-06-01

    Full Text Available The use of photodynamic therapy (PDT and development of novel photosensitizers (PSs for cancer treatment have received more and more attention nowadays. In the present work, five benzo[a]phenoxazinium derivatives have been prepared and evaluated for their in vitro anticancer photodynamic activity for the first time. They are red light absorbers and show low fluorescence quantum yield. Of these compounds, PS4 exhibited a higher quantum yield for reactive oxygen species (ROS generation. The assays with cells in vitro showed that PS1 and PS4 were not significantly toxic in the dark, but was robustly toxic against the murine breast adenocarcinoma cells 4T1 and normal murine fibroblast cells NIH-3T3 upon photoactivation. More interestingly, PS5 was particularly selective towards 4T1 cancer cells and nearly non-phototoxic to non-cancerous NIH-3T3 cells. The results described in this report suggest that these new benzo[a]phenoxazinium derivatives are potential candidates as PSs for anticancer PDT. Further investigation of benzo[a]phenoxaziniums for anticancer PDT is warranted.

  14. ATM suppresses SATB1-induced malignant progression in breast epithelial cells.

    Directory of Open Access Journals (Sweden)

    Ellen Ordinario

    Full Text Available SATB1 drives metastasis when expressed in breast tumor cells by radically reprogramming gene expression. Here, we show that SATB1 also has an oncogenic activity to transform certain non-malignant breast epithelial cell lines. We studied the non-malignant MCF10A cell line, which is used widely in the literature. We obtained aliquots from two different sources (here we refer to them as MCF10A-1 and MCF10A-2, but found them to be surprisingly dissimilar in their responses to oncogenic activity of SATB1. Ectopic expression of SATB1 in MCF10A-1 induced tumor-like morphology in three-dimensional cultures, led to tumor formation in immunocompromised mice, and when injected into tail veins, led to lung metastasis. The number of metastases correlated positively with the level of SATB1 expression. In contrast, SATB1 expression in MCF10A-2 did not lead to any of these outcomes. Yet DNA copy-number analysis revealed that MCF10A-1 is indistinguishable genetically from MCF10A-2. However, gene expression profiling analysis revealed that these cell lines have significantly divergent signatures for the expression of genes involved in oncogenesis, including cell cycle regulation and signal transduction. Above all, the early DNA damage-response kinase, ATM, was greatly reduced in MCF10A-1 cells compared to MCF10A-2 cells. We found the reason for reduction to be phenotypic drift due to long-term cultivation of MCF10A. ATM knockdown in MCF10A-2 and two other non-malignant breast epithelial cell lines, 184A1 and 184B4, enabled SATB1 to induce malignant phenotypes similar to that observed for MCF10A-1. These data indicate a novel role for ATM as a suppressor of SATB1-induced malignancy in breast epithelial cells, but also raise a cautionary note that phenotypic drift could lead to dramatically different functional outcomes.

  15. Umbilical cord bloods hematopoietic stem cells ex vivo expansion (the literature review

    Directory of Open Access Journals (Sweden)

    T. V. Shamanskaya

    2012-01-01

    Full Text Available Umbilical cord blood (CB is now an attractive source of hematopoietic stem cells (HSCs for transplantation in pediatric and adult patients with various malignant and non-malignant diseases. However, its clinical application is limited by low cells numbers in graft, which correlates with delayed engraftment, an extension of time to platelets and neutrophils recovery and increasing risk of infectious complications. Several strategies have been suggested to overcome this limitation, one of which is obtaining a sufficient number of hematopoietic progenitor cells by ex vivo expansion. Literature review about CB HSCs expansion in given article is presented.

