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Sample records for cell line glycoproteins

  1. Distinct P-glycoprotein precursors are overproduced in independently isolated drug-resistant cell lines.

    Science.gov (United States)

    Greenberger, L M; Lothstein, L; Williams, S S; Horwitz, S B

    1988-06-01

    A family of P-glycoproteins are overproduced in multidrug-resistant cells derived from the murine macrophage-like line J774.2. To determine whether individual family members are overproduced in response to different drugs, the P-glycoprotein precursors in several independently isolated cell lines, which were selected for resistance to vinblastine or taxol, were compared. Individual cell lines selected with vinblastine overproduced P-glycoprotein precursors of either 120 or 125 kDa. Taxol-selected cell lines overproduced either the 125-kDa precursor or both precursors simultaneously. Two similar but distinct peptide maps for the mature P-glycoproteins were observed. These maps corresponded to each precursor regardless of the drug used for selection. One vinblastine-resistant cell line switched from the 125- to the 120-kDa precursor when grown in increasing concentrations of drug. This change coincided with the overexpression of a distinct subset of mRNA species that code for P-glycoprotein. It is concluded that precursor expression is not drug-specific. These data suggest that individual overproduced P-glycoprotein family members are translated as distinct polypeptides. The results may help to explain the diversity in the multidrug-resistant phenotype. PMID:2897689

  2. Dual regulation of P-glycoprotein expression by Trichostatin A in cancer cell lines

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    Balaguer Trinidad

    2012-07-01

    Full Text Available Abstract Background It has been reported that the histone deacetylase inhibitor (iHDAc trichostatin A (TSA induces an increase in MDR1 gene transcription (ABCB1. This result would compromise the use of iHDACs in combination with other cytotoxic agents that are substrates of P-glycoprotein (Pgp. It has also been reported the use of alternative promoters by the ABCB1 gene and the existence of a translational control of Pgp protein. Finally, the ABCB1 gene is located in a genetic locus with the nested gene RUNDC3B in the complementary DNA strand, raising the possibility that RUNDC3B expression could interfere with ABCB1 alternative promoter regulation. Methods A combination of RT-PCR, real time RT-PCR, Western blot and drug accumulation assays by flow cytometry has been used in this study. Results The iHDACs-induced increase in MDR1 mRNA levels is not followed by a subsequent increase in Pgp protein levels or activity in several pancreatic and colon carcinoma cell lines, suggesting a translational control of Pgp in these cell lines. In addition, the MDR1 mRNA produced in these cell lines is shorter in its 5′ end that the Pgp mRNA produced in cell lines expressing Pgp protein. The different size of the Pgp mRNA is due to the use of alternative promoters. We also demonstrate that these promoters are differentially regulated by TSA. The translational blockade of Pgp mRNA in the pancreatic carcinoma cell lines could be related to alterations in the 5′ end of the MDR1 mRNA in the Pgp protein expressing cell lines. In addition, we demonstrate that the ABCB1 nested gene RUNDC3B expression although upregulated by TSA is independent of the ABCB1 alternative promoter used. Conclusions The results show that the increase in MDR1 mRNA expression after iHDACs treatment is clinically irrelevant since this mRNA does not render an active Pgp protein, at least in colon and pancreatic cancer cell lines. Furthermore, we demonstrate that TSA in fact, regulates

  3. Resistance to paclitaxel in a cisplatin-resistant ovarian cancer cell line is mediated by P-glycoprotein.

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    Britta Stordal

    Full Text Available The IGROVCDDP cisplatin-resistant ovarian cancer cell line is also resistant to paclitaxel and models the resistance phenotype of relapsed ovarian cancer patients after first-line platinum/taxane chemotherapy. A TaqMan low-density array (TLDA was used to characterise the expression of 380 genes associated with chemotherapy resistance in IGROVCDDP cells. Paclitaxel resistance in IGROVCDDP is mediated by gene and protein overexpression of P-glycoprotein and the protein is functionally active. Cisplatin resistance was not reversed by elacridar, confirming that cisplatin is not a P-glycoprotein substrate. Cisplatin resistance in IGROVCDDP is multifactorial and is mediated in part by the glutathione pathway and decreased accumulation of drug. Total cellular glutathione was not increased. However, the enzyme activity of GSR and GGT1 were up-regulated. The cellular localisation of copper transporter CTR1 changed from membrane associated in IGROV-1 to cytoplasmic in IGROVCDDP. This may mediate the previously reported accumulation defect. There was decreased expression of the sodium potassium pump (ATP1A, MRP1 and FBP which all have been previously associated with platinum accumulation defects in platinum-resistant cell lines. Cellular localisation of MRP1 was also altered in IGROVCDDP shifting basolaterally, compared to IGROV-1. BRCA1 was also up-regulated at the gene and protein level. The overexpression of P-glycoprotein in a resistant model developed with cisplatin is unusual. This demonstrates that P-glycoprotein can be up-regulated as a generalised stress response rather than as a specific response to a substrate. Mechanisms characterised in IGROVCDDP cells may be applicable to relapsed ovarian cancer patients treated with frontline platinum/taxane chemotherapy.

  4. Sulphation of proteins secreted by a human hepatoma-derived cell line. Sulphation of N-linked oligosaccharides on alpha 2HS-glycoprotein.

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    Hortin, G; Green, E D; Baenziger, J U; Strauss, A W

    1986-01-01

    Several human glycoproteins, including alpha 1-antitrypsin, alpha 1-acid glycoprotein, transferrin, caeruloplasmin and alpha 2HS-glycoprotein, synthesized by the hepatoma-derived cell line HepG2 were observed to contain covalently linked sulphate. These proteins were estimated to contain about 0.1 mol of sulphate/mol of protein. The most abundant of the sulphated glycoproteins, alpha 2HS-glycoprotein, was analysed in detail. All of the sulphate on this protein was attached to N-linked oligosaccharides which contained sialic acid and resisted release by endoglycosidase H. Several independent analytical approaches established that approx. 10% of the molecules of alpha 2HS-glycoprotein contained sulphate. Our results suggest that a number of human plasma proteins contain small amounts of sulphate linked to oligosaccharides. Images Fig. 1. Fig. 2. Fig. 3. PMID:3017304

  5. Temozolomide Resistance in Glioblastoma Cell Lines: Implication of MGMT, MMR, P-Glycoprotein and CD133 Expression.

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    Gloria Perazzoli

    Full Text Available The use of temozolomide (TMZ has improved the prognosis for glioblastoma multiforme patients. However, TMZ resistance may be one of the main reasons why treatment fails. Although this resistance has frequently been linked to the expression of O6-methylguanine-DNA methyltransferase (MGMT it seems that this enzyme is not the only molecular mechanism that may account for the appearance of drug resistance in glioblastoma multiforme patients as the mismatch repair (MMR complex, P-glycoprotein, and/or the presence of cancer stem cells may also be implicated.Four nervous system tumor cell lines were used to analyze the modulation of MGMT expression and MGMT promoter methylation by TMZ treatment. Furthermore, 5-aza-2'-deoxycytidine was used to demethylate the MGMT promoter and O(6-benzylguanine to block GMT activity. In addition, MMR complex and P-glycoprotein expression were studied before and after TMZ exposure and correlated with MGMT expression. Finally, the effect of TMZ exposure on CD133 expression was analyzed.Our results showed two clearly differentiated groups of tumor cells characterized by low (A172 and LN229 and high (SF268 and SK-N-SH basal MGMT expression. Interestingly, cell lines with no MGMT expression and low TMZ IC50 showed a high MMR complex expression, whereas cell lines with high MGMT expression and high TMZ IC50 did not express the MMR complex. In addition, modulation of MGMT expression in A172 and LN229 cell lines was accompanied by a significant increase in the TMZ IC50, whereas no differences were observed in SF268 and SK-N-SH cell lines. In contrast, P-glycoprotein and CD133 was found to be unrelated to TMZ resistance in these cell lines.These results may be relevant in understanding the phenomenon of TMZ resistance, especially in glioblastoma multiforme patients laking MGMT expression, and may also aid in the design of new therapeutic strategies to improve the efficacy of TMZ in glioblastoma multiforme patients.

  6. Reversion of P-Glycoprotein-Mediated Multidrug Resistance in Human Leukemic Cell Line by Diallyl Trisulfide

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    Qing Xia

    2012-01-01

    Full Text Available Multidrug resistance (MDR is the major obstacle in chemotherapy, which involves multiple signaling pathways. Diallyl trisulfide (DATS is the main sulfuric compound in garlic. In the present study, we aimed to explore whether DATS could overcome P-glycoprotein-(P-gp-mediated MDR in K562/A02 cells, and to investigate whether NF-κB suppression is involved in DATS-induced reversal of MDR. MTT assay revealed that cotreatment with DATS increased the response of K562/A02 cells to adriamycin (the resistance reversal fold was 3.79 without toxic side effects. DATS could enhance the intracellular concentration of adriamycin by inhibiting the function and expression of P-gp, as shown by flow cytometry, RT-PCR, and western blot. In addition, DATS resulted in more K562/A02 cell apoptosis, accompanied by increased expression of caspase-3. The expression of NF-κB/p65 (downregulation was significantly linked to the drug-resistance mechanism of DATS, whereas the expression of IκBα was not affected by DATS. Our findings demonstrated that DATS can serve as a novel, nontoxic modulator of MDR, and can reverse the MDR of K562/A02 cells in vitro by increasing intracellular adriamycin concentration and inducing apoptosis. More importantly, we proved for the first time that the suppression of NF-κB possibly involves the molecular mechanism in the course of reversion by DATS.

  7. Reversion of p-glycoprotein-mediated multidrug resistance in human leukemic cell line by diallyl trisulfide.

    Science.gov (United States)

    Xia, Qing; Wang, Zhi-Yong; Li, Hui-Qing; Diao, Yu-Tao; Li, Xiao-Li; Cui, Jia; Chen, Xue-Liang; Li, Hao

    2012-01-01

    Multidrug resistance (MDR) is the major obstacle in chemotherapy, which involves multiple signaling pathways. Diallyl trisulfide (DATS) is the main sulfuric compound in garlic. In the present study, we aimed to explore whether DATS could overcome P-glycoprotein-(P-gp-)mediated MDR in K562/A02 cells, and to investigate whether NF-κB suppression is involved in DATS-induced reversal of MDR. MTT assay revealed that cotreatment with DATS increased the response of K562/A02 cells to adriamycin (the resistance reversal fold was 3.79) without toxic side effects. DATS could enhance the intracellular concentration of adriamycin by inhibiting the function and expression of P-gp, as shown by flow cytometry, RT-PCR, and western blot. In addition, DATS resulted in more K562/A02 cell apoptosis, accompanied by increased expression of caspase-3. The expression of NF-κB/p65 (downregulation) was significantly linked to the drug-resistance mechanism of DATS, whereas the expression of IκBα was not affected by DATS. Our findings demonstrated that DATS can serve as a novel, nontoxic modulator of MDR, and can reverse the MDR of K562/A02 cells in vitro by increasing intracellular adriamycin concentration and inducing apoptosis. More importantly, we proved for the first time that the suppression of NF-κB possibly involves the molecular mechanism in the course of reversion by DATS. PMID:22919419

  8. The inhibitory and combinative mechanism of HZ08 with P-glycoprotein expressed on the membrane of Caco-2 cell line

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    Zhang, Yanyan; Hu, Yahui; Feng, Yidong; Kodithuwakku, Nandani Darshika; Fang, Weirong [State Key Laboratory of Natural Medicines, Department of Physiology, China Pharmaceutical University, Nanjing 210009 (China); Li, Yunman, E-mail: yunmanlicpu@hotmail.com [State Key Laboratory of Natural Medicines, Department of Physiology, China Pharmaceutical University, Nanjing 210009 (China); Huang, Wenlong [Center of Drug Discovery, China Pharmaceutical University, Nanjing 210009 (China)

    2014-01-15

    Recently, the research and development of agents to reverse the phenomenon of multidrug resistance has been an attractive goal as well as a key approach to elevating the clinical survival of cancer patients. Although three generations of P-glycoprotein modulators have been identified, poor clearance and metabolism render these agents too toxic to be used in clinical application. HZ08, which has been under investigation for several years, shows a dramatic reversal effect with low cytotoxicity. For the first time, we aimed to describe the interaction between HZ08 and P-glycoprotein in Caco-2 cell line in which P-glycoprotein is overexpressed naturally. Cytotoxicity and multidrug resistance reversal assays, together with flow cytometry, fluorescence microscopy and siRNA interference as well as Caco-2 monolayer transport model were employed in this study to evaluate the interaction between HZ08 and P-glycoprotein. This study revealed that HZ08 was capable of reversing adriamycin resistance mediated by P-glycoprotein as a result of intracellular enhancement of adriamycin accumulation, which was found to be superior to verapamil. In addition, we confirmed that HZ08 suppressed the transport of Rhodamine123 in the Caco-2 monolayer model but had little effect on P-glycoprotein expression. The transport of HZ08 was diminished by P-glycoprotein inhibitors (verapamil and LY335979) and its accumulation was increased via siRNA targeting MDR1 in Caco-2 cells. Furthermore, considering the binding site of P-glycoprotein, verapamil performed as a competitive inhibitor with HZ08. In conclusion, as a P-glycoprotein substrate, HZ08 inhibited P-glycoprotein activity and may share the same binding site of verapamil to P-glycoprotein. - Highlights: • The cytotoxicity and reversing effect of HZ08 was measured in Caco-2 cell line. • HZ08 inhibited the transport of Rhodamine123 across Caco-2 cell monolayer. • The efflux ratio of HZ08 was dropped when combined with P-glycoprotein

  9. Establishment and characterization of an MDCK cell line stably-transfected with chicken Abcb1 encoding P-glycoprotein.

    Science.gov (United States)

    Sun, Yong; Guo, Tingting; Guo, Dawei; Guo, Li; Chen, Li; Zhang, Yu; Wang, Liping

    2016-06-01

    Chicken P-glycoprotein (chP-gp), encoded by Abcb1, determines the bioavailability because of its effect on pharmacokinetics of various drugs. However, comprehensive studies on chP-gp are still limited. In this study, the chicken full-length cDNA was first successfully cloned and then stably expressed in MDCK cell line. The open reading frame of chicken Abcb1 consists of 3864 nucleotides, encoding for a 1287-amino acid protein. Sequence alignments analysis showed that chicken P-gp had high identities with the homologues of turkey (95%), human (72%), pig (72%), rat (71%) and cattle (68%). The efflux ratio of rhodamine123 (Rho123, a human P-gp substrate) in chAbcb1 transfected MDCK cells was significantly higher than that in the wild type MDCK cell (6.24 vs 1.64, P<0.05), suggesting a good transporting function of chicken P-gp overexpressed in the transfected cell. Importantly, MDCK-chAbcb1 cells, unlike Caco-2 cells, exhibited biphasic saturation kinetics in transporting Rho123. In conclusion, an MDCK cell line stably expressing chAbcb1 was successfully established, which could provide a new cell model to screen its substrates and inhibitors and study the drug-drug interaction medicated via chicken P-gp. PMID:27234533

  10. Collagen promotes sustained glycoprotein VI signaling in platelets and cell lines

    NARCIS (Netherlands)

    Tomlinson, M. G.; Calaminus, S. D.; Berlanga, O.; Bori-Sanz, T.; Meyaard, L.; Watson, S. P.; Auger, J.M.

    2007-01-01

    Background: Glycoprotein (GP)VI is the major signaling receptor for collagen on platelets and signals via the associated FcR-gamma-chain, which has an immunoreceptor tyrosine-containing activation motif (ITAM). Objective: To determine why GPVI-FcR gamma signals poorly, or not at all, in response to

  11. Comparison of an immunoperoxidase "sandwich" staining method and western blot detection of P-glycoprotein in human cell lines and sarcomas.

    OpenAIRE

    Tóth, K.; Vaughan, M. M.; Slocum, H. K.; Fredericks, W. J.; Chen, Y.F.; Arredondo, M. A.; Harstrick, A.; Karakousis, C.; Baker, R. M.; Rustum, Y M

    1992-01-01

    The applicability of a multilayer immunoperoxidase "sandwich" method (IpS) developed by Chan14 for the amplified detection of P-glycoprotein (Pgp) was investigated. The authors examined 15 formalin-fixed cell lines, as well as formalin-fixed, paraffin-embedded sections from single biopsies of 46 sarcomas. The cell lines included sensitive and multidrug resistant sublines (KB, A2780, MCF-7, HeLa) with various relative degrees of resistance to doxorubicin (Dox). The sarcoma biopsy specimens wer...

  12. Effect of recombinant human interleukin-11 on expressions of interleukin-11 receptor α-chain and glycoprotein 130 in intestinal epithelium cell line-6 after neutron irradiation

    OpenAIRE

    Wang, Rui-Juan; Peng, Rui-Yun; Fu, Kai-Fei; Gao, Ya-Bing; Han, Rui-Gang; Hu, Wen-Hua; Luo, Qing-Liang; Ma, Jun-Jie

    2006-01-01

    AIM: To explore the effect of recombinant human interleukin-11 (rhIL-11) on the expressions of interleukin-11 receptor α-chain (IL-11Rα) and an additional signal transducer glycoprotein 130 (gp130) in intestinal epithelium cell line-6 (IEC-6) after neutron irradiation.

  13. TRANSFECTED MDCK CELL LINE WITH ENHANCED EXPRESSION OF CYP3A4 AND P-GLYCOPROTEIN AS A MODEL TO STUDY THEIR ROLE IN DRUG TRANSPORT AND METABOLISM

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    Kwatra, Deep; Budda, Balasubramanyam; Vadlapudi, Aswani Dutt; Vadlapatla, Ramya Krishna; Pal, Dhananjay; Ashim K. Mitra

    2012-01-01

    The aim of this study was to characterize and utilize MDCK cell line expressing CYP3A4 and P-glycoprotein as an in vitro model for evaluating drug-herb and drug-drugs of abuse interactions. MDCK cell line simultaneously expressing P-gp and CYP3A4 (MMC) was developed and characterized by using expression and activity studies. Cellular transport study of 200 μM cortisol was performed to determine their combined activity. The study was carried across MDCK-WT, MDCK-MDR1 and MMC cell lines. Simila...

  14. A transgenic Bm cell line of piggyBac transposon-derived targeting expression of humanized glycoproteins through N-glycosylation.

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    Hu, Jia-Biao; Zhang, Peng; Wang, Mei-Xian; Zhou, Fang; Niu, Yan-Shan; Miao, Yun-Gen

    2012-08-01

    Glycoproteins have been implicated in a wide variety of important biochemical and biological functions, including protein stability, immune function, enzymatic function, cellular adhesion and others. Unfortunately, there is no therapeutic protein produced in insect system to date, due to the expressed glycoproteins are paucimannosidic N-glycans, rather than the complex, terminally sialylated N-glycans in mammalian cells. In this paper, we cloned the necessary genes in glycosylation of mammalian cells, such as N-acetylglucosaminyltransferase II (Gn-TII), galactosyltransferases (Gal-Ts), 2,6-Sial-T (ST6 GalII)and 2,3-Sial-T (ST3GalIII), and transformed them to silkworm genome of BmN cell line through transgenesis to establish a transgenic Bm cell line of piggyBac transposon-derived targeting expression of humanized glycoproteins. The study supplied a new insect cell line which is practically to produce "bisected" complex N-glycans like in mammalian cells. PMID:22699878

  15. Distinct N-Glycan Glycosylation of P-glycoprotein Isolated from the Human Uterine Sarcoma Cell Line MES-SA/D×5

    OpenAIRE

    Greer, D.A.; Ivey, S.

    2007-01-01

    The uterine sarcoma human cell line MES-SA/D×5 overexpresses the MDR1 gene product, P-glycoprotein (Pgp). Pgp is a heavily glycosylated, ATP-dependent drug efflux pump expressed in many human cancers. There are more than 50 known isoforms of Pgp, which complicates the characterization of Pgp glycans because each isoform could present a different glycome. The contribution of these oligosaccharides to the structure and function of Pgp remains unclear. We identified distinct Pgp glycans recogniz...

  16. Expression of the glycoprotein of viral haemorrhagic septicaemia virus (VHSV) on the surface of the fish cell line RTG-P1 induces type 1 interferon expression in neighbouring cells

    DEFF Research Database (Denmark)

    Acosta, F.; Collet, B.; Lorenzen, Niels;

    2006-01-01

    In the present study using a luciferase/Mx promoter reporter system, it was shown that the rainbow trout gonad cell line (RTG-P1), a fibroblastic cell line, produces IFN when transfected with a plasmid encoding the glycoprotein of VHSV but not with plasmid vector alone. Only a small percentage of...

  17. Alpha-2 Heremans Schmid Glycoprotein (AHSG) Modulates Signaling Pathways in Head and Neck Squamous Cell Carcinoma Cell Line SQ20B

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    Thompson, Pamela D.; Sakwe, Amos [Department of Biochemistry and Cancer Biology, Meharry Medical College, Nashville, TN 37208 (United States); Koumangoye, Rainelli [Division of Surgical Oncology and Endocrine Surgery, Vanderbilt University Medical Center, Nashville, TN 37232 (United States); Yarbrough, Wendell G. [Division of Otolaryngology, Departments of Surgery and Pathology and Yale Cancer Center, Yale University, New Haven, CT 06520 (United States); Ochieng, Josiah [Department of Biochemistry and Cancer Biology, Meharry Medical College, Nashville, TN 37208 (United States); Marshall, Dana R., E-mail: dmarshall@mmc.edu [Department of Pathology, Anatomy and Cell Biology, Meharry Medical College, Nashville, TN 37208 (United States)

    2014-02-15

    This study was performed to identify the potential role of Alpha-2 Heremans Schmid Glycoprotein (AHSG) in Head and Neck Squamous Cell Carcinoma (HNSCC) tumorigenesis using an HNSCC cell line model. HNSCC cell lines are unique among cancer cell lines, in that they produce endogenous AHSG and do not rely, solely, on AHSG derived from serum. To produce our model, we performed a stable transfection to down-regulate AHSG in the HNSCC cell line SQ20B, resulting in three SQ20B sublines, AH50 with 50% AHSG production, AH20 with 20% AHSG production and EV which is the empty vector control expressing wild-type levels of AHSG. Utilizing these sublines, we examined the effect of AHSG depletion on cellular adhesion, proliferation, migration and invasion in a serum-free environment. We demonstrated that sublines EV and AH50 adhered to plastic and laminin significantly faster than the AH20 cell line, supporting the previously reported role of exogenous AHSG in cell adhesion. As for proliferative potential, EV had the greatest amount of proliferation with AH50 proliferation significantly diminished. AH20 cells did not proliferate at all. Depletion of AHSG also diminished cellular migration and invasion. TGF-β was examined to determine whether levels of the TGF-β binding AHSG influenced the effect of TGF-β on cell signaling and proliferation. Whereas higher levels of AHSG blunted TGF-β influenced SMAD and ERK signaling, it did not clearly affect proliferation, suggesting that AHSG influences on adhesion, proliferation, invasion and migration are primarily due to its role in adhesion and cell spreading. The previously reported role of AHSG in potentiating metastasis via protecting MMP-9 from autolysis was also supported in this cell line based model system of endogenous AHSG production in HNSCC. Together, these data show that endogenously produced AHSG in an HNSCC cell line, promotes in vitro cellular properties identified as having a role in tumorigenesis. Highlights: • Head

  18. Alpha-2 Heremans Schmid Glycoprotein (AHSG) Modulates Signaling Pathways in Head and Neck Squamous Cell Carcinoma Cell Line SQ20B

    International Nuclear Information System (INIS)

    This study was performed to identify the potential role of Alpha-2 Heremans Schmid Glycoprotein (AHSG) in Head and Neck Squamous Cell Carcinoma (HNSCC) tumorigenesis using an HNSCC cell line model. HNSCC cell lines are unique among cancer cell lines, in that they produce endogenous AHSG and do not rely, solely, on AHSG derived from serum. To produce our model, we performed a stable transfection to down-regulate AHSG in the HNSCC cell line SQ20B, resulting in three SQ20B sublines, AH50 with 50% AHSG production, AH20 with 20% AHSG production and EV which is the empty vector control expressing wild-type levels of AHSG. Utilizing these sublines, we examined the effect of AHSG depletion on cellular adhesion, proliferation, migration and invasion in a serum-free environment. We demonstrated that sublines EV and AH50 adhered to plastic and laminin significantly faster than the AH20 cell line, supporting the previously reported role of exogenous AHSG in cell adhesion. As for proliferative potential, EV had the greatest amount of proliferation with AH50 proliferation significantly diminished. AH20 cells did not proliferate at all. Depletion of AHSG also diminished cellular migration and invasion. TGF-β was examined to determine whether levels of the TGF-β binding AHSG influenced the effect of TGF-β on cell signaling and proliferation. Whereas higher levels of AHSG blunted TGF-β influenced SMAD and ERK signaling, it did not clearly affect proliferation, suggesting that AHSG influences on adhesion, proliferation, invasion and migration are primarily due to its role in adhesion and cell spreading. The previously reported role of AHSG in potentiating metastasis via protecting MMP-9 from autolysis was also supported in this cell line based model system of endogenous AHSG production in HNSCC. Together, these data show that endogenously produced AHSG in an HNSCC cell line, promotes in vitro cellular properties identified as having a role in tumorigenesis. Highlights: • Head

  19. Comparison of P-glycoprotein expression in cell lines and xenogragraft sections using I-125 MRK-16 monoclonal antibody (MAB)

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    Mehta, B.M.; Kostakoglu, L.; Levchenko, A. [Kettering Cancer, New York, NY (United States)] [and others

    1994-05-01

    P-glycoprotein (Pgp) is known to be associated with multidrug resistance (MDR). Quantitation of P-glycoprotein expression may permit appropriate therapy depending on Pgp expression in tumors. The present study was undertaken to evaluate the utility of quantitative autoradiography (QAR) in the quantification of MDR using MRK-16, a murine IgG mAb reactive against Pgp. Balb/c mice were xenografted with colchicine resistant BE(2)C/CHC cells. Animals with established tumors were sacrificed, and 8 {mu}m tumor sections were prepared. Mab MRK-16 was labeled with I-125 (150 {mu}Ci/0.625 nmole) by the iodogen method and subsequently purified by size exclusion chromatography. Consecutive tumor sections were incubated overnight at 4{degrees}C with serial dilutions of I-125 MRK-16. Similarly cell suspensions containing 1 X 10{sup 7} cells per ml were also incubated with serial dilutions. QAR analysis of tissue sections of BE(2)C/CHC tumors growing as xenografts in nude mice, determined the binding affinity (K{sub a}) for MRK-16 to be 1 x 10{sup 9} L/M and the number of binding sites (B{sub max}) to be 137, 700 per cell (222 picomols/g); it compared very well with the K{sub a} value of 5 x 10{sup 8} L/M and the B{sub max} value of 130,000 per cell (217 picomols/g) obtained from binding analysis with cell suspensions.

  20. Astragaloside Ⅳ reduces the expression level of P-glycoprotein in multidrug-resistant human hepatic cancer cell lines.

    Science.gov (United States)

    Wang, Pei-Pei; Xu, Du-Juan; Huang, Can; Wang, Wei-Ping; Xu, Wen-Ke

    2014-06-01

    Astragaloside is a saponin widely used in traditional Chinese medicine and has been reported to be a potent multidrug resistance (MDR) reversal agent. The present study investigated the role of astragaloside Ⅳ (ASIV) in the regulation of P-glycoprotein (P-gp, encoded by the mdr1 gene) and its effect on the reversal of MDR. The activity of ASIV was evaluated using human hepatic cancer cells Bel-7402 and the corresponding 5-fluorouracil (5-FU) resistant cells Bel-7402/FU. ASIV (0.08 mg/ml) potentiated the cytotoxicity of 5-FU which was demonstrated using the MTT assay on Bel-7402/FU cells. ASIV reduced the expression of P-gp as was revealed by immunocytochemistry. Accumulation and efflux studies with the P-gp substrate, rhodamine 123 (Rh123), demonstrated that ASIV inhibited P-gp-mediated drug efflux. Furthermore, it was demonstrated that ASⅣ enhanced the drug accumulation of 5-FU using a high performance liquid chromatography (HPLC) assay for drug resistant cells. Furthermore, ASIV may downregulate the expression of P-gp, which was examined using western blot analysis and polymerase chain reaction. In conclusion, the results of the present study indicated that ASIV reverses the drug resistance of Bel-7402/FU cells by downregulating the expression of mdr1. ASIV may represent a potent modulator of P-gp-mediated MDR in hepatic cancer therapy. PMID:24676670

  1. Quercetin as a Potential Modulator of P-Glycoprotein Expression and Function in Cells of Human Pancreatic Carcinoma Line Resistant to Daunorubicin

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    Piotr Dziegiel

    2010-02-01

    Full Text Available P-glycoprotein (P-gp is one of the ABC transporters responsible for the resistance of several tumours to successful chemotherapy. Numerous agents are capable of interfering with the P-gp-mediated export of drugs but unfortunately most of them produce serious side effects. Some plant polyphenols, including the flavonol quercetin (Q, manifest anti-neoplastic activity mainly due to their influence on cell cycle control and apoptosis. Reports are also available which show that Q may intensify action of cytostatic drugs and suppress the multidrug resistance (MDR phenomenon. The study aimed at determination if Q sensitizes cells resistant to daunorubicin (DB through its effect on P-gp expression and action. The experiments were conducted on two cell lines of human pancreatic carcinoma, resistant to DB EPP85-181RDB and sensitive EPP85-181P as a comparison. Cells of both lines were exposed to selected concentrations of Q and DB, and then membranous expression of P-gp and its transport function were examined. The influence on expression of gene for P-gp (ABCB1 was also investigated. Results of the studies confirmed that Q affects expression and function of P-gp in a concentration-dependent manner. Moreover it decreased expression of ABCB1. Thus, Q may be considered as a potential modulator of P-gp.

  2. Generation and efficacy evaluation of recombinant classical swine fever virus E2 glycoprotein expressed in stable transgenic mammalian cell line.

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    Rong-Hong Hua

    Full Text Available Classical swine fever virus (CSFV is the causative agent of classical swine fever (CSF, which is a highly contagious swine disease that causes significant economic loses to the pig industry worldwide. The envelope E2 glycoprotein of CSFV is the most important viral antigen in inducing protective immune response against CSF. In this study, we generated a mammalian cell clone (BCSFV-E2 that could stably produce a secreted form of CSFV E2 protein (mE2. The mE2 protein was shown to be N-linked glycosylated and formed a homodimer. The vaccine efficacy of mE2 was evaluated by immunizing pigs. Twenty-five 6-week-old Landrace piglets were randomly divided into five groups. Four groups were intramuscularly immunized with mE2 emulsified in different adjuvants twice at four-week intervals. One group was used as the control group. All mE2-vaccinated pigs developed CSFV-neutralizing antibodies two weeks after the first vaccination with neutralizing antibody titers ranging from 1:40 to 1:320. Two weeks after the booster vaccination, the neutralizing antibody titers increased greatly and ranged from 1:10,240 to 1:81,920. At 28 weeks after the booster vaccine was administered, the neutralizing antibody titers ranged from 1:80 to 1:10240. At 32 weeks after the first vaccination, pigs in all the groups were challenged with a virulent CSFV strain at a dose of 1 × 10(5 TCID50. At two weeks after the challenge, all the mE2-immunized pigs survived and exhibited no obvious symptoms of CSF. The neutralizing antibody titer at this time was 20,480. Unvaccinated pigs in the control group exhibited symptoms of CSF 3-4 days after challenge and were euthanized from 7-9 days after challenge when the pigs became moribund. These results indicate that the mE2 is a good candidate for the development of a safe and effective CSFV subunit vaccine.

  3. Cathepsins B and L activate Ebola but not Marburg virus glycoproteins for efficient entry into cell lines and macrophages independent of TMPRSS2 expression.

    Science.gov (United States)

    Gnirss, Kerstin; Kühl, Annika; Karsten, Christina; Glowacka, Ilona; Bertram, Stephanie; Kaup, Franziska; Hofmann, Heike; Pöhlmann, Stefan

    2012-03-01

    Ebola (EBOV) and Marburg virus (MARV) cause severe hemorrhagic fever. The host cell proteases cathepsin B and L activate the Zaire ebolavirus glycoprotein (GP) for cellular entry and constitute potential targets for antiviral intervention. However, it is unclear if different EBOV species and MARV equally depend on cathepsin B/L activity for infection of cell lines and macrophages, important viral target cells. Here, we show that cathepsin B/L inhibitors markedly reduce 293T cell infection driven by the GPs of all EBOV species, independent of the type II transmembrane serine protease TMPRSS2, which cleaved but failed to activate EBOV-GPs. Similarly, a cathepsin B/L inhibitor blocked macrophage infection mediated by different EBOV-GPs. In contrast, MARV-GP-driven entry exhibited little dependence on cathepsin B/L activity. Still, MARV-GP-mediated entry was efficiently blocked by leupeptin. These results suggest that cathepsins B/L promote entry of EBOV while MARV might employ so far unidentified proteases for GP activation. PMID:22222211

  4. Development and characterization of P-glycoprotein 1 (Pgp1, ABCB1)-mediated doxorubicin-resistant PLHC-1 hepatoma fish cell line

    International Nuclear Information System (INIS)

    The development of the multidrug resistance (MDR) phenotype in mammals is often mediated by the overexpression of the P-glycoprotein1 (Pgp, ABCB1) or multidrug resistance-associated protein (MRP)-like ABC transport proteins. A similar phenomenon has also been observed and considered as an important part of the multixenobiotic resistance (MXR) defence system in aquatic organisms. We have recently demonstrated the presence of ABC transporters in the widely used in vitro fish model, the PLHC-1 hepatoma cell line. In the present study we were able to select a highly resistant PLHC-1 sub-clone (PLHC-1/dox) by culturing the wild-type cells in the presence of 1 μM doxorubicin. Using quantitative PCR a 42-fold higher expression of ABCB1 gene was determined in the PLHC-1/dox cells compared to non-selected wild-type cells (PLHC-1/wt). The efflux rates of model fluorescent Pgp1 substrates rhodamine 123 and calcein-AM were 3- to 4-fold higher in the PLHC-1/dox in comparison to the PLHC-1/wt cells. PLHC-1/dox were 45-fold more resistant to doxorubicin cytotoxicity than PLHC-1/wt. Similarly to mammalian cell lines, typical cross-resistance to cytotoxicity of other chemotherapeutics such as daunorubicin, vincristine, vinblastine, etoposide and colchicine, occurred. Furthermore, cyclosporine A, verapamil and PSC833, specific inhibitors of Pgp1 transport activity, completely reversed resistance of PLHC-1/dox cells to all tested drugs, resulting in EC50 values similar to the EC50 values found for PLHC-1/wt. In contrast, MK571, a specific inhibitor of MRP type of efflux transporters, sensitized PLHC-1/dox cells, neither to doxorubicin, nor to any other of the chemotherapeutics used in the study. These data demonstrate for the first time that a specific Pgp1-mediated doxorubicin resistance mechanism is present in the PLHC-1 fish hepatoma cell line. In addition, the fact that low micromolar concentrations of specific inhibitors may completely reverse a highly expressed doxorubicin

  5. Effect of recombinant human interleukin-11 on expressions of interleukin-11 receptor α-chain and glycoprotein 130 in intestinal epithelium cell line-6 after neutron irradiation

    Institute of Scientific and Technical Information of China (English)

    Rui-Juan Wang; Rui-Yun Peng; Kai-Fei Fu; Ya-Bing Gao; Rui-Gang Han; Wen-Hua Hu; Qing-Liang Luo; Jun-Jie Ma

    2006-01-01

    AIM: To explore the effect of recombinant human interleukin-11 (rhIL-11) on the expressions of interleukin-11 receptor α-chain (IL-11Rα) and an additional signal transducer glycoprotein 130 (gp130) in intestinal epithelium cell line-6 (IEC-6) after neutron irradiation.METHODS: Cultured IEC-6 cells were exposed to 4.0Gy neutron and treated with 100 ng/mL rhIL-11 12 h prior to or immediately after irradiation. The apoptosis and necrosis rates and expressions of IL-11Rα and gp130 were observed by flow cytometry, immunohistochemistry, Western blot and image analysis.RESULTS: The apoptosis rate of IEC-6 cells was increased by irradiation at 6 h (P < 0.01), IL-11 stimulation resulted in a decreased apoptosis rate in irradiated IEC-6 cells (P < 0.05). In normal control IEC-6 cells, intense immunoreactivity of IL-11Rα was located within the cell membrane and cytoplasm. The level of IL-11Rα expression significantly decreased at 6 h after irradiation (P < 0.01) and restored at 24 h after irradiation. In IEC-6 cells treated with both radiation and rhIL-11, the level of IL-11Rα expression was higher than that of irradiated cells (P < 0.05). When it came to gp130 protein, it was located in the cytoplasm of IEC-6 cells. After irradiation, we found a progressive decrease in the expression of gp130 protein (P < 0.05) in 48 hours post-radiation, while in rhIL-11-stimulated cells, it came back to normal level at 24 h after irradiation and decreased at 48 h, but was still higher than that of only irradiated cells (P < 0.05).CONCLUSION: rhIL-11 can protect IEC-6 cells from neutron irradiation. The protective effect of rhIL-11 might be connected with its ability to up-regulate the expressions of specific ligand-binding subunit IL-11Rα and signal-transducing subunit gp130.

  6. Inhibitors of glycoprotein processing alter T-cell proliferative responses to antigen and to interleukin 2.

    OpenAIRE

    Wall, K A; Pierce, J D; Elbein, A D

    1988-01-01

    Most of the cell-surface molecules involved in T-cell immune responses are N-linked glycoproteins. We have investigated the effects of inhibitors of glycoprotein processing on specific T-cell functions, with the dual aims of examining the functional role of carbohydrate and of testing the usefulness of such compounds as immunomodulators. Treatment of a cloned murine helper T-cell line with these inhibitors differentially affects the proliferative response of the cell, depending upon the natur...

  7. Glycoprotein component of plant cell walls

    International Nuclear Information System (INIS)

    The primary wall surrounding most dicotyledonous plant cells contains a hydroxyproline-rich glycoprotein (HRGP) component named extensin. A small group of glycopeptides solubilized from isolated cell walls by proteolysis contained a repeated pentapeptide glycosylated by tri- and tetraarabinosides linked to hydroxyproline and, by galactose, linked to serine. Recently, two complementary approaches to this problem have provided results which greatly increase the understanding of wall extensin. In this paper the authors describe what is known about the structure of soluble extensin secreted into the walls of the carrot root cells

  8. Replacement of the cytoplasmic domain alters sorting of a viral glycoprotein in polarized cells.

    OpenAIRE

    Puddington, L; Woodgett, C; Rose, J. K.

    1987-01-01

    The envelope glycoprotein (G protein) of vesicular stomatitis virus (VSV) is transported to the basolateral plasma membrane of polarized epithelial cells, whereas the hemagglutinin glycoprotein (HA protein) of influenza virus is transported to the apical plasma membrane. To determine if the cytoplasmic domain of VSV G protein might be important in directing G protein to the basolateral membrane, we derived polarized Madin-Darby canine kidney cell lines expressing G protein or G protein with i...

  9. Pneumocystis carinii major surface glycoprotein induces interleukin-8 and monocyte chemoattractant protein-1 release from a human alveolar epithelial cell line

    DEFF Research Database (Denmark)

    Benfield, T L; Lundgren, Bettina; Shelhamer, J H;

    1999-01-01

    BACKGROUND: The major surface glycoprotein (MSG) is an abundant, immunogenic glycoprotein located on the surface of Pneumocystis carinii. Little is known about the proinflammatory effects of MSG. DESIGN: We have investigated the effect of human MSG on the secretion of the chemokines interleukin 8...... in response to MSG stimulation occurred by 4 h and persisted throughout 8 h of stimulation. CONCLUSION: These findings suggest that MSG can alter alveolar epithelial cytokine release and may be capable of modulating the local inflammatory response in this manner....

  10. An unusual dependence of human herpesvirus-8 glycoproteins-induced cell-to-cell fusion on heparan sulfate

    Energy Technology Data Exchange (ETDEWEB)

    Tiwari, Vaibhav [Department of Ophthalmology, University of Illinois at Chicago, Chicago, IL 60612 (United States); Department of Microbiology and Immunology, University of Illinois at Chicago, Chicago, IL 60612 (United States); Department of Basic Medical Sciences, College of Osteopathic Medicine of the Pacific and College of Optometry, Western University of Health Sciences, Pomona, CA 91766 (United States); Darmani, Nissar A.; Thrush, Gerald R. [Department of Basic Medical Sciences, College of Osteopathic Medicine of the Pacific and College of Optometry, Western University of Health Sciences, Pomona, CA 91766 (United States); Shukla, Deepak, E-mail: dshukla@uic.edu [Department of Ophthalmology, University of Illinois at Chicago, Chicago, IL 60612 (United States); Department of Microbiology and Immunology, University of Illinois at Chicago, Chicago, IL 60612 (United States)

    2009-12-18

    Human herpesvirus-8 (HHV-8) is known to interact with cell surface heparan sulfate (HS) for entry into a target cell. Here we investigated the role of HS during HHV-8 glycoproteins-induced cell fusion. Interestingly, the observed fusion demonstrated an unusual dependence on HS as evident from following lines of evidence: (1) a significant reduction in cell-to-cell fusion occurred when target cells were treated with heparinase; (2) in a competition assay, when the effector cells expressing HHV-8 glycoproteins were challenged with soluble HS, cell-to-cell fusion was reduced; and, (3) co-expression of HHV-8 glycoproteins gH-gL on target cells resulted in inhibition of cell surface HS expression. Taken together, our results indicate that cell surface HS can play an additional role during HHV-8 pathogenesis.

  11. An unusual dependence of human herpesvirus-8 glycoproteins-induced cell-to-cell fusion on heparan sulfate

    International Nuclear Information System (INIS)

    Human herpesvirus-8 (HHV-8) is known to interact with cell surface heparan sulfate (HS) for entry into a target cell. Here we investigated the role of HS during HHV-8 glycoproteins-induced cell fusion. Interestingly, the observed fusion demonstrated an unusual dependence on HS as evident from following lines of evidence: (1) a significant reduction in cell-to-cell fusion occurred when target cells were treated with heparinase; (2) in a competition assay, when the effector cells expressing HHV-8 glycoproteins were challenged with soluble HS, cell-to-cell fusion was reduced; and, (3) co-expression of HHV-8 glycoproteins gH-gL on target cells resulted in inhibition of cell surface HS expression. Taken together, our results indicate that cell surface HS can play an additional role during HHV-8 pathogenesis.

  12. Cell wall O-glycoproteins and N-glycoproteins: aspects of biosynthesis and function

    Science.gov (United States)

    Nguema-Ona, Eric; Vicré-Gibouin, Maïté; Gotté, Maxime; Plancot, Barbara; Lerouge, Patrice; Bardor, Muriel; Driouich, Azeddine

    2014-01-01

    Cell wall O-glycoproteins and N-glycoproteins are two types of glycomolecules whose glycans are structurally complex. They are both assembled and modified within the endomembrane system, i.e., the endoplasmic reticulum (ER) and the Golgi apparatus, before their transport to their final locations within or outside the cell. In contrast to extensins (EXTs), the O-glycan chains of arabinogalactan proteins (AGPs) are highly heterogeneous consisting mostly of (i) a short oligo-arabinoside chain of three to four residues, and (ii) a larger β-1,3-linked galactan backbone with β-1,6-linked side chains containing galactose, arabinose and, often, fucose, rhamnose, or glucuronic acid. The fine structure of arabinogalactan chains varies between, and within plant species, and is important for the functional activities of the glycoproteins. With regards to N-glycans, ER-synthesizing events are highly conserved in all eukaryotes studied so far since they are essential for efficient protein folding. In contrast, evolutionary adaptation of N-glycan processing in the Golgi apparatus has given rise to a variety of organism-specific complex structures. Therefore, plant complex-type N-glycans contain specific glyco-epitopes such as core β,2-xylose, core α1,3-fucose residues, and Lewisa substitutions on the terminal position of the antenna. Like O-glycans, N-glycans of proteins are essential for their stability and function. Mutants affected in the glycan metabolic pathways have provided valuable information on the role of N-/O-glycoproteins in the control of growth, morphogenesis and adaptation to biotic and abiotic stresses. With regards to O-glycoproteins, only EXTs and AGPs are considered herein. The biosynthesis of these glycoproteins and functional aspects are presented and discussed in this review. PMID:25324850

  13. Capsosiphon fulvescens glycoprotein inhibits AGS gastric cancer cell proliferation by downregulating Wnt-1 signaling

    OpenAIRE

    Kim, Young-Min; KIM, IN-HYE; NAM, TAEK-JEONG

    2013-01-01

    Previously, we examined various apoptosis pathways in the AGS gastric cancer cell line using Capsosiphon fulvescens glycoprotein (Cf-GP). In this study, we focused on the downregulation of the Wnt-1 signaling pathway and cell cycle arrest. Upregulation of the Wnt signaling pathway has been observed in various cancer cells. The Wnt signal ligand acts in both canonical and non-canonical pathways. Among them, Wnt-1 was dependent on the canonical pathway. Here, we show inhibition of Wnt-1 signali...

  14. Proteomic dataset for altered glycoprotein expression upon GALNT3 knockdown in ovarian cancer cells.

    Science.gov (United States)

    Sheta, Razan; Roux-Dalvai, Florence; Woo, Christina M; Fournier, Frédéric; Bourassa, Sylvie; Bertozzi, Carolyn R; Droit, Arnaud; Bachvarov, Dimcho

    2016-09-01

    This article contains raw and processed data related to research published in "Role of the polypeptide N-acetylgalactosaminyltransferase 3 in ovarian cancer progression: possible implications in abnormal mucin O-glycosylation" [1]. The data presented here was obtained with the application of a bioorthogonal chemical reporter strategy analyzing differential glycoprotein expression following the knock-down (KD) of the GALNT3 gene in the epithelial ovarian cancer (EOC) cell line A2780s. LC-MS/MS mass spectrometry analysis was then performed and the processed data related to the identified glycoproteins show that several hundred proteins are differentially expressed between control and GALNT3 KD A2780s cells. The obtained data also uncover numerous novel glycoproteins; some of which could represent new potential EOC biomarkers and/or therapeutic targets. PMID:27331112

  15. Glucocorticoid-regulated and constitutive trafficking of proteolytically processed cell surface-associated glycoproteins in wild type and variant rat hepatoma cells

    International Nuclear Information System (INIS)

    Glucocorticoids regulate the trafficking of mouse mammary tumor virus (MMTV) glycoproteins to the cell surface in the rat hepatoma cell line M1.54, but not in the immunoselected sorting variant CR4. To compare the localization of MMTV glycoproteins to another proteolytically processed glycoprotein, both wild type M1.54 cells and variant CR4 cells were transfected with a human insulin receptor (hIR) expression vector, pRSVhIR. The production of cell surface hIR was monitored in dexamethasone-treated and -untreated wild type M1.54 and variant CR4 cells by indirect immunofluorescence, direct plasma membrane immunoprecipitation, and by [125I] insulin binding. In both wild type and variant rat hepatoma cells, hIR were localized at the cell surface in the presence or in the absence of 1 microM dexamethasone. In contrast, the glucocorticoid-regulated trafficking of cell surface MMTV glycoproteins occurred only in wild type M1.54 cells. We conclude that the hIR, which undergoes posttranslational processing reactions similar to MMTV glycoproteins, does not require glucocorticoids to be transported to the plasma membrane and is representative of a subset of cell surface glycoproteins whose trafficking is constitutive in rat hepatoma cells. Thus, MMTV glycoproteins and hIR provide specific cell surface markers to characterize the glucocorticoid-regulated and constitutive sorting pathways

  16. EFFECTS OF AMIODARONE, CYCLOSPORINE-A, AND PSC-833 ON THE CYTOTOXICITY OF MITOXANTRONE, DOXORUBICIN, AND VINCRISTINE IN NON-P-GLYCOPROTEIN HUMAN SMALL-CELL LUNG-CANCER CELL-LINES

    NARCIS (Netherlands)

    VANDERGRAAF, WTA; DEVRIES, EGE; TIMMERBOSSCHA, H; MEERSMA, GJ; MESANDER, G; VELLENGA, E; MULDER, NH

    1994-01-01

    The multidrug resistance (MDR) modulators amiodarone (AM), cyclosporin A (CsA), and PSC 833 were tested for their potential to modulate cytotoxicity of doxorubicin (DOX), vincristine (VCR), and mitoxantrone (MX) in a sensitive human small cell lung carcinoma cell line GLC4 in its DOX-resistant non-P

  17. Engineered CHO cells for production of diverse, homogeneous glycoproteins

    DEFF Research Database (Denmark)

    Yang, Zhang; Wang, Shengjun; Halim, Adnan; Schulz, Morten Alder; Frodin, Morten; Rahman, Shamim H.; Vester-Christensen, Malene Bech; Behrens, Carsten; Kristensen, Claus; Vakhrushev, Sergey Y.; Bennett, Eric Paul; Wandall, Hans H.; Clausen, Henrik

    2015-01-01

    genes controlling N-glycosylation in CHO cells and constructed a design matrix that facilitates the generation of desired glycosylation, such as human-like alpha 2,6-linked sialic acid capping. This engineering approach will aid the production of glycoproteins with improved properties and therapeutic...

  18. Nipah virus infection and glycoprotein targeting in endothelial cells

    Directory of Open Access Journals (Sweden)

    Maisner Andrea

    2010-11-01

    Full Text Available Abstract Background The highly pathogenic Nipah virus (NiV causes fatal respiratory and brain infections in animals and humans. The major hallmark of the infection is a systemic endothelial infection, predominantly in the CNS. Infection of brain endothelial cells allows the virus to overcome the blood-brain-barrier (BBB and to subsequently infect the brain parenchyma. However, the mechanisms of NiV replication in endothelial cells are poorly elucidated. We have shown recently that the bipolar or basolateral expression of the NiV surface glycoproteins F and G in polarized epithelial cell layers is involved in lateral virus spread via cell-to-cell fusion and that correct sorting depends on tyrosine-dependent targeting signals in the cytoplasmic tails of the glycoproteins. Since endothelial cells share many characteristics with epithelial cells in terms of polarization and protein sorting, we wanted to elucidate the role of the NiV glycoprotein targeting signals in endothelial cells. Results As observed in vivo, NiV infection of endothelial cells induced syncytia formation. The further finding that infection increased the transendothelial permeability supports the idea of spread of infection via cell-to-cell fusion and endothelial cell damage as a mechanism to overcome the BBB. We then revealed that both glycoproteins are expressed at lateral cell junctions (bipolar, not only in NiV-infected primary endothelial cells but also upon stable expression in immortalized endothelial cells. Interestingly, mutation of tyrosines 525 and 542/543 in the cytoplasmic tail of the F protein led to an apical redistribution of the protein in endothelial cells whereas tyrosine mutations in the G protein had no effect at all. This fully contrasts the previous results in epithelial cells where tyrosine 525 in the F, and tyrosines 28/29 in the G protein were required for correct targeting. Conclusion We conclude that the NiV glycoprotein distribution is responsible for

  19. Glycoprotein H of herpes simplex virus type 1 requires glycoprotein L for transport to the surfaces of insect cells

    NARCIS (Netherlands)

    Westra, DF; Glazenburg, KL; Harmsen, MC; Tiran, A; Scheffer, AJ; Welling, GW; The, TH; WellingWester, S

    1997-01-01

    In mammalian cells, formation of heterooligomers consisting of the glycoproteins H and L (gH and gL) of herpes simplex virus type 1 is essential for the cell-to-cell spread of virions and for the penetration of virions into cells. We examined whether formation of gH1/gL1 heterooligomers and cell sur

  20. Studies on glycoproteins produced by wild type and wheat germ agglutinin-resistant B16 mouse melanoma cells

    International Nuclear Information System (INIS)

    Two variants of B16 mouse melanoma cells have been selected in serum-free medium for their resistance to toxic levels of wheat germ agglutinin isolation 1 (WGA). Chromosome analysis and characteristic melanin production showed that the variants are derived from the parent mouse melanoma cell lines. However, the two variants were less tumorigenic in mice compared to the parent B16 mouse melanoma cells. The variants showed a marked decrease in cell agglutination with WGA. Cell agglutination with recin and peanut lectin was not different between the three cell lines, but the two variants showed a slight increase in agglutination with concanavalin A. The binding of 125I-labeled wheat germ agglutinin to the two variant cells was reduced compared to that of the parent cell. Glycoproteins secreted or shed by the three lines were isolated after growth in serum-free medium in the presence of [3He]glucosamine and bovine serum albumin (1%). These metabolically labeled products were fractionated on the basis of their interaction with WGA-Sepharose (2 mg/ml). The WGA-Sepharose affinity chromatographic data suggested a decrease in WGA-binding glycoprotein(s) secreted to the medium by the two variants. The WGA-bound glycoproteins from the two variants upon SDS-PAGE revealed three bands of approximate molecular weights, 92,000, 56,000, and 42,000, none of which were present in the parent cell line (50,000 molecular weight)

  1. Synthesis of cell envelope glycoproteins of Cryptococcus laurentii.

    Science.gov (United States)

    Schutzbach, John; Ankel, Helmut; Brockhausen, Inka

    2007-05-21

    Fungi of the genus Cryptococcus are encapsulated basidiomycetes that are ubiquitously found in the environment. These organisms infect both lower and higher animals. Human infections that are common in immune-compromised individuals have proven difficult to cure or even control with currently available antimycotics that are quite often toxic to the host. The virulence of Cryptococcus has been linked primarily to its polysaccharide capsule, but also to cell-bound glycoproteins. In this review, we show that Cryptococcus laurentii is an excellent model for studies of polysaccharide and glycoprotein synthesis in the more pathogenic relative C. neoformans. In particular, we will discuss the structure and biosynthesis of O-linked carbohydrates on cell envelope glycoproteins of C. laurentii. These O-linked structures are synthesized by at least four mannosyltransferases, two galactosyltransferases, and at least one xylosyltransferase that have been characterized. These glycosyltransferases have no known homologues in human tissues. Therefore, enzymes involved in the synthesis of cryptococcal glycoproteins, as well as related enzymes involved in capsule synthesis, are potential targets for the development of specific inhibitors for treatment of cryptococcal disease. PMID:17316583

  2. siRNA-based targeting of antiapoptotic genes can reverse chemoresistance in P-glycoprotein expressing chondrosarcoma cells

    Directory of Open Access Journals (Sweden)

    Yang Jay

    2009-05-01

    Full Text Available Abstract Background High expression of P-glycoprotein is one of the well-known mechanisms of chemoresistance in chondrosarcomas. However, the role of antiapoptotic proteins, a common mechanism responsible for chemoresistance in other tumors, has not been well studied in chondrosarcomas. We examined the importance of P-glycoprotein and antiapoptotic proteins in the chemoresistance to doxorubicin of two Grade II chondrosarcoma cell lines, JJ012 and SW1353. Results We confirmed that both chondrosarcoma cell types expressed P-glycoprotein and antiapoptotic proteins (Bcl-2, Bcl-xL and XIAP. siRNA knockdown as well as pharmacologic inhibitors of cell survival proteins (Bcl-2, Bcl-xL and XIAP enhanced apoptosis of chemoresistant chondrosarcoma cells by up to 5.5 fold at 0.1 μmol and 5.5 fold at 1 μmol doxorubicin. These chemosensitizing effects were comparable to those of P-glycoprotein inhibition by siRNA or pharmacologic inhibitor. Conclusion These findings suggest that antiapoptotic proteins play a significant role in the chemoresistance of chondrosarcoma cells independent of P-glycoprotein. Based on the results, a new siRNA-based therapeutic strategy targeting antiapoptotic genes can be designed to overcome the chemoresistance of chondrosarcomas which is often conferred by P-glycoprotein.

  3. Evidence for P-Glycoprotein Involvement in Cell Volume Regulation Using Coulter Sizing in Flow Cytometry

    Directory of Open Access Journals (Sweden)

    Jennifer Pasquier

    2015-06-01

    Full Text Available The regulation of cell volume is an essential function that is coupled to a variety of physiological processes such as receptor recycling, excitability and contraction, cell proliferation, migration, and programmed cell death. Under stress, cells undergo emergency swelling and respond to such a phenomenon with a regulatory volume decrease (RVD where they release cellular ions, and other osmolytes as well as a concomitant loss of water. The link between P-glycoprotein, a transmembrane transporter, and cell volume regulation is controversial, and changes in cells volume are measured using microscopy or electrophysiology. For instance, by using the patch-clamp method, our team demonstrated that chloride currents activated in the RVD were more intense and rapid in a breast cancer cell line overexpressing the P-glycoprotein (P-gp. The Cell Lab Quanta SC is a flow cytometry system that simultaneously measures electronic volume, side scatter and three fluorescent colors; altogether this provides unsurpassed population resolution and accurate cell counting. Therefore, here we propose a novel method to follow cellular volume. By using the Coulter-type channel of the cytometer Cell Lab Quanta SC MPL (multi-platform loading, we demonstrated a role for the P-gp during different osmotic treatments, but also a differential activity of the P-gp through the cell cycle. Altogether, our data strongly suggests a role of P-gp in cell volume regulation.

  4. Baculovirus Coinfection Strategy for Improved Galactosylation of Recombinant Glycoprotein Produced by Insect Cell Culture

    Science.gov (United States)

    Ney, Yap Wei; Rahman, Badarulhisam Abdul; Aziz, Azila Abdul

    Baculovirus Expression Vector System (BEVS) is widely used for the production of recombinant glycoproteins, but it is not ideal for pharmaceutical glycoprotein production due to incomplete glycosylation. The factors that ensure successful glycosylation are the presence of sufficient amount of glycosyltransferases, sugar nucleotides as the substrate donor and the recombinant protein as the substrate acceptor. In this study, we analyzed the galactosylation process by the introduction of ß-1,4galactosyltransferase (ß-1,4GalT) as the glycosyltransferase of interest and uridine-5`-diphosphogalactose (UDP-Gal) as the substrate donor. Recombinant human transferrin (rhTf) as a model protein was used as the substrate acceptor. Insect cell lines have been reported to produce a small amount of ß-1,4GalT and thus insufficient for effective galactosylation. In this study, we developed a method to produce galactosylated rhTf and optimized the expression of rhTf with better N-glycan quality. Recombinant ß-1,4GalT was introduced during protein expression by the coinfection of the BEVS with baculovirus carrying bovine ß-1,4GalT. To evaluate the extent of galactosylation by the coinfection strategy, a binding assay was established. In this binding assay, glycoprotein acceptor was absorbed onto ELISA plate surface. A lectin known as Ricinus communis agglutinin-I (RCA-I) labeled with peroxidase, was added and allowed to recognize Gal ß1>4GlcNAc group on the N-glycan of the glycoprotein, followed by appropriate color reaction measurements. Coexpression between rhTf and ß-1,4GalT did not show encouraging results due to the reduction of UDP-Gal upon baculovirus infection. This interesting finding suggested that the introduction of ß-1,4GalT alone was not sufficient for successful galactosylation. Alternatively, post harvest glycosylation method strategy seems to be a promising technique in the improvement of glycoprotein quality.

  5. Broad target cell selectivity of Kaposi's sarcoma-associated herpesvirus glycoprotein-mediated cell fusion and virion entry

    International Nuclear Information System (INIS)

    The molecular mechanism of Kaposi's sarcoma-associated herpesvirus (KSHV, human herpesvirus 8) entry is poorly understood. We tested a broad variety of cell types of diverse species and tissue origin for their ability to function as targets in a quantitative reporter gene assay for KSHV-glycoprotein-mediated cell fusion. Several human, non-human primate, and rabbit cell lines were efficient targets, whereas rodent and all human lymphoblastoid cell lines were weak targets. Parallel findings were obtained with a virion entry assay using a recombinant KSHV encoding a reporter gene. No correlation was observed between target cell activity and surface expression of α3β1 integrin, a proposed KSHV receptor. We hypothesize that target cell permissiveness in both the cell fusion and virion entry assays reflects the presence of a putative KSHV fusion-entry receptor

  6. Inhibition of human immunodeficiency virus type-1 (HIV-1 glycoprotein-mediated cell-cell fusion by immunor (IM28

    Directory of Open Access Journals (Sweden)

    Akoume Marie-Yvonne

    2005-02-01

    Full Text Available Abstract Background Immunor (IM28, an analog of dehydroepiandrosterone (DHEA, inhibits human immunodeficiency virus type-1 (HIV-1 by inhibiting reverse transcriptase. We assessed the ability of IM28 to inhibit the cell-cell fusion mediated by HIV envelope glycoprotein in an in vitro system. For this purpose, we co-cultured TF228.1.16, a T-cell line expressing stably HIV-1 glycoprotein envelopes, with an equal number of 293/CD4+, another T cell line expressing CD4, and with the SupT1 cell line with or without IM28. Results In the absence of IM28, TF228.1.16 fused with 293/CD4+, inducing numerous large syncytia. Syncytia appeared more rapidly when TF228.1.16 was co-cultured with SupT1 cells than when it was co-cultured with the 293/CD4+ cell line. IM28 (1.6 – 45 μg/ml completely inhibits cell-cell fusion. IM28 also prevented the development of new syncytia in infected cells and protected naive SupT1 cells from HIV-1 infection. Evaluation of 50% inhibitory dose (IC50 of IM28 revealed a decrease in HIV-1 replication with an IC50 of 22 mM and 50% cytotoxicity dose (CC50 as determined on MT2 cells was 75 mM giving a selectivity index of 3.4 Conclusions These findings suggest that IM28 exerts an inhibitory action on the env proteins that mediate cell-cell fusion between infected and healthy cells. They also suggest that IM28 interferes with biochemical processes to stop the progression of existing syncytia. This property may lead to the development of a new class of therapeutic drug.

  7. Tomato spotted wilt virus glycoproteins exhibit trafficking and localization signals that are functional in mammalian cells

    NARCIS (Netherlands)

    Kikkert, M.; Verschoor, A.; Kormelink, R.; Rottier, P.; Goldbach, R.

    2001-01-01

    The glycoprotein precursor (G1/G2) gene of tomato spotted wilt virus (TSWV) was expressed in BHK cells using the Semliki Forest virus expression system. The results reveal that in this cell system, the precursor is efficiently cleaved and the resulting G1 and G2 glycoproteins are transported from th

  8. DUSP1 induces paclitaxel resistance through the regulation of p-glycoprotein expression in human ovarian cancer cells.

    Science.gov (United States)

    Kang, Yu-Seon; Seok, Hyun-Jeong; Jeong, Eun-Jeong; Kim, Yuna; Yun, Seok-Joong; Min, Jeong-Ki; Kim, Sun Jin; Kim, Jang-Seong

    2016-09-01

    The heterogeneity and genetic instability of ovarian cancer cells often lead to the development of drug resistance, closely related with the increased cancer-related mortality. In this study, we investigated the role of dual-specificity phosphatase 1 (DUSP1) in the development of the resistance in human ovarian cancer cells against paclitaxel. Overexpression of DUSP1 in HeyA8 human ovarian cancer cells (HeyA8-DUSP1) up-regulated the expression of the drug efflux pump, p-glycoprotein. Consequently, HeyA8-DUSP1 cells are highly resistant to paclitaxel, with the resistance comparable to that of a multi-drug resistance cell line (HeyA8-MDR). Moreover, over expression of DUSP1 significantly increased the activation of p38 MAPK, leaving the activation of ERK1/2 and JNK1/2 unaffected. Pharmacological suppression of p38 MAPK activity prevents the up-regulation of p-glycoprotein expression and the consequent resistance against paclitaxel in HeyA8-DUSP1 cells. By contrast, HeyA8-MDR cells expressed a significantly higher level of DUSP1, but treatment with small interference RNA against DUSP1 significantly suppressed the expression of p-glycoprotein and the resistance against paclitaxel in HeyA8-MDR cells. Ectopic expression of MKK3, an upstream activator of p38 MAPK, significantly up-regulated the expression of p-glycoprotein and increased the consequent resistance against paclitaxel in HeyA8 cells. Collectively, these data indicated that DUSP1 may induce the resistance against paclitaxel through the p38 MAPK-mediated overexpression of p-glycoprotein in human ovarian cancer cells. PMID:27422607

  9. The putative P-gp inhibitor telmisartan does not affect the transcellular permeability and cellular uptake of the calcium channel antagonist verapamil in the P-glycoprotein expressing cell line MDCK II MDR1

    DEFF Research Database (Denmark)

    Saaby, Lasse; Tfelt-Hansen, Peer; Brodin, Birger

    2015-01-01

    Verapamil is used in high doses for the treatment of cluster headache. Verapamil has been described as a P-glycoprotein (P-gp, ABCB1) substrate. We wished to evaluate in vitro whether co administration of a P-gp inhibitor with verapamil could be a feasible strategy for increasing CNS uptake of...

  10. Glycoproteins of coated pits, cell junctions, and the entire cell surface revealed by monoclonal antibodies and immunoelectron microscopy

    OpenAIRE

    1983-01-01

    Topographical descriptions of three major plasma membrane glycoproteins of murine 3T3 cells were obtained by immunoelectron microscopy with monoclonal antibodies. A glycoprotein of Mr 80,000 was distributed throughout the total cell surface. A second of Mr 90,000 was concentrated in coated pits, and a third of Mr 100,000 was localized at cell junctions.

  11. Phosphorylation of the multidrug resistance associated glycoprotein

    International Nuclear Information System (INIS)

    Drug-resistant cell lines derived from the mouse macrophage-like cell line J774.2 express the multidrug resistant phenotype which includes the overexpression of a membrane glycoprotein (130-140 kilodaltons). Phosphorylation of this resistant-specific glycoprotein (P-glycoprotein) in intact cells and in cell-free membrane fractions has been studied. The phosphorylated glycoprotein can be immunoprecipitated by a rabbit polyclonal antibody specific for the glycoprotein. Phosphorylation studies done with partially purified membrane fractions derived from colchicine-resistant cells indicated that (a) phosphorylation of the glycoprotein in 1 mM MgCl2 was enhanced a minimum of 2-fold by 10 μM cAMP and (b) the purified catalytic subunit of the cAMP-dependent protein kinase (protein kinase A) phosphorylated partially purified glycoprotein that was not phosphorylated by [γ-32P]ATP alone, suggesting that autophosphorylation was not involved. These results indicate that the glycoprotein is a phosphoprotein and that at least one of the kinases responsible for its phosphorylation is a membrane-associated protein kinase A. The state of phosphorylation of the glycoprotein, which is a major component of the multidrug resistance phenotype, may be related to the role of the glycoprotein in maintaining drug resistance

  12. Phosphorylation of the multidrug resistance associated glycoprotein.

    Science.gov (United States)

    Mellado, W; Horwitz, S B

    1987-11-01

    Drug-resistant cell lines derived from the mouse macrophage-like cell line J774.2 express the multidrug resistance phenotype which includes the overexpression of a membrane glycoprotein (130-140 kilodaltons). Phosphorylation of this resistant-specific glycoprotein (P-glycoprotein) in intact cells and in cell-free membrane fractions has been studied. The phosphorylated glycoprotein can be immunoprecipitated by a rabbit polyclonal antibody specific for the glycoprotein. Phosphorylation studies done with partially purified membrane fractions derived from colchicine-resistant cells indicated that (a) phosphorylation of the glycoprotein in 1 mM MgCl2 was enhanced a minimum of 2-fold by 10 microM cAMP and (b) the purified catalytic subunit of the cAMP-dependent protein kinase (protein kinase A) phosphorylated partially purified glycoprotein that was not phosphorylated by [gamma-32P]ATP alone, suggesting that autophosphorylation was not involved. These results indicate that the glycoprotein is a phosphoprotein and that at least one of the kinases responsible for its phosphorylation is a membrane-associated protein kinase A. The state of phosphorylation of the glycoprotein, which is a major component of the multidrug resistance phenotype, may be related to the role of the glycoprotein in maintaining drug resistance. PMID:3427052

  13. Integrated Proteomic and Glycoproteomic Analyses of Prostate Cancer Cells Reveal Glycoprotein Alteration in Protein Abundance and Glycosylation.

    Science.gov (United States)

    Shah, Punit; Wang, Xiangchun; Yang, Weiming; Toghi Eshghi, Shadi; Sun, Shisheng; Hoti, Naseruddin; Chen, Lijun; Yang, Shuang; Pasay, Jered; Rubin, Abby; Zhang, Hui

    2015-10-01

    Prostate cancer is the most common cancer among men in the U.S. and worldwide, and androgen-deprivation therapy remains the principal treatment for patients. Although a majority of patients initially respond to androgen-deprivation therapy, most will eventually develop castration resistance. An increased understanding of the mechanisms that underline the pathogenesis of castration resistance is therefore needed to develop novel therapeutics. LNCaP and PC3 prostate cancer cell lines are models for androgen-dependence and androgen-independence, respectively. Herein, we report the comparative analysis of these two prostate cancer cell lines using integrated global proteomics and glycoproteomics. Global proteome profiling of the cell lines using isobaric tags for relative and absolute quantitation (iTRAQ) labeling and two- dimensional (2D) liquid chromatography-tandem MS (LC-MS/MS) led to the quantification of 8063 proteins. To analyze the glycoproteins, glycosite-containing peptides were isolated from the same iTRAQ-labeled peptides from the cell lines using solid phase extraction followed by LC-MS/MS analysis. Among the 1810 unique N-linked glycosite-containing peptides from 653 identified N-glycoproteins, 176 glycoproteins were observed to be different between the two cell lines. A majority of the altered glycoproteins were also observed with changes in their global protein expression levels. However, alterations in 21 differentially expressed glycoproteins showed no change at the protein abundance level, indicating that the glycosylation site occupancy was different between the two cell lines. To determine the glycosylation heterogeneity at specific glycosylation sites, we further identified and quantified 1145 N-linked glycopeptides with attached glycans in the same iTRAQ-labeled samples. These intact glycopeptides contained 67 glycan compositions and showed increased fucosylation in PC3 cells in several of the examined glycosylation sites. The increase in

  14. Glucocorticoid-Dependent Complementation of a Hepatoma Cell Variant Defective in Viral Glycoprotein Sorting

    Science.gov (United States)

    John, Nancy J.; Bravo, Deborah A.; Haffar, Omar K.; Firestone, Gary L.

    1988-02-01

    We have utilized the rat hepatoma (HTC) cell sorting variant CR4 to examine the glucocorticoid-regulated pathways that localize mouse mammary tumor virus glycoproteins to the cell surface. The defective sorting of cell surface mouse mammary tumor virus glycoproteins in CR4 cells was complemented after fusion with either normal rat hepatocytes or uninfected HTC cells. Indirect immunofluorescence of transient heterokaryons revealed that the regulated localization of mouse mammary tumor virus glycoproteins was dependent upon glucocorticoid treatment and required de novo RNA and protein synthesis. Thus, a glucocorticoid-regulated trafficking activity, unrelated to mouse mammary tumor virus sequences, which is induced in both adult rat liver and cultured hepatoma cells, can act in trans to mediate an intracellular sorting pathway for membrane glycoproteins.