  16. Investigating potential exogenous tumor initiating and promoting factors for Cutaneous T-Cell Lymphomas (CTCL), a rare skin malignancy

    DEFF Research Database (Denmark)

    Litvinov, Ivan V.; Shtreis, Anna; Kobayashi, Kenneth

    2016-01-01

    -Cell lymphotropic virus type 1 (HTLV1), Epstein-Barr virus (EBV), and herpes simplex virus (HSV). In this report, we review recent evidence evaluating the involvement of these agents in cancer initiation/progression. Most importantly, recent molecular experimental evidence documented for the first time that S....... aureus can activate oncogenic STAT3 signaling in malignant T cells. Specifically, S. aureus Enterotoxin type A (SEA) was recently shown to trigger non-malignant infiltrating T cells to release IL-2 and other cytokines. These signals upon binging to their cognate receptors on malignant T cells...

  17. Aequorin chimeras as valuable tool in the measurement of Ca2+ concentration during cadmium injury

    International Nuclear Information System (INIS)

    Biagioli, M.; Pinton, P.; Scudiero, R.; Ragghianti, M.; Bucci, S.; Rizzuto, R.

    2005-01-01

    The ability of cadmium to disrupt calcium homeostasis has been known since a long time, but the precise cellular targets of its toxic action are still debated. A great problem in the interpretation of data has been associated with the ability of cadmium to strongly bind traditional calcium probes. Aequorin, the well-characterized calcium-sensitive photoprotein, was used as intracellular calcium indicator during cadmium injury in NIH 3T3 murine fibroblasts. NIH 3T3 cells were transfected with a cDNA construct containing aequorin fused to a truncated glutamate receptor, which directs the probe to the outer surface of intracellular membranes. At first, we tested if different cadmium concentrations were able to modify the rate of light emission by aequorin showing that cadmium concentrations 2+ /Ca 2+ interference. To directly investigate the role of Cd 2+ in Ca 2+ homeostasis, we have started to selectively measure the free Ca 2+ concentration in different cell compartments. Here, we report that cadmium reduces the transient free calcium signal after stimulation of cells with bradykinin. Further studies are in progress to clarify the role of mitochondria and endoplasmic reticulum in cadmium-induced alterations of Ca 2+ homeostasis in order to link signal transduction modifications with the onset of apoptosis induced by cadmium exposure

  18. Study on cloning of the Egr-1 promoter and its radiation-inducible property

    International Nuclear Information System (INIS)

    Wu Congmei; Li Xiuyi; Liu Shuzheng

    2001-01-01

    Objective: Egr-1 promoter was amplified and Egr-PGL plasmid was constructed to study the expression of luciferase in transfected NIH3T3 cells after different doses of X-ray irradiation. Methods: Egr-1p was amplified by PCR and inserted into PGL3-E vector. The expression of luciferase induced by X-ray was studied by counting the light of luciferase and substrate. Results: Egr-1 cDNA was obtained by PCR and was sequenced. The results indicated the sequence was almost correct. The Egr-1p was connected with PGL3 vector and was detected by electrophoresis. The constructs were used to transfect mouse NIH3T3 cells to characterize the regulatory function of Egr-1p after exposure to X-ray irradiation. The results indicate that the expression of luciferase of all groups irradiated is higher than that of 0 Gy group. The expression of groups irradiated is about 5-7.5 times greater than that of 0 Gy group. Conclusion: Egr-1p obtained can induce the expression of its downstream gene after different doses of X-ray irradiation. Low dose irradiation also can do it and it is may be more important in tumor gene therapy

  19. The Use of Xanthan Gum as Vaccine Adjuvant: An Evaluation of Immunostimulatory Potential in BALB/c Mice and Cytotoxicity In Vitro

    Directory of Open Access Journals (Sweden)