  15. Genetic transfer of non-P-glycoprotein-mediated multidrug resistance (MDR) in somatic cell fusion : Dissection of a compound MDR phenotype

    NARCIS (Netherlands)

    EIJDEMS, EWHM; BORST, P; JONGSMA, APM; de Jong, Steven; DEVRIES, EGE; VANGROENIGEN, M; VERSANTVOORT, CHM; NIEUWINT, AWM; BAAS, F

    1992-01-01

    A non-P-glycoprotein-mediated mechanism of multidrug resistance (non-Pgp MDR) bas been identified in doxorubicin-selected sublines of the human non-small cell lung carcinoma cell lines SW-1573. These sublines are cross-resistant to daunorubicin, VP16-213, Vinca alkaloids, colchicine, gramicidin D, a

  16. Secretion of N- and O-linked Glycoproteins from 4T1 Murine Mammary Carcinoma Cells.

    Science.gov (United States)

    Phang, Wai-Mei; Tan, Aik-Aun; Gopinath, Subash C B; Hashim, Onn H; Kiew, Lik Voon; Chen, Yeng

    2016-01-01

    Breast cancer is one of the most common cancers that affect women globally and accounts for ~23% of all cancers diagnosed in women. Breast cancer is also one of the leading causes of death primarily due to late stage diagnoses and a lack of effective treatments. Therefore, discovering protein expression biomarkers is mandatory for early detection and thus, critical for successful therapy. Two-dimensional electrophoresis (2D-E) coupled with lectin-based analysis followed by mass spectrometry were applied to identify potential biomarkers in the secretions of a murine mammary carcinoma cell line. Comparisons of the protein profiles of the murine 4T1 mammary carcinoma cell line and a normal murine MM3MG mammary cell line indicated that cadherin-1 (CDH), collagenase 3 (MMP-13), Viral envelope protein G7e (VEP), Gag protein (GAG) and Hypothetical protein LOC433182 (LOC) were uniquely expressed by the 4T1 cells, and pigment epithelium-derived factor (PEDF) was exclusively secreted by the MM3MG cells. Further analysis by a lectin-based study revealed that aberrant O-glycosylated CDH, N-glycosylated MMP-13 and LOC were present in the 4T1 medium. These differentially expressed N- and O-linked glycoprotein candidates, which were identified by combining lectin-based analysis with 2D-E, could serve as potential diagnostic and prognostic markers for breast cancer. PMID:27226773

  17. Secretion of N- and O-linked Glycoproteins from 4T1 Murine Mammary Carcinoma Cells

    Science.gov (United States)

    Phang, Wai-Mei; Tan, Aik-Aun; Gopinath, Subash C.B.; Hashim, Onn H.; Kiew, Lik Voon; Chen, Yeng

    2016-01-01

    Breast cancer is one of the most common cancers that affect women globally and accounts for ~23% of all cancers diagnosed in women. Breast cancer is also one of the leading causes of death primarily due to late stage diagnoses and a lack of effective treatments. Therefore, discovering protein expression biomarkers is mandatory for early detection and thus, critical for successful therapy. Two-dimensional electrophoresis (2D-E) coupled with lectin-based analysis followed by mass spectrometry were applied to identify potential biomarkers in the secretions of a murine mammary carcinoma cell line. Comparisons of the protein profiles of the murine 4T1 mammary carcinoma cell line and a normal murine MM3MG mammary cell line indicated that cadherin-1 (CDH), collagenase 3 (MMP-13), Viral envelope protein G7e (VEP), Gag protein (GAG) and Hypothetical protein LOC433182 (LOC) were uniquely expressed by the 4T1 cells, and pigment epithelium-derived factor (PEDF) was exclusively secreted by the MM3MG cells. Further analysis by a lectin-based study revealed that aberrant O-glycosylated CDH, N-glycosylated MMP-13 and LOC were present in the 4T1 medium. These differentially expressed N- and O-linked glycoprotein candidates, which were identified by combining lectin-based analysis with 2D-E, could serve as potential diagnostic and prognostic markers for breast cancer.

  18. The three-dimensional structure of the cell wall glycoprotein of Chlorogonium elongatum.

    Science.gov (United States)

    Shaw, P J; Hills, G J

    1984-06-01

    The green alga Chlorogonium elongatum, a member of the Volvocales, possesses a crystalline cell wall composed of hydroxyproline-rich glycoprotein similar to the primary cell wall glycoproteins of higher plants. Electron microscopy and computer image processing have been used to determine the crystal structure of the Chlorogonium cell wall in three dimensions to a resolution of 2.0 nm. The structure is composed of heterologous dimers. Each subunit of the dimer comprises a long, thin spacer domain and a large globular domain, which is the site of the intra- and inter-dimer interactions. There are also sites of intersubunit interactions at the opposite ends of the rod domains. We suggest that the rods are composed predominantly of glycosylated polyproline helix, as has been suggested for higher plant cell wall glycoproteins and has been shown for the cell wall glycoprotein of Chlamydomonas reinhardtii, which is closely related to Chlorogonium. PMID:6490737

  19. Glycoprotein IIIa Preparation for T-cell Stimulation

    Directory of Open Access Journals (Sweden)

    GR Anani Sarab

    2005-10-01

    Full Text Available Chronic immune thrombocytopenic purpura (ITP is an autoimmune disease characterized by antiplatelet autoantibodis. The major target of the anti platelet antibodies is platelet membrane glycoprotein IIb/IIIa. In order to characterize the immunodominant epitopes in the structure of GPIIIa, the extracellular portions of GPIIIa will be expressed and purified. These antigens will be tested for antigenicity in further investigation. The first segment of GPIIIa which was considered for expression as a recombinant glutathion S-transferase (GST fusion proteins included IIIa22-262 which encompass amino acid residue 22-262 of the 762 amino acids of GPIIIa. A segment of GPIIIa complementary DNA (cDNA was subcloned into the 39 end of the Schistosoma japonicum GST gene in the bacterial expression plasmid vector, pGEX 6P-1 (Amersham Pharmacia Biotech. In summary, the expression plasmid vector, pGEX 6P-1 containing segment IIIa22-262 was introduced to E.coli. Saturated overnight culture was used and the bacterial cells were grown to log-phase. IPTG was added to the culture to induce overexpression of fusion protein and the cells were grown for an additional 1-3 hours. Bacterial lysate containing recombinant protein was prepared by sonication. The fusion protein was purified from total cell extract using glutathione-agarose beads. Specificity of the GST-fusion proteins was confirmed on Immunoblot probed with rabbit anti-GST polyclonal antibodies. PreScission Protease was used to remove the GST tag. Protein extract and purified products were analyzed by SDS gel electrophoresis. The recombinant GST-fusion protein IIIa22-262 was successfully expressed and purified in large quantities but the yield of the IIIa22-262 peptide after enzyme treatment was low. When a good yield is fully obtained, the purified protein segment will be used for T-cell stimulation in culture.

  20. A 220-kilodalton glycoprotein in yeast extract inhibits Staphylococcus aureus adherence to human endothelial cells.

    OpenAIRE

    Elliott, D.A.; Hatcher, V B; Lowy, F D

    1991-01-01

    A 220-kDa glycoprotein from yeast extract causes a twofold decrease in S. aureus adherence to human endothelial cells in vitro. Medium constituents can have a significant effect on bacterial adherence interactions.

  1. Capsosiphon fulvescens glycoprotein inhibits AGS gastric cancer cell proliferation by downregulating Wnt-1 signaling.

    Science.gov (United States)

    Kim, Young-Min; Kim, In-Hye; Nam, Taek-Jeong

    2013-11-01

    Previously, we examined various apoptosis pathways in the AGS gastric cancer cell line using Capsosiphon fulvescens glycoprotein (Cf-GP). In this study, we focused on the downregulation of the Wnt-1 signaling pathway and cell cycle arrest. Upregulation of the Wnt signaling pathway has been observed in various cancer cells. The Wnt signal ligand acts in both canonical and non-canonical pathways. Among them, Wnt-1 was dependent on the canonical pathway. Here, we show inhibition of Wnt-1 signaling, β-catenin and transcription factors in AGS cells via Cf-GP. First, we examined the Frizzled receptor and Wnt-1 signal-related proteins including Axin, LRP, β-catenin, APC and GSK-3β. In addition, the expression levels of transcription factors Tcf/LEF were determined by western blot analysis and RT-PCR. Based on the data, we confirmed downregulation of the Wnt-1 signaling pathway by Cf-GP. Also, we determined the expression levels of cell cycle-related proteins cyclin D and c-myc, and looked for cell cycle arrest by cell cycle test analysis. We found that AGS cells arrested in the G0/G1 phase by Cf-GP. These results provide a mechanism of AGS cell inhibition through the downregulation of Wnt-1 signaling by Cf-GP. PMID:23982808

  2. Overexpression of P-glycoprotein induces acquired resistance to imatinib in chronic myelogenous leukemia cells

    Institute of Scientific and Technical Information of China (English)

    Xing-Xiang Peng; Amit K. Tiwari; Hsiang-Chun Wu; Zhe-Sheng Chen

    2012-01-01

    Imatinib,a breakpoint cluster region (BCR)-Abelson murine leukemia (ABL) tyrosine kinase inhibitor (TKI),has revolutionized the treatment of chronic myelogenous leukemia (CML).However,development of multidrug resistance(MDR) limits the use of imatinib.In the present study,we aimed to investigate the mechanisms of cellular resistance to imatinib in CML.Therefore,we established an imatinib-resistant human CML cell line (K562-imatinib) through a stepwise selection process.While characterizing the phenotype of these cells,we found that K562-imatinib cells were 124.6-fold more resistant to imatinib than parental K562 cells.In addition,these cells were cross-resistant to second- and third-generation BCR-ABL TKIs.Western blot analysis and reverse transcription-polymerase chain reaction(RT-PCR) demonstrated that P-glycoprotein (P-gp) and MDR1 mRNA levels were increased in K562-imatinib cells.In addition,accumulation of [14C]6-mercaptopurine (6-MP) was decreased,whereas the ATP-dependent efflux of [14C] 6-MP and [3H]methotrexate transport were increased in K562-imatinib cells.These data suggest that the overexpression of P-gp may play a crucial role in acquired resistance to imatinib in CML K562-imatinib cells.

  3. The putative P-gp inhibitor telmisartan does not affect the transcellular permeability and cellular uptake of the calcium channel antagonist verapamil in the P-glycoprotein expressing cell line MDCK II MDR1

    DEFF Research Database (Denmark)

    Saaby, Lasse; Tfelt-Hansen, Peer; Brodin, Birger

    2015-01-01

    Verapamil is used in high doses for the treatment of cluster headache. Verapamil has been described as a P-glycoprotein (P-gp, ABCB1) substrate. We wished to evaluate in vitro whether co administration of a P-gp inhibitor with verapamil could be a feasible strategy for increasing CNS uptake of...... verapamil. Fluxes of radiolabelled verapamil across MDCK II MDR1 monolayers were measured in the absence and presence of the putative P-gp inhibitor telmisartan (a clinically approved drug compound). Verapamil displayed a vectorial basolateral-toapical transepithelial efflux across the MDCK II MDR1...... monolayers with a permeability of 5.7 9 105 cm sec1 compared to an apical to basolateral permeability of 1.3 9 105 cm sec-1. The efflux could be inhibited with the P-gp inhibitor zosuquidar. Zosuquidar (0.4 lmol/L) reduced the efflux ratio (PB-A/PA-B) for verapamil 4.6–1.6. The presence of telmisartan...

  4. Interactions of bovine viral diarrhoea virus glycoprotein E(rns) with cell surface glycosaminoglycans.

    Science.gov (United States)

    Iqbal, M; Flick-Smith, H; McCauley, J W

    2000-02-01

    Recombinant E(rns) glycoprotein of bovine viral diarrhoea virus (BVDV) has been tagged with a marker epitope or linked to an immunoglobulin Fc tail and expressed in insect and mammalian cell lines. The product was shown to be functional, both having ribonuclease activity and binding to a variety of cells that were permissive and non-permissive for replication of BVDV. Addition of soluble E(rns) to the medium blocked replication of BVDV in permissive cells. Binding of epitope-tagged E(rns) to permissive calf testes (CTe) cells was abolished and virus infection was reduced when cells were treated with heparinases I or III. E(rns) failed to bind to mutant Chinese hamster ovary (CHO) cells that lacked glycosaminoglycans (pgsA-745 cells) or heparan sulphate (pgsD-677 cells) but bound to normal CHO cells. E(rns) also bound to heparin immobilized on agarose and could be eluted by heparin and by a high concentration of salt. Flow cytometric analysis of E(rns) binding to CTe cell cultures showed that glycosaminoglycans such as heparin, fucoidan and dermatan sulphate all inhibit binding but dextran sulphate, keratan sulphate, chondroitin sulphate and mannan fail to inhibit binding. The low molecular mass polysulphonated inhibitor suramin also inhibited binding to CTe cells but poly-L-lysine did not. Furthermore, suramin, the suramin analogue CPD14, fucoidan and pentosan polysulphate inhibited the infectivity of virus. It is proposed that binding of E(rns) to cells is through an interaction with glycosaminoglycans and that BVDV may bind to cells initially through this interaction. PMID:10644844

  5. Altered intracellular pH regulation in cells with high levels of P-glycoprotein expression.

    Science.gov (United States)

    Young, Gregory; Reuss, Luis; Altenberg, Guillermo A

    2011-01-01

    P-glycoprotein is an ATP-binding-cassette transporter that pumps many structurally unrelated drugs out of cells through an ATP-dependent mechanism. As a result, multidrug-resistant cells that overexpress P-glycoprotein have reduced intracellular steady-state levels of a variety of chemotherapeutic agents. In addition, increased cytosolic pH has been a frequent finding in multidrug-resistant cells that express P-glycoprotein, and it has been proposed that this consequence of P-glycoprotein expression may contribute to the lower intracellular levels of chemotherapeutic agents. In these studies, we measured intracellular pH and the rate of acid extrusion in response to an acid load in two cells with very different levels of P-glycoprotein expression: V79 parental cells and LZ-8 multidrug resistant cells. Compared to the wild-type V79 cells, LZ-8 cells have a lower intracellular pH and a slower recovery of intracellular pH after an acid load. The data also show that LZ-8 cells have reduced ability to extrude acid, probably due to a decrease in Na(+)/H(+) exchanger activity. The alterations in intracellular pH and acid extrusion in LZ-8 cells are reversed by 24-h exposure to the multidrug-resistance modulator verapamil. The lower intracellular pH in LZ-8 indicates that intracellular alkalinization is not necessary for multidrug resistance. The reversal by verapamil of the decreased acid-extrusion suggests that P-glycoprotein can affect other membrane transport mechanism. PMID:22003434

  6. Phosphatidylinositol-anchored glycoproteins of PC12 pheochromocytoma cells and brain

    Energy Technology Data Exchange (ETDEWEB)

    Margolis, R.K.; Goossen, B.; Margolis, R.U.

    1988-05-03

    PC12 pheochromocytoma cells and cultures of early postnatal rat cerebellium were labeled with (/sup 3/H)glucosamine, (/sup 3/H)fucose, (/sup 3/H)leucine, (/sup 3/H)ethanolamine, or sodium (/sup 35/S)sulfate and treated with a phosphatidylinositol-specific phospholipase C. Enzyme treatment of (/sup 3/H) glucosamine- or (/sup 3/H)fucose-labeled PC12 cells led to a 15-fold increase in released glycoproteins. On sodium dodecyl sulfate-polyacrylamide gel ectrophoresis, most of the released material migrated as a broad band with an apparent molecular size of 32,000 daltons (Da), which was specifically immunoprecipitated by a monoclonal antibody to the Thy-l glycoprotein. A second glycoprotein, with an apparent molecular size of 158,000 Da, was also released. After treatment with endo-..beta..-galactosidase, 40-45% of the (/sup 3/H)glucosamine of (/sup 3/H)fucose radioactivity in the phospholipase-released glycoproteins was converted to products of disaccharide size, and the molecular size of the 158-kDa glycoprotein decreased to 145 kDa, demonstrating that it contains fucosylated poly-(N-acetyllactosaminyl) oligosaccharides. The phospholipase also released labeled Thy-1 and the 158-kDa glycoprotein from PC12 cells cultured in the presence of (/sup 3/H)ethanolamine, which specifically labels this component of the phosphatidylinositol membrane-anchoring sequence,while in the lipid-free protein residue of cells not treated with phospholipase, Thy-1 and a doublet at 46/48 kDa were the only labeled proteins. Sulfated glycoproteins of 155, 132/134, 61, and 21 kDa are the predominant species released by phospholipase, which does not affect a major 44-kDa protein seen in (/sup 3/H)ethanolamine-labeled brain cultures. The 44-48- and 155/158-kDa proteins may be common to both PC12 cells and brain.

  7. Phosphatidylinositol-anchored glycoproteins of PC12 pheochromocytoma cells and brain

    International Nuclear Information System (INIS)

    PC12 pheochromocytoma cells and cultures of early postnatal rat cerebellium were labeled with [3H]glucosamine, [3H]fucose, [3H]leucine, [3H]ethanolamine, or sodium [35S]sulfate and treated with a phosphatidylinositol-specific phospholipase C. Enzyme treatment of [3H] glucosamine- or [3H]fucose-labeled PC12 cells led to a 15-fold increase in released glycoproteins. On sodium dodecyl sulfate-polyacrylamide gel ectrophoresis, most of the released material migrated as a broad band with an apparent molecular size of 32,000 daltons (Da), which was specifically immunoprecipitated by a monoclonal antibody to the Thy-l glycoprotein. A second glycoprotein, with an apparent molecular size of 158,000 Da, was also released. After treatment with endo-β-galactosidase, 40-45% of the [3H]glucosamine of [3H]fucose radioactivity in the phospholipase-released glycoproteins was converted to products of disaccharide size, and the molecular size of the 158-kDa glycoprotein decreased to 145 kDa, demonstrating that it contains fucosylated poly-(N-acetyllactosaminyl) oligosaccharides. The phospholipase also released labeled Thy-1 and the 158-kDa glycoprotein from PC12 cells cultured in the presence of [3H]ethanolamine, which specifically labels this component of the phosphatidylinositol membrane-anchoring sequence,while in the lipid-free protein residue of cells not treated with phospholipase, Thy-1 and a doublet at 46/48 kDa were the only labeled proteins. Sulfated glycoproteins of 155, 132/134, 61, and 21 kDa are the predominant species released by phospholipase, which does not affect a major 44-kDa protein seen in [3H]ethanolamine-labeled brain cultures. The 44-48- and 155/158-kDa proteins may be common to both PC12 cells and brain

  8. Involvement of cell surface phosphatidylinositol-anchored glycoproteins in cell-cell adhesion of chick embryo myoblasts

    OpenAIRE

    1989-01-01

    During myogenesis myoblasts fuse to form multinucleate cells that express muscle-specific proteins. A specific cell-cell adhesion process precedes lipid bilayer union during myoblast fusion (Knudsen, K. A., and A. F. Horwitz. 1977. Dev. Biol. 58:328-338) and is mediated by cell surface glycoproteins (Knudsen, K. A., 1985. J. Cell Biol. 101:891- 897). In this paper we show that myoblast adhesion and myotube formation are inhibited by treating fusion-competent myoblasts with phosphatidylinosito...

  9. Radiosensitivity of mesothelioma cell lines

    International Nuclear Information System (INIS)

    The present study was carried out in order to examine the radiosensitivity of malignant pleural mesothelioma cell lines. Cell kinetics, radiation-induced delay of the cell cycle and DNA ploidy of the cell lines were also determined. For comparison an HeLa and a human foetal fibroblast cell line were simultaneously explored. Six previously cytogenetically and histologically characterized mesothelioma tumor cell lines were applied. A rapid tiazolyl blue microtiter (MTT) assay was used to analyze radiosensitivity and cell kinetics and DNA ploidy of the cultured cells were determined by flow cytometry. The survival fraction after a dose of 2 Gy (SF2), parameters α and β of the linear quadratic model (LQ-model) and mean inactivation dose (DMID) were also estimated. The DNA index of four cell lines equaled 1.0 and two cell lines equaled 1.5 and 1.6. Different mesothelioma cell lines showed a great variation in radiosensitivity. Mean survival fraction after a radiation dose of 2 Gy (SF2) was 0.60 and ranged from 0.36 to 0.81 and mean α value was 0.26 (range 0.48-0.083). The SF2 of the most sensitive diploid mesothelioma cell line was 0.36: Less than that of the foetal fibroblast cell line (0.49). The survival fractions (0.81 and 0.74) of the two most resistant cell lines, which also were aneuploid, were equal to that of the HeLa cell line (0.78). The α/β ratios of the most sensitive cell lines were almost an order of magnitude greater than those of the two most resistant cell lines. Radiation-induced delay of the most resistant aneuploid cell line was similar to that of HeLa cells but in the most sensitive (diploid cells) there was practically no entry into the G1 phase following the 2 Gy radiation dose during 36 h. (orig.)

  10. Expression of bovine herpesvirus 1 glycoproteins gI and gIII in transfected murine cells

    International Nuclear Information System (INIS)

    Genes encoding two of the major glycoproteins of bovine herpesvirus 1 (BHV-1), gI and gIII, were cloned into the eucaryotic expression vectors pRSVcat and pSV2neo and transfected into murine LMTK- cells, and cloned cell lines were established. The relative amounts of gI or gIII expressed from the two vectors were similar. Expression of gI was cell associated and localized predominantly in the perinuclear region, but nuclear and plasma membrane staining was also observed. Expression of gI was additionally associated with cell fusion and the formation of polykaryons and giant cells. Expression of gIII was localized predominantly in the nuclear and plasma membranes. Radioimmunoprecipitation in the presence or absence of tunicamycin revealed that the recombinant glycoproteins were proteolytically processed and glycosylated and had molecular weights similar to those of the forms of gI and gIII expressed in BHV-1 infected bovine cells. However, both recombinant glycoproteins were glycosylated to a lesser extent than were the forms found in BHV-1 infected bovine cells. For gI, a deficiency in N-linked glycosylated of the amino-terminal half of the protein was identified; for gIII, a deficiency in O-linked glycosylation was implicated. The reactivity pattern of a panel of gI- and gIII-specific monoclonal antibodies, including six which recognize conformation-dependent epitopes, was found to be unaffected by the glycosylation differences and was identical for transfected of BHV-1-infected murine cells. Use of the transfected cells as targets in immune-mediated cytotoxicity assays demonstrated the functional recognition of recombinant gI and gIII by murine antibody and cytotoxic T lymphocytes

  11. Interaction of herpes simplex virus glycoprotein gC with mammalian cell surface molecules.

    OpenAIRE

    Tal-Singer, R; Peng, C.; Ponce de Leon, M; Abrams, W R; Banfield, B W; Tufaro, F; Cohen, G H; Eisenberg, R J

    1995-01-01

    The entry of herpes simplex virus (HSV) into mammalian cells is a multistep process beginning with an attachment step involving glycoproteins gC and gB. A second step requires the interaction of glycoprotein gD with a cell surface molecule. We explored the interaction between gC and the cell surface by using purified proteins in the absence of detergent. Truncated forms of gC and gD, gC1(457t), gC2(426t), and gD1(306t), lacking the transmembrane and carboxyl regions were expressed in the bacu...

  12. Effect of the ionophore monensin on herpes simplex virus type 1-induced cell fusion, glycoprotein synthesis, and virion infectivity.

    Science.gov (United States)

    Kousoulas, K G; Bzik, D J; Person, S

    1983-01-01

    The ionophore monensin inhibited the formation of mature, fully glycosylated glycoproteins gB, gC, and gD during herpes simplex virus type 1 infection of human embryonic lung cells. Underglycosylated forms, including the apparent high-mannose precursor forms of the major glycoproteins, appeared. Monensin inhibited virus-induced cell fusion. Infectious virions produced in the presence of monensin appeared to contain predominantly underglycosylated glycoproteins. PMID:6307921

  13. Cell-surface expression of a mutated Epstein–Barr virus glycoprotein B allows fusion independent of other viral proteins

    OpenAIRE

    McShane, Marisa P.; Longnecker, Richard

    2004-01-01

    Epstein–Barr virus (EBV) infects human B lymphocytes and epithelial cells. We have compared the requirements for EBV glycoprotein-induced cell fusion between Chinese hamster ovary effecter cells and human B lymphoblasts or epithelial cells by using a virus-free cell fusion assay. EBV-encoded gB, gH, gL, and gp42 glycoproteins were required for efficient B cell fusion, whereas EBV gB, gH, and gL glycoproteins were required for Chinese hamster ovary effecter cell fusion with epithelial cell lin...

  14. Biology of SNU Cell Lines

    OpenAIRE

    Ku, Ja-Lok; Park, Jae-Gahb

    2005-01-01

    SNU (Seoul National University) cell lines have been established from Korean cancer patients since 1982. Of these 109 cell lines have been characterized and reported, i.e., 17 colorectal carcinoma, 12 hepatocellular carcinoma, 11 gastric carcinoma, 12 uterine cervical carcinoma, 17 B-lymphoblastoid cell lines derived from cancer patients, 5 ovarian carcinoma, 3 malignant mixed Mllerian tumor, 6 laryngeal squamous cell carcinoma, 7 renal cell carcinoma, 9 brain tumor, 6 biliary tract, and 4 pa...

  15. Characterisation of non-P-glycoprotein multidrug-resistant Ehrlich ascites tumour cells selected for resistance to mitoxantrone

    DEFF Research Database (Denmark)

    Nielsen, D; Eriksen, J; Maare, C;

    2000-01-01

    (i) value for P-glycoprotein-positive cells. However, whereas verapamil (50 microM) inhibited the ATPase activity of EHR2/MITOX microsomes, it stimulated the ATPase activity of microsomes derived from P-glycoprotein-positive cells. In conclusion, the resistance in EHR2/MITOX was multifactorial and appeared...

  16. Selection of mutant Chinese hamster ovary cells altered glycoproteins by means of tritiated fucose suicide.

    OpenAIRE

    Hirschberg, C B; Baker, R.M.; Perez, M.; Spencer, L A; Watson, D

    1981-01-01

    Mutant Chinese hamster ovary cells altered in glycoproteins have been isolated by selecting for ability to survive exposure to [6-3H]fucose. Mutagenized wild-type cells were permitted to incorporate [3H]fucose to approximately 1 cpm of trichloroacetic acid-insoluble radioactivity per cell and then frozen for several days to accumulate radiation damage. The overall viability of the population was reduced by 5- to 50-fold. Four consecutive selection cycles were carried out. The surviving cells ...

  17. The expression of two P-glycoprotein (pgp) genes in transgenic Caenorhabditis elegans is confined to intestinal cells.

    OpenAIRE

    Lincke, C R; Broeks, A; the, I; Plasterk, R H; Borst, P

    1993-01-01

    P-glycoproteins can cause multidrug resistance in mammalian tumor cells by active extrusion of cytotoxic drugs. The natural function of these evolutionarily conserved, membrane-bound ATP binding transport proteins is unknown. In mammals, P-glycoproteins are abundantly present in organs associated with the digestive tract. We have studied the tissue-specific expression of Caenorhabditis elegans P-glycoprotein genes pgp-1 and pgp-3 by transformation of nematodes with pgp-lacZ gene fusion constr...

  18. Sialic Acids on Varicella-Zoster Virus Glycoprotein B Are Required for Cell-Cell Fusion.

    Science.gov (United States)

    Suenaga, Tadahiro; Matsumoto, Maki; Arisawa, Fuminori; Kohyama, Masako; Hirayasu, Kouyuki; Mori, Yasuko; Arase, Hisashi

    2015-08-01

    Varicella-zoster virus (VZV) is a member of the human Herpesvirus family that causes varicella (chicken pox) and zoster (shingles). VZV latently infects sensory ganglia and is also responsible for encephalomyelitis. Myelin-associated glycoprotein (MAG), a member of the sialic acid (SA)-binding immunoglobulin-like lectin family, is mainly expressed in neural tissues. VZV glycoprotein B (gB) associates with MAG and mediates membrane fusion during VZV entry into host cells. The SA requirements of MAG when associating with its ligands vary depending on the specific ligand, but it is unclear whether the SAs on gB are involved in the association with MAG. In this study, we found that SAs on gB are essential for the association with MAG as well as for membrane fusion during VZV infection. MAG with a point mutation in the SA-binding site did not bind to gB and did not mediate cell-cell fusion or VZV entry. Cell-cell fusion and VZV entry mediated by the gB-MAG interaction were blocked by sialidase treatment. N-glycosylation or O-glycosylation inhibitors also inhibited the fusion and entry mediated by gB-MAG interaction. Furthermore, gB with mutations in N-glycosylation sites, i.e. asparagine residues 557 and 686, did not associate with MAG, and the cell-cell fusion efficiency was low. Fusion between the viral envelope and cellular membrane is essential for host cell entry by herpesviruses. Therefore, these results suggest that SAs on gB play important roles in MAG-mediated VZV infection. PMID:26105052

  19. Targeting HIV-1 Envelope Glycoprotein Trimers to B Cells by Using APRIL Improves Antibody Responses

    OpenAIRE

    Melchers M; Bontjer I; Tong T; Chung NP; Klasse PJ; Eggink D; Montefiori DC; Gentile M; Cerutti A; Olson WC; Berkhout B; Binley JM; Moore JP; Sanders RW

    2012-01-01

    An HIV-1 vaccine remains elusive, in part because various factors limit the quantity and quality of the antibodies raised against the viral envelope glycoprotein complex (Env). We hypothesized that targeting Env vaccines directly to B cells, by fusing them to molecules that bind and activate these cells, would improve Env-specific antibody responses. Therefore, we fused trimeric Env gp140 to A PRoliferation-Inducing Ligand (APRIL), B-cell Activating Factor (BAFF), and CD40 Ligand (CD40L). The...

  20. Effect of carbenoxolone on the synthesis of glycoproteins and DNA in rat gastric epithelial cells.

    OpenAIRE

    van Huis, G A; Kramer, M.F.

    1981-01-01

    The influence of carbenoxolone on the synthesis of glycoproteins in the surface mucous cells and the production of new cells in the rat gastric mucosa was studied by means of a vascular perfusion system. The rate of incorporation of tritiated galactose, glucosamine, serine, and sulphate in surface mucous cells, studied by autoradiography, was not affected by the addition of carbenoxolone to the drinking water. The sugar composition (determined by gas-liquid chromatography) of the gastric glyc...

  1. Pluripotent stem cell lines

    OpenAIRE

    Yu, Junying; Thomson, James A.

    2008-01-01

    The derivation of human embryonic stem cells 10 years ago ignited an explosion of public interest in stem cells, yet this achievement depended on prior decades of research on mouse embryonic carcinoma cells and embryonic stem cells. In turn, the recent derivation of mouse and human induced pluripotent stem cells depended on the prior studies on mouse and human embryonic stem cells. Both human embryonic stem cells and induced pluripotent stem cells can self-renew indefinitely in vitro while ma...

  2. Structure of a Pestivirus Envelope Glycoprotein E2 Clarifies Its Role in Cell Entry

    Directory of Open Access Journals (Sweden)

    Kamel El Omari

    2013-01-01

    Full Text Available Enveloped viruses have developed various adroit mechanisms to invade their host cells. This process requires one or more viral envelope glycoprotein to achieve cell attachment and membrane fusion. Members of the Flaviviridae such as flaviviruses possess only one envelope glycoprotein, E, whereas pestiviruses and hepacivirus encode two glycoproteins, E1 and E2. Although E2 is involved in cell attachment, it has been unclear which protein is responsible for membrane fusion. We report the crystal structures of the homodimeric glycoprotein E2 from the pestivirus bovine viral diarrhea virus 1 (BVDV1 at both neutral and low pH. Unexpectedly, BVDV1 E2 does not have a class II fusion protein fold, and at low pH the N-terminal domain is disordered, similarly to the intermediate postfusion state of E2 from sindbis virus, an alphavirus. Our results suggest that the pestivirus and possibly the hepacivirus fusion machinery are unlike any previously observed.

  3. Intercellular transfer of P-glycoprotein in human blood-brain barrier endothelial cells is increased by histone deacetylase inhibitors.

    Science.gov (United States)

    Noack, Andreas; Noack, Sandra; Buettner, Manuela; Naim, Hassan Y; Löscher, Wolfgang

    2016-01-01

    The blood-brain barrier (BBB) controls the entry of compounds into the brain, thereby regulating brain homeostasis. Efflux transporters such as P-glycoprotein (Pgp) significantly contribute to BBB function. Multiple signaling pathways modulate the expression and activity of Pgp in response to xenobiotics and disease. A non-genetic way of intercellular transfer of Pgp occurs in cancer cells, but whether this also occurs in non-cancer cells such as endothelial cells that form the BBB is not known. A human brain endothelial cell line (hCMEC/D3) was used to study whether cell-to-cell Pgp transfer occurs during co-culturing with Pgp-EGFP expressing hCMEC/D3 cells. The Pgp-EGFP fusion protein was transferred from donor to recipient cells by cell-to-cell contact and Pgp-EGFP enriched vesicles, which were exocytosed by donor cells and endocytosed by adherent recipient cells. Flow cytometry experiments with the Pgp substrate eFLUXX-ID Gold demonstrated that the transferred Pgp is functional in the recipient cells. Exposure of the donor cells with inhibitors of histone deacetylases (HDACs) resulted in an enhanced intercellular Pgp transfer. Non-genetic transfer of a resistance phenotype and its regulation by HDACs is a novel mechanism of altering BBB functionality. This mechanism may have important implications for understanding drug-induced alterations in Pgp expression and activity. PMID:27375084

  4. Glycosylation Engineering of Glycoproteins

    Science.gov (United States)

    Sadamoto, Reiko; Nishimura, Shin-Ichiro

    Naturally occurring glycosylation of glycoproteins varies in glycosylation site and in the number and structure of glycans. The engineering of well-defined glycoproteins is an important technology for the preparation of pharmaceutically relevant glycoproteins and in the study of the relationship between glycans and proteins on a structure-function level. In pharmaceutical applications of glycoproteins, the presence of terminal sialic acids on glycans is particularly important for the in vivo circulatory half life, since sialic acid-terminated glycans are not recognized by asialoglycoprotein receptors. Therefore, there have been a number of attempts to control or modify cellular metabolism toward the expression of glycoproteins with glycosylation profiles similar to that of human glycoproteins. In this chapter, recent methods for glycoprotein engineering in various cell culture systems (mammalian cells, plant, yeast, and E. coli) and advances in the chemical approach to glycoprotein formation are described.

  5. P-Glycoprotein Induction Ameliorates Colistin Induced Nephrotoxicity in Cultured Human Proximal Tubular Cells.

    Directory of Open Access Journals (Sweden)

    Sun-hyo Lee

    Full Text Available The pathogenesis of colistin induced nephrotoxicity is poorly understood. Currently there are no effective therapeutic or prophylactic agents available. This study was aimed to determine the mechanism of colistin induced nephrotoxicity and to determine whether P-glycoprotein (P-gp induction could prevent colistin induced nephrotoxicity. Colistin induced cell toxicity in cultured human proximal tubular cells in both dose and time dependent manner. Colistin provoked ROS in a dose dependent manner as measured by DCF-DA. To investigate apoptosis, caspase 3/7 activity was determined. Caspase 3/7 activity was increased dose dependently (25, 50, 100 μg/ml at 6 h. Autophagosome formation was assessed by measuring LC3- II/LC3-I ratio. The ratio of LC3-II to LC3- I was increased at 2 h (25 μg/ml. Suppression of autophagosome formation increased colistin induced nephrotoxicity. The expression of P-gp and the cell toxicity was determined in colistin with or without dexamethasone (P-gp inducer and verapamil (selective P-gp inhibitor. Colistin itself suppressed the expression of P-gp. P-gp expression and activity decreased colistin induced nephrotoxicity with dexamethasone treatment. In addition induced P-gp transporter was shown to improve the efflux effect on colistin treated HK2 cell line, which was demonstrated by calcein-AM fluorescence accumulation assay. The increased activity could be blocked by N-acetylcysteine. In conclusion, colistin induces nephrotoxicity by suppressing P-gp. Induction of P-gp could ameliorate colistin induced nephrotoxicity by decreasing apoptosis.

  6. The cell surface expressed nucleolin is a glycoprotein that triggers calcium entry into mammalian cells

    International Nuclear Information System (INIS)

    Nucleolin is an ubiquitous nucleolar phosphoprotein involved in fundamental aspects of transcription regulation, cell proliferation and growth. It has also been described as a shuttling molecule between nucleus, cytosol and the cell surface. Several studies have demonstrated that surface nucleolin serves as a receptor for various extracellular ligands implicated in cell proliferation, differentiation, adhesion, mitogenesis and angiogenesis. Previously, we reported that nucleolin in the extranuclear cell compartment is a glycoprotein containing N- and O-glycans. In the present study, we show that glycosylation is an essential requirement for surface nucleolin expression, since it is prevented when cells are cultured in the presence of tunicamycin, an inhibitor of N-glycosylation. Accordingly, surface but not nuclear nucleolin is radioactively labeled upon metabolic labeling of cells with [3H]glucosamine. Besides its well-demonstrated role in the internalization of specific ligands, here we show that ligand binding to surface nucleolin could also induce Ca2+ entry into cells. Indeed, by flow cytometry, microscopy and patch-clamp experiments, we show that the HB-19 pseudopeptide, which binds specifically surface nucleolin, triggers rapid and intense membrane Ca2+ fluxes in various types of cells. The use of several drugs then indicated that Store-Operated Ca2+ Entry (SOCE)-like channels are involved in the generation of these fluxes. Taken together, our findings suggest that binding of an extracellular ligand to surface nucleolin could be involved in the activation of signaling pathways by promoting Ca2+ entry into cells

  7. Two-dimensional analysis of metabolically and cell surface radiolabeled proteins of some human lymphoid and myeloid leukemia cell lines. II

    International Nuclear Information System (INIS)

    Cell surface glycoproteins, radiolabelled by the sodium metaperiodate/tritiated borohydride technique, and cell phosphoproteins, metabolically radiolabelled with 32P-orthophosphate were analyzed by two-dimensional electrophoretic analysis in some myeloid and lymphoid leukemia cell lines. Some markedly expressed major glycoproteins were predominant in some of the cell lines (such as 95k and 100k glycoproteins with marked charge heterogeneity in non-T, non-B acute lymphoblastic leukemia cell lines NALM 6 and NALM 16), but markedly quantitatively reduced in other examined cell lines, such as lymphoblastoid cell line UHKT 34/2. 32P-orthophosphate radiolabelled phosphoprotein two-dimensional patterns of the examined lymphoid leukemia cell lines were essentially similar, with some minor differences, in examined lymphoid and myeloid leukemia cell lines, such as marked expression of a series of large phosphoproteins in the molecular weight range 80-100k in lymphoid cell lines and almost complete absence of these phosphoproteins on the examined myeloid leukemia cell lines. Another configuration of acidic phosphoproteins (30-35k) exhibited individual cell line variability and differences between both individual myeloid leukemia cell lines and between the lymphoid and myeloid cell lines examined. (author) 2 figs., 15 refs

  8. P-Glycoprotein Induction Ameliorates Colistin Induced Nephrotoxicity in Cultured Human Proximal Tubular Cells

    OpenAIRE

    Lee, Sun-hyo; Kim, Jin-Sun; Ravichandran, Kameswaran; Gil, Hyo-Wook; SONG, HO-YEON; Hong, Sae-Yong

    2015-01-01

    The pathogenesis of colistin induced nephrotoxicity is poorly understood. Currently there are no effective therapeutic or prophylactic agents available. This study was aimed to determine the mechanism of colistin induced nephrotoxicity and to determine whether P-glycoprotein (P-gp) induction could prevent colistin induced nephrotoxicity. Colistin induced cell toxicity in cultured human proximal tubular cells in both dose and time dependent manner. Colistin provoked ROS in a dose dependent man...

  9. Doxycycline Induces Expression of P Glycoprotein in MCF-7 Breast Carcinoma Cells

    OpenAIRE

    Mealey, Katrina L; Barhoumi, Rola; Burghardt, Robert C.; Safe, Stephen; Kochevar, Deborah T.

    2002-01-01

    P-glycoprotein (P-gp) overexpression by tumor cells imparts resistance to multiple antineoplastic chemotherapeutic agents (multiple drug resistance). Treatment of tumor cells with chemotherapeutic agents such as anthracyclines, epipodophyllotoxins, and Vinca alkaloids results in induction of P-gp expression. This study was performed to determine if clinically relevant antimicrobial drugs (i.e., drugs that are used to treat bacterial infections in cancer patients) other than antineoplastic age...

  10. Impact of small molecules immunosuppressants on P-glycoprotein activity and T-cell function

    OpenAIRE

    Llaudó Vallmajor, Inés; Cassis, L.; Torras Ambròs, Joan; Bestard Matamoros, Oriol; Franquesa, M.; Cruzado, Josep Ma.; Cerezo, G.; Castaño Boldú, Esther; Pétriz, J; Herrero Fresneda, Immaculada; Grinyo Boira, Josep M.; Lloberas Blanch, Núria

    2012-01-01

    Purpose. P-glycoprotein (Pgp) is a member of the ABC-transporter family that transports substances across cellular membranes acting as an efflux pump extruding drugs out of the cells. Pgp plays a key role on the pharmacokinetics of several dr ugs. Herein, we have studied the effects of immunosuppressants on Pgp function, assessing rhodamine-123 (Rho123) uptake and efflux in different T- cell subsets. Methods. Different immunosuppressants such as Cyclosporine (CsA), Rapamycin (Rapa) and Tacrol...

  11. Transient axonal glycoprotein-1 induces apoptosis-related gene expression without triggering apoptosis in U251 glioma cells

    Institute of Scientific and Technical Information of China (English)

    Haigang Chang; Xiaodan Jiang; Shanshan Song; Zhongcan Chen; Yaxiao Wang; Lujun Yang; Mouxuan Du; Yiquan Ke; Ruxiang Xu; Baozhe Jin

    2014-01-01

    Previous studies show that transient axonal glycoprotein-1, a ligand of amyloid precursor pro-tein, increases the secretion of amyloid precursor protein intracellular domain and is involved in apoptosis in Alzheimer’s disease. In this study, we examined the effects of transient axonal glyco-protein-1 on U251 glioma cells. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay showed that transient axonal glycoprotein-1 did not inhibit the proliferation of U251 cells, but promoted cell viability. The terminal deoxynucleotidyl transferase dUTP nick end labeling assay showed that transient axonal glycoprotein-1 did not induce U251 cell apoptosis. Real-time PCR revealed that transient axonal glycoprotein-1 substantially upregulated levels of amyloid precursor protein intracellular C-terminal domain, and p53 and epidermal growth factor recep-tor mRNA expression. Thus, transient axonal glycoprotein-1 increased apoptosis-related gene expression in U251 cells without inducing apoptosis. Instead, transient axonal glycoprotein-1 promoted the proliferation of these glioma cells.

  12. 3,3′,4,4′,5-Pentachlorobiphenyl Inhibits Drug Efflux Through P-Glycoprotein in KB-3 Cells Expressing Mutant Human P-Glycoprotein

    Directory of Open Access Journals (Sweden)

    Hiroshi Fujise

    2004-01-01

    Full Text Available The effects on the drug efflux of 3,3′,4,4′,5-pentachlorobiphenyl (PCB-126, the most toxic of all coplanar polychlorinated biphenyls (Co-PCBs, were examined in KB-3 cells expressing human wild-type and mutant P-glycoprotein in which the 61st amino acid was substituted for serine or phenylalanine (KB3-Phe61. In the cells expressing P-glycoproteins, accumulations of vinblastine and colchicine decreased form 85% to 92% and from 62% to 91%, respectively, and the drug tolerances for these chemicals were increased. In KB3-Phe61, the decreases in drug accumulation were inhibited by adding PCB-126 in a way similar to that with cyclosporine A: by adding 1 μM PCB-126, the accumulations of vinblastine and colchicine increased up to 3.3- and 2.3-fold, respectively. It is suggested that PCB-126 decreased the drug efflux by inhibiting the P-glycoprotein in KB3-Phe61. Since there were various P-glycoproteins and many congeners of Co-PCBs, this inhibition has to be considered a new cause of the toxic effects of Co-PCBs.

  13. Separation of bivalent anti-T cell immunotoxin from Pichia pastoris glycoproteins by borate anion exchange.

    Science.gov (United States)

    Woo, Jung Hee; Neville, David M

    2003-08-01

    A major problem encountered in the large-scale purification of the bivalent anti-T cell immunotoxin, A-dmDT390-bisFv(G4S), from Pichia pastoris supernatants was the presence of host glycoproteins exhibiting similar charge, size, and hydrophobicity characteristics. We overcame this problem by employing borate anion exchange chromatography. The borate anion has an affinity for carbohydrates and imparts negative charges to these structures. We found that at a concentration of sodium borate between 50 and 100 mM, the nonglycosylated immunotoxin did not bind to Poros 50 HQ anion exchanger resin, but glycoproteins, including aggregates related to the immunotoxin, did. By using this property of the immunotoxin in the presence of sodium borate, we successfully developed a 3-step purification procedure: (i) Butyl-650M hydrophobic interaction chromatography, (ii) Poros 50 HQ anion exchange chromatography in the presence of borate, and (iii) HiTrap Q anion exchange chromatography. The final preparation exhibited a purity of greater than 98% and a yield of greater than 50% from the supernatant. Previously, boronic acid resins have been used to separate glycoproteins from proteins. However, combining borate anion with conventional anion exchange resins accomplishes the separation of the immunotoxin from glycoproteins and eliminates the need to evaluate nonstandard resins with respect to good manufacturing practice guidelines. PMID:12951782

  14. Expression of the human immunodeficiency virus envelope glycoprotein is restricted to basolateral surfaces of polarized epithelial cells

    International Nuclear Information System (INIS)

    Polarized epithelial cells exhibit apical (lumenal) and basolateral (serosal) membrane domains that are separated by circumferential tight junctions. In such cells, enveloped viruses that mature by budding at cell surfaces are released at particular membrane domains. The authors have used a vaccinia virus recombinant to investigate the site of surface expression of the human immunodeficiency virus type 1 envelope glycoprotein in Madin-Darby canine kidney cells. Cells were infected with the vaccinia virus recombinant, and surface expression of the glycoprotein was analyzed by indirect immunofluorescence, 125I-protein A binding, and immunoelectron microscopy. The glycoprotein appeared exclusively at the basolateral surface as early as 2 h postinfection and reached a maximum level at 8 h postinfection. The gp120 glycoprotein was found to be secreted efficiently into culture medium, and this secretion occurred exclusively at the basolateral surface

  15. P-glycoprotein is expressed and causes resistance to chemotherapy in EBV-positive T-cell lymphoproliferative diseases

    International Nuclear Information System (INIS)

    Epstein–Barr virus-positive T-cell lymphoproliferative diseases (EBV-T-LPDs) are rare lymphomas with poor prognosis. Although chemotherapeutic strategies such as CHOP have been often selected, they have exhibited only limited efficacy. To clarify the mechanism of chemoresistance, we examined P-glycoprotein (P-gp) expression. P-gp acts as an energy-dependent efflux pump that excretes drugs from the cytoplasm, resulting in low-intracellular drug concentrations and poor sensitivity to chemotherapy. We examined P-gp expression in EBV-positive cells by immunohistochemistry staining in three patients of EBV-T-LPDs and the expression was detected in all patients. We also examined mdr1 mRNA expression by reverse-transcriptase polymerase-chain reaction (RT-PCR) in EBV-positive tumor cells from these patients and additional three patients. The expression was detected in all examined patients. In five EBV-T-LPDs patients, P-gp function was detected by Rhodamine-123 efflux assay in these cells. The efflux was inhibited by treatment with a P-gp inhibitor, cyclosporine A (CsA). We also examined and detected P-gp expression in EBV-positive T-cell lines SNT8 and SNT16 established from EBV-T-LPDs patients, by RT-PCR and western blotting. The function was also detected by Rhodamine-123 efflux in these cell lines. Inhibition and knock down of P-gp by CsA and siRNA, respectively, enhanced etoposide- and doxorubicin-induced cell death in the EBV-positive T-cell lines. Finally, we infected the T-cell line MOLT4 with EBV, and found that mdr1 mRNA expression and Rhodamine 123 efflux were upregulated after infection. These results indicated that enhanced P-gp expression contributed to the chemoresistance of EBV-T-LPDs

  16. Autoradiographic detection of RNA and glycoprotein synthesis in epithelial cells of the oviduct in ovulated heifers

    International Nuclear Information System (INIS)

    Heifers (n=9) of the Black-Pied Holstein-Friesian breed were slaughtered on the 3rd, 6th and 9th day of the synchronized cycle, respectively (heat=day 0). Tissue samples from the ampullar part of the oviduct were collected immediately after slaughter and treated for histoautoradiographic (ARG) analyses. Investigated autoradiograms revealed an intensive synthesis of RNA in both cilliated as well as in secretory cells of the oviduct epithelium. The intensity of RNA synthesis remains comparable during the early luteal phase from the 3rd to the 9th day of the cycle. The glycoproteins, synthesized first in the Golgi apparatus in the supranuclear region, were seen to move to the apical region of the epithelial cells later on. The most intensive autoradiographic reaction of newly synthesized glycoproteins was detected on the 3rd day of the cycle without an evident change on the 6th day but decreasing on the 9th day

  17. Expression and Purification of E2 Glycoprotein from Insect Cells (Sf9) for Use in Serology.

    Science.gov (United States)

    Chua, Chong Long; Sam, I-Ching; Chan, Yoke Fun

    2016-01-01

    Chikungunya virus (CHIKV) is a mosquito-borne arbovirus which poses a major threat to global public health. Definitive CHIKV diagnosis is crucial, especially in distinguishing the disease from dengue virus, which co-circulates in endemic areas and shares the same mosquito vectors. Laboratory diagnosis is mainly based on serological or molecular approaches. The E2 glycoprotein is a good candidate for serological diagnosis since it is the immunodominant antigen during the course of infection, and reacts with seropositive CHIKV sera. In this chapter, we describe the generation of stable clone Sf9 (Spodoptera frugiperda) cells expressing secreted, soluble, and native recombinant CHIKV E2 glycoprotein. We use direct plasmid expression in insect cells, rather than the traditional technique of generating recombinant baculovirus. This recombinant protein is useful for serological diagnosis of CHIKV infection. PMID:27233260

  18. Membrane glycoprotein folding, oligomerization and intracellular transport: effects of dithiothreitol in living cells

    OpenAIRE

    Braakman, L.J.; Tatu, U.; Helenius, A

    1993-01-01

    Using influenza hemagglutinin (HA0) and vesicular stomatitis virus G protein as model proteins, we have analyzed the effects of dithiothreitol (DTT) on conformational maturation and transport of glycoproteins in the secretory pathway of living cells. While DTT caused reduction of folding intermediates and misfolded proteins in the endoplasmic reticulum (ER), it did not affect molecules that had already acquired a mature trimeric conformation, whether present in the ER or elsewhere. The conver...

  19. The effect of the state of differentiation on labeling of epidermal cell surface glycoproteins

    International Nuclear Information System (INIS)

    Epidermal cells were grown in a medium in which the Ca++ concentration controlled the stage of differentiation. Cell surface molecules of differentiated and undifferentiated cells were compared by lactoperoxidase-catalyzed iodination, by the interaction with 125I-lectins, and by the metabolic incorporation of L-(3H)-fucose. Molecular weights of the labeled components were determined by SDS-PAGE and autoradiography. After lactoperoxidase iodination, most of the radioactivity was found in polypeptide bands of 79,000, 65,000 and 56,000 daltons. The 79,000 band is the most intense for undifferentiated cells but disappears as differentiation proceeds. The 56,000 band is present in normal cells at all stages of differentiation but is absent from neoplastic cells. Glycoproteins reacted with 125I-lectins were found at 180,000, 130,000 and 85,000 daltons. The 130,000 band was the most prominent for differentiated cells labeled with wheat germ agglutinin but was essentially absent from the undifferentiated cells. With Ricinus communis agglutinin, this band was weaker for undifferentiated than for differentiated cells but was the most intense for both. After metabolic incorporation of tritiated fucose, radioactive glycoproteins were found at 130,000 and 85,000 daltons, with comparable intensities for differentiated and undifferentiated cells

  20. High-Throughput Selection of Retrovirus Producer Cell Lines Leads to Markedly Improved Efficiency of Germ Line-Transmissible Insertions in Zebra Fish

    OpenAIRE

    Chen, Wenbiao; Burgess, Shawn; Golling, Greg; Amsterdam, Adam; Hopkins, Nancy

    2002-01-01

    Vesicular stomatitis virus glycoprotein G-pseudotyped mouse retroviral vectors have been used as mutagens for a large-scale insertional mutagenesis screen in the zebra fish. To reproducibly generate high-titer virus stocks, we devised a method for rapidly selecting cell lines that can yield high-titer viruses and isolated a producer cell line that yields virus at a high titer on zebra fish embryos. Virus produced from this line, designated GT virus, is nontoxic following injection of zebra fi...

  1. Cell lines and Salmonella

    NARCIS (Netherlands)

    Jonge R de; Hendriks H; Garssen J; Universteit Utrecht, afdeling; MGB; LPI

    2001-01-01

    In human gastrointestinal disease caused by Salmonella, transepithelial migration of neutrophils follows the attachment of bacteria to epithelial tissue. This migration of neutrophils is stimulated by the release of chemokines, including interleukin-8 (Il -8), from the epithelial cells. We have dev

  2. Phosphorylation of the multidrug resistant associated glycoprotein (p-glycoprotein): Preparation and characterization of 7-acetyltaxol

    International Nuclear Information System (INIS)

    To assess the role of phosphorylation in P-glycoprotein function, phosphorylation of P-glycoprotein in intact cells and in cell-free membrane fractions has been studied. Results obtained with cell-free membrane fractions indicate that P-glycoprotein is a substrate for a membrane-associated protein kinase A (PK-A). To assess whether P-glycoprotein was phosphorylated in vivo by PK-A, MDR cells were incubated with [32P]Pi in the presence or absence of 100 uM 8Br-cAMP. The tryptic phosphopeptides of six P-glycoproteins from five independently derived MDR cell lines were analyzed by HPLC. A similar analysis carried out with two other P-glycoproteins (from J7.V3-1 and the lower band of J7.T1-50) demonstrated a major phosphopeptide with a retention time of 26 min. Fraction 26 was resolved as a single phosphopeptide by 2-D mapping. The phosphorylation of fraction 26 which was derived from P-glycoprotein in J7.V3-1 or the J7.T1-50 lower band was enhanced when the cells were treated with 8BrcAMP

  3. Phosphorylation of the multidrug resistant associated glycoprotein (p-glycoprotein): Preparation and characterization of 7-acetyltaxol

    Energy Technology Data Exchange (ETDEWEB)

    Mellado, W.

    1988-01-01

    To assess the role of phosphorylation in P-glycoprotein function, phosphorylation of P-glycoprotein in intact cells and in cell-free membrane fractions has been studied. Results obtained with cell-free membrane fractions indicate that P-glycoprotein is a substrate for a membrane-associated protein kinase A (PK-A). To assess whether P-glycoprotein was phosphorylated in vivo by PK-A, MDR cells were incubated with ({sup 32}P)Pi in the presence or absence of 100 uM 8Br-cAMP. The tryptic phosphopeptides of six P-glycoproteins from five independently derived MDR cell lines were analyzed by HPLC. A similar analysis carried out with two other P-glycoproteins (from J7.V3-1 and the lower band of J7.T1-50) demonstrated a major phosphopeptide with a retention time of 26 min. Fraction 26 was resolved as a single phosphopeptide by 2-D mapping. The phosphorylation of fraction 26 which was derived from P-glycoprotein in J7.V3-1 or the J7.T1-50 lower band was enhanced when the cells were treated with 8BrcAMP.

  4. Thyroid cell lines in research on goitrogenesis.

    Science.gov (United States)

    Gerber, H; Peter, H J; Asmis, L; Studer, H

    1991-12-01

    Thyroid cell lines have contributed a lot to the understanding of goitrogenesis. The cell lines mostly used in thyroid research are briefly discussed, namely the rat thyroid cell lines FRTL and FRTL-5, the porcine thyroid cell lines PORTHOS and ARTHOS, The sheep thyroid cell lines OVNIS 5H and 6H, the cat thyroid cell lines PETCAT 1 to 4 and ROMCAT, and the human thyroid cell lines FTC-133 and HTh 74. Chinese hamster ovary (CHO) cells and COS-7 cells, stably transfected with TSH receptor cDNA and expressing a functional TSH receptor, are discussed as examples for non-thyroidal cells, transfected with thyroid genes. PMID:1726925

  5. [Hydroxyproline: Rich glycoproteins of the plant and cell wall

    Energy Technology Data Exchange (ETDEWEB)

    Varner, J.E.

    1993-01-01

    Since xylem tissue includes the main cell types which are lignified, we are interested in gene expression of glycine-rich proteins and proline-rich proteins, and other proteins which are involved in secondary cell wall thickening during xylogenesis. Since the main feature of xylogenesis is the deposition of additional wall components, study of the mechanism of xylogenesis will greatly advance our knowledge of the synthesis and assembly of wall macromolecules. We are using the in vitro xylogenesis system from isolated Zinnia mesophyll cells to isolate genes which are specifically expressed during xylogenesis. We have used subtractive hybridization methods to isolate a number of cDNA clones for differentially regulated genes from the cells after hormonal induction. So far, we have partially characterized 18 different cDNA clones from 239 positive clones. These differentially regulated genes can be divided into three sets according to the characteristics of gene expression in the induction medium and the control medium. The first set is induced in both the induction medium and the control medium without hormones. The second set is induced mainly in the induction medium and in the control medium with the addition of NAA alone. Two of thesegenes are exclusively induced by auxin. The third set of genes is induced mainly in the induction medium. Since these genes are not induced by either auxin or cytokinin alone, they may be directly involved in the process of xylogenesis. Our experiments on the localization of H[sub 2]O[sub 2] production reinforce the earlier ideas of others that H[sub 2]O[sub 2] is involved in normal lignification.

  6. Expression of P-glycoprotein in excised human nasal mucosa and optimized models of RPMI 2650 cells.

    Science.gov (United States)

    Dolberg, Anne M; Reichl, Stephan

    2016-07-11

    To assess the transmucosal drug transport in the development of medications for intranasal administration, cellular in vitro models are preferred over the use of animal tissues due to inter-species variations and ethical concerns. With regard to the distribution of active agents and multidrug resistance, the ABC transporter P-glycoprotein plays a major role in several mammalian tissues. The present study compares the expression of this efflux pump in optimized in vitro models based on the human RPMI 2650 cell line with specimens of human turbinate mucosa. The presence of the ABCB1 gene was investigated at the mRNA and protein levels using RT-PCR and Western blot analysis in differently cultured RPMI 2650 cells and excised human nasal epithelium. Furthermore, the localization and activity of P-gp was examined by immunohistochemical staining and functionality assays using different substrates in both in vitro and ex vivo models. Both mRNA and protein expression of P-gp was found in all studied models. Furthermore, transporter functionality was detected in both RPMI 2650 cell culture models and excised human mucosa. The results demonstrated a highly promising comparability between RPMI 2650 models and explants of human nasal tissue concerning the influence of MDR1 on drug disposition. The RPMI 2650 cell line might become a useful tool in preclinical trials to improve reproducibility and achieve greater applicability to humans of experimental data regarding passive diffusion and active efflux of drug candidates. PMID:27155589

  7. Genetic analysis of the SARS-coronavirus spike glycoprotein functional domains involved in cell-surface expression and cell-to-cell fusion

    International Nuclear Information System (INIS)

    The SARS-coronavirus (SARS-CoV) is the etiological agent of severe acute respiratory syndrome (SARS). The SARS-CoV spike (S) glycoprotein mediates membrane fusion events during virus entry and virus-induced cell-to-cell fusion. To delineate functional domains of the SARS-CoV S glycoprotein, single point mutations, cluster-to-lysine and cluster-to-alanine mutations, as well as carboxyl-terminal truncations were investigated in transient expression experiments. Mutagenesis of either the coiled-coil domain of the S glycoprotein amino terminal heptad repeat, the predicted fusion peptide, or an adjacent but distinct region, severely compromised S-mediated cell-to-cell fusion, while intracellular transport and cell-surface expression were not adversely affected. Surprisingly, a carboxyl-terminal truncation of 17 amino acids substantially increased S glycoprotein-mediated cell-to-cell fusion suggesting that the terminal 17 amino acids regulated the S fusogenic properties. In contrast, truncation of 26 or 39 amino acids eliminating either one or both of the two endodomain cysteine-rich motifs, respectively, inhibited cell fusion in comparison to the wild-type S. The 17 and 26 amino-acid deletions did not adversely affect S cell-surface expression, while the 39 amino-acid truncation inhibited S cell-surface expression suggesting that the membrane proximal cysteine-rich motif plays an essential role in S cell-surface expression. Mutagenesis of the acidic amino-acid cluster in the carboxyl terminus of the S glycoprotein as well as modification of a predicted phosphorylation site within the acidic cluster revealed that this amino-acid motif may play a functional role in the retention of S at cell surfaces. This genetic analysis reveals that the SARS-CoV S glycoprotein contains extracellular domains that regulate cell fusion as well as distinct endodomains that function in intracellular transport, cell-surface expression, and cell fusion

  8. Reversal of acquired resistance to adriamycin in CHO cells by tamoxifen and 4-hydroxy tamoxifen: role of drug interaction with alpha 1 acid glycoprotein.

    OpenAIRE

    Chatterjee, M.; Harris, A. L.

    1990-01-01

    Tamoxifen and 4-OH tamoxifen were used to reverse multidrug resistance (MDR) in CHO cells with acquired resistance to adriamycin (CHO-Adrr). Because alpha 1 acid glycoprotein (AAG) can bind a range of calcium channel blockers that also reverse MDR and rises in malignancy, its interactions with tamoxifen and 4-OH tamoxifen were also studied. Tamoxifen decreased the IC50 of 10 microM adriamycin 4.8-fold in the parent CHO-K1 cell line and 16-fold in CHO-Adrr. Similarly 4-OH tamoxifen decreased t...

  9. Profiling of Concanavalin A-Binding Glycoproteins in Human Hepatic Stellate Cells Activated with Transforming Growth Factor-β1

    Directory of Open Access Journals (Sweden)

    Yannan Qin

    2014-11-01

    Full Text Available Glycoproteins play important roles in maintaining normal cell functions depending on their glycosylations. Our previous study indicated that the abundance of glycoproteins recognized by concanavalin A (ConA was increased in human hepatic stellate cells (HSCs following activation by transforming growth factor-β1 (TGF-β1; however, little is known about the ConA-binding glycoproteins (CBGs of HSCs. In this study, we employed a targeted glycoproteomics approach using lectin-magnetic particle conjugate-based liquid chromatography-tandem mass spectrometry to compare CBG profiles between LX-2 HSCs with and without activation by TGF-β1, with the aim of discovering novel CBGs and determining their possible roles in activated HSCs. A total of 54 and 77 proteins were identified in the quiescent and activated LX-2 cells, respectively. Of the proteins identified, 14.3% were glycoproteins and 73.3% were novel potential glycoproteins. Molecules involved in protein processing in the endoplasmic reticulum (e.g., calreticulin and calcium signaling (e.g., 1-phosphatidylinositol-4,5-bisphosphate phosphodiesterase β-2 [PLCB2] were specifically identified in activated LX-2 cells. Additionally, PLCB2 expression was upregulated in the cytoplasm of the activated LX-2 cells, as well as in the hepatocytes and sinusoidal cells of liver cirrhosis tissues. In conclusion, the results of this study may aid future investigations to find new molecular mechanisms involved in HSC activation and antifibrotic therapeutic targets.

  10. Enhanced production and isotope enrichment of recombinant glycoproteins produced in cultured mammalian cells

    International Nuclear Information System (INIS)

    NMR studies of post-translationally modified proteins are complicated by the lack of an efficient method to produce isotope enriched recombinant proteins in cultured mammalian cells. We show that reducing the glucose concentration and substituting glutamate for glutamine in serum-free medium increased cell viability while simultaneously increasing recombinant protein yield and the enrichment of non-essential amino acids compared to culture in unmodified, serum-free medium. Adding dichloroacetate, a pyruvate dehydrogenase kinase inhibitor, further improves cell viability, recombinant protein yield, and isotope enrichment. We demonstrate the method by producing partially enriched recombinant Thy1 glycoprotein from Lec1 Chinese hamster ovary (CHO) cells using U-13C-glucose and 15N-glutamate as labeled precursors. This study suggests that uniformly 15N,13C-labeled recombinant proteins may be produced in cultured mammalian cells starting from a mixture of labeled essential amino acids, glucose, and glutamate.

  11. Automation of cell line development

    OpenAIRE

    Lindgren, Kristina; Salmén, Andréa; Lundgren, Mats; Bylund, Lovisa; Ebler, Åsa; Fäldt, Eric; Sörvik, Lina; Fenge, Christel; Skoging-Nyberg, Ulrica

    2009-01-01

    An automated platform for development of high producing cell lines for biopharmaceutical production has been established in order to increase throughput and reduce development costs. The concept is based on the Cello robotic system (The Automation Partnership) and covers screening for colonies and expansion of static cultures. In this study, the glutamine synthetase expression system (Lonza Biologics) for production of therapeutic monoclonal antibodies in Chinese hamster ovary cells was used ...

  12. eEF-2 Phosphorylation Down-Regulates P-Glycoprotein Over-Expression in Rat Brain Microvessel Endothelial Cells.

    Directory of Open Access Journals (Sweden)

    Xing Hua Tang

    Full Text Available We investigated whether glutamate, NMDA receptors, and eukaryote elongation factor-2 kinase (eEF-2K/eEF-2 regulate P-glycoprotein expression, and the effects of the eEF-2K inhibitor NH125 on the expression of P-glycoprotein in rat brain microvessel endothelial cells (RBMECs.Cortex was obtained from newborn Wistar rat brains. After surface vessels and meninges were removed, the pellet containing microvessels was resuspended and incubated at 37°C in culture medium. Cell viability was assessed by the MTT assay. RBMECs were identified by immunohistochemistry with anti-vWF. P-glycoprotein, phospho-eEF-2, and eEF-2 expression were determined by western blot analysis. Mdr1a gene expression was analyzed by RT-PCR.Mdr1a mRNA, P-glycoprotein and phospho-eEF-2 expression increased in L-glutamate stimulated RBMECs. P-glycoprotein and phospho-eEF-2 expression were down-regulated after NH125 treatment in L-glutamate stimulated RBMECs.eEF-2K/eEF-2 should have played an important role in the regulation of P-glycoprotein expression in RBMECs. eEF-2K inhibitor NH125 could serve as an efficacious anti-multidrug resistant agent.

  13. Differential overexpression of three mdr gene family members in multidrug-resistant J774.2 mouse cells. Evidence that distinct P-glycoprotein precursors are encoded by unique mdr genes.

    Science.gov (United States)

    Hsu, S I; Lothstein, L; Horwitz, S B

    1989-07-15

    A hallmark of the multidrug-resistant phenotype is the overproduction of a family of 130-180-kDa integral membrane phosphoglycoproteins collectively called P-glycoprotein. Gene-specific hybridization probes were derived from three classes of mouse P-glycoprotein cDNAs. These probes revealed the differential amplification and/or transcriptional activation of three distinct but closely related mdr genes (mdr1a, mdr1b, and mdr2) in independently selected multidrug-resistant J774.2 mouse cell lines. Overexpression of mdr1a and mdr1b was found to correlate, in general, with the differential overproduction of either a 120- or 125-kDa P-glycoprotein precursor, respectively. This same correlation was observed in a single cell line during the course of stepwise selection for resistance to vinblastine in which a switch in gene expression from mdr1b to mdr1a resulted in a switch from the 125- to 120-kDa P-glycoprotein precursor. These findings suggest that differential overexpression of distinct mdr genes which encode unique P-glycoprotein isoforms is a possible mechanism for generating diversity in the multidrug-resistant phenotype. PMID:2473069

  14. The multidrug transporter, P-glycoprotein, actively mediates cholesterol redistribution in the cell membrane

    OpenAIRE

    Garrigues, Alexia; Escargueil, Alexandre E.; Orlowski, Stéphane

    2002-01-01

    P-glycoprotein (P-gp) is a plasma membrane ATP-binding cassette transporter, responsible for multidrug resistance in tumor cells. P-gp catalyzes the ATP hydrolysis-dependent efflux of numerous amphiphilic compounds of unrelated chemical structures. In the absence of any identified substrate, P-gp exhibits an apparently futile, basal ATPase activity. By using native membrane vesicles containing high amounts of P-gp, we show here that (i) this basal ATPase activity is tightly dependent on the p...

  15. Distinct Glycoprotein O Complexes Arise in a Post-Golgi Compartment of Cytomegalovirus-Infected Cells

    OpenAIRE

    Theiler, Regan N.; Compton, Teresa

    2002-01-01

    Human cytomegalovirus (CMV) glycoproteins H, L, and O (gH, gL, and gO, respectively) form a heterotrimeric disulfide-bonded complex that participates in the fusion of the viral envelope with the host cell membrane. During virus maturation, this complex undergoes a series of intracellular assembly and processing events which are not entirely defined (M. T. Huber and T. Compton, J. Virol. 73:3886-3892, 1999). Here, we demonstrate that gO does not undergo the same posttranslational processing in...

  16. Transient expression of Fc-fused human glycoprotein 130 in Expi293F suspension cells.

    Science.gov (United States)

    Zhao, Xiaozhi; Chen, Wei; Ge, Liyuan; Jiang, Wei; Tang, Bo; Zhang, Qing; Xu, Xiaoyu; Wang, Chong; Cao, Lin; Guo, Hongqian

    2016-08-01

    Human glycoprotein 130 (gp130) is a signal-transducing receptor for interleukin 6 (IL-6), whose signaling plays a critical role in chronic inflammation and cancer. The soluble form of gp130 specifically inhibits IL-6 trans-signaling. However, achieving high-level expression of a large glycoprotein such as gp130 is difficult. Here, we designed and constructed one Fc-gp130-pcDNA mammalian expression vector, with the mouse IgG2a Fc fragment added to the N-terminus of human gp130, which greatly increased the secretion of recombinant gp130 protein from Expi293F suspension cells. Recombinant fusion Fc-gp130 was easily and efficiently purified from the supernatant of transfected cells by one-step affinity chromatography. Moreover, Fc-gp130 could automatically form dimers by the disulfide bond. Fc-gp130 was confirmed as a more efficient IL-6 trans-signaling blocker by its higher biological activity against signal transducer and activator of transcription 3 (STAT3). This purified active Fc-gp130 could be used to develop valuable therapeutic agents against inflammatory diseases and cancers. PMID:27113713

  17. Sulphation of N-linked oligosaccharides of vesicular stomatitis and influenza virus envelope glycoproteins: host cell specificity, subcellular localization and identification of substituted saccharides.