    Rodrigo Andrade Schuch

    2017-01-01

    Full Text Available The successful production of new, safe, and effective vaccines that generate immunological memory is directly related to adjuvant feature, which is responsible for increasing and/or modulating the immune response. Several compounds display adjuvant activity, including carbohydrates. These compounds play important roles in the immune response, as well as having biocompatible properties in vaccine formulations. One such carbohydrate is xanthan gum, a polysaccharide that is produced by the plant-pathogenic bacterium Xanthomonas spp., which has adjuvant attributes. This study evaluated the immune response induced by xanthan gum associated with ovalbumin in BALB/c mice, which were subcutaneously immunized, in terms of antibody production (IgG1, IgG2a, IgG2b, and IgG3, and assessed the levels of IFN-γ in the splenocyte culture using indirect ELISA. Furthermore, we investigated in vitro cytotoxicity of xanthan in the embryo fibroblasts cell line of the NIH/3T3 mouse by MTT assay and propidium iodide uptake assay. The mice immunized with ovalbumin plus xanthan gum exhibited higher antibody IgG1 responses than control groups. Furthermore, the xanthan polysaccharide was capable of increasing the immunogenicity of antigens by producing IFN-γ and did not exhibit cytotoxicity effects in NIH/3T3 mouse fibroblast cells, considered a promising candidate for vaccine adjuvant.

  20. The Use of Xanthan Gum as Vaccine Adjuvant: An Evaluation of Immunostimulatory Potential in BALB/c Mice and Cytotoxicity In Vitro.

    Science.gov (United States)

    Schuch, Rodrigo Andrade; Oliveira, Thaís Larré; Collares, Thaís Farias; Monte, Leonardo Garcia; Inda, Guilherme Roig; Dellagostin, Odir Antonio; Vendruscolo, Claire Tondo; Moreira, Angelita da Silveira; Hartwig, Daiane Drawanz

    2017-01-01

    The successful production of new, safe, and effective vaccines that generate immunological memory is directly related to adjuvant feature, which is responsible for increasing and/or modulating the immune response. Several compounds display adjuvant activity, including carbohydrates. These compounds play important roles in the immune response, as well as having biocompatible properties in vaccine formulations. One such carbohydrate is xanthan gum, a polysaccharide that is produced by the plant-pathogenic bacterium Xanthomonas spp., which has adjuvant attributes. This study evaluated the immune response induced by xanthan gum associated with ovalbumin in BALB/c mice, which were subcutaneously immunized, in terms of antibody production (IgG1, IgG2a, IgG2b, and IgG3), and assessed the levels of IFN- γ in the splenocyte culture using indirect ELISA. Furthermore, we investigated in vitro cytotoxicity of xanthan in the embryo fibroblasts cell line of the NIH/3T3 mouse by MTT assay and propidium iodide uptake assay. The mice immunized with ovalbumin plus xanthan gum exhibited higher antibody IgG1 responses than control groups. Furthermore, the xanthan polysaccharide was capable of increasing the immunogenicity of antigens by producing IFN- γ and did not exhibit cytotoxicity effects in NIH/3T3 mouse fibroblast cells, considered a promising candidate for vaccine adjuvant.

  1. Selective transcription and cellular proliferation induced by PDGF require histone deacetylase activity

    International Nuclear Information System (INIS)

    Catania, Annunziata; Iavarone, Carlo; Carlomagno, Stella M.; Chiariello, Mario

    2006-01-01

    Histone deacetylases (HDACs) are key regulatory enzymes involved in the control of gene expression and their inhibition by specific drugs has been widely correlated to cell cycle arrest, terminal differentiation, and apoptosis. Here, we investigated whether HDAC activity was required for PDGF-dependent signal transduction and cellular proliferation. Exposure of PDGF-stimulated NIH3T3 fibroblasts to the HDAC inhibitor trichostatin A (TSA) potently repressed the expression of a group of genes correlated to PDGF-dependent cellular growth and pro-survival activity. Moreover, we show that TSA interfered with STAT3-dependent transcriptional activity induced by PDGF. Still, neither phosphorylation nor nuclear translocation and DNA-binding in vitro and in vivo of STAT3 were affected by using TSA to interfere with PDGF stimulation. Finally, TSA treatment resulted in the suppression of PDGF-dependent cellular proliferation without affecting cellular survival of NIH3T3 cells. Our data indicate that inhibition of HDAC activity antagonizes the mitogenic effect of PDGF, suggesting that these drugs may specifically act on the expression of STAT-dependent, PDGF-responsive genes

  2. Caveolin-1 interacts with the Gag precursor of murine leukaemia virus and modulates virus production

    Directory of Open Access Journals (Sweden)

    Koester Mario

    2006-09-01

    Full Text Available Abstract Background Retroviral Gag determines virus assembly at the plasma membrane and the formation of virus-like particles in intracellular multivesicular bodies. Thereby, retroviruses exploit by interaction with cellular partners the cellular machineries for vesicular transport in various ways. Results The retroviral Gag precursor protein drives assembly of murine leukaemia viruses (MLV at the plasma membrane (PM and the formation of virus like particles in multivesicular bodies (MVBs. In our study we show that caveolin-1 (Cav-1, a multifunctional membrane-associated protein, co-localizes with Gag in a punctate pattern at the PM of infected NIH 3T3 cells. We provide evidence that Cav-1 interacts with the matrix protein (MA of the Gag precursor. This interaction is mediated by a Cav-1 binding domain (CBD within the N-terminus of MA. Interestingly, the CBD motif identified within MA is highly conserved among most other γ-retroviruses. Furthermore, Cav-1 is incorporated into MLV released from NIH 3T3 cells. Overexpression of a GFP fusion protein containing the putative CBD of the retroviral MA resulted in a considerable decrease in production of infectious retrovirus. Moreover, expression of a dominant-negative Cav-1 mutant affected retroviral titres significantly. Conclusion This study demonstrates that Cav-1 interacts with MLV Gag, co-localizes with Gag at the PM and affects the production of infectious virus. The results strongly suggest a role for Cav-1 in the process of virus assembly.

  3. Evaluation of bacterial nanocellulose-based uniform wound dressing for large area skin transplantation

    Energy Technology Data Exchange (ETDEWEB)

    Fu, Lina [Department of Biomedical Engineering, College of Life Science and Technology, Huazhong University of Science and Technology, Wuhan 430074 (China); National Engineering Research Center for Nano-Medicine, Huazhong University of Science and Technology, Wuhan 430074 (China); Zhou, Ping [Institute of Organ Transplant of Tongji Hospital, Huazhong University of Science and Technology, Wuhan (China); Zhang, Shengmin [Advanced Biomaterials and Tissue Engineering Center, Huazhong University of Science and Technology, Wuhan (China); Yang, Guang, E-mail: yang_sunny@yahoo.com [Department of Biomedical Engineering, College of Life Science and Technology, Huazhong University of Science and Technology, Wuhan 430074 (China); National Engineering Research Center for Nano-Medicine, Huazhong University of Science and Technology, Wuhan 430074 (China)

    2013-07-01

    Bacterial nanocellulose (BNC) was biosynthesized by Gluconacetobacter xylinus. The surface area, physicochemical structure and morphology of the materials were characterized. Here provides a method for an efficient production of uniform BNC, which is beneficial for the fast characterization and evaluation of BNC. In vitro cytotoxicity of the materials was evaluated by the proliferation, the adhesion, the viability and the morphology of NIH/3T3 cells. Low cytotoxicity of the BNC was observed, and micrographs demonstrate a good proliferation and adhesion of NIH/3T3 cells on BNC. Large area full-thickness skin defects were made on the back of C57BL/6 mice in animal surgery. The wounds were transplanted with BNC films and the results compared to those in a control group. The rehabilitation of the wound surfaces and the pathological sections of mice were investigated and are discussed. Histological examinations demonstrated faster and better healing effect and lower inflammatory response in the BNC group than those in the control group. Preliminary results on wound dressings from BNC show a curative effect promoting the healing of epithelial tissue. BNC is a promising natural polymer with medical applications in wound dressings. - Highlights: • BNC is expected to be a promising material in wound healing and skin transplantation. • We studied surface area, physicochemical structures and morphology of uniform BNC. • Cyto-evaluation results of BNC-based wound dressing show a good biocompatibility. • Large area skin transplantation experiments suggest a good performance of healing.