    Science.gov (United States)

    Karaivanova, V K; Spiro, R G

    1998-02-01

    The presence of sulphate groups on various saccharide residues of N-linked carbohydrate units has now been observed in a number of glycoproteins. To explore the cell specificity of this post-translational modification, we evaluated sulphate incorporation into virus envelope glycoproteins by a variety of cells, since it is believed that assembly of their N-linked oligosaccharides is to a large extent dependent on the enzymic machinery of the host. Employing the vesicular stomatitis virus (VSV) envelope glycoprotein (G protein) as a model, we noted that the addition of [35S]sulphate substituents into its complex carbohydrate units occurred in Madin-Darby canine kidney (MDCK), Madin-Darby bovine kidney, LLC-PK1 and BHK-21 cell lines but was not detectable in BRL 3A, BW5147.3, Chinese hamster ovary, HepG2, NRK-49F, IEC-18, PtK1 or 3T3 cells. The sulphate groups were exclusively located on C-3 of galactose [Gal(3-SO4)] and/or C-6 of N-acetylglucosamine [GlcNAc(6-SO4)] residues in the N-acetyllactosamine sequence of the branch chains. Moreover, we observed that the pronounced host-cell-dependence of the terminal galactose sulphation was reflected by the 3'-phosphoadenosine 5'-phosphosulphate:Gal-3-O-sulphotransferase activity assayed in vitro. Comparative studies carried out on the haemagglutinin of the influenza virus envelope formed by MDCK and LLC-PK1 cells indicated that sulphate in this glycoprotein was confined to its complex N-linked oligosaccharides where it occurred as Gal(3-SO4) and GlcNAc(6-SO4) on peripheral chains as well as on the mannose-substituted N-acetylglucosamine of the core. Since sulphation in both internal and peripheral locations of the virus glycoproteins was found to be arrested by the alpha1-->2 mannosidase inhibitor, kifunensine, as well as by the intracellular migration block imposed by brefeldin A, it was concluded that this modification is a late biosynthetic event which most likely takes place in the trans-Golgi network. PMID:9445377

  18. Ebola virus glycoprotein-mediated anoikis of primary human cardiac microvascular endothelial cells

    International Nuclear Information System (INIS)

    Ebola virus glycoprotein (EGP) has been implicated for the induction of cytotoxicity and injury in vascular cells. On the other hand, EGP has also been suggested to induce massive cell rounding and detachment from the plastic surface by downregulating cell adhesion molecules without causing cytotoxicity. In this study, we have examined the cytotoxic role of EGP in primary endothelial cells by transduction with a replication-deficient recombinant adenovirus expressing EGP (Ad-EGP). Primary human cardiac microvascular endothelial cells (HCMECs) transduced with Ad-EGP displayed loss of cell adhesion from the plastic surface followed by cell death. Transfer of conditioned medium from EGP-transduced HCMEC into naive cells did not induce loss of adhesion or cell death, suggesting that EGP needs to be expressed intracellularly to exert its cytotoxic effect. Subsequent studies suggested that HCMEC death occurred through apoptosis. Results from this study shed light on the EGP-induced anoikis in primary human cardiac endothelial cells, which may have significant pathological consequences

  19. A cell surface integral membrane glycoprotein of 85,000 mol wt (gp85) associated with triton X-100-insoluble cell skeleton

    OpenAIRE

    1984-01-01

    The Triton X-100-insoluble skeleton of baby hamster kidney BHK cells consists of the nucleus, intermediate-size filaments, and actin fibers. By transmission electron microscopy, membrane fragments were found to be associated with these insoluble structures. When radioiodinated or [3H]glucosamine-labeled cells were extracted with 0.5% Triton, most plasma membrane glycoproteins were solubilized except for a glycoprotein with a molecular weight of 85,000 (gp85) that remained associated with the ...

  20. Adenovirus vector infection of non-small-cell lung cancer cells is a trigger for multi-drug resistance mediated by P-glycoprotein.

    Science.gov (United States)

    Tomono, Takumi; Kajita, Masahiro; Yano, Kentaro; Ogihara, Takuo

    2016-08-01

    P-glycoprotein (P-gp) is an ATP-binding cassette protein involved in cancer multi-drug resistance (MDR). It has been reported that infection with some bacteria and viruses induces changes in the activities of various drug-metabolizing enzymes and transporters, including P-gp. Although human adenoviruses (Ad) cause the common cold, the effect of Ad infection on MDR in cancer has not been established. In this study, we investigated whether Ad infection is a cause of MDR in A549, H441 and HCC827 non-small-cell lung cancer (NSCLC) cell lines, using an Ad vector system. We found that Ad vector infection of NSCLC cell lines induced P-gp mRNA expression, and the extent of induction was dependent on the number of Ad vector virus particles and the infection time. Heat-treated Ad vector, which is not infectious, did not alter P-gp mRNA expression. Uptake experiments with doxorubicin (DOX), a P-gp substrate, revealed that DOX accumulation was significantly decreased in Ad vector-infected A549 cells. The decrease of DOX uptake was blocked by verapamil, a P-gp inhibitor. Our results indicated that Ad vector infection of NSCLC cells caused MDR mediated by P-gp overexpression. The Ad vector genome sequence is similar to that of human Ad, and therefore human Ad infection of lung cancer patients may lead to chemoresistance in the clinical environment. PMID:27286705

  1. Multidrug-resistance gene (P-glycoprotein) is expressed by endothelial cells at blood-brain barrier sites

    Energy Technology Data Exchange (ETDEWEB)

    Cordon-Cardo, C.; O' Brien, J.P.; Casals, D.; Biedler, J.L.; Melamed, M.R.; Bertino, J.R. (Memorial Sloan-Kettering Cancer Center, New York, NY (USA)); Rittman-Grauer, L. (Hybritech, Inc., San Diego, CA (USA))

    1989-01-01

    Endothelial cells of human capillary blood vessels at the blood-brain and other blood-tissue barrier sites express P-glycoprotein as detected by mouse monoclonal antibodies against the human multidrug-resistance gene product. This pattern of endothelial cell expression may indicate a physiological role for P-glycoprotein in regulating the entry of certain molecules into the central nervous system and other anatomic compartments, such as the testes. These tissues, which limit the access of systemic drugs, are known pharmacologic sanctuaries for metastatic cancer. P-glycoprotein expression in capillary endothelium of brain and testes and not other tissues (i.e., kidney and placenta) may in part explain this phenomenon and could have important implications in cancer chemotherapy.

  2. Rift Valley fever virus incorporates the 78 kDa glycoprotein into virions matured in mosquito C6/36 cells.

    Science.gov (United States)

    Weingartl, Hana M; Zhang, Shunzhen; Marszal, Peter; McGreevy, Alan; Burton, Lynn; Wilson, William C

    2014-01-01

    Rift Valley fever virus (RVFV), genus Phlebovirus, family Bunyaviridae is a zoonotic arthropod-borne virus able to transition between distant host species, causing potentially severe disease in humans and ruminants. Viral proteins are encoded by three genomic segments, with the medium M segment coding for four proteins: nonstructural NSm protein, two glycoproteins Gn and Gc and large 78 kDa glycoprotein (LGp) of unknown function. Goat anti-RVFV polyclonal antibody and mouse monoclonal antibody, generated against a polypeptide unique to the LGp within the RVFV proteome, detected this protein in gradient purified RVFV ZH501 virions harvested from mosquito C6/36 cells but not in virions harvested from the mammalian Vero E6 cells. The incorporation of LGp into the mosquito cell line - matured virions was confirmed by immune-electron microscopy. The LGp was incorporated into the virions immediately during the first passage in C6/36 cells of Vero E6 derived virus. Our data indicate that LGp is a structural protein in C6/36 mosquito cell generated virions. The protein may aid the transmission from the mosquitoes to the ruminant host, with a possible role in replication of RVFV in the mosquito host. To our knowledge, this is a first report of different protein composition between virions formed in insect C6/36 versus mammalian Vero E6 cells. PMID:24489907

  3. Rift Valley fever virus incorporates the 78 kDa glycoprotein into virions matured in mosquito C6/36 cells.

    Directory of Open Access Journals (Sweden)

    Hana M Weingartl

    Full Text Available Rift Valley fever virus (RVFV, genus Phlebovirus, family Bunyaviridae is a zoonotic arthropod-borne virus able to transition between distant host species, causing potentially severe disease in humans and ruminants. Viral proteins are encoded by three genomic segments, with the medium M segment coding for four proteins: nonstructural NSm protein, two glycoproteins Gn and Gc and large 78 kDa glycoprotein (LGp of unknown function. Goat anti-RVFV polyclonal antibody and mouse monoclonal antibody, generated against a polypeptide unique to the LGp within the RVFV proteome, detected this protein in gradient purified RVFV ZH501 virions harvested from mosquito C6/36 cells but not in virions harvested from the mammalian Vero E6 cells. The incorporation of LGp into the mosquito cell line - matured virions was confirmed by immune-electron microscopy. The LGp was incorporated into the virions immediately during the first passage in C6/36 cells of Vero E6 derived virus. Our data indicate that LGp is a structural protein in C6/36 mosquito cell generated virions. The protein may aid the transmission from the mosquitoes to the ruminant host, with a possible role in replication of RVFV in the mosquito host. To our knowledge, this is a first report of different protein composition between virions formed in insect C6/36 versus mammalian Vero E6 cells.

  4. Effects of P-Glycoprotein and Its Inhibitors on Apoptosis in K562 Cells

    Directory of Open Access Journals (Sweden)

    Yaqiong Zu

    2014-08-01

    Full Text Available P-glycoprotein (P-gp is a major factor in multidrug resistance (MDR which is a serious obstacle in chemotherapy. P-gp has also been implicated in causing apoptosis of tumor cells, which was shown to be another important mechanism of MDR recently. To study the influence of P-gp in tumor cell apoptosis, K562/A cells (P-gp+ and K562/S cells (P-gp− were subjected to doxorubicin (Dox, serum withdrawal, or independent co-incubation with multiple P-gp inhibitors, including valspodar (PSC833, verapamil (Ver and H108 to induce apoptosis. Apoptosis was simultaneously detected by apoptotic rate, cell cycle by flow cytometry and cysteine aspartic acid-specific protease 3 (caspase 3 activity by immunoassay. Cytotoxicity and apoptosis induced by PSC833 were evaluated through an MTT method and apoptosis rate, and cell cycle combined with caspase 3 activity, respectively. The results show that K562/A cells are more resistant to apoptosis and cell cycle arrest than K562/S cells after treatment with Dox or serum deprivation. The apoptosis of K562/A cells increased after co-incubation with each of the inhibitors of P-gp. P-gp inhibitors also enhanced cell cycle arrest in K562/A cell. PSC833 most strikingly decreased viability and led to apoptosis and S phase arrest of cell cycle in K562/A cells. Our study demonstrates that P-gp inhibits the apoptosis of tumor cells in addition to participating in the efflux of intracellular chemotherapy drugs. The results of the caspase 3 activity assay also suggest that the role of P-gp in apoptosis avoidance is caspase-related.

  5. Signal transduction in endothelial cells by the angiogenesis inhibitor histidine-rich glycoprotein targets focal adhesions

    International Nuclear Information System (INIS)

    Histidine-rich glycoprotein (HRGP) is an abundant heparin-binding plasma protein. We have shown that a fragment released from the central histidine/proline-rich (His/Pro-rich) domain of HRGP blocks endothelial cell migration in vitro and vascularization and growth of murine fibrosarcoma in vivo. The minimal active HRGP domain exerting the anti-angiogenic effect was recently narrowed down to a 35 amino acid peptide, HRGP330, derived from the His/Pro-rich domain of HRGP. By use of a signal transduction antibody array representing 400 different signal transduction molecules, we now show that HRGP and the synthetic peptide HRGP330 specifically induce tyrosine phosphorylation of focal adhesion kinase and its downstream substrate paxillin in endothelial cells. HRGP/HRGP330 treatment of endothelial cells induced disruption of actin stress fibers, a process reversed by treatment of cells with the FAK inhibitor geldanamycin. In addition, VEGF-mediated endothelial cell tubular morphogenesis in a three-dimensional collagen matrix was inhibited by HRGP and HRGP330. In contrast, VEGF-induced proliferation was not affected by HRGP or HRGP330, demonstrating the central role of cell migration during tube formation. In conclusion, our data show that HRGP targets focal adhesions in endothelial cells, thereby disrupting the cytoskeletal organization and the ability of endothelial cells to assemble into vessel structures

  6. Functional processing and secretion of Chikungunya virus E1 and E2 glycoproteins in insect cells

    Directory of Open Access Journals (Sweden)

    Goldbach Rob W

    2011-07-01

    Full Text Available Abstract Background Chikungunya virus (CHIKV is a mosquito-borne, arthrogenic Alphavirus that causes large epidemics in Africa, South-East Asia and India. Recently, CHIKV has been transmitted to humans in Southern Europe by invading and now established Asian tiger mosquitoes. To study the processing of envelope proteins E1 and E2 and to develop a CHIKV subunit vaccine, C-terminally his-tagged E1 and E2 envelope glycoproteins were produced at high levels in insect cells with baculovirus vectors using their native signal peptides located in CHIKV 6K and E3, respectively. Results Expression in the presence of either tunicamycin or furin inhibitor showed that a substantial portion of recombinant intracellular E1 and precursor E3E2 was glycosylated, but that a smaller fraction of E3E2 was processed by furin into mature E3 and E2. Deletion of the C-terminal transmembrane domains of E1 and E2 enabled secretion of furin-cleaved, fully processed E1 and E2 subunits, which could then be efficiently purified from cell culture fluid via metal affinity chromatography. Confocal laser scanning microscopy on living baculovirus-infected Sf21 cells revealed that full-length E1 and E2 translocated to the plasma membrane, suggesting similar posttranslational processing of E1 and E2, as in a natural CHIKV infection. Baculovirus-directed expression of E1 displayed fusogenic activity as concluded from syncytia formation. CHIKV-E2 was able to induce neutralizing antibodies in rabbits. Conclusions Chikungunya virus glycoproteins could be functionally expressed at high levels in insect cells and are properly glycosylated and cleaved by furin. The ability of purified, secreted CHIKV-E2 to induce neutralizing antibodies in rabbits underscores the potential use of E2 in a subunit vaccine to prevent CHIKV infections.

  7. Defining the N-linked glycosylation site of Hantaan virus envelope glycoproteins essential for cell fusion.

    Science.gov (United States)

    Zheng, Feng; Ma, Lixian; Shao, Lihua; Wang, Gang; Chen, Fengzhe; Zhang, Ying; Yang, Song

    2007-02-01

    The Hantaan virus (HTNV) is an enveloped virus that is capable of inducing low pH-dependent cell fusion. We molecularly cloned the viral glycoprotein (GP) and nucleocapsid (NP) cDNA of HTNV and expressed them in Vero E6 cells under the control of a CMV promoter. The viral gene expression was assessed using an indirect immunofluorescence assay and immunoprecipitation. The transfected Vero E6 cells expressing GPs, but not those expressing NP, fused and formed a syncytium following exposure to a low pH. Monoclonal antibodies (MAbs) against envelope GPs inhibited cell fusion, whereas MAbs against NP did not. We also investigated the N-linked glycosylation of HTNV GPs and its role in cell fusion. The envelope GPs of HTNV are modified by N-linked glycosylation at five sites: four sites on G1 (N134, N235, N347, and N399) and one site on G2 (N928). Site-directed mutagenesis was used to construct eight GP gene mutants, including five single N-glycosylation site mutants and three double-site mutants, which were then expressed in Vero E6 cells. The oligosaccharide chain on residue N928 of G2 was found to be crucial for cell fusion after exposure to a low pH. These results suggest that G2 is likely to be the fusion protein of HTNV. PMID:17342054

  8. The glycoprotein-hormones activin A and inhibin A interfere with dendritic cell maturation

    Directory of Open Access Journals (Sweden)

    Reichardt Holger M

    2008-05-01

    Full Text Available Abstract Background Pregnancy represents an exclusive situation in which the immune and the endocrine system cooperate to prevent rejection of the embryo by the maternal immune system. While immature dendritic cells (iDC in the early pregnancy decidua presumably contribute to the establishment of peripheral tolerance, glycoprotein-hormones of the transforming growth factor beta (TGF-beta family including activin A (ActA and inhibin A (InA are candidates that could direct the differentiation of DCs into a tolerance-inducing phenotype. Methods To test this hypothesis we generated iDCs from peripheral-blood-monocytes and exposed them to TGF-beta1, ActA, as well as InA and Dexamethasone (Dex as controls. Results Both glycoprotein-hormones prevented up-regulation of HLA-DR during cytokine-induced DC maturation similar to Dex but did not influence the expression of CD 40, CD 83 and CD 86. Visualization of the F-actin cytoskeleton confirmed that the DCs retained a partially immature phenotype under these conditions. The T-cell stimulatory capacity of DCs was reduced after ActA and InA exposure while the secretion of cytokines and chemokines was unaffected. Conclusion These findings suggest that ActA and InA interfere with selected aspects of DC maturation and may thereby help preventing activation of allogenic T-cells by the embryo. Thus, we have identified two novel members of the TGF-beta superfamily that could promote the generation of tolerance-inducing DCs.

  9. Interlab Cell Line Collection: Bioresource of Established Human and Animal Cell Lines

    OpenAIRE

    Parodi, Barbara; Aresu, Ottavia; Visconti, Paola; Manniello, Maria Assunta; Strada, Paolo

    2015-01-01

    The Interlab Cell Line Collection (ICLC) was established in 1994 as a core facility of the National Institute of Cancer Research. It supplies: human and animal cell lines; Short Tandem Repeat (STR) profiling of human cell lines; quality control service; mycoplasma detection and eradication service; safe deposit service and patent deposit service of cell lines and hybridomas. The catalogue of services is on-line, and the cell lines are distributed all over the world. 

  10. Differential profiling studies of N-linked glycoproteins in glioblastoma cancer stem cells upon treatment with γ-secretase inhibitor.

    Science.gov (United States)

    Dai, Lan; Liu, Yashu; He, Jintang; Flack, Callie G; Talsma, Caroline E; Crowley, Jessica G; Muraszko, Karin M; Fan, Xing; Lubman, David M

    2011-10-01

    We have recently demonstrated that Notch pathway blockade by γ-secretase inhibitor (GSI) depletes cancer stem cells (CSCs) in Glioblastoma Multiforme (GBM) through reduced proliferation and induced apoptosis. However, the detailed mechanism by which the manipulation of Notch signal induces alterations on post-translational modifications such as glycosylation has not been investigated. Herein, we present a differential profiling work to detect the change of glycosylation pattern upon drug treatment in GBM CSCs. Rapid screening of differential cell surface glycan structures has been performed by lectin microarray on live cells followed by the detection of N-linked glycoproteins from cell lysates using multi-lectin chromatography and label-free quantitative mass spectrometry analysis. A total of 51 and 52 glycoproteins were identified in the CSC- and GSI-treated groups, respectively, filtered by a combination of decoy database searching and Trans-Proteomic Pipeline (TPP) processing. Although no significant changes were detected from the lectin microarray experiment, 7 differentially expressed glycoproteins with high confidence were captured after the multi-lectin column including key enzymes involved in glycan processing. Functional annotations of the altered glycoproteins suggest a phenotype transformation of CSCs toward a less tumorigenic form upon GSI treatment. PMID:21898824

  11. Intercellular transfer of P-glycoprotein in human blood-brain barrier endothelial cells is increased by histone deacetylase inhibitors

    OpenAIRE

    Andreas Noack; Sandra Noack; Manuela Buettner; Naim, Hassan Y.; Wolfgang Löscher

    2016-01-01

    The blood–brain barrier (BBB) controls the entry of compounds into the brain, thereby regulating brain homeostasis. Efflux transporters such as P-glycoprotein (Pgp) significantly contribute to BBB function. Multiple signaling pathways modulate the expression and activity of Pgp in response to xenobiotics and disease. A non-genetic way of intercellular transfer of Pgp occurs in cancer cells, but whether this also occurs in non-cancer cells such as endothelial cells that form the BBB is not kno...

  12. A 160-kilodalton epithelial cell surface glycoprotein recognized by plant lectins that inhibit the adherence of Actinomyces naeslundii.

    OpenAIRE

    Brennan, M J; Cisar, J O; Sandberg, A L

    1986-01-01

    The adherence of Actinomyces naeslundii to human epithelial (KB) cells is mediated by the interaction of a fimbrial lectin on this oral bacterium with epithelial cell receptors exposed by sialidase. The D-galactose- and N-acetyl-D-galactosamine-reactive plant lectins from peanut and from Bauhinia purpurea inhibit this interaction. This report describes the partial purification and characterization of a 160-kilodalton (kDa) cell surface glycoprotein which is the principal receptor for these le...

  13. The Glycoproteins of All Filovirus Species Use the Same Host Factors for Entry into Bat and Human Cells but Entry Efficiency Is Species Dependent.

    Directory of Open Access Journals (Sweden)

    Markus Hoffmann

    Full Text Available Ebola and marburgviruses, members of the family Filoviridae, can cause severe hemorrhagic fever in humans. The ongoing Ebola virus (EBOV disease epidemic in Western Africa claimed more than 11,300 lives and was associated with secondary cases outside Africa, demonstrating that filoviruses pose a global health threat. Bats constitute an important natural reservoir of filoviruses, including viruses of the recently identified Cuevavirus genus within the Filoviridae family. However, the interactions of filoviruses with bat cells are incompletely understood. Here, we investigated whether filoviruses employ different strategies to enter human and bat cells. For this, we examined host cell entry driven by glycoproteins (GP from all filovirus species into cell lines of human and fruit bat origin. We show that all GPs were able to mediate entry into human and most fruit bat cell lines with roughly comparable efficiency. In contrast, the efficiency of entry into the cell line EidNi/41 derived from a straw-colored fruit bat varied markedly between the GPs of different filovirus species. Furthermore, inhibition studies demonstrated that filoviruses employ the same host cell factors for entry into human, non-human primate and fruit bat cell lines, including cysteine proteases, two pore channels and NPC1 (Niemann-Pick C1 molecule. Finally, processing of GP by furin and the presence of the mucin-like domain in GP were dispensable for entry into both human and bat cell lines. Collectively, these results show that filoviruses rely on the same host cell factors for entry into human and fruit bat cells, although the efficiency of the usage of these factors might differ between filovirus species.

  14. The Glycoproteins of All Filovirus Species Use the Same Host Factors for Entry into Bat and Human Cells but Entry Efficiency Is Species Dependent.

    Science.gov (United States)

    Hoffmann, Markus; González Hernández, Mariana; Berger, Elisabeth; Marzi, Andrea; Pöhlmann, Stefan

    2016-01-01

    Ebola and marburgviruses, members of the family Filoviridae, can cause severe hemorrhagic fever in humans. The ongoing Ebola virus (EBOV) disease epidemic in Western Africa claimed more than 11,300 lives and was associated with secondary cases outside Africa, demonstrating that filoviruses pose a global health threat. Bats constitute an important natural reservoir of filoviruses, including viruses of the recently identified Cuevavirus genus within the Filoviridae family. However, the interactions of filoviruses with bat cells are incompletely understood. Here, we investigated whether filoviruses employ different strategies to enter human and bat cells. For this, we examined host cell entry driven by glycoproteins (GP) from all filovirus species into cell lines of human and fruit bat origin. We show that all GPs were able to mediate entry into human and most fruit bat cell lines with roughly comparable efficiency. In contrast, the efficiency of entry into the cell line EidNi/41 derived from a straw-colored fruit bat varied markedly between the GPs of different filovirus species. Furthermore, inhibition studies demonstrated that filoviruses employ the same host cell factors for entry into human, non-human primate and fruit bat cell lines, including cysteine proteases, two pore channels and NPC1 (Niemann-Pick C1 molecule). Finally, processing of GP by furin and the presence of the mucin-like domain in GP were dispensable for entry into both human and bat cell lines. Collectively, these results show that filoviruses rely on the same host cell factors for entry into human and fruit bat cells, although the efficiency of the usage of these factors might differ between filovirus species. PMID:26901159

  15. A lack of Adriamycin (ADR) resistance in Chinese hamster ovary (CHO) cells overexpressing P-glycoprotein (Pgp) following in vitro exposure to fractionated X-irradiation

    International Nuclear Information System (INIS)

    Using x-ray pretreated CHO cells, the authors demonstrated differing accumulation of adriamycin and vincristine in cells overexpressing P-glycoprotein. Response was also varied by the addition of calcium channel antagonist verapamil. (author)

  16. High-resolution electron microscopy of glycoproteins: the crystalline cell wall of Lobomonas.

    Science.gov (United States)

    Roberts, K; Shaw, P J; Hills, G J

    1981-10-01

    Lobomonas piriformis is a member of an order of green algae (Volvocales) that have crystalline glycoprotein cell walls. As part of a program of investigation of these glycoproteins and their architecture we have studied the cell wall of Lobomonas by a variety of chemical, electron-microscopical and image-analysis techniques. Lobomonas and Vitreochlamys incisa show a very similar structure in their cell walls and represent I of the 4 classes into which all the structures of the wall of these algae that we have so far examined fall. The 2 classes that we have previously studied in detail, represented by Chlamydomonas reinhardii and chlorogonium elongatum, have a crystalline component of the wall that is a more or less smooth continuous surface overlying an amorphous inner wall layer. Although Lobomonas also has this 2-layer structure, the crystalline layer consists of distinct plates, each of which is built around a single, very coherent crystal lattice. The polar nature of the architecture of the cell wall is shown by sectioning and by examination of the cell-wall surface by metal-shadowing of carbon replicas, both of intact cells and of isolated cell-wall plates. There are great similarities in chemical composition between the glycoproteins of the cell wall of C. reinhardii and those of Lobomonas. Both has a large content of hydroxyproline in their amino acid composition and a sugar/hydroxyproline ratio of about 6.0, and both contain sugar sulphates. Lobomonas however has a large glucose content, whereas Chlamydamonas has almost none. Electron micrographs of walls stained with methylamine tungstate and shadowed specimens show that the Lobomonas crystal structure is entirely different from that of C. Reinhardii, and that there is a distinctly different structure in the centre of the plates from that at their edges, although the transition between the 2 areas occurs with no distortion of the crystal lattice. Computer image analysis has been used to calculate

  17. Ferulic acid reverses ABCB1-mediated paclitaxel resistance in MDR cell lines.

    Science.gov (United States)

    Muthusamy, Ganesan; Balupillai, Agilan; Ramasamy, Karthikeyan; Shanmugam, Mohana; Gunaseelan, Srithar; Mary, Beaulah; Prasad, N Rajendra

    2016-09-01

    Multidrug resistance (MDR) remains a major obstacle in cancer chemotherapy. The use of the dietary phytochemicals as chemosensitizing agents to enhance the efficacy of conventional cytostatic drugs has recently gained the attention as a plausible approach for overcoming the drug resistance. The aim of this study was to investigate whether a naturally occurring diet-based phenolic acid, ferulic acid, could sensitize paclitaxel efficacy in ABCB1 overexpressing (P-glycoprotein) colchicine selected KB Ch(R)8-5 cell line. In vitro drug efflux assays demonstrated that ferulic acid inhibits P-glycoprotein transport function in drug resistant KB Ch(R)8-5 cell lines. However, ferulic acid significantly downregulates ABCB1 expression in a concentration dependent manner. Cytotoxicity assay reveals that ferulic acid decreased paclitaxel resistance in KBCh(R)8-5 and HEK293/ABCB1 cells, which indicates its chemosensitizing potential. Clonogenic cell survival assay and apoptotic morphological staining further confirm the chemosensitizing potential of ferulic acid in drug resistant KB Ch(R)8-5 cell lines. Ferulic acid treatment enhances paclitaxel mediated cell cycle arrest and upregulates paclitaxel-induced apoptotic signaling in KB resistant cells. Hence, it has been concluded that downregulation of ABCB1 and subsequent induction of paclitaxel-mediated cell cycle arrest and apoptotic signaling may be the cause for the chemosensitizing potential of ferulic acid in P-gp overexpressing cell lines. PMID:27262378

  18. Host cell P-glycoprotein is essential for cholesterol uptake and replication of Toxoplasma gondii.

    Science.gov (United States)

    Bottova, Iveta; Hehl, Adrian B; Stefanić, Sasa; Fabriàs, Gemma; Casas, Josefina; Schraner, Elisabeth; Pieters, Jean; Sonda, Sabrina

    2009-06-26

    P-glycoprotein (P-gp) is a membrane-bound efflux pump that actively exports a wide range of compounds from the cell and is associated with the phenomenon of multidrug resistance. However, the role of P-gp in normal physiological processes remains elusive. Using P-gp-deficient fibroblasts, we showed that P-gp was critical for the replication of the intracellular parasite Toxoplasma gondii but was not involved in invasion of host cells by the parasite. Importantly, we found that the protein participated in the transport of host-derived cholesterol to the intracellular parasite. T. gondii replication in P-gp-deficient host cells not only resulted in reduced cholesterol content in the parasite but also altered its sphingolipid metabolism. In addition, we found that different levels of P-gp expression modified the cholesterol metabolism in uninfected fibroblasts. Collectively our findings reveal a key and previously undocumented role of P-gp in host-parasite interaction and suggest a physiological role for P-gp in cholesterol trafficking in mammalian cells. PMID:19389707

  19. Host Cell P-glycoprotein Is Essential for Cholesterol Uptake and Replication of Toxoplasma gondii*

    Science.gov (United States)

    Bottova, Iveta; Hehl, Adrian B.; Štefanić, Saša; Fabriàs, Gemma; Casas, Josefina; Schraner, Elisabeth; Pieters, Jean; Sonda, Sabrina

    2009-01-01

    P-glycoprotein (P-gp) is a membrane-bound efflux pump that actively exports a wide range of compounds from the cell and is associated with the phenomenon of multidrug resistance. However, the role of P-gp in normal physiological processes remains elusive. Using P-gp-deficient fibroblasts, we showed that P-gp was critical for the replication of the intracellular parasite Toxoplasma gondii but was not involved in invasion of host cells by the parasite. Importantly, we found that the protein participated in the transport of host-derived cholesterol to the intracellular parasite. T. gondii replication in P-gp-deficient host cells not only resulted in reduced cholesterol content in the parasite but also altered its sphingolipid metabolism. In addition, we found that different levels of P-gp expression modified the cholesterol metabolism in uninfected fibroblasts. Collectively our findings reveal a key and previously undocumented role of P-gp in host-parasite interaction and suggest a physiological role for P-gp in cholesterol trafficking in mammalian cells. PMID:19389707

  20. Inflammatory response of endothelial cells to hepatitis C virus recombinant envelope glycoprotein 2 protein exposure

    Directory of Open Access Journals (Sweden)

    Ana Carolina Urbaczek

    2014-09-01

    Full Text Available The hepatitis C virus (HCV encodes approximately 10 different structural and non-structural proteins, including the envelope glycoprotein 2 (E2. HCV proteins, especially the envelope proteins, bind to cell receptors and can damage tissues. Endothelial inflammation is the most important determinant of fibrosis progression and, consequently, cirrhosis. The aim of this study was to evaluate and compare the inflammatory response of endothelial cells to two recombinant forms of the HCV E2 protein produced in different expression systems (Escherichia coli and Pichia pastoris. We observed the induction of cell death and the production of nitric oxide, hydrogen peroxide, interleukin-8 and vascular endothelial growth factor A in human umbilical vein endothelial cells (HUVECs stimulated by the two recombinant E2 proteins. The E2-induced apoptosis of HUVECs was confirmed using the molecular marker PARP. The apoptosis rescue observed when the antioxidant N-acetylcysteine was used suggests that reactive oxygen species are involved in E2-induced apoptosis. We propose that these proteins are involved in the chronic inflammation caused by HCV.

  1. Overcoming of P-glycoprotein mediated vincristine resistance of L1210/VCR mouse leukemic cells could be induced by pentoxifylline but not by theophylline and caffeine

    International Nuclear Information System (INIS)

    Effects of xanthine derivatives (pentoxifylline (PTX), caffeine, theophylline, 1-methyl-3-isobutylxanthine) on P-glycoprotein mediated vincristine resistance of L1210/VCR mouse leukemic cell sub-line were studied. From the applied xanthines only PTX was found to reverse the vincristine resistance of the above cells. Moreover, only PTX, but not other xanthine, increased the accumulation of [3H]vincristine by L1210/VCR cells. Thus it may be concluded that PTX-induced reversal of vincristine (VCR) resistance could not be explained from the point of known pharmacological effects of PTX that are common for other xanthines such as inhibition of phosphodiesterase activity, calcium mobilizing effect, inhibition of tumor necrosis factor α (TNF), etc. (author)

  2. Cannabidiol changes P-gp and BCRP expression in trophoblast cell lines

    OpenAIRE

    Valeria Feinshtein; Offer Erez; Zvi Ben-Zvi; Noam Erez; Tamar Eshkoli; Boaz Sheizaf; Eyal Sheiner; Mahmud Huleihel; Gershon Holcberg

    2013-01-01

    Objectives. Marijuana is the most commonly used illicit drug during pregnancy. Due to high lipophilicity, cannabinoids can easily penetrate physiological barriers like the human placenta and jeopardize the developing fetus. We evaluated the impact of cannabidiol (CBD), a major non-psychoactive cannabinoid, on P-glycoprotein (P-gp) and Breast Cancer Resistance Protein (BCRP) expression, and P-gp function in a placental model, BeWo and Jar choriocarcinoma cell lines (using P-gp induced MCF7 cel...

  3. Generation of rabbit pluripotent stem cell lines

    OpenAIRE

    Tancos, Z.; Nemes, C.; Polgar, Z.; Gocza, E; Daniel, N; Stout, T. A. E.; P. Maraghechi; Pirity, M.K.; Osteil, P.; Tapponnier, Y.; Markossian, S.; Godet, M.; Afanassieff, M.; Bosze, Z.; Duranthon, V

    2012-01-01

    Pluripotent stem cells have the capacity to divide indefinitely and to differentiate into all somatic cells and tissue lines. They can be genetically manipulated in vitro by knocking genes in or out, and therefore serve as an excellent tool for gene function studies and for the generation of models for some human diseases. Since 1981, when the first mouse embryonic stem cell (ESC) line was generated, many attempts have been made to generate pluripotent stem cell lines from other species. Comp...

  4. Marine sponge-derived sipholane triterpenoids reverse P-glycoprotein (ABCB1)-mediated multidrug resistance in cancer cells

    OpenAIRE

    Abraham, Ioana; Jain, Sandeep; Wu, Chung-pu; Khanfar, Mohammad A.; Kuang, Yehong; Dai, Chun-ling; Shi, Zhi; Chen, Xiang; FU, LIWU; Suresh V Ambudkar; Sayed, Khalid El; Chen, Zhe-Sheng

    2010-01-01

    Previously, we reported sipholenol A, a sipholane triterpenoid from the Red Sea sponge Callyspongia siphonella, as a potent reversal of multidrug resistance (MDR) in cancer cells that overexpressed P-glycoprotein (P-gp). Through extensive screening of several related sipholane triterpenoids that have been isolated from the same sponge, we identified sipholenone E, sipholenol L and siphonellinol D as potent reversals of MDR in cancer cells. These compounds enhanced the cytotoxicity of several ...

  5. Human Cytomegalovirus Glycoprotein B Contains Autonomous Determinants for Vectorial Targeting to Apical Membranes of Polarized Epithelial Cells

    OpenAIRE

    Tugizov, Sharof; Maidji, Ekaterina; Xiao, Jianqiao; Zheng, Zhenwei; Pereira, Lenore

    1998-01-01

    We previously reported that human cytomegalovirus (CMV) glycoprotein B (gB) is vectorially transported to apical membranes of CMV-infected polarized human retinal pigment epithelial cells propagated on permeable filter supports and that virions egress predominantly from the apical membrane domain. In the present study, we investigated whether gB itself contains autonomous information for apical transport by expressing the molecule in stably transfected Madine-Darby canine kidney (MDCK) cells ...

  6. Glycoprotein J of infectious laryngotracheitis virus is required for efficient egress of infectious virions from cells.

    Science.gov (United States)

    Mundt, Alice; Mundt, Egbert; Hogan, Robert J; García, Maricarmen

    2011-11-01

    Glycoprotein J (gJ) of infectious laryngotracheitis virus (ILTV) represents a major viral antigen and is dispensable for replication in cell culture and chickens. We generated gJ deletion mutants derived from the United States Department of Agriculture standard challenge strain (USDA-ch), a GFP-expressing mutant GΔgJ, a gJ deletion mutant void of any foreign DNA insertion (BΔgJ) and a gJ rescue mutant gJR with US5 restored. GΔgJ, BΔgJ and gJR were characterized in cell culture and embryonated eggs. Entry kinetic assays showed that the gJ deletion mutants did not differ in their entry kinetics from gJR. Replication kinetics strongly indicated that gJ plays an important role during egress of the virus. Differences in the abilities of the mutants to replicate in chorioallantoic membranes of chicken embryos and to release infectious virus into the allantoic fluid supported a function of gJ during the egress of ILTV from infected cells. PMID:21752963

  7. Expression of Ebolavirus glycoprotein on the target cells enhances viral entry

    Directory of Open Access Journals (Sweden)

    Manicassamy Balaji

    2009-06-01

    Full Text Available Abstract Background Entry of Ebolavirus to the target cells is mediated by the viral glycoprotein GP. The native GP exists as a homotrimer on the virions and contains two subunits, a surface subunit (GP1 that is involved in receptor binding and a transmembrane subunit (GP2 that mediates the virus-host membrane fusion. Previously we showed that over-expression of GP on the target cells blocks GP-mediated viral entry, which is mostly likely due to receptor interference by GP1. Results In this study, using a tetracycline inducible system, we report that low levels of GP expression on the target cells, instead of interfering, specifically enhance GP mediated viral entry. Detailed mapping analysis strongly suggests that the fusion subunit GP2 is primarily responsible for this novel phenomenon, here referred to as trans enhancement. Conclusion Our data suggests that GP2 mediated trans enhancement of virus fusion occurs via a mechanism analogous to eukaryotic membrane fusion processes involving specific trans oligomerization and cooperative interaction of fusion mediators. These findings have important implications in our current understanding of virus entry and superinfection interference.

  8. Radioautographic study of the synthesis and migration of glycoproteins in the cells of the rat adrenal medulla

    International Nuclear Information System (INIS)

    Rats were injected intravenously with (3H) fucose to study the synthesis and migration of glycoproteins into adrenaline-storing and noradrenaline-storing cells of the adrenal medulla and to evaluate the fate of this radioactive sugar in both serum and adrenal-medulla at various time intervals. Radioactivity was decreased in serum by 50% between 5 and 20 min after the injection and by a hundred fold with 1 h. There was a sharp decrease in the radioactivity of the adrenal-medulla between 5 and 20 min after the injection and a slight, continuous decrease thereafter. The adrenal-medullae were fixed 5 min, 20 min, 1 h and 4 h after intravenous injection of [3H] fucose, and radiautographs were analysed quantitatively after development in Microdol X. Kinetic analysis showed that, in both cell types, glycoprotein synthesis is completed in the Golgi complex and glycoproteins migrate subsequently to the secretory granules and to the cell coat. This analysis also revealed that [3H] fucose moves much more rapidly in the Golgi complex of noradrenaline-storing cells than in that of adrenaline-storing cells and appears much earlier in the secretory granules of the former cell type

  9. Modulation of P-glycoprotein function and multidrug resistance in cancer cells by Thai plant extracts.

    Science.gov (United States)

    Takano, M; Kakizoe, S; Kawami, M; Nagai, J; Patanasethnont, D; Sripanidkulchai, B; Yumoto, R

    2014-11-01

    The effects of ethanol extracts from Thai plants belonging to the families of Annonaceae, Rutaceae, and Zingiberaceae on P-glycoprotein (P-gp) function and multidrug resistance were examined in paclitaxel-resistant HepG2 (PR-HepG2) cells. All the extracts tested, significantly increased the accumulation of [3H]paclitaxel, a P-gp substrate, in the cells. Among nine extracts, Z01 and Z02, extracts from Curcuma comosa and Kaempferia marginata (Zingiberaceae family), respectively, potently increased the accumulation. In addition, Z01 and Z02 increased the accumulation of other P-gp substrates, rhodamine 123 and doxorubicin, in PR-HepG2 cells in a concentration-dependent manner. Increased accumulation of rhodamine 123 and doxorubicin by Z01 and Z02 was also confirmed by confocal laser scanning microscopy. The effect of Z01 and Z02 pretreatment on the expression of MDR1 mRNA was also examined. The expression of MDR1 mRNA was not affected by the treatment of PR-HepG2 cells with these extracts for 48 hours. Cytotoxicity of paclitaxel was examined by XTT and protein assays in the absence and presence of Z02. Z02 potentiated the cytotoxicity of paclitaxel in PR-HepG2 cells. These results suggest that Curcuma comosa and Kaempferia marginata belonging to Zingiberaceae are useful sources to search for new P-gp modulator(s) that can be used to overcome multidrug resistance of cancer cells. PMID:25985578

  10. Human monoclonal antibody directed against an envelope glycoprotein of human T-cell leukemia virus type I.

    OpenAIRE

    Matsushita, S; Robert-Guroff, M; Trepel, J. (Jane); Cossman, J; Mitsuya, H; Broder, S

    1986-01-01

    We report the production and characterization of a human monoclonal antibody reactive against the major envelope glycoprotein of human T-cell leukemia virus type I (HTLV-I), a virus linked to the etiology of adult T-cell leukemia. We exposed lymph-node cells derived from a patient with adult T-cell leukemia to the Epstein-Barr virus in vitro and obtained a B-cell clone (designated 0.5 alpha) by a limiting dilution technique. The secreted product of 0.5 alpha is a monoclonal antibody (also des...

  11. Cell Surface Glycoprotein of Reactive Stromal Fibroblasts as a Potential Antibody Target in Human Epithelial Cancers

    Science.gov (United States)

    Garin-Chesa, Pilar; Old, Lloyd J.; Rettig, Wolfgang J.

    1990-09-01

    The F19 antigen is a cell surface glycoprotein (M_r, 95,000) of human sarcomas and proliferating, cultured fibroblasts that is absent from resting fibroblasts in normal adult tissues. Normal and malignant epithelial cells are also F19^-. The present immunohistochemical study describes induction of F19 in the reactive mesenchyme of epithelial tumors. F19^+ fibroblasts were found in primary and metastatic carcinomas, including colorectal (18 of 18 cases studied), breast (14/14), ovarian (21/21), bladder (9/10), and lung carcinomas (13/13). In contrast, the stroma of benign colorectal adenomas, fibrocystic disease and fibroadenomas of breast, benign prostate hyperplasia, in situ bladder carcinomas, and benign ovarian tumors showed no or only moderate numbers of F19^+ fibroblasts. Analysis of dermal incision wounds revealed that F19 is strongly induced during scar formation. Comparison of F19 with the extracellular matrix protein tenascin, a putative marker of tumor mesenchyme, showed a cellular staining pattern for F19 vs. the extracellular matrix pattern for tenascin and widespread expression of tenascin in F19^- normal tissues and benign tumors. Our results suggest that the F19^+ phenotype correlates with specialized fibroblast functions in wound healing and malignant tumor growth. Because of its abundance in tumor mesenchyme, F19 may serve as a target for antibodies labeled with radioisotopes or toxic agents, or inflammatogenic antibodies, in carcinoma patients.

  12. Varicella-Zoster Virus glycoprotein expression differentially induces the unfolded protein response in infected cells.

    Directory of Open Access Journals (Sweden)

    John Earl Carpenter

    2014-07-01

    Full Text Available Varicella-zoster virus (VZV is a human herpesvirus that spreads to children as varicella or chicken pox. The virus then establishes latency in the nervous system and re-emerges, typically decades later, as zoster or shingles. We have reported previously that VZV induces autophagy in infected cells as well as exhibiting evidence of the Unfolded Protein Response (UPR: XBP1 splicing, a greatly expanded Endoplasmic Reticulum (ER and CHOP expression. Herein we report the results of a UPR specific PCR array that measures the levels of mRNA of 84 different components of the UPR in VZV infected cells as compared to tunicamycin treated cells as a positive control and uninfected, untreated cells as a negative control. Tunicamycin is a mixture of chemicals that inhibits N-linked glycosylation in the ER with resultant protein misfolding and the UPR. We found that VZV differentially induces the UPR when compared to tunicamycin treatment. For example, tunicamycin treatment moderately increased (8 fold roughly half of the array elements while downregulating only three (one ERAD and two FOLD components. VZV infection on the other hand upregulated 33 components including a little described stress sensor CREB-H (64 fold as well as ER membrane components INSIG and gp78, which modulate cholesterol synthesis while downregulating over 20 components mostly associated with ERAD and FOLD. We hypothesize that this expression pattern is associated with an expanding ER with downregulation of active degradation by ERAD and apoptosis as the cell attempts to handle abundant viral glycoprotein synthesis.

  13. Varicella-zoster virus glycoprotein expression differentially induces the unfolded protein response in infected cells.

    Science.gov (United States)

    Carpenter, John E; Grose, Charles

    2014-01-01

    Varicella-zoster virus (VZV) is a human herpesvirus that spreads to children as varicella or chicken pox. The virus then establishes latency in the nervous system and re-emerges, typically decades later, as zoster or shingles. We have reported previously that VZV induces autophagy in infected cells as well as exhibiting evidence of the Unfolded Protein Response (UPR): XBP1 splicing, a greatly expanded Endoplasmic Reticulum (ER) and CHOP expression. Herein we report the results of a UPR specific PCR array that measures the levels of mRNA of 84 different components of the UPR in VZV infected cells as compared to tunicamycin treated cells as a positive control and uninfected, untreated cells as a negative control. Tunicamycin is a mixture of chemicals that inhibits N-linked glycosylation in the ER with resultant protein misfolding and the UPR. We found that VZV differentially induces the UPR when compared to tunicamycin treatment. For example, tunicamycin treatment moderately increased (8-fold) roughly half of the array elements while downregulating only three (one ERAD and two FOLD components). VZV infection on the other hand upregulated 33 components including a little described stress sensor CREB-H (64-fold) as well as ER membrane components INSIG and gp78, which modulate cholesterol synthesis while downregulating over 20 components mostly associated with ERAD and FOLD. We hypothesize that this expression pattern is associated with an expanding ER with downregulation of active degradation by ERAD and apoptosis as the cell attempts to handle abundant viral glycoprotein synthesis. PMID:25071735

  14. Human Cell Line and Tissue Sample Authentication

    OpenAIRE

    Ewing, Margaret M.; McLaren, Robert S.; Hebble, Kathryn D.; Ready, Kim; Storts, Douglas R.; Hooper, Kyle

    2013-01-01

    Background: Short Tandem Repeat (STR) genotyping analysis is a proven technology for uniquely identifying virtually all human samples. STR genotyping was adopted as the preferred technology for identification of human tissue culture cell lines by the ATCC Standards Development Organization (ASN-0002: Authentication of Human Cell Lines: Standardization of STR Profiling). We developed new automation-compatible protocols/systems for generating STR profiles from human cell lines or tissue samples...

  15. Expression of P-glycoprotein and multidrug resistance associated protein in Ehrlich ascites tumor cells after fractionated irradiation

    International Nuclear Information System (INIS)

    Purpose: To characterize irradiated murine tumor cells with respect to drug resistance, drug kinetics, and ATPase activity, and to evaluate the possible role of P-glycoprotein (PGP) and murine multidrug resistance associated protein (Mrp1) in the drug-resistant phenotype of these cells. Methods and Materials: Sensitive Ehrlich ascites tumor cells (EHR2) were in vitro exposed to fractionated irradiation (60 Gy). Western blot analysis was performed for determination of PGP and Mrp1, reverse transcriptase-polymerase chain reaction (RT-PCR) for determination of mdr1a + b mRNA, and semiquantitative RT-PCR for Mrp1 mRNA. The clonogenic assay was applied to investigate sensitivity, whereas the steady-state drug accumulation of daunorubicin (DNR), 3H-vincristine (VCR), and 3H-etoposide (VP16) was measured by spectrofluorometry and scintillation counting, respectively. For determining of ATPase activity, the release of inorganic phosphate from ATP was quantified using a colorimetric method. Results: Compared with EHR2, the irradiated cell line EHR2/irr showed increased expression of PGP (threefold), Mrp1 (eightfold), and Mrp1 mRNA (sixfold), and a slight reduction of mdr1b mRNA, whereas mdr1a was present in EHR2 but could not be detected in EHR2/irr. EHR2/irr developed sixfold resistance to VP16, twofold resistance to vincristine, but remained sensitive to DNR. Addition of the PGP inhibitor, verapamil (VER) or depletion of glutathione by buthionine sulfoximine (BSO) partly reversed the resistance in EHR2/irr. In EHR2/irr, the steady-state accumulation of 3H-VCR and 3H-VP16 was significantly decreased as compared with EHR2, whereas the accumulation of DNR was unchanged. The ATPase activity of plasma membrane vesicles prepared from EHR2/irr cells was similar to that of wild-type EHR2 cells. The ATPase activity was neither stimulated by vinblastine nor VER. Conclusion: Irradiation induced a multidrug-resistant phenotype in sensitive tumor cells. This phenotype was

  16. The cell-wall glycoproteins of the green alga Scenedesmus obliquus. The predominant cell-wall polypeptide of Scenedesmus obliquus is related to the cell-wall glycoprotein gp3 of Chlamydomonas reinhardtii.

    Science.gov (United States)

    Voigt, Jürgen; Stolarczyk, Adam; Zych, Maria; Malec, Przemysław; Burczyk, Jan

    2014-02-01

    The green alga Scenedesmus obliquus contains a multilayered cell wall, ultrastructurally similar to that of Chlamydomonas reinhardtii, although its proportion of hydroxyproline is considerably lower. Therefore, we have investigated the polypeptide composition of the insoluble and the chaotrope-soluble wall fractions of S. obliquus. The polypeptide pattern of the chaotrope-soluble wall fraction was strongly modified by chemical deglycosylation with anhydrous hydrogen fluoride (HF) in pyridine indicating that most of these polypeptides are glycosylated. Polypeptide constituents of the chaotrope-soluble cell-wall fraction with apparent molecular masses of 240, 270, 265, and 135 kDa cross-reacted with a polyclonal antibody raised against the 100 kDa deglycosylation product of the C. reinhardtii cell-wall glycoprotein GP3B. Chemical deglycosylation of the chaotrope-soluble wall fraction resulted in a 135 kDa major polypeptide and a 106 kDa minor component reacting with the same antibody. This antibody recognized specific peptide epitopes of GP3B. When the insoluble wall fraction of S. obliquus was treated with anhydrous HF/pyridine, three polypeptides with apparent molecular masses of 144, 135, and 65 kDa were solubilized, which also occured in the deglycosylated chaotrope-soluble wall fraction. These findings indicate that theses glycoproteins are cross-linked to the insoluble wall fraction via HF-sensitive bonds. PMID:24388513

  17. Re-evaluation of the role of P-glycoprotein in in vitro drug permeability studies with the bovine brain microvessel endothelial cells.

    Science.gov (United States)

    Hakkarainen, Jenni J; Rilla, Kirsi; Suhonen, Marjukka; Ruponen, Marika; Forsberg, Markus M

    2014-03-01

    1.  Currently available in vitro blood-brain barrier models all have recognized restrictions. In addition to leakiness, inconsistent data about P-glycoprotein mediated efflux limit the attractiveness of the primary bovine brain microvessel endothelial cells (BBMECs). Therefore, we re-evaluated the role of P-glycoprotein mediated efflux with two culture conditions in BBMECs for prediction of drug permeability of potential P-glycoprotein substrates. 2.  BBMECs were monocultured on filters on petri dishes and on filter inserts, and expression and localization of P-glycoprotein were compared by using western blot and confocal microscopy, respectively. The functionality of P-glycoprotein was assessed by using cellular uptake, calcein-AM and bidirectional transport assays. 3.  P-glycoprotein expression was higher in BBMECs cultured on filter inserts decreasing the permeability of digoxin and paclitaxel, but not the permeability of vinblastine. However, the monocultured BBMECs were not able to demonstrate efflux in the bidirectional transport assays. Under certain culture conditions, occludin may not be correctly located, perhaps explaining in part the leakiness of BBMECs. 4.  In conclusion, BBMECs, despite possessing a functional P-glycoprotein, under certain culture conditions may not be a suitable in vitro model for the bidirectional transport assays and for predicting the permeability of drugs and xenobiotics that are potential P-glycoprotein substrates. PMID:23924297

  18. Reverse Genetics Demonstrates that Proteolytic Processing of the Ebola Virus Glycoprotein Is Not Essential for Replication in Cell Culture

    OpenAIRE

    Neumann, Gabriele; Feldmann, Heinz; Watanabe, Shinji; Lukashevich, Igor; Kawaoka, Yoshihiro

    2002-01-01

    Ebola virus, a prime example of an emerging pathogen, causes fatal hemorrhagic fever in humans and in nonhuman primates. Identification of major determinants of Ebola virus pathogenicity has been hampered by the lack of effective strategies for experimental mutagenesis. Here we exploit a reverse genetics system that allows the generation of Ebola virus from cloned cDNA to engineer a mutant Ebola virus with an altered furin recognition motif in the glycoprotein (GP). When expressed in cells, t...

  19. The major surface glycoprotein (gp63) from Leishmania major and Leishmania donovani cleaves CD4 molecules on human T cells

    DEFF Research Database (Denmark)

    Hey, A S; Theander, T G; Hviid, L;

    1994-01-01

    The effect of Leishmania major and L. donovani surface protease gp63 on surface markers on human T cells was studied using fluorescence-activated flow cytometry. Purified gp63 (63,000 m.w. glycoprotein) at concentrations above 10 micrograms/ml completely inhibited binding of six different anti-CD4...... in interfering with the induction of the immune response and thus disease progression in Leishmania infections....

  20. Cationic amphiphilic drugs enhance entry of lentiviral particles pseudotyped with rabies virus glycoprotein into non-neuronal cells.

    Science.gov (United States)

    Klintworth, Anneke; Nolden, Tobias; Westhaus, Sandra; Rohrmann, Katrin; David, Sascha; Manns, Michael P; Finke, Stefan; Ciesek, Sandra; von Hahn, Thomas

    2015-12-01

    Amiodarone and other cationic amphiphilic drugs (CADs) inhibit cell entry by diverse human pathogenic viruses including Filoviruses, Dengue virus and Japanese encephalitis virus. They are thus considered potential broad spectrum antiviral agents. Here we report the unexpected finding that amiodarone and other CADs markedly enhance rabies virus (RABV) glycoprotein- (GP-) mediated cell entry of pseudotyped lentiviruses into non-neuronal cells but not in neuronal cells. Increased cell entry can also be elicited when CADs are added several hours after pseudoviral attachment. Perturbing endosomal processing with phosphoinosite-3-kinase inhibitors wortmannin and LY294002 mimics the effects of CADs on RABV GP-mediated cell entry. Thus, CADs may enhance RABV GP-mediated cell entry of pseudotyped lentiviruses by promoting a late step of the pseudoviral cell entry process, possibly release from an endosomal compartment into the cytosol. In contrast to the pseudotyped lentiviruses, infection by fully infectious RABV was not enhanced by CADs, indicating, that the observed stimulation of RABV GP mediated lentivirus entry also depended on the used lentivirus vector backbone. In conclusion, we show that while CADs inhibit cell entry of diverse viruses they can also have a paradoxical enhancing effect on the ability of a viral glycoprotein to mediate cell entry depending on the cellular and viral context. Although, we show CAD-mediated enhancement of entry only for pseudoviruses, but not fully infectious RABV, the potential to unexpectedly enhance viral entry should be taken into account when considering use of CADs as antiviral agents. PMID:26542648

  1. Nitric oxide mediates increased P-glycoprotein activity in interferon-{gamma}-stimulated human intestinal cells.

    Science.gov (United States)

    Dixit, Santosh G; Zingarelli, Basilia; Buckley, Donna J; Buckley, Arthur R; Pauletti, Giovanni M

    2005-03-01

    Patients with refractory inflammatory bowel disease (IBD) exhibit increased expression of intestinal P-glycoprotein (P-gp) as well as elevated luminal IFN-gamma and nitric oxide (NO) levels. Using the in vitro Caco-2 cell culture model, we investigated whether these pathological mediators associated with the etiology of IBD affect functional activity of intestinal efflux systems. IFN-gamma reduced cellular uptake of cyclosporin A (CysA) but not methotrexate (MTX) in a time- and concentration-dependent manner. Simultaneously, P-gp expression increased by approximately twofold. Coincubation with the inducible NO synthase inhibitor l-N(6)-(1-iminoethyl)lysine (l-NIL) dramatically reduced production of intracellular NO in response to IFN-gamma stimulus. The presence of l-NIL also abrogated the cytokine-mediated increase in P-gp expression and function suggesting that NO is required for IFN-gamma-mediated activation of this efflux system. Exposure of Caco-2 cells to the chemical NO donor S-nitroso-N-acetylpenicillamine (SNAP) produced a concentration-dependent decrease in intracellular CysA accumulation that was paralleled by an increase in P-gp expression. Both IFN-gamma and SNAP enhanced DNA binding of NF-kappaB, whereas inclusion of l-NIL dramatically decreased this cytokine-induced effect on NF-kappaB binding. These results suggest that NO mediates IFN-gamma-induced increase in expression and function of intestinal P-gp in the human Caco-2 cell culture model by altering DNA binding of NF-kappaB, which may enhance transcription of the ABCB1 gene encoding for this efflux system. PMID:15486347

  2. Expression of recombinant myelin-associated glycoprotein in primary Schwann cells promotes the initial investment of axons by myelinating Schwann cells

    OpenAIRE

    1990-01-01

    Myelin-associated glycoprotein (MAG) is an integral membrane protein expressed by myelinating glial cells that occurs in two developmentally regulated forms with different carboxyterminal cytoplasmic domains (L- MAG and S-MAG). To investigate the role of MAG in myelination a recombinant retrovirus was used to introduce a MAG cDNA (L-MAG form) into primary Schwann cells in vitro. Stably infected populations of cells were obtained that constitutively expressed MAG at the cell surface without th...

  3. Receptor-Targeted Nipah Virus Glycoproteins Improve Cell-Type Selective Gene Delivery and Reveal a Preference for Membrane-Proximal Cell Attachment.

    Directory of Open Access Journals (Sweden)

    Ruben R Bender

    2016-06-01

    Full Text Available Receptor-targeted lentiviral vectors (LVs can be an effective tool for selective transfer of genes into distinct cell types of choice. Moreover, they can be used to determine the molecular properties that cell surface proteins must fulfill to act as receptors for viral glycoproteins. Here we show that LVs pseudotyped with receptor-targeted Nipah virus (NiV glycoproteins effectively enter into cells when they use cell surface proteins as receptors that bring them closely enough to the cell membrane (less than 100 Å distance. Then, they were flexible in receptor usage as demonstrated by successful targeting of EpCAM, CD20, and CD8, and as selective as LVs pseudotyped with receptor-targeted measles virus (MV glycoproteins, the current standard for cell-type specific gene delivery. Remarkably, NiV-LVs could be produced at up to two orders of magnitude higher titers compared to their MV-based counterparts and were at least 10,000-fold less effectively neutralized than MV glycoprotein pseudotyped LVs by pooled human intravenous immunoglobulin. An important finding for NiV-LVs targeted to Her2/neu was an about 100-fold higher gene transfer activity when particles were targeted to membrane-proximal regions as compared to particles binding to a more membrane-distal epitope. Likewise, the low gene transfer activity mediated by NiV-LV particles bound to the membrane distal domains of CD117 or the glutamate receptor subunit 4 (GluA4 was substantially enhanced by reducing receptor size to below 100 Å. Overall, the data suggest that the NiV glycoproteins are optimally suited for cell-type specific gene delivery with LVs and, in addition, for the first time define which parts of a cell surface protein should be targeted to achieve optimal gene transfer rates with receptor-targeted LVs.

  4. Histidine-rich glycoprotein does not interfere with interactions between antithrombin III and heparin-like compounds on vascular endothelial cells

    International Nuclear Information System (INIS)

    The role of histidine-rich glycoprotein in controlling heparin-like compounds on the endothelial cell surface is still unclear. The effects of this heparin-neutralizing protein on the interaction between antithrombin III and cultured porcine aortic endothelial cells were examined. Displacement of 125I-labeled antithrombin III specifically bound to endothelial cells by unlabeled histidine-rich glycoprotein was much less potent than that by unlabeled antithrombin III. One hundred-fold molar excess of histidine-rich glycoprotein displaced specific 125I-antithrombin III binding only by 20%. Furthermore, the endothelial cell-mediated acceleration of thrombin inactivation by antithrombin III was diminished by protamine sulfate, but was not affected by histidine-rich glycoprotein even at a histidine-rich glycoprotein/antithrombin III molar ratio of approximately 7:1. These data indicate that histidine-rich glycoprotein does not interfere with the interaction of endothelial cell heparin-like compounds with antithrombin III. Thus, it may not play an important role in the modulation of anticoagulant activity of endothelial cells in vivo, suggesting that the commonly accepted view of the probable function of this protein is erroneous

  5. Progesterone interacts with P-glycoprotein in multidrug-resistant cells and in the endometrium of gravid uterus.

    Science.gov (United States)

    Yang, C P; DePinho, S G; Greenberger, L M; Arceci, R J; Horwitz, S B

    1989-01-15

    P-Glycoprotein (P-GP) plays a pivotal role in maintaining the multidrug-resistant (MDR) phenotype. This membrane glycoprotein is overproduced in MDR cells and the endometrium of the mouse gravid uterus (Arceci, R.J., Croop, J.M., Horwitz, S.B., and Housman, D. (1988) Proc. Natl. Acad. Sci. U.S.A. 85, 4350-4354). This latter observation and an interest in endogenous substrates for P-GP led to a study of the interaction of steroids with P-GP found in the endometrium of the mouse gravid uterus and in MDR cells derived from the murine macrophage-like cell J774.2. [3H]Azidopine labeling of P-GP from these two sources was inhibited by various steroids, particularly progesterone. Progesterone also markedly inhibited [3H]vinblastine binding to membrane vesicles prepared from MDR cells, enhanced vinblastine accumulation in MDR cells, and increased the sensitivity of MDR cells to vinblastine. In addition, we have demonstrated that the hydrophobicity of a steroid is important in determining its effect on inhibition of drug binding to P-GP. It is concluded that progesterone, a relatively nontoxic endogenous steroid, interacts with P-GP and is capable of reversing drug resistance in MDR cells. PMID:2562956

  6. Grifola frondosa Glycoprotein GFG-3a Arrests S phase, Alters Proteome, and Induces Apoptosis in Human Gastric Cancer Cells.

    Science.gov (United States)

    Cui, Fengjie; Zan, Xinyi; Li, Yunhong; Sun, Wenjing; Yang, Yan; Ping, Lifeng

    2016-01-01

    GFG-3a is a novel glycoprotein previously purified from the fermented mycelia of Grifola frondosa with novel sugar compositions and protein sequencing. The present study aims to investigate its effects on the cell cycle, differential proteins expression, and apoptosis of human gastric cancer SGC-7901 cells. Our findings revealed that GFG-3a induced the cell apoptosis and arrested cell cycle at S phase. GFG-3a treatment resulted in the differential expression of 21 proteins in SGC-7901 cells by upregulating 10 proteins including RBBP4 associated with cell cycle arrest and downregulating 11 proteins including RUVBL1, NPM, HSP90AB1, and GRP78 involved in apoptosis and stress response. qRT-PCR and Western blot analysis also suggested that GFG-3a could increase the expressions of Caspase-8/-3, p53, Bax, and Bad while decrease the expressions of Bcl2, Bcl-xl, PI3K, and Akt1. These results indicated that the stress response, p53-dependent mitochondrial-mediated, Caspase-8/-3-dependent, and PI3k/Akt pathways were involved in the GFG-3a-induced apoptosis process in SGC-7901 cells. These findings might provide a basis to prevent or treat human gastric cancer with GFG-3a and understand the tumor-inhibitory molecular mechanisms of mushroom glycoproteins. PMID:27040446

  7. P-glycoprotein and breast cancer resistance protein in acute myeloid leukaemia cells treated with the Aurora-B Kinase Inhibitor barasertib-hQPA

    International Nuclear Information System (INIS)

    Aurora kinases play an essential role in orchestrating chromosome alignment, segregation and cytokinesis during mitotic progression, with both aurora-A and B frequently over-expressed in a variety of human malignancies. Over-expression of the ABC drug transporter proteins P-glycoprotein (Pgp) and Breast cancer resistance protein (BCRP) is a major obstacle for chemotherapy in many tumour types with Pgp conferring particularly poor prognosis in acute myeloid leukaemia (AML). Barasertib-hQPA is a highly selective inhibitor of aurora-B kinase that has shown tumouricidal activity against a range tumour cell lines including those of leukaemic AML origin. Effect of barasertib-hQPA on the pHH3 biomarker and cell viability was measured in a panel of leukaemic cell lines and 37 primary AML samples by flow cytometry. Pgp status was determined by flow cytometry and BCRP status by flow cytometry and real-time PCR. In this study we report the creation of the cell line OCI-AML3DNR, which over-expresses Pgp but not BCRP or multidrug resistance-associated protein (MRP), through prolonged treatment of OCI-AML3 cells with daunorubicin. We demonstrate that Pgp (OCI-AML3DNR and KG-1a) and BCRP (OCI-AML6.2) expressing AML cell lines are less sensitive to barasertib-hQPA induced pHH3 inhibition and subsequent loss of viability compared to transporter negative cell lines. We also show that barasertib-hQPA resistance in these cell lines can be reversed using known Pgp and BCRP inhibitors. We report that barasertib-hQPA is not an inhibitor of Pgp or BCRP, but by using 14[C]-barasertib-hQPA that it is effluxed by these transporters. Using phosphoHistone H3 (pHH3) as a biomarker of barasertib-hQPA responsiveness in primary AML blasts we determined that Pgp and BCRP positive primary samples were less sensitive to barasertib-hQPA induced pHH3 inhibition (p = <0.001) than samples without these transporters. However, we demonstrate that IC50 inhibition of pHH3 by barasertib-hQPA was achieved in

  8. P-glycoprotein and breast cancer resistance protein in acute myeloid leukaemia cells treated with the Aurora-B Kinase Inhibitor barasertib-hQPA

    Directory of Open Access Journals (Sweden)

    Russell Nigel H

    2011-06-01

    Full Text Available Abstract Background Aurora kinases play an essential role in orchestrating chromosome alignment, segregation and cytokinesis during mitotic progression, with both aurora-A and B frequently over-expressed in a variety of human malignancies. Over-expression of the ABC drug transporter proteins P-glycoprotein (Pgp and Breast cancer resistance protein (BCRP is a major obstacle for chemotherapy in many tumour types with Pgp conferring particularly poor prognosis in acute myeloid leukaemia (AML. Barasertib-hQPA is a highly selective inhibitor of aurora-B kinase that has shown tumouricidal activity against a range tumour cell lines including those of leukaemic AML origin. Methods Effect of barasertib-hQPA on the pHH3 biomarker and cell viability was measured in a panel of leukaemic cell lines and 37 primary AML samples by flow cytometry. Pgp status was determined by flow cytometry and BCRP status by flow cytometry and real-time PCR. Results In this study we report the creation of the cell line OCI-AML3DNR, which over-expresses Pgp but not BCRP or multidrug resistance-associated protein (MRP, through prolonged treatment of OCI-AML3 cells with daunorubicin. We demonstrate that Pgp (OCI-AML3DNR and KG-1a and BCRP (OCI-AML6.2 expressing AML cell lines are less sensitive to barasertib-hQPA induced pHH3 inhibition and subsequent loss of viability compared to transporter negative cell lines. We also show that barasertib-hQPA resistance in these cell lines can be reversed using known Pgp and BCRP inhibitors. We report that barasertib-hQPA is not an inhibitor of Pgp or BCRP, but by using 14[C]-barasertib-hQPA that it is effluxed by these transporters. Using phosphoHistone H3 (pHH3 as a biomarker of barasertib-hQPA responsiveness in primary AML blasts we determined that Pgp and BCRP positive primary samples were less sensitive to barasertib-hQPA induced pHH3 inhibition (p = 50 inhibition of pHH3 by barasertib-hQPA was achieved in 94.6% of these samples after 1

  9. Metabolism of cobalamin bound to transcobalamin II and to glycoproteins that bind Cbl in HepG2 cells (human hepatoma)

    International Nuclear Information System (INIS)

    The binding, internalization, processing and release of labeled cyanocobalamin (CN[57Co]Cbl) bound to human transcobalamin II (TC II) were studied in HepG2 cells, a line of hepatocytes derived from a human hepatoma. The cells bound the TC II-Cbl by specific, high affinity receptors. Within the cell, the CN-Cbl was promptly freed from TC II and the CN-Cbl converted to more active forms including adenosyl Cbl (AdoCbl) and methyl Cbl (MeCbl). Whereas free labeled Cbl was still present at 72 hours after entry, the cells also bound Cbl to an intracellular binder (ICB) presumed to represent the holo enzymes dependent on Cbl. At levels of TC II that saturated the receptors for TC II-Cbl, much of the Cbl entering the cells remained free and was converted to AdoCbl. Under these circumstances the cells released free Cbl, mostly AdoCbl. Human R type binders of Cbl, which are glycoproteins and some having a terminal galactose, were bound by the HepG2 cells. Cbl bound to R binder was internalized and converted to coenzyme forms of Cbl, but the process was much less effective than when the Cbl entered via the TC II receptor system. It was concluded that the receptors for R-Cbl were unlikely to contribute to the physiologic transport of Cbl in man, but may function in some yet unknown way

  10. Biphasic regulation of P-glycoprotein function and expression by NO donors in Caco-2 cells

    Institute of Scientific and Technical Information of China (English)

    Ru DUAN; Nan HU; Hai-yan LIU; Jia LI; Hai-fang GUO; Can LIU; Li LIU; Xiao-dong LIU

    2012-01-01

    Aim:To investigate the effects of nitric oxide (NO) donors on the function and expression of P-glycoprotein (P-gp) in Caco-2 cells.Methods:Caco-2 cells were exposed to NO donors for designated times.P-gp function and expression were assessed using Rhodamine123 uptake assay and Western blotting,respectively.Intracellular reactive oxygen species (iROS) and intracellular reactive nitrogen species (iRNS) levels were measured using ROS and RNS assay kits,respectively.Results:Exposure of Caco-2 cells to 0.1 or 2 mmol/L of sodium nitroprusside (SNP) affected the function and expression of P-gp in concentration- and time-dependent manners.A short-term (4 h) exposure reduced P-gp function and expression accompanied with significantly increased levels of iROS and iRNS.In contrast,a long-term (24 h) exposure stimulated the P-gp function and expression.The stimulatory effects of 2 mmol/L SNP was less profound as compared to those caused by 0.1 mmol/L SNP.The other NO donors SIN-1 and SNAP showed similar effects.Neither the NO scavenger PTIO (2 mmol/L) nor soluble guanylate cyclase inhibitor ODQ (50 μmol/L) reversed the SNP-induced alteration of P-gp function.On the other hand,free radical scavengers ascorbate,glutathione and uric acid (2 mmol/L for each),PKC inhibitor chelerythrine (5 μmol/L),PI3K/Akt inhibitor wortmannin (1 pmol/L) and p38 MAPK inhibitor SB203580 (10 μmol/L) reversed the upregulation of P-gp function by the long-term exposure to SNP,but these agents had no effect on the impaired P-gp function following the short-term exposure to SNP.Conclusion:NO donors time-dependently regulate P-gp function and expression in Caco-2 cells:short-term exposure impairs P-gp function and expression,whereas long-term exposure stimulates P-gp function and expression.The regulation occurs via a NO-independent mechanism.