  4. Evaluation of bacterial nanocellulose-based uniform wound dressing for large area skin transplantation

    International Nuclear Information System (INIS)

    Fu, Lina; Zhou, Ping; Zhang, Shengmin; Yang, Guang

    2013-01-01

    Bacterial nanocellulose (BNC) was biosynthesized by Gluconacetobacter xylinus. The surface area, physicochemical structure and morphology of the materials were characterized. Here provides a method for an efficient production of uniform BNC, which is beneficial for the fast characterization and evaluation of BNC. In vitro cytotoxicity of the materials was evaluated by the proliferation, the adhesion, the viability and the morphology of NIH/3T3 cells. Low cytotoxicity of the BNC was observed, and micrographs demonstrate a good proliferation and adhesion of NIH/3T3 cells on BNC. Large area full-thickness skin defects were made on the back of C57BL/6 mice in animal surgery. The wounds were transplanted with BNC films and the results compared to those in a control group. The rehabilitation of the wound surfaces and the pathological sections of mice were investigated and are discussed. Histological examinations demonstrated faster and better healing effect and lower inflammatory response in the BNC group than those in the control group. Preliminary results on wound dressings from BNC show a curative effect promoting the healing of epithelial tissue. BNC is a promising natural polymer with medical applications in wound dressings. - Highlights: • BNC is expected to be a promising material in wound healing and skin transplantation. • We studied surface area, physicochemical structures and morphology of uniform BNC. • Cyto-evaluation results of BNC-based wound dressing show a good biocompatibility. • Large area skin transplantation experiments suggest a good performance of healing

  5. Cell membrane softening in human breast and cervical cancer cells

    Science.gov (United States)

    Händel, Chris; Schmidt, B. U. Sebastian; Schiller, Jürgen; Dietrich, Undine; Möhn, Till; Kießling, Tobias R.; Pawlizak, Steve; Fritsch, Anatol W.; Horn, Lars-Christian; Briest, Susanne; Höckel, Michael; Zink, Mareike; Käs, Josef A.

    2015-08-01

    Biomechanical properties are key to many cellular functions such as cell division and cell motility and thus are crucial in the development and understanding of several diseases, for instance cancer. The mechanics of the cellular cytoskeleton have been extensively characterized in cells and artificial systems. The rigidity of the plasma membrane, with the exception of red blood cells, is unknown and membrane rigidity measurements only exist for vesicles composed of a few synthetic lipids. In this study, thermal fluctuations of giant plasma membrane vesicles (GPMVs) directly derived from the plasma membranes of primary breast and cervical cells, as well as breast cell lines, are analyzed. Cell blebs or GPMVs were studied via thermal membrane fluctuations and mass spectrometry. It will be shown that cancer cell membranes are significantly softer than their non-malignant counterparts. This can be attributed to a loss of fluid raft forming lipids in malignant cells. These results indicate that the reduction of membrane rigidity promotes aggressive blebbing motion in invasive cancer cells.