  11. Difference in Membrane Repair Capacity Between Cancer Cell Lines and a Normal Cell Line.

    Science.gov (United States)

    Frandsen, Stine Krog; McNeil, Anna K; Novak, Ivana; McNeil, Paul L; Gehl, Julie

    2016-08-01

    Electroporation-based treatments and other therapies that permeabilize the plasma membrane have been shown to be more devastating to malignant cells than to normal cells. In this study, we asked if a difference in repair capacity could explain this observed difference in sensitivity. Membrane repair was investigated by disrupting the plasma membrane using laser followed by monitoring fluorescent dye entry over time in seven cancer cell lines, an immortalized cell line, and a normal primary cell line. The kinetics of repair in living cells can be directly recorded using this technique, providing a sensitive index of repair capacity. The normal primary cell line of all tested cell lines exhibited the slowest rate of dye entry after laser disruption and lowest level of dye uptake. Significantly, more rapid dye uptake and a higher total level of dye uptake occurred in six of the seven tested cancer cell lines (p electroporation. Viability in the primary normal cell line (98 % viable cells) was higher than in the three tested cancer cell lines (81-88 % viable cells). These data suggest more effective membrane repair in normal, primary cells and supplement previous explanations why electroporation-based therapies and other therapies permeabilizing the plasma membrane are more effective on malignant cells compared to normal cells in cancer treatment. PMID:27312328

  12. Herpes simplex virus (HSV)-specific proliferative and cytotoxic T-cell responses in humans immunized with an HSF type 2 glycoprotein subunit vaccine

    International Nuclear Information System (INIS)

    Studies were undertaken to determine whether immunization of humans with a herpes simplex virus type 2 (HSV-2) glycoprotein-subunit vaccine would result in the priming of both HSV-specific proliferating cells and cytotoxic T cells. Peripheral blood lymphocytes (PBL) from all eight vaccinees studied responded by proliferating after stimulation with HSV-2, HSV-1, and glycoprotein gB-1. The PBL of five of these eight vaccinees proliferated following stimulation with gD-2, whereas stimulation with Gd-1 resulted in relatively low or no proliferative responses. T-cell clones were generated from HSV-2-stimulated PBL of three vaccinees who demonstrated strong proliferative responses to HSV-1 and HSV-2. Of 12 clones studied in lymphoproliferative assays, 9 were found to be cross-reactive for HSV-1 and HSV-2. Of the approximately 90 T-cell clones isolated, 14 demonstrated HSV-specific cytotoxic activity. Radioimmunoprecipitation-polyacrylamide gel electrophoresis analyses confirmed that the vaccinees had antibodies only to HSV glycoproteins, not to proteins which are absent in the subunit vaccine, indicating that these vaccinees had not become infected with HSV. Immunization of humans with an HSV-2 glycoprotein-subunit vaccine thus results in the priming of T cells that proliferate in response to stimulation with HSV and its glycoproteins and T cells that have cytotoxic activity against HSV-infected cells. Such HSV-specific memory T cells were detected as late as 2 years following the last boost with the subunit vaccine

  13. Standards for Cell Line Authentication and Beyond

    Science.gov (United States)

    Cole, Kenneth D.; Plant, Anne L.

    2016-01-01

    Different genomic technologies have been applied to cell line authentication, but only one method (short tandem repeat [STR] profiling) has been the subject of a comprehensive and definitive standard (ASN-0002). Here we discuss the power of this document and why standards such as this are so critical for establishing the consensus technical criteria and practices that can enable progress in the fields of research that use cell lines. We also examine other methods that could be used for authentication and discuss how a combination of methods could be used in a holistic fashion to assess various critical aspects of the quality of cell lines. PMID:27300367

  14. In vitro development of chemotherapy and targeted therapy drug-resistant cancer cell lines: A practical guide with case studies

    Directory of Open Access Journals (Sweden)

    Martina eMcDermott

    2014-03-01

    Full Text Available The development of a drug-resistant cell line can take from 3-18 months. However, little is published on the methodology of this development process. This article will discuss key decisions to be made prior to starting resistant cell line development; the choice of parent cell line, dose of selecting agent, treatment interval and optimising the dose of drug for the parent cell line. Clinically-relevant drug-resistant cell lines are developed by mimicking the conditions cancer patients experience during chemotherapy and cell lines display between 2-8 fold resistance compared to their parental cell line. Doses of drug administered are low, and a pulsed treatment strategy is often used where the cells recover in drug-free media. High-level laboratory models are developed with the aim of understanding potential mechanisms of resistance to chemotherapy agents. Doses of drug are higher and escalated over time. It is common to have difficulty developing stable clinically-relevant drug-resistant cell lines. A comparative selection strategy of multiple cell lines or multiple chemotherapeutic agents mitigates this risk and gives insight into which agents or type of cell line develops resistance easily. Successful selection strategies from our research are presented. Pulsed-selection produced platinum or taxane-resistant large cell lung cancer (H1299, H460 and temozolomide-resistant melanoma (Malme-3M and HT144 cell lines. Continuous selection produced lapatinib-resistant breast cancer cell line (HCC1954. Techniques for maintaining drug-resistant cell lines are outlined including; maintaining cells with chemotherapy, pulse treating with chemotherapy or returning to master drug-resistant stocks. The heterogeneity of drug-resistant models produced from the same parent cell line with the same chemotherapy agent is explored with reference to P-glycoprotein. Heterogeneity in drug-resistant cell lines reflects the heterogeneity that can occur in clinical drug

  15. Establishment of a hamster lymphoma cell line

    Directory of Open Access Journals (Sweden)

    Abe,Shinji

    1974-08-01

    Full Text Available The establishment of a hamster lymphoma cell line was attempted. Simple mincing and trypsinization of lymphoma tissue resulted in a high degree of cell degeneration. The ascitic tumor cells produced by intraperitoneal transplantation of lymphoma tissue gave a better result. These ascitic cells grew and were cultured successively in medium consisting of RPMI 1640 and 20% fetal calf serum. Cells were round and grew in suspension. Accelerated cell growth was observed one month after starting the culture. In the stained preparations, cells were lymphoblastic. Cells were transplantable into new-born hamsters and produced tumors, but not in young adult hamsters.

  16. Near neighbour analysis of variant cell lines derived from the promyeloid cell line HL60.

    OpenAIRE

    Bunce, C. M.; Lord, J M; Wong, A K; Brown, G.

    1988-01-01

    The human promyeloid cell line H60 can be induced to differentiate towards either neutrophils or monocytes. Variant cell lines, derived from HL60, which show reduced capacities for neutrophil and monocyte differentiation can be arranged in a developmental sequence which suggests that the potentials for neutrophil and monocyte differentiation are expressed sequentially by HL60 cells in this order. Analysis of the patterns of total cellular phosphoproteins within HL60 and 5 variant cell lines, ...

  17. A cell surface integral membrane glycoprotein of 85,000 mol wt (gp85) associated with triton X-100-insoluble cell skeleton.

    Science.gov (United States)

    Tarone, G; Ferracini, R; Galetto, G; Comoglio, P

    1984-08-01

    The Triton X-100-insoluble skeleton of baby hamster kidney BHK cells consists of the nucleus, intermediate-size filaments, and actin fibers. By transmission electron microscopy, membrane fragments were found to be associated with these insoluble structures. When radioiodinated or [3H]glucosamine-labeled cells were extracted with 0.5% Triton, most plasma membrane glycoproteins were solubilized except for a glycoprotein with a molecular weight of 85,000 (gp85) that remained associated with the insoluble skeletons. Immunoprecipitation with a specific antiserum indicated that the gp85 is not a proteolytic degradation product of fibronectin, an extracellular matrix glycoprotein insoluble in detergent. A monoclonal antibody of BHK cells specific for gp85 was produced. Immunofluorescence analysis with this monoclonal antibody indicated that gp85 is not associated with the extracellular matrix, but is confined to the cell membrane. Both in fixed and unfixed intact cells, fluorescence was concentrated in dots preferentially aligned in streaks on the cell surface. Gp85 was found to behave as an integral membrane protein interacting with the hydrophobic core of the lipid bilayer since it was extracted from membrane preparations by ionic detergents such as SDS, but not by 0.1 N NaOH (pH 12) in the absence of detergents, a condition known to release peripheral molecules. Association of gp85 with the cell skeleton was unaffected by increasing the Triton concentration up to 5%, but it was affected when actin filaments were dissociated or when a protein-denaturing agent (6 M urea) was used in the presence of Triton, suggesting that protein-protein interactions are involved in the association of gp85 with the cell skeleton. We conclude that gp85 is an integral plasma membrane glycoprotein that might have a role in cell surface-cytoskeleton interaction. PMID:6378925

  18. Susceptibility of cell lines to avian viruses

    Directory of Open Access Journals (Sweden)

    Simoni Isabela Cristina

    1999-01-01

    Full Text Available The susceptibility of the five cell lines - IB-RS-2, RK-13, Vero, BHK-21, CER - to reovirus S1133 and infectious bursal disease virus (IBDV vaccine GBV-8 strain was studied to better define satisfactory and sensitive cell culture systems. Cultures were compared for presence of CPE, virus titers and detection of viral RNA. CPE and viral RNA were detected in CER and BHK-21 cells after reovirus inoculation and in RK-13 cell line after IBDV inoculation and with high virus titers. Virus replication by production of low virus titers occurred in IB-RS-2 and Vero cells with reovirus and in BHK-21 cell line with IBDV.

  19. Identification of P-Glycoprotein and Transport Mechanism of Paclitaxel in Syncytiotrophoblast Cells

    OpenAIRE

    Lee, Na-Young; Lee, Ha-Eun; Kang, Young-Sook

    2014-01-01

    When chemotherapy is administered during pregnancy, it is important to consider the fetus chemotherapy exposure, because it may lead to fetal consequences. Paclitaxel has become widely used in the metastatic and adjuvant settings for woman with cancer including breast and ovarian cancer. Therefore, we attempted to clarify the transport mechanisms of paclitaxel through blood-placenta barrier using rat conditionally immortalized syncytiotrophoblast cell lines (TR-TBTs). The uptake of paclitaxel...

  20. Characterisation and Manipulation of Docetaxel Resistant Prostate Cancer Cell Lines

    LENUS (Irish Health Repository)

    O'Neill, Amanda J

    2011-10-07

    Abstract Background There is no effective treatment strategy for advanced castration-resistant prostate cancer. Although Docetaxel (Taxotere®) represents the most active chemotherapeutic agent it only gives a modest survival advantage with most patients eventually progressing because of inherent or acquired drug resistance. The aims of this study were to further investigate the mechanisms of resistance to Docetaxel. Three Docetaxel resistant sub-lines were generated and confirmed to be resistant to the apoptotic and anti-proliferative effects of increasing concentrations of Docetaxel. Results The resistant DU-145 R and 22RV1 R had expression of P-glycoprotein and its inhibition with Elacridar partially and totally reversed the resistant phenotype in the two cell lines respectively, which was not seen in the PC-3 resistant sublines. Resistance was also not mediated in the PC-3 cells by cellular senescence or autophagy but multiple changes in pro- and anti-apoptotic genes and proteins were demonstrated. Even though there were lower basal levels of NF-κB activity in the PC-3 D12 cells compared to the Parental PC-3, docetaxel induced higher NF-κB activity and IκB phosphorylation at 3 and 6 hours with only minor changes in the DU-145 cells. Inhibition of NF-κB with the BAY 11-7082 inhibitor reversed the resistance to Docetaxel. Conclusion This study confirms that multiple mechanisms contribute to Docetaxel resistance and the central transcription factor NF-κB plays an immensely important role in determining docetaxel-resistance which may represent an appropriate therapeutic target.

  1. Identification of a highly conserved hydroxyproline-rich glycoprotein in the cell walls of Chlamydomonas reinhardtii and two other Volvocales.

    Science.gov (United States)

    Adair, W S; Appel, H

    1989-10-01

    The unicellular alga Chlamydomonas reinhardtii Dang, has a cell wall made entirely from hydroxyproline-rich glycoproteins (HRGPs). We recently employed a quantiative in vitro reconstitution system (Adair et al. 1987, J. Cell Biol. 105, 2373-2382) to assign outer-wall HRGPs of C. reinhardtii to specific sublayers, and describe the major interactions responsible for their assembly. Some of these interactions appear to involve relatively conserved HRGP domains, as evidenced by interspecific cell-wall reconstitution between C. reinhardtii and two multicellular Volvocales (Volvoxcarteri lyengar and Gonium pectorale Müller). In the present report we provide biochemical and immunological evidence that the outer cell-walls of V. carteri and G. pectorale both contain prominent HRGPs closely related to C. reinhardtii GP2. Identification of conserved GP2 homologues indicates a molecular basis for interspecific reconstitution and provides a useful avenue for characterization of HRGP domains mediating cell-wall formation in these algae. PMID:24201668

  2. Amplification of rabies virus-induced stimulation of human T-cell lines and clones by antigen-specific antibodies.

    OpenAIRE

    Celis, E; Wiktor, T J; Dietzschold, B.; Koprowski, H

    1985-01-01

    The effect of antigen-specific antibodies on the response of human T-cell lines and clones to rabies virus was studied. Plasmas from rabies-immune vaccine recipients, but not those from nonimmune individuals, enhanced the proliferative response of rabies-reactive T cells to whole inactivated virus or to the purified glycoprotein and nucleocapsid from the rabies virion. Rabies-immune plasma also increased the antigen-induced production of gamma interferon by the rabies-specific T-cell lines. E...

  3. Plant-originated glycoprotein (24 kDa) has an inhibitory effect on proliferation of BNL CL.2 cells in response to di(2-ethylhexyl)phthalate.

    Science.gov (United States)

    Lee, Jin; Lim, Kye-Taek

    2011-08-01

    Di(2-ethylhexyl)phthalate (DEHP) is one of the many environmental chemicals that are widely used in polyvinyl chloride products, vinyl flooring, food packaging and infant toys. They cause cell proliferation or dysfunction of human liver. The purpose of this study is to investigate the inhibitory effect of a glycoprotein (24 kDa) isolated from Zanthoxylum piperitum DC (ZPDC) on proliferation of liver cell in the DEHP-induced BNL CL. 2 cells. [³H]-thymidine incorporation, intracellular reactive oxygen species (ROS), intracellular Ca²⁺ mobilization and activity of protein kinase C (PKC) were measured using radioactivity and fluorescence method respectively. The expression of mitogen-activated protein kinases [extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK)], activator protein (AP)-1 (c-Jun and c-Fos), proliferating cell nuclear antigen (PCNA) and cell cycle-related factors (cyclin D1/cyclin-dependent kinase [CDK] 4) were evaluated using Western blotting or electrophoretic mobility shift assay. The results in this study showed that the levels of [³H]-thymidine incorporation, intracellular ROS, intracellular Ca²⁺ mobilization and activity of PKCα were inhibited by ZPDC glycoprotein (100 µg/ml) in the DEHP-induced BNL CL. 2 cells. Also, activities of ERK, JNK and AP-1 were reduced by ZPDC glycoprotein (100 µg/ml). With regard to cell proliferation, activities of PCNA and cyclin D1/CDK4 were significantly suppressed at treatment with ZPDC glycoprotein (100 µg/ml) in the presence of DEHP. Taken together, these findings suggest that ZPDC glycoprotein significantly normalized activities of PCNA and cyclin D1/CDK4, which relate to cell proliferation factors. Thus, ZPDC glycoprotein appears to be one of the compounds derived from natural products that are able to inhibit cell proliferation in the phthalate-induced BNL CL. 2 cells. PMID:21721021

  4. The equine herpesvirus 1 glycoprotein gp21/22a, the herpes simplex virus type 1 gM homolog, is involved in virus penetration and cell-to-cell spread of virions.

    OpenAIRE

    Osterrieder, N.; Neubauer, A.; Brandmuller, C; Braun, B.; Kaaden, O R; Baines, J D

    1996-01-01

    Experiments to analyze the function of the equine herpesvirus 1 (EHV-1) glycoprotein gM homolog were conducted. To this end, an Rk13 cell line (TCgM) that stably expressed EHV-1 gM was constructed. Proteins with apparent M(r)s of 46,000 to 48,000 and 50,000 to 55,000 were detected in TCgM cells with specific anti-gM antibodies, and the gM protein pattern was indistinguishable from that in cells infected with EHV-1 strain RacL11. A viral mutant (L11deltagM) bearing an Escherichia coli lacZ gen...

  5. Induction of actin disruption and downregulation of P-glycoprotein expression by solamargine in multidrug-resistant K562/A02 cells

    Institute of Scientific and Technical Information of China (English)

    LI Xia; ZHAO Ying; JI Mei; LIU Shan-shan; CUI Min; LOU Hong-xiang

    2011-01-01

    Background Solamargine (SM), a steroidal glycoalkaloid isolated from the Chinese herb Solarium incanum, has been shown to inhibit the growth of some cancer cell lines and induce significant apoptosis. However, the effects of SM on multidrug-resistant (MDR) cells and the molecular mechanisms involved are poorly understood. The purpose of this study was to evaluate the anti-MDR effects of SM and the associated mechanisms in MDR K562/A02 cells.Methods The cytotoxicity of SM was measured by 3-(4,5-dimethylthiazol)-2,5-diphenyltetrazolium bromide (MTT) assay. The 14',6-diamidino-2-phenylindole (DAPI) nuclear staining and flow cytometry were used to detect SM-induced apoptosis. The mRNA expression of P-glycoprotein (P-gp) was investigated by real-time PCR (RT-PCR). Western blotting was used to determine the expression of Bcl-2, Bax, and actin. The changes in the morphology of actin were examined with immunofluorescence staining.Results MTT results showed that SM effectively killed the MDR sublines K562/A02, KB/VCR, and H460/paclitaxel (Taxol), and their parental cell lines K562, KB, and H460 to an equivalent or more sensitive degree. Based on the results by flow cytometry and immunostaining, the pro-apoptotic effects of SM were observed in MDR K562/A02 cells. Furthermore, the RT-PCR results showed that SM induced the downregulation of MDR1 mRNA. In addition, the expression of P-gp and actin was decreased in the SM-treated cells, as measured by western blotting and immunostaining.Conclusions These results demonstrate that SM effectively triggers apoptosis in MDR tumor cells, which is associated with actin disruption and downregulation of MDR1 expression. This compound may merit further investigation as a potential therapeutic agent that bypasses the MDR mechanism for the treatment of MDR tumors.

  6. Chimeric antigen receptor (CAR)-engineered T cells redirected against hepatitis C virus (HCV) E2 glycoprotein

    Science.gov (United States)

    Sautto, Giuseppe A; Wisskirchen, Karin; Clementi, Nicola; Castelli, Matteo; Diotti, Roberta A; Graf, Julia; Clementi, Massimo; Burioni, Roberto; Protzer, Ulrike; Mancini, Nicasio

    2016-01-01

    Objective The recent availability of novel antiviral drugs has raised new hope for a more effective treatment of hepatitis C virus (HCV) infection and its severe sequelae. However, in the case of non-responding or relapsing patients, alternative strategies are needed. To this end we have used chimeric antigen receptors (CARs), a very promising approach recently used in several clinical trials to redirect primary human T cells against different tumours. In particular, we designed the first CARs against HCV targeting the HCV/E2 glycoprotein (HCV/E2). Design Anti-HCV/E2 CARs were composed of single-chain variable fragments (scFvs) obtained from a broadly cross-reactive and cross-neutralising human monoclonal antibody (mAb), e137, fused to the intracellular signalling motif of the costimulatory CD28 molecule and the CD3ζ domain. Activity of CAR-grafted T cells was evaluated in vitro against HCV/E2-transfected cells as well as hepatocytes infected with cell culture-derived HCV (HCVcc). Results In this proof-of-concept study, retrovirus-transduced human T cells expressing anti-HCV/E2 CARs were endowed with specific antigen recognition accompanied by degranulation and secretion of proinflammatory and antiviral cytokines, such as interferon γ, interleukin 2 and tumour necrosis factor α. Moreover, CAR-grafted T cells were capable of lysing target cells of both hepatic and non-hepatic origin expressing on their surface the HCV/E2 glycoproteins of the most clinically relevant genotypes, including 1a, 1b, 2a, 3a, 4 and 5. Finally, and more importantly, they were capable of lysing HCVcc-infected hepatocytes. Conclusions Clearance of HCV-infected cells is a major therapeutic goal in chronic HCV infection, and adoptive transfer of anti-HCV/E2 CARs-grafted T cells represents a promising new therapeutic tool. PMID:25661083

  7. Dynamics of synthetic activity of RNA and glycoproteins in epithel cells of endometrium in heifers after ovulation

    International Nuclear Information System (INIS)

    Synchronized heifers (n=9) of Black Pied HF breed were slaughtered on 3rd, 6th and 9th day of sexual cycle (first day of estrus = 0). Excisions from basal part (A) and functional part (B) of uteri were taken immediately after killing and were processed for autoradiographic analyses. The samples of endometrium were incubated for 20 minutes in isotonic medium with 100 micro Ci uridine (5 -3H) additive with specific activity 740 GBq/mM (UVVVR Prague) to study the RNA synthesis. The endometrium samples were incubated for 60 minutes, and 240 minutes in isotonic medium with 100 micro Ci L-(6-3 H) fucose with specific activity 0.55-1.1 TBqImM (Amersham Int., G.B.) for autoradiographic analysis of the glycoprotein synthesis. The samples were fixed, dehydrated and embedded in Epon 812 after the incubation. The prepared cuts were covered with photographic emulsion and stored in dark box in a refrigerator at 5øC. They were developed in the developer ORWO D 19, stained with methylene blue and examined through the light microscope after one month exposition. We found out by the autoradiographic analysis that the activity of RNA synthesis in cells of the surface epithel is of rising tendency from 3rd to 9th day. The intensity of RNA synthesis does not change in the functional zone during the early lutheal phase, it rises in the basal layer on 6th day, but on 9th day it is the same as on 3rd day. The autoradiographical analysis showed that the activity of RNA synthesis in cells of the surface epithel is of rising tendency from 3rd to 9th day The intensity of RNA synthesis in functional zone does not change during the early lutheal phase, it rises in the basal layer on 6th day, but on 9th day it is the same as on 3rd day. The glycoproteins are synthetised mainly by the Golgi apparatus in supranuclear sphere in the cells of surface epithel and in glandular cells. The glycoproteins were not observed in apical regions of cells on 3rd day of cycle, however, they are intensively

  8. Expression of P-glycoprotein and multidrug resistance associated protein in Ehrlich ascites tumor cells after fractionated irradiation

    DEFF Research Database (Denmark)

    Nielsen, D; Maare, C; Eriksen, J;

    2001-01-01

    ATPase activity was neither stimulated by vinblastine nor VER. CONCLUSION: Irradiation induced a multidrug-resistant phenotype in sensitive tumor cells. This phenotype was characterized by increased expression of Mrp1 mRNA, Mrp1, and PGP but decreased expression of mdr1a + b mRNA. The influence of......PURPOSE: To characterize irradiated murine tumor cells with respect to drug resistance, drug kinetics, and ATPase activity, and to evaluate the possible role of P-glycoprotein (PGP) and murine multidrug resistance associated protein (Mrp1) in the drug-resistant phenotype of these cells. METHODS AND...... MATERIALS: Sensitive Ehrlich ascites tumor cells (EHR2) were in vitro exposed to fractionated irradiation (60 Gy). Western blot analysis was performed for determination of PGP and Mrp1, reverse transcriptase-polymerase chain reaction (RT-PCR) for determination of mdr1a + b mRNA, and semiquantitative RT...

  9. Molecular and immuno-characteristics of immunoglobulin-like glycoproteins in cancer cell-expressed biomarker, CA215.

    Science.gov (United States)

    Lee, Gregory; Cheung, Anthony P; Li, Bo; Ge, Bixia; Chow, Po-Ming

    2012-01-01

    RP215 monoclonal antibody (Mab) was shown to recognize a specific carbohydrate-associated epitope found in cancer cell-expressed glycoproteins, known as CA215. The membrane-bound and soluble forms of CA215 were detected in almost all of the cancer cells in humans, but rarely found in normal tissues. Through MALDI-TOF MS analysis, it has been reported previously that as much as 40% of the detected tryptic peptides of CA215 showed high degrees of sequence homology to those found in immunoglobulin heavy chains. The cancer cell-derived immunoglobulins were further purified from CA215 by affinity column-linked with goat anti-human IgG for molecular characterizations. Semi-quantitative RT-PCR was used to determine the mRNA levels of various immunoglobulin genes expressed by cancer cells of single or multi-cell origins and compared with those found in normal human serum. The stability of CA215 was investigated under different experimental conditions. It was observed that the RP215-specific epitope in CA215 is stable at neutral pH, in human serum or in mice (half life of 5-18 days), but unstable at extreme pH's (pH ≤ 2.0; pH ≥ 12.0) or high temperatures. Enzyme immunoassays were performed with several secondary antibody probes related to human IgG. It was demonstrated that cancer cell-expressed immunoglobulins with RP215-specific epitope have much lower immunoactivity than that of normal human IgG (≤ 5%), despite the fact that both showed almost identical amino acid sequence in the respective Fc region reported previously. This could be the result of aberrant glycosylation of CA215 in cancer cells. Aberrant glycosylation of glycoproteins may have important biological implications on the proliferation of cancer cells in vitro or in vivo. PMID:22417288

  10. Equine herpesvirus type 1 (EHV-1) glycoprotein K is required for efficient cell-to-cell spread and virus egress

    International Nuclear Information System (INIS)

    The function of the equine herpesvirus type 1 (EHV-1) glycoprotein K (gK) homologue was investigated. Deletion of 88% of the UL53-homologous open reading frame in EHV-1 strain RacH resulted in a severe growth defect of the gK-negative virus (HΔgK) as reflected by a significant decrease in the production of infectious virus progeny on RK13 cells. The HΔgK virus induced only minute plaques, was unable to form syncytia, and its penetration efficiency into RK13 cells was reduced by approximately 40%. To further analyze gK function and intracellular trafficking, gK of strain RacH was replaced by a C-terminally truncated gK-green fluorescent protein fusion protein (gK-GFP). The generated recombinant virus was shown to replicate well on non-complementing cells, and virus penetration and syncytium formation were comparable to parental RacH. A reduction in plaque size and slightly decreased intra- and extracellular virus titers, however, were observed. The gK-GFP fusion protein was expressed with early-late kinetics, and multiple forms of the protein exhibiting Mrs between 50,000 and 85,000 were detected by Western blot analysis. The various gK-GFP forms were shown to be N-glycosylated, associated with membranes of the Golgi apparatus, and were incorporated into extracellular virions. Complete processing of gK-GFP was only observed within the context of viral infection. From the results, we concluded that EHV-1 gK is required for efficient virus growth in vitro and that the carboxy-terminal amino acids are not required for its function, because the gK-GFP fusion protein was able to complement for EHV-1 growth in the absence of authentic gK

  11. Pregnancy-specific Glycoprotein 1 Induces Endothelial Tubulogenesis through Interaction with Cell Surface Proteoglycans*

    OpenAIRE

    Lisboa, Felipe A.; Warren, James; Sulkowski, Gisela; Aparicio, Marta; David, Guido; Zudaire, Enrique; Dveksler, Gabriela S.

    2010-01-01

    Pregnancy-specific β1 glycoproteins (PSGs) are the most abundant fetal proteins in the maternal bloodstream in late pregnancy. They are secreted by the syncytiotrophoblast and are detected around day 14 postfertilization. There are 11 human PSG genes, which encode a family of proteins exhibiting significant conservation at the amino acid level. We and others have proposed that PSGs have an immune modulatory function. In addition, we recently postulated that they are proangiogenic due to their...

  12. Keratinocyte growth factor-2 stimulates P-glycoprotein expression and function in intestinal epithelial cells

    OpenAIRE

    Saksena, Seema; Priyamvada, Shubha; Kumar, Anoop; Akhtar, Maria; Soni, Vikas; Anbazhagan, Arivarasu Natarajan; Alakkam, Anas; Alrefai, Waddah A.; Dudeja, Pradeep K.; Gill, Ravinder K.

    2013-01-01

    Intestinal P-glycoprotein (Pgp/multidrug resistance 1), encoded by the ATP-binding cassette B1 gene, is primarily involved in the transepithelial efflux of toxic metabolites and xenobiotics from the mucosa into the gut lumen. Reduced Pgp function and expression has been shown to be associated with intestinal inflammatory disorders. Keratinocyte growth factor-2 (KGF2) has emerged as a potential target for modulation of intestinal inflammation and maintenance of gut mucosal integrity. Whether K...

  13. Analysis of glycoprotein E-selectin ligANDs on human and mouse marrow cells enriched for hematopoietic stem/progenitor cells

    KAUST Repository

    Merzaban, Jasmeen

    2011-06-09

    Although well recognized that expression of E-selectin on marrow microvessels mediates osteotropism of hematopoietic stem/progenitor cells (HSPCs), our knowledge regarding the cognate E-selectin ligand(s) on HSPCs is incomplete. Flow cytometry using E-selectin-Ig chimera (E-Ig) shows that human marrow cells enriched for HSPCs (CD34+ cells) display greater E-selectin binding than those obtained from mouse (lin-/Sca-1+/c-kit+ [LSK] cells). To define the relevant glycoprotein E-selectin ligands, lysates from human CD34+ and KG1a cells and from mouse LSK cells were immunoprecipitated using E-Ig and resolved byWestern blot using E-Ig. In both human and mouse cells, E-selectin ligand reactivity was observed at ∼ 120- to 130-kDa region, which contained two E-selectin ligands, the P-selectin glycoprotein ligand- 1 glycoform "CLA," and CD43. Human, but not mouse, cells displayed a prominent ∼ 100-kDa band, exclusively comprising the CD44 glycoform "HCELL."E-Ig reactivity was most prominent on CLA in mouse cells and on HCELL in human cells. To further assess HCELL\\'s contribution to E-selectin adherence, complementary studies were performed to silence (via CD44 siRNA) or enforce its expression (via exoglycosylation). Under physiologic shear conditions, CD44/HCELL-silenced human cells showed striking decreases (> 50%) in E-selectin binding. Conversely, enforced HCELL expression of LSK cells profoundly increased E-selectin adherence, yielding > 3-fold more marrow homing in vivo. These data define the key glycoprotein E-selectin ligands of human and mouse HSPCs, unveiling critical species-intrinsic differences in both the identity and activity of these structures. © 2011 by The American Society of Hematology.

  14. Clearance and binding of radiolabeled glycoproteins by cells of the murine mononuclear phagocyte system

    International Nuclear Information System (INIS)

    The clearance and binding of radiolabeled lactoferrin and fast α2-macroglobulin were studied. Both glycoproteins cleared rapidly following intravenous injection in mice, and both bound specifically to discrete receptors on murine peritoneal macrophages. The simultaneous presence of excess, unlabeled ligands specific for receptors recognizing terminal fucose, mannose, N-acetylglucosamine or galactose residues did not inhibit the clearance or binding of either lactoferrin or fast-α2M. The clearance and binding of enzymatically defucosylated lactoferrin was indistinguishable from native lactoferrin, indicating that terminal α(1-3)-linked fucose on lactoferrin is not necessary for receptor recognition. The clearance and binding of two fast -α2M forms, α2M-trypsin and α2M-MeNH2 cross compete with each other. Saturation binding studies indicated that the total binding of mannosyl -BSA, fusocyl-BSA, and N-acetylglucosaminyl-BSA to macrophages activated by BCG was approximately 15% of the levels observed with inflammatory macrophages elicited by thioglycollate broth. Cross-competition binding studies demonstrated a common surface receptor mediated binding of all three neoglycoprotein ligands and was identical to the receptor on mononuclear phagocytes that binds mannosyl- and N-acetylglucosaminyl-terminated glycoproteins. These results suggest that difference between discrete states of macrophage function may be correlated with selective changes in levels of the surface receptor for mannose-containing glycoproteins

  15. Natural lignans from Arctium lappa modulate P-glycoprotein efflux function in multidrug resistant cancer cells.

    Science.gov (United States)

    Su, Shan; Cheng, Xinlai; Wink, Michael

    2015-02-15

    Arctium lappa is a well-known traditional medicinal plant in China (TCM) and Europe that has been used for thousands of years to treat arthritis, baldness or cancer. The plant produces lignans as secondary metabolites which have a wide range of bioactivities. Yet, their ability to reverse multidrug resistance (MDR) in cancer cells has not been explored. In this study, we isolated six lignans from A. lappa seeds, namely arctigenin, matairesinol, arctiin, (iso)lappaol A, lappaol C, and lappaol F. The MDR reversal potential of the isolated lignans and the underlying mechanism of action were studied using two MDR cancer cell lines, CaCo2 and CEM/ADR 5000 which overexpress P-gp and other ABC transporters. In two-drug combinations of lignans with the cytotoxic doxorubicin, all lignans exhibited synergistic effects in CaCo2 cells and matairesinol, arctiin, lappaol C and lappaol F display synergistic activity in CEM/ADR 5000 cells. Additionally, in three-drug combinations of lignans with the saponin digitonin and doxorubicin MDR reversal activity was even stronger enhanced. The lignans can increase the retention of the P-gp substrate rhodamine 123 in CEM/ADR 5000 cells, indicating that lignans can inhibit the activity of P-gp. Our study provides a first insight into the potential chemosensitizing activity of a series of natural lignans, which might be candidates for developing novel adjuvant anticancer agents. PMID:25765837

  16. Therapeutic implications of an enriched cancer stem-like cell population in a human osteosarcoma cell line

    Directory of Open Access Journals (Sweden)

    Martins-Neves Sara R

    2012-04-01

    Full Text Available Abstract Background Osteosarcoma is a bone-forming tumor of mesenchymal origin that presents a clinical pattern that is consistent with the cancer stem cell model. Cells with stem-like properties (CSCs have been identified in several tumors and hypothesized as the responsible for the relative resistance to therapy and tumor relapses. In this study, we aimed to identify and characterize CSCs populations in a human osteosarcoma cell line and to explore their role in the responsiveness to conventional therapies. Methods CSCs were isolated from the human MNNG/HOS cell line using the sphere formation assay and characterized in terms of self-renewal, mesenchymal stem cell properties, expression of pluripotency markers and ABC transporters, metabolic activity and tumorigenicity. Cell's sensitivity to conventional chemotherapeutic agents and to irradiation was analyzed and related with cell cycle-induced alterations and apoptosis. Results The isolated CSCs were found to possess self-renewal and multipotential differentiation capabilities, express markers of pluripotent embryonic stem cells Oct4 and Nanog and the ABC transporters P-glycoprotein and BCRP, exhibit low metabolic activity and induce tumors in athymic mice. Compared with parental MNNG/HOS cells, CSCs were relatively more resistant to both chemotherapy and irradiation. None of the treatments have induced significant cell-cycle alterations and apoptosis in CSCs. Conclusions MNNG/HOS osteosarcoma cells contain a stem-like cell population relatively resistant to conventional chemotherapeutic agents and irradiation. This resistant phenotype appears to be related with some stem features, namely the high expression of the drug efflux transporters P-glycoprotein and BCRP and their quiescent nature, which may provide a biological basis for resistance to therapy and recurrence commonly observed in osteosarcoma.

  17. Therapeutic implications of an enriched cancer stem-like cell population in a human osteosarcoma cell line

    International Nuclear Information System (INIS)

    Osteosarcoma is a bone-forming tumor of mesenchymal origin that presents a clinical pattern that is consistent with the cancer stem cell model. Cells with stem-like properties (CSCs) have been identified in several tumors and hypothesized as the responsible for the relative resistance to therapy and tumor relapses. In this study, we aimed to identify and characterize CSCs populations in a human osteosarcoma cell line and to explore their role in the responsiveness to conventional therapies. CSCs were isolated from the human MNNG/HOS cell line using the sphere formation assay and characterized in terms of self-renewal, mesenchymal stem cell properties, expression of pluripotency markers and ABC transporters, metabolic activity and tumorigenicity. Cell's sensitivity to conventional chemotherapeutic agents and to irradiation was analyzed and related with cell cycle-induced alterations and apoptosis. The isolated CSCs were found to possess self-renewal and multipotential differentiation capabilities, express markers of pluripotent embryonic stem cells Oct4 and Nanog and the ABC transporters P-glycoprotein and BCRP, exhibit low metabolic activity and induce tumors in athymic mice. Compared with parental MNNG/HOS cells, CSCs were relatively more resistant to both chemotherapy and irradiation. None of the treatments have induced significant cell-cycle alterations and apoptosis in CSCs. MNNG/HOS osteosarcoma cells contain a stem-like cell population relatively resistant to conventional chemotherapeutic agents and irradiation. This resistant phenotype appears to be related with some stem features, namely the high expression of the drug efflux transporters P-glycoprotein and BCRP and their quiescent nature, which may provide a biological basis for resistance to therapy and recurrence commonly observed in osteosarcoma

  18. Efflux of rhodamine from CD56+ cells as a surrogate marker for reversal of P-glycoprotein-mediated drug efflux by PSC 833

    DEFF Research Database (Denmark)

    Robey, R; Bakke, S; Stein, W;

    1999-01-01

    The expression of high levels of P-glycoprotein (Pgp) in circulating mononuclear cells allowed us to use an ex vivo assay as a surrogate measure of Pgp antagonism. Efflux of rhodamine from CD56(+) cells was measured before the start of PSC 833 and at varying times thereafter. Patients receiving P...