  6. Integration of growth factor signals at the c-fos serum response element.

    Science.gov (United States)

    Price, M A; Hill, C; Treisman, R

    1996-04-29

    A transcription factor ternary complex composed of serum response factor (SRF) and a second factor, ternary complex factor (TCF), mediates the response of the c-fos Serum Response Element to growth factors and mitogens. In NIH3T3 fibroblasts, TCF binding is required for transcriptional activation by the SRE in response to activation of the Ras-Raf-ERK pathway. We compared the properties of three members of the TCF family, Elk-1, SAP-1 and SAP-2 (ERP/NET). Although all the proteins contain sequences required for ternary complex formation with SRF, only Elk-1 and SAP-1 appear to interact with the c-fos SRE efficiently in vivo. Each TCF contains a C-terminal activation domain capable of transcriptional activation in response to activation of the Ras-Raf-ERK pathway, and this is dependent on the integrity of S/T-P motifs conserved between all the TCF family members. In contrast, activation of the SRE by whole serum and the mitogenic phospholipid LPA requires SRF binding alone. Constitutively activated members of the Rho subfamily of Ras-like GTPases are also capable of inducing activation of the SRE in the absence of TCF; unlike activated Ras itself, these proteins do not activate the TCFs in NIH3T3 cells. At the SRE, SRF- and TCF-linked signalling pathways act synergistically to potentiate transcription.

  7. Improved penetration of wild ginseng extracts into the skin using low-temperature atmospheric pressure plasma

    Science.gov (United States)

    Nam, Seoul Hee; Hae Choi, Jeong; Song, Yeon Suk; Lee, Hae-June; Hong, Jin-Woo; Kim, Gyoo Cheon

    2018-04-01

    Wild ginseng (WG) is a well-known traditional medicinal plant that grows in natural environments in deep mountains. WG has been thought to exert potent physiological and medicinal effects, and, recently, its use in skin care has attracted much interest. This study investigated the efficient penetration of WG extracts into the skin by means of low-temperature atmospheric pressure plasma (LTAPP), and its effects on the skin at the cellular and tissue levels. NIH3T3 mouse embryonic fibroblasts and HRM-2 hairless mice were used to confirm the improved absorption of WG extracts into the skin using LTAPP. The gene expression levels in NIH3T3 cells and morphological changes in skin tissues after WG treatment were monitored using both in vitro and in vivo experiments. Although WG extracts did not show any significant effects on proliferative activity and cytotoxicity, at a concentration of 1:800, it significantly increased the expression of fibronectin and vascular endothelial growth factor. In the in vivo study, the combinational treatment of LTAPP and WG markedly induced the expression of fibronectin and integrin α6, and it thickened. Our results showed that LTAPP treatment safely and effectively accelerated the penetration of the WG extracts into the skin, thereby increasing the effects of WG on the skin.

  8. Intermittent Fluorescence Oscillations in Lipid Droplets in a Live Normal and Lung Cancer Cell: Time-Resolved Confocal Microscopy.

    Science.gov (United States)

    Chowdhury, Rajdeep; Amin, Md Asif; Bhattacharyya, Kankan

    2015-08-27

    Intermittent structural oscillation in the lipid droplets of live lung cells is monitored using time-resolved confocal microscopy. Significant differences are observed between the lung cancer cell (A549) and normal (nonmalignant) lung cell (WI38). For this study, the lipid droplets are covalently labeled with a fluorescent dye, coumarin maleimide (7-diethylamino-3-(4-maleimido-phenyl)-4-methylcoumarin, CPM). The number of lipid droplets in the cancer cell is found to be ∼20-fold higher than that in the normal (nonmalignant) cell. The fluctuation in the fluorescence intensity of the dye (CPM) is attributed to the red-ox processes and periodic formation/rupture of the S-CPM bond. The amount of reactive oxygen species (ROS) is much higher in a cancer cell. This is manifested in faster oscillations (0.9 ± 0.3 s) in cancer cells compared to that in the normal cells (2.8 ± 0.7 s). Solvation dynamics in the lipid droplets of cancer cells is slower compared to that in the normal cell.

  9. Fourier analysis of the cell shape of paired human urothelial cell lines of the same origin but of different grades of transformation.

    Science.gov (United States)

    Ostrowski, K; Dziedzic-Goclawska, A; Strojny, P; Grzesik, W; Kieler, J; Christensen, B; Mareel, M

    1986-01-01

    The rationale of the present investigation is the observations made by many authors of changes in the molecular structure of the cell surface during the multistep process of malignant transformation. These changes may influence cell-matrix and cell-cell interactions and thereby cause changes in cell adhesiveness and cell shape. The aim of the present work was to investigate whether the development of various grades of transformation in vivo and in vitro of human urothelial cells is accompanied by significant changes in cell shape as measured by Fourier analysis. The following transformation grades (TGr) have been defined (Christensen et al. 1984; Kieler 1984): TGr I = nonmalignant, mortal cell lines that grow independently of fibroblasts and have a prolonged life span. TGr II = nonmalignant cell lines with an infinite life span. TGr III = malignant and immortal cell lines that grow invasively in co-cultures with embryonic chick heart fragments and possess tumorigenic properties after s.c. injection into nude mice. Comparisons of 4 pairs of cell lines were performed; each pair was of the same origin. Two pairs--each including a TGr I cell line (Hu 961b and Hu 1703S) compared to a TGr III cell line (Hu 961a or Hu 1703He)--were derived from two transitional cell carcinomas (TCC) containing a heterogeneous cell population. Two additional cell lines classified as TGr II (HCV-29 and Hu 609) were compared to two TGr III sublines (HCV-29T and Hu 609T, respectively) which arose by "spontaneous" transformation during propagation in vitro of the respective maternal TGr II-cell lines.(ABSTRACT TRUNCATED AT 250 WORDS)

  10. Breast Cancer Cells in Three-dimensional Culture Display an Enhanced Radioresponse after Coordinate Targeting of Integrin ?5?1 and Fibronectin

    Energy Technology Data Exchange (ETDEWEB)

    Nam, Jin-Min; Onodera, Yasuhito; Bissell, Mina J; Park, Catherine C

    2010-04-07

    Tactics to selectively enhance cancer radioresponse are of great interest. Cancer cells actively elaborate and remodel their extracellular matrix (ECM) to aid in survival and progression. Previous work has shown that {beta}1-integrin inhibitory antibodies can enhance the growth-inhibitory and apoptotic responses of human breast cancer cell lines to ionizing radiation, either when cells are cultured in three-dimensional laminin-rich ECM (3D lrECM) or grown as xenografts in mice. Here, we show that a specific {alpha} heterodimer of {beta}1-integrin preferentially mediates a prosurvival signal in human breast cancer cells that can be specifically targeted for therapy. 3D lrECM culture conditions were used to compare {alpha}-integrin heterodimer expression in malignant and nonmalignant cell lines. Under these conditions, we found that expression of {alpha}5{beta}1-integrin was upregulated in malignant cells compared with nonmalignant breast cells. Similarly, we found that normal and oncofetal splice variants of fibronectin, the primary ECM ligand of {alpha}5{beta}1-integrin, were also strikingly upregulated in malignant cell lines compared with nonmalignant acini. Cell treatment with a peptide that disrupts the interactions of {alpha}5{beta}1-integrin with fibronectin promoted apoptosis in malignant cells and further heightened the apoptotic effects of radiation. In support of these results, an analysis of gene expression array data from breast cancer patients revealed an association of high levels of {alpha}5-integrin expression with decreased survival. Our findings offer preclinical validation of fibronectin and {alpha}5{beta}1-integrin as targets for breast cancer therapy.

  11. STAT3-mediated constitutive expression of SOCS-3 in cutaneous T-cell lymphoma

    DEFF Research Database (Denmark)

    Brender, C; Nielsen, M; Kaltoft, K

    2001-01-01

    ) obtained from affected skin from a patient with mycosis fungoides (MF) and from peripheral blood from a patient with Sezary syndrome (SS). In contrast, constitutive SOCS-3 expression is not found in the leukemic Jurkat T-cell line, the MOLT-4 acute lymphoblastic leukemia cell line, and the monocytic......, it has been hypothesized that an aberrant SOCS expression plays a role in neoplastic transformation. This study reports on a constitutive SOCS-3 expression in cutaneous T-cell lymphoma (CTCL) cell lines. SOCS-3 protein is constitutively expressed in tumor cell lines (but not in nonmalignant T cells...... leukemic cell line U937. Expression of SOCS-3 coincides with a constitutive activation of STAT3 in CTCL tumor cells, and stable transfection of CTCL tumor cells with a dominant negative STAT3 strongly inhibits SOCS-3 expression, whereas transfection with wild-type STAT3 does not. Moreover, the reduced SOCS...

  12. Bioengineering Embryonic Stem Cell Microenvironments for the Study of Breast Cancer

    Directory of Open Access Journals (Sweden)

    Yubing Xie

    2011-11-01

    Full Text Available Breast cancer is the most prevalent disease amongst women worldwide and metastasis is the main cause of death due to breast cancer. Metastatic breast cancer cells and embryonic stem (ES cells display similar characteristics. However, unlike metastatic breast cancer cells, ES cells are nonmalignant. Furthermore, embryonic microenvironments have the potential to convert metastatic breast cancer cells into a less invasive phenotype. The creation of in vitro embryonic microenvironments will enable better understanding of ES cell-breast cancer cell interactions, help elucidate tumorigenesis, and lead to the restriction of breast cancer metastasis. In this article, we will present the characteristics of breast cancer cells and ES cells as well as their microenvironments, importance of embryonic microenvironments in inhibiting tumorigenesis, convergence of tumorigenic and embryonic signaling pathways, and state of the art in bioengineering embryonic microenvironments for breast cancer research. Additionally, the potential application of bioengineered embryonic microenvironments for the prevention and treatment of invasive breast cancer will be discussed.

  13. Exposure to a specific time-varying electromagnetic field inhibits cell proliferation via cAMP and ERK signaling in cancer cells.

    Science.gov (United States)

    Buckner, Carly A; Buckner, Alison L; Koren, Stan A; Persinger, Michael A; Lafrenie, Robert M

    2018-04-01

    Exposure to specific electromagnetic field (EMF) patterns can affect a variety of biological systems. We have shown that exposure to Thomas-EMF, a low-intensity, frequency-modulated (25-6 Hz) EMF pattern, inhibited growth and altered cell signaling in malignant cells. Exposure to Thomas-EMF for 1 h/day inhibited the growth of malignant cells including B16-BL6 mouse melanoma cells, MDA-MB-231, MDA-MB-468, BT-20, and MCF-7 human breast cancer and HeLa cervical cancer cells but did not affect non-malignant cells. The Thomas-EMF-dependent changes in cell proliferation were mediated by adenosine 3',5'-cyclic monophosphate (cAMP) and extracellular-signal-regulated kinase (ERK) signaling pathways. Exposure of malignant cells to Thomas-EMF transiently changed the level of cellular cAMP and promoted ERK phosphorylation. Pharmacologic inhibitors (SQ22536) and activators (forskolin) of cAMP production both blocked the ability of Thomas-EMF to inhibit cell proliferation, and an inhibitor of the MAP kinase pathway (PD98059) was able to partially block Thomas-EMF-dependent inhibition of cell proliferation. Genetic modulation of protein kinase A (PKA) in B16-BL6 cells also altered the effect of Thomas-EMF on cell proliferation. Cells transfected with the constitutively active form of PKA (PKA-CA), which interfered with ERK phosphorylation, also interfered with the Thomas-EMF effect on cell proliferation. The non-malignant cells did not show any EMF-dependent changes in cAMP levels, ERK phosphorylation, or cell growth. These data indicate that exposure to the specific Thomas-EMF pattern can inhibit the growth of malignant cells in a manner dependent on contributions from the cAMP and MAP kinase pathways. Bioelectromagnetics. 39;217-230, 2018. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.