  19. Palmitoylation of the cysteine-rich endodomain of the SARS-coronavirus spike glycoprotein is important for spike-mediated cell fusion

    International Nuclear Information System (INIS)

    The SARS-coronavirus (SARS-CoV) is the etiological agent of the severe acute respiratory syndrome (SARS). The SARS-CoV spike (S) glycoprotein mediates membrane fusion events during virus entry and virus-induced cell-to-cell fusion. The cytoplasmic portion of the S glycoprotein contains four cysteine-rich amino acid clusters. Individual cysteine clusters were altered via cysteine-to-alanine amino acid replacement and the modified S glycoproteins were tested for their transport to cell-surfaces and ability to cause cell fusion in transient transfection assays. Mutagenesis of the cysteine cluster I, located immediately proximal to the predicted transmembrane, domain did not appreciably reduce cell-surface expression, although S-mediated cell fusion was reduced by more than 50% in comparison to the wild-type S. Similarly, mutagenesis of the cysteine cluster II located adjacent to cluster I reduced S-mediated cell fusion by more than 60% compared to the wild-type S, while cell-surface expression was reduced by less than 20%. Mutagenesis of cysteine clusters III and IV did not appreciably affect S cell-surface expression or S-mediated cell fusion. The wild-type S was palmitoylated as evidenced by the efficient incorporation of 3H-palmitic acid in wild-type S molecules. S glycoprotein palmitoylation was significantly reduced for mutant glycoproteins having cluster I and II cysteine changes, but was largely unaffected for cysteine cluster III and IV mutants. These results show that the S cytoplasmic domain is palmitoylated and that palmitoylation of the membrane proximal cysteine clusters I and II may be important for S-mediated cell fusion

  20. Evidence that muscle cells do not express the histidine-rich glycoprotein associated with AMP deaminase but can internalise the plasma protein

    Directory of Open Access Journals (Sweden)

    A.R.M. Sabbatini

    2011-02-01

    Full Text Available Histidine-rich glycoprotein (HRG is synthesized by liver and is present at relatively high concentration in the plasma of vertebrates. We have previously described the association of a HRG-like molecule to purified rabbit skeletal muscle AMP deaminase (AMPD. We also provided the first evidence for the presence of a HRG-like protein in human skeletal muscle where a positive correlation between HRG content and total determined AMPD activity has been shown. In the present paper we investigate the origin of skeletal muscle HRG. The screening of a human skeletal muscle cDNA expression library using an anti-HRG antibody failed to reveal any positive clone. The RT-PCR analysis, performed on human skeletal muscle RNA as well as on RNA from the rhabdomyosarcoma (RD cell line, failed to show any mRNA specific for the plasma HRG or for the putative muscle variant. When the RD cells were incubated with human plasma HRG, a time-dependent increase of the HRG immunoreactivity was detected both at the plasma membrane level and intracellularly. The internalisation of HRG was inhibited by the addition of heparin. The above data strongly suggest that skeletal muscle cells do not synthesize the muscle variant of HRG but instead can actively internalise it from plasma.

  1. Characterization of naturally acquired multiple-drug resistance of Yoshida rat ascites hepatoma AH66 cell line.

    Science.gov (United States)

    Miyamoto, K; Wakabayashi, D; Minamino, T; Nomura, M; Wakusawa, S; Nakamura, S

    1996-01-01

    Characteristics of multiple-drug resistance of rat ascites hepatoma AH66, a cell line induced by dimethylaminoazobenzene and established as a transplantable tumor, were compared with those of AH66F, a drug sensitive line obtained from AH66. The AH66 cell line was resistant to vinblastine, adriamycin, SN-38 an active form of camptothesine, etoposide, and clorambucil by 10-fold or more than the AH66F cell line. The resistance of AH66 cells to vinblastine, adriamycin, and SN-38 was closely related to P-glycoprotein overexpression in the plasma membrane, because the resistance was significantly inhibited by verapamil. AH66 cells contained much glutahione and had a high activity of glutathione S-transferase P-form (GST-P), compared with AH66F cells, and resistance to clorambucil was decreased by treatment with buthionine sulfoximine, an inhibitor of glutathione synthesis. AH66 cells have a similar topoisomerase I activity, but about 6 times lower topoisomerase II activity than AH66F cells. Therefore, the resistance to etoposide and a part of the resistance to adriamycin of AH66 cells seems to depend upon this low topoisomerase II activity. These results, show that the AH66 cell line has high multiple-drug resistance compared with the AH66F cell line, by several mechanisms. Consequently, the AH66 and AH66F cell lines are useful to study naturally acquired multiple-drug resistance of hepatomas. PMID:8702243

  2. Herpesvirus 6 Glycoproteins B (gB), gH, gL, and gQ Are Necessary and Sufficient for Cell-to-Cell Fusion

    OpenAIRE

    Tanaka, Yuki; Suenaga, Tadahiro; Matsumoto, Misako; Seya, Tsukasa; Arase, Hisashi

    2013-01-01

    The human herpesvirus 6 (HHV-6) envelope glycoprotein gH/gL/gQ1/gQ2 complex associates with host cell CD46 as its cellular receptor. Although gB has been suggested to be involved in HHV-6 infection, its function in membrane fusion has remained unclear. Here, we have developed an HHV-6A (strain GS)and HHV-6B (strain Z29) virus-free cell-to-cell fusion assay and demonstrate that gB and the gH/gL/gQ1/gQ2 complex are the minimum components required for membrane fusion by HHV-6.

  3. Impact of BCRP/MXR, MRP1 and MDR1/P-Glycoprotein on thermoresistant variants of atypical and classical multidrug resistant cancer cells

    DEFF Research Database (Denmark)

    Stein, Ulrike; Lage, Hermann; Jordan, Andreas;

    2002-01-01

    The impact of the ABC transporters breast cancer resistance protein/mitoxantrone resistance associated transporter (BCRP/MXR), multidrug resistance-associated protein 1 (MRP1) and multidrug resistance gene-1/P-glycoprotein (MDR1/PGP) on the multidrug resistance (MDR) phenotype in chemoresistance...... and thermoresistance was investigated in the parental human gastric carcinoma cell line EPG85-257P, the atypical MDR subline EPG85-257RNOV, the classical MDR subline EPG85-257RDB and their thermoresistant counterparts EPG85-257P-TR, EPG85-257RNOV-TR and EPG85-257RDB-TR. Within the atypical MDR subline...... EPG85-257RNOV expression of BCRP/MXR and of MRP1 were clearly enhanced (vs. parental and classical MDR lines). MDR1/PGP expression was distinctly elevated in the classical MDR subline EPG85-257RDB (vs. parental and atypical MDR sublines). In all thermoresistant counterparts basal expression of BCRP...

  4. Plant Cell Lines in Cell Morphogenesis Research

    Czech Academy of Sciences Publication Activity Database

    Seifertová, Daniela; Klíma, Petr; Pařezová, Markéta; Petrášek, Jan; Zažímalová, Eva; Opatrný, Z.

    Vol. 1080. New York: Humana Press, 2014 - (Žárský, V.; Cvrčková, F.), s. 215-229. (Methods in Molecular Biology). ISBN 978-1-62703-643-6 R&D Projects: GA ČR(CZ) GAP305/11/0797; GA ČR(CZ) GAP305/11/2476 Institutional support: RVO:61389030 Keywords : BY-2 * VBI-0 * Suspension-cultured cells Subject RIV: EB - Genetics ; Molecular Biology

  5. Stressor-dependent Alterations in Glycoprotein 130: Implications for Glial Cell Reactivity, Cytokine Signaling and Ganglion Cell Health in Glaucoma

    Science.gov (United States)

    Echevarria, FD; Walker, CC; Abella, SK; Won, M; Sappington, RM

    2013-01-01

    Objective: The interleukin-6 (IL-6) family of cytokines is associated with retinal ganglion cell (RGC) survival and glial reactivity in glaucoma. The purpose of this study was to evaluate glaucoma-related changes in glycoprotein-130 (gp130), the common signal transducer of the IL-6 family of cytokines, as they relate to RGC health, glial reactivity and expression of IL-6 cytokine family members. Methods: For all experiments, we examined healthy retina (young C57), aged retina (aged C57), retina predisposed to glaucoma (young DBA/2) and retina with IOP-induced glaucoma (aged DBA/2). We determined retinal gene expression of gp130 and IL-6 family members, using quantitative PCR, and protein expression of gp130, using multiplex ELISA. For protein localization and cell-specific expression, we performed co-immunolabeling for gp130 and cell type-specific markers. We used quantitative microscopy to measure layer-specific expression of gp130 and its relationships to astrocyte and Müller glia reactivity and RGC axonal transport, as determined by uptake and transport of cholera toxin β-subunit (CTB). Results: Gene expression of gp130 was elevated with all glaucoma-related stressors, but only normal aging increased protein levels. In healthy retina, gp130 localized primarily to the inner retina, where it was expressed by astrocytes, Müller cells and RGCs. Layer-specific analysis of gp130 expression revealed increased expression in aging retina and decreased expression in glaucomatous retina that was eccentricity-dependent. These glaucoma-related changes in gp130 expression correlated with the level of GFAP and glutamine synthetase expression, as well as axonal transport in RGCs. The relationships between gp130, glial reactivity and RGC health could impact signaling by many IL-6 family cytokines, which exhibited overall increased expression in a stressor-dependent manner. Conclusions: Glaucoma-related stressors, including normal aging, glaucoma predisposition and IOP

  6. P-Glycoprotein/MDR1 regulates pokemon gene transcription through p53 expression in human breast cancer cells.

    Science.gov (United States)

    He, Shengnan; Liu, Feng; Xie, Zhenhua; Zu, Xuyu; Xu, Wei; Jiang, Yuyang

    2010-01-01

    P-glycoprotein (Pgp), encoded by the multidrug resistance 1 (MDR1) gene, is an efflux transporter and plays an important role in pharmacokinetics. In this study, we demonstrated that the pokemon promoter activity, the pokemon mRNA and protein expression can be significantly inhibited by Pgp. Chromatin immunoprecipitation assay showed that Pgp can bind the pokemon prompter to repress pokemon transcription activity. Furthermore, Pgp regulated pokemon transcription activity through expression of p53 as seen by use of p53 siRNA transfected MCF-7 cells or p53 mutated MDA-MB-231 cells. Moreover, p53 was detected to bind with Pgp in vivo using immunoprecipitation assay. Taken together, we conclude that Pgp can regulate the expression of pokemon through the presence of p53, suggesting that Pgp is a potent regulator and may offer an effective novel target for cancer therapy. PMID:20957096

  7. P-Glycoprotein/MDR1 Regulates Pokemon Gene Transcription Through p53 Expression in Human Breast Cancer Cells

    Directory of Open Access Journals (Sweden)

    Wei Xu

    2010-08-01

    Full Text Available P-glycoprotein (Pgp, encoded by the multidrug resistance 1 (MDR1 gene, is an efflux transporter and plays an important role in pharmacokinetics. In this study, we demonstrated that the pokemon promoter activity, the pokemon mRNA and protein expression can be significantly inhibited by Pgp. Chromatin immunoprecipitation assay showed that Pgp can bind the pokemon prompter to repress pokemon transcription activity. Furthermore, Pgp regulated pokemon transcription activity through expression of p53 as seen by use of p53 siRNA transfected MCF-7 cells or p53 mutated MDA-MB-231 cells. Moreover, p53 was detected to bind with Pgp in vivo using immunoprecipitation assay. Taken together, we conclude that Pgp can regulate the expression of pokemon through the presence of p53, suggesting that Pgp is a potent regulator and may offer an effective novel target for cancer therapy.

  8. The leukocyte common antigen (CD45) on human pre-B leukemia cells: variant glycoprotein form expression during the cell exposure to phorbol ester is blocked by a nonselective protein kinase inhibitor H7

    International Nuclear Information System (INIS)

    The human pre-B acute lymphoblastic leukemia cell line REH6 was utilized for characterization of CD45 glycoprotein by monoclonal antibodies (mAb) recognizing four distinct CD45 antigen specificities, i.e. nonrestricted CD45, restricted, CD45RA, CD45RB and CD45R0. Immunoprecipitation revealed two antigen specificities on REH6 cells of m.w. 220 kDa and 190 kDa, both presenting wide range of isoelectric point pI∼6.0-7.5. Nonrestricted CD45 epitopes were not affected by the sialyl acid cleavage with sodium meta-periodate or neuraminidase, but were sensitive to both, tunicamycin, the N-glycosylation inhibitor and monensin, an inhibitor of protein transport through the Golgi compartment. O-sialoglycoprotein endopeptidase from Pasteurella haemolytica A1 partially cleaved CD45RA and CD45RB epitopes, while nonrestricted CD45 determinants were not affected by this enzyme. Limited proteolysis of this antigen resulted in the appearance of 160-180 kDa peptide domains which retained CD45 epitopes. Further, the treatment of cells with phorbol myristate acetate (PMA) induced marked down-regulation of 220 and 190 kDa isoforms and the appearance of new 210, 180 and 170 kDa variant glycoprotein forms which were not found on parental cells. This PMA effect was not accompanied by the programmed cell death and was markedly blocked by a nonselective protein kinase (PK) inhibitor iso-quinoline sulfonamide H7. Modulation of CD45 by phorbol esters might serve as an in vitro model for an additional insight into the function of CD45 in hematopoietic cells. (author)

  9. Cancer stem cell-like cells from a single cell of oral squamous carcinoma cell lines

    Energy Technology Data Exchange (ETDEWEB)

    Felthaus, O. [Department of Operative Dentistry and Periodontology, University of Regensburg (Germany); Department of Oral and Maxillofacial Surgery, University of Regensburg (Germany); Ettl, T.; Gosau, M.; Driemel, O. [Department of Oral and Maxillofacial Surgery, University of Regensburg (Germany); Brockhoff, G. [Department of Gynecology and Obstetrics, University of Regensburg (Germany); Reck, A. [Department of Oral and Maxillofacial Surgery, University of Regensburg (Germany); Zeitler, K. [Institute of Pathology, University of Regensburg (Germany); Hautmann, M. [Department of Radiotherapy, University of Regensburg (Germany); Reichert, T.E. [Department of Oral and Maxillofacial Surgery, University of Regensburg (Germany); Schmalz, G. [Department of Operative Dentistry and Periodontology, University of Regensburg (Germany); Morsczeck, C., E-mail: christian.morsczeck@klinik.uni-regensburg.de [Department of Operative Dentistry and Periodontology, University of Regensburg (Germany)

    2011-04-01

    Research highlights: {yields} Four oral squamous cancer cell lines (OSCCL) were analyzed for cancer stem cells (CSCs). {yields} Single cell derived colonies of OSCCL express CSC-marker CD133 differentially. {yields} Monoclonal cell lines showed reduced sensitivity for Paclitaxel. {yields} In situ CD133{sup +} cells are slow cycling (Ki67-) indicating a reduced drug sensitivity. {yields} CD133{sup +} and CSC-like cells can be obtained from single colony forming cells of OSCCL. -- Abstract: Resistance of oral squamous cell carcinomas (OSCC) to conventional chemotherapy or radiation therapy might be due to cancer stem cells (CSCs). The development of novel anticancer drugs requires a simple method for the enrichment of CSCs. CSCs can be enriched from OSCC cell lines, for example, after cultivation in serum-free cell culture medium (SFM). In our study, we analyzed four OSCC cell lines for the presence of CSCs. CSC-like cells could not be enriched with SFM. However, cell lines obtained from holoclone colonies showed CSC-like properties such as a reduced rate of cell proliferation and a reduced sensitivity to Paclitaxel in comparison to cells from the parental lineage. Moreover, these cell lines differentially expressed the CSC-marker CD133, which is also upregulated in OSCC tissues. Interestingly, CD133{sup +} cells in OSCC tissues expressed little to no Ki67, the cell proliferation marker that also indicates reduced drug sensitivity. Our study shows a method for the isolation of CSC-like cell lines from OSCC cell lines. These CSC-like cell lines could be new targets for the development of anticancer drugs under in vitro conditions.

  10. Cancer stem cell-like cells from a single cell of oral squamous carcinoma cell lines

    International Nuclear Information System (INIS)

    Research highlights: → Four oral squamous cancer cell lines (OSCCL) were analyzed for cancer stem cells (CSCs). → Single cell derived colonies of OSCCL express CSC-marker CD133 differentially. → Monoclonal cell lines showed reduced sensitivity for Paclitaxel. → In situ CD133+ cells are slow cycling (Ki67-) indicating a reduced drug sensitivity. → CD133+ and CSC-like cells can be obtained from single colony forming cells of OSCCL. -- Abstract: Resistance of oral squamous cell carcinomas (OSCC) to conventional chemotherapy or radiation therapy might be due to cancer stem cells (CSCs). The development of novel anticancer drugs requires a simple method for the enrichment of CSCs. CSCs can be enriched from OSCC cell lines, for example, after cultivation in serum-free cell culture medium (SFM). In our study, we analyzed four OSCC cell lines for the presence of CSCs. CSC-like cells could not be enriched with SFM. However, cell lines obtained from holoclone colonies showed CSC-like properties such as a reduced rate of cell proliferation and a reduced sensitivity to Paclitaxel in comparison to cells from the parental lineage. Moreover, these cell lines differentially expressed the CSC-marker CD133, which is also upregulated in OSCC tissues. Interestingly, CD133+ cells in OSCC tissues expressed little to no Ki67, the cell proliferation marker that also indicates reduced drug sensitivity. Our study shows a method for the isolation of CSC-like cell lines from OSCC cell lines. These CSC-like cell lines could be new targets for the development of anticancer drugs under in vitro conditions.

  11. Comparison of Haptoglobin and Alpha1-Acid Glycoprotein Glycosylation in the Sera of Small Cell and Non-Small Cell Lung Cancer Patients

    Directory of Open Access Journals (Sweden)

    Mirosława Ferens-Sieczkowska

    2013-08-01

    Full Text Available Introduction: Cancer-related carbohydrate epitopes, which are regarded as potential diagnostic and prognostic biomarkers, are carried on the main acute phase proteins. It is not clear, however, if the glycosylation profile is similar in different glycoproteins, or it is protein specific to some extent. The aim of the study was to compare fucosylation, α2,3 sialylation and expression of sialyl-Lewisx epitopes (sLex in the serum as a whole, AGP and haptoglobin of small cell (SCLC and non-small cell lung cancer (NSCLC patients with respect to healthy subjects as well as the cancer stage and its histological type.Material and Methods: Thirty-three NSCLC, 13 SCLC patients and 20 healthy volunteers were included in the study. Carbohydrate epitopes were detected by means of their reactivity with specific lectins and monoclonal anti-sLex antibodies in direct or dual-ligand ELISA tests.Results: Significantly increased fucosylation was found in total serum in both cancer groups and in NSCLC haptoglobin. No difference was observed in SCLC haptoglobin or α1-acid glycoprotein in both cancer groups. Also α2,3 sialylation was elevated in total serum, but not in α1-acid glycoprotein. This type of sialylation was undetectable in haptoglobin by means of MAA reactivity, in both healthy and cancer subjects. Complete sLex antigens were overexpressed in total NSCLC serum and SCLC AGP, and their level was considerably lowered in cancer haptoglobin.Discussion: Typical acute phase proteins, haptoglobin and AGP, exhibit different glycosylation profiles in lung cancer. Alterations observed in haptoglobin reflected the disease process better than those in AGP. Comparison of haptoglobin and AGP glycosylation to that observed in total serum suggests that some efficient carriers of disease-altered glycoproteins still remain unidentified.

  12. DNA hypomethylation upregulates expression of the MGAT3 gene in HepG2 cells and leads to changes in N-glycosylation of secreted glycoproteins.

    Science.gov (United States)

    Klasić, Marija; Krištić, Jasminka; Korać, Petra; Horvat, Tomislav; Markulin, Dora; Vojta, Aleksandar; Reiding, Karli R; Wuhrer, Manfred; Lauc, Gordan; Zoldoš, Vlatka

    2016-01-01

    Changes in N-glycosylation of plasma proteins are observed in many types of cancer, nevertheless, few studies suggest the exact mechanism involved in aberrant protein glycosylation. Here we studied the impact of DNA methylation on the N-glycome in the secretome of the HepG2 cell line derived from hepatocellular carcinoma (HCC). Since the majority of plasma glycoproteins originate from the liver, the HepG2 cells represent a good model for glycosylation changes in HCC that are detectable in blood, which is an easily accessible analytic material in a clinical setting. Two different concentrations of 5-aza-2'-deoxycytidine (5-aza-2dC) differentially affected global genome methylation and induced different glycan changes. Around twenty percent of 84 glyco-genes analysed changed expression level after the 5-aza-2dC treatment as a result of global genome hypomethylation. A correlation study between the changes in glyco-gene expression and the HepG2 glycosylation profile suggests that the MGAT3 gene might be responsible for the glycan changes consistently induced by both doses of 5-aza-2dC. Core-fucosylated tetra-antennary structures were decreased in quantity likely as a result of hypomethylated MGAT3 gene promoter followed by increased expression of this gene. PMID:27073020

  13. Effect of the infectious laryngotracheitis virus (ILTV) glycoprotein G on virus attachment, penetration, growth curve and direct cell-to-cell spread

    Institute of Scientific and Technical Information of China (English)

    SUN; Zhaogang; ZHANG; Manfu

    2005-01-01

    The secreted alphaherpesvirus glycoprotein G (gG) works differently from other proteins. Analysis of the role of ILTV gG in virus attachment, penetration, direct cell-to-cell spread (CTCS) and the growth curve showed that gG or its antibody had no effect on ILTV attachment and penetration and that the gG antibody reduced the virus plaque size and the one-step growth curve on chicken embryo liver (CEL) cells, but gG did not affect the virus plaque size or the one-step growth curve on CEL cells. Laser scanning confocal microscopy (LSCM) detection showed that ILTV gG is located in the perinuclear region and the membrane of the CEL cells. These results suggested that ILTV gG might contribute to direct cell-to-cell transmission.

  14. Reversion effects of curcumin on multidrug resistance of MNNG/HOS human osteosarcoma cells in vitro and in vivo through regulation of P-glycoprotein

    Institute of Scientific and Technical Information of China (English)

    SI Meng; ZHAO Jie; LI Xin; TIAN Ji-guang; LI Yong-gang; LI Jian-min

    2013-01-01

    Background P-glycoprotein (P-gp) encoded by ATP-binding cassette sub-family B member 1 (ABCB1) gene is a kind of ATP-dependent drug transporter,which plays important roles in multidrug resistance (MDR) of human cancers,such as osteosarcoma.Curcumin is a natural phenolic coloring compound originating from the rhizomes of Curcuma longa,which is proved to possess antitumor biological activities including reversion of MDR.However,the effect and molecular mechanisms of curcumin to osteosarcoma MDR remain unclear.Methods We established a human osteosarcoma drug-resistant cell line MNNG/HOS/MTX by pulse exposure to methotrexate (MTX) and verified that the new cell lines were cross-resistant to other anticancer agents.Then,according to the cytotoxicity assay,we reversed MDR of MNNG/HOS/MTX by 30 μmol/L curcumin,and detected the mechanisms of curcumin reversing MDR through Real-time PCR,Western blotting assay,and Rhodamine123 (Rh123)transport test.Finally,we evaluated the effect of curcumin reversing MDR in vivo by MNNG/HOS/MTX cells xenograft-nude mice model.Results MNNG/HOS/MTX was proved to be a human osteosarcoma MDR cell line.MTT tumor chemosensitivity test indicates that 30 μmol/L curcumin attenuates the half maximal inhibitory concentration (IC50) and resistance index (RI)to MTX,diamminedichloroplatinum (DDP),adriamycin (ADM),ifosfamide (IFO),and epirubicin (EPI) in MNNG/HOS/MTX cells (P <0.05).Real-time PCR and Western blotting assays demonstrated that curcumin down-regulated P-gp expression of MNNG/HOS/MTX cells.Rh123 transport test showed that curcumin inhibited the transport function of P-gp in vitro.In vivo studies showed that curcumin displayed the features of sensitizing antitumor drugs and inhibiting the proliferation,invasion,and metastasis of osteosarcoma MDR cells.Conclusion Down-regulation of P-gp and inhibition of the function of P-gp efflux pump may contribute to MDR reversion induced by curcumin in vitro and in vivo.

  15. Rhesus and Human Cytomegalovirus Glycoprotein L Are Required for Infection and Cell-to-Cell Spread of Virus but Cannot Complement Each Other▿

    OpenAIRE

    Bowman, J. Jason; Lacayo, Juan C.; Burbelo, Peter; Fischer, Elizabeth R.; Cohen, Jeffrey I.

    2010-01-01

    Rhesus cytomegalovirus (RhCMV), the homolog of human cytomegalovirus (HCMV), serves as a model for understanding the pathogenesis of HCMV and for developing candidate vaccines. In order to develop a replication-defective virus as a vaccine candidate, we constructed RhCMV with glycoprotein L (gL) deleted. RhCMV gL was essential for viral replication, and virus with gL deleted could only replicate in cells expressing RhCMV gL. Noncomplementing cells infected with RhCMV with gL deleted released ...

  16. Studies on secretory glycoproteins in the rat exocrine pancreas. III. Intracellular transport of fucose-labeled proteins as studied by cell fractionation.

    Science.gov (United States)

    Völkl, A; Schick, J; Adler, G; Kern, H F

    1978-10-01

    The transcellular movement of fucosylated glycoproteins has been studied in vitro using rat pancreatic lobules and cell fractionation procedures, and has been compared with the well established pathway of secretory proteins. Using tritiated leucine as pulse label for the latter, their translocation from the rough endoplasmatic reticulum into the Golgi complex and finally into zymogen granules could be followed. In the case of glycoproteins, 14C-fucose was incorporated mainly into the smooth microsomal fraction (representative of the Golgi complex) and only one third of this specific activity was transported into the zymogen granule fraction. A detailed analysis of this fraction after separation of the content of zymogen granules from their membranes revealed a predominant labeling of membrane glycoproteins by 14C-fucose. In comparison, leucine-labeled bulk proteins were found almost exclusively in the zymogen granule content fraction, with little radioactivity in the membrane fraction. The data indicate a concomitant synthesis of fucosylated glycoproteins destined in part for the zymogen granule membrane and to a greater amount associated with the smooth microsomal fraction. The results are discussed in the light of recent findings indicating that about 40% of the proteins in the zymogen granule membrane are made up of one major glycoprotein which could be involved in the mechanism of exocytosis. PMID:214234

  17. Comparative uptake of Tc-99m sestamibi and Tc-99m tetrofosmin in cancer cells and tissue expressing P-Glycoprotein or multidrug resistance associated protein

    International Nuclear Information System (INIS)

    99mTc-sestamibi(MIBI) and 99mTc-tetrofosmin have been used as substrates for P-glycoprotein (Pgp) and multidrug resistance associated protein (MRP), which are closely associated with multidrug resistance of the tumors. To understand different handling of radiotracers in cancer cell lines expressing Pgp and MRP, we compared cellular uptakes of 99mTc-MIBI and 99mTc-tetrofosmin. The effects of cyclosporin A (CsA), well-known multidrug resistant reversing agent, on the uptake of both tracers were also compared. HCT15/CL02 human colorectal cancer cells for Pgp expressing cells, and human non-small cell lung cancer A549 cells for MRP expressing cells, were used for in vitro and in vivo studies. RT-PCR, western blot analysis and immunohistochemistry were used for detection of Pgp and MRP. MDR-reversal effect with CsA was evaluated at different drug concentrations after incubation with MIBI or tetrofosmin. Radioactivities of supernatant and pellet were measured with gamma well counter. Tumoral uptake of the tracers were measured from tumor bearing nude mice treated with or without CsA. RT-PCR, western blot analysis of the cells and immunochemical staining revealed selective expression of Pgp and MRP for HCT15/CL02 and A549 cells, respectively. There were no significant difference in cellular uptakes of both tracers in HCT15/CL02 cells, but MIBI uptake was slightly higher than that of tetrofosmin in A549 cells. Co-incubation with CsA resulted in a increase in cellular uptakes of MIBI and tetrofosmin. Uptake of MIBI or tetrofosmin in HCT15/CL02 cells was increased by 10-and 2.4-fold, and by 7.5 and 6.3-fold in A549 cells, respectively. Percentage increase of MIBI was higher than that of tetrofosmin with CsA for both cells (ρ < 0.05). In vivo biodistribution study showed that MIBI (114% at 10 min, 257% at 60 min, 396% at 24C min) and tetrofosmin uptake (110% at 10 min, 205% at 60 min, 410% at 240 min) were progressively increased by the time, up to 240 min with CsA. But

  18. Comparative uptake of Tc-99m sestamibi and Tc-99m tetrofosmin in cancer cells and tissue expressing P-Glycoprotein or multidrug resistance associated protein

    Energy Technology Data Exchange (ETDEWEB)

    Cho, Jung Ah; Lee, Jae Tae; Yoo, Jung Ah [School of Medicine, Kyungpook National University, Daegu (Korea, Republic of)] (and others)

    2005-02-15

    {sup 99m}Tc-sestamibi(MIBI) and {sup 99m}Tc-tetrofosmin have been used as substrates for P-glycoprotein (Pgp) and multidrug resistance associated protein (MRP), which are closely associated with multidrug resistance of the tumors. To understand different handling of radiotracers in cancer cell lines expressing Pgp and MRP, we compared cellular uptakes of {sup 99m}Tc-MIBI and {sup 99m}Tc-tetrofosmin. The effects of cyclosporin A (CsA), well-known multidrug resistant reversing agent, on the uptake of both tracers were also compared. HCT15/CL02 human colorectal cancer cells for Pgp expressing cells, and human non-small cell lung cancer A549 cells for MRP expressing cells, were used for in vitro and in vivo studies. RT-PCR, western blot analysis and immunohistochemistry were used for detection of Pgp and MRP. MDR-reversal effect with CsA was evaluated at different drug concentrations after incubation with MIBI or tetrofosmin. Radioactivities of supernatant and pellet were measured with gamma well counter. Tumoral uptake of the tracers were measured from tumor bearing nude mice treated with or without CsA. RT-PCR, western blot analysis of the cells and immunochemical staining revealed selective expression of Pgp and MRP for HCT15/CL02 and A549 cells, respectively. There were no significant difference in cellular uptakes of both tracers in HCT15/CL02 cells, but MIBI uptake was slightly higher than that of tetrofosmin in A549 cells. Co-incubation with CsA resulted in a increase in cellular uptakes of MIBI and tetrofosmin. Uptake of MIBI or tetrofosmin in HCT15/CL02 cells was increased by 10-and 2.4-fold, and by 7.5 and 6.3-fold in A549 cells, respectively. Percentage increase of MIBI was higher than that of tetrofosmin with CsA for both cells ({rho} < 0.05). In vivo biodistribution study showed that MIBI (114% at 10 min, 257% at 60 min, 396% at 24C min) and tetrofosmin uptake (110% at 10 min, 205% at 60 min, 410% at 240 min) were progressively increased by the time, up to

  19. P-glycoprotein in adriamycin-resistant cells functions as an efflux pump for benzopyrene, a chemical carcinogen

    Energy Technology Data Exchange (ETDEWEB)

    Chao Yeh, G.; Poore, C.M.; Lopaczynska, J.; Phang, J.M. (NCI-FCRDC, Frederick, MD (United States))

    1991-03-15

    The physiological function of multidrug resistant gene (MDR 1) coded P-glycoprotein 170 (P-gp) in normal tissues remains unknown. The authors propose that P-gp functions as an efflux pump in normal tissues for benzopyrene and other xenobiotic substances. To examine their hypothesis the authors used a series of MDR human breast cancer MCF-7 cells with increasing degrees of drug resistance, expression of MDR and levels of P-gp. First, they found the IC{sub 50} for benzopyrene is linearly correlated with the levels of P-gp at different stages of adriamycin resistant MCF-7 cells. Using P-gp ({sup 3}H)azidopine labeling as a measurement of P-gp they found benzopyrene competes for labeling of P-gp. Finally, they directly measured cellular efflux of benzopyrene with adherent cell laser cytometry and found that resistant cells expressing high levels of P-gp showed rapid efflux of benzopyrene. By contrast, drug sensitive wild type cells with undetectable P-gp showed negligible efflux. They conclude that P-gp can function as an efflux pump for benzopyrene and suggest that P-gp may be a cellular mechanism for resistance to carcinogens.

  20. Cellular radiosensitivity of small-cell lung cancer cell lines

    DEFF Research Database (Denmark)

    Krarup, M; Poulsen, H S; Spang-Thomsen, M

    1997-01-01

    PURPOSE: The objective of this study was to determine the radiobiological characteristics of a panel of small-cell lung cancer (SCLC) cell lines by use of a clonogenic assay. In addition, we tested whether comparable results could be obtained by employing a growth extrapolation method based on the...

  1. Probing the O-glycoproteome of Gastric Cancer Cell Lines for Biomarker Discovery

    DEFF Research Database (Denmark)

    Vieira Campos, Diana Alexandra; Freitas, Daniela; Gomes, Joana; Magalhães, Ana; Steentoft, Catharina; Gomes, Catarina; Vester-Christensen, Malene B; Ferreira, José Alexandre; Afonso, Luis P; Santos, Lúcio L; de Sousa, João Pinto; Mandel, Ulla; Clausen, Henrik; Vakhrushev, Sergey Y; Reis, Celso A

    2015-01-01

    Circulating O-glycoproteins shed from cancer cells represent important serum biomarkers for diagnostic and prognostic purposes. We have recently shown that selective detection of cancer-associated aberrant glycoforms of circulating O-glycoprotein biomarkers can increase specificity of cancer biom...

  2. Relation between the regulation of DNA synthesis and the production of two secreted glycoproteins by 12-O-tetradecanoylphorbol-13-acetate in 3T3 cells and in phorbol ester nonresponsive 3T3 variants

    International Nuclear Information System (INIS)

    12-O-tetradecanoylphorbol-13-acetate (TPA), a potent tumor promoter, acts similarly to growth factors by selectively increasing the rate of production of the secreted proteins, mitogen regulated protein (MRP) and major excreted protein (MEP) by murine 3T3 cells. MRP, a 34 kilodalton (kDa) glycoprotein, is a member of the prolactin-growth hormone family of proteins. MEP, a 39 kDa glycoprotein, is a lysosomal thiol protease that is also secreted. The aim of this investigation was to determine the relation between increases in MRP and MEP production and the initiation of DNA synthesis in response to mitogens. The TNR-9 cell line is a variant of 3T3 cells in which growth factors, but not TPA and teleocidin, stimulate DNA synthesis and cell division. Using [35S]methionine to metabolically label proteins and SDS polyacrylamide gel electrophoresis to resolve the proteins, they found that growing cultures of 3T3 and TNR-9 cells responded equally well to TPA and teleocidin with increased rates of production of MRP and MEP. By contrast, the responses of quiescent TNR-9 cells to these tumor promoters in the increased production of MRP and MEP was greatly diminished compared with quiescent 3T3 cells. In summary, the ability to TPA and teleocidin to increase the rate of production of MRP and MEP correlated with the ability of these tumor promoters to stimulated DNA synthesis in quiescent 3T3 and TNR-9 cells. Evidently the biochemical condition that distinguishes TNR-9 from 3T3 cells and that limits the ability of tumor promoters to stimulate the production of MEP and MRP, and perhaps also DNA synthesis in TNR-9 cells occurs only when cells are quiescent

  3. Multidrug resistance in tumour cells: characterisation of the multidrug resistant cell line K562-Lucena 1

    Directory of Open Access Journals (Sweden)

    VIVIAN M. RUMJANEK

    2001-03-01

    Full Text Available Multidrug resistance to chemotherapy is a major obstacle in the treatment of cancer patients. The best characterised mechanism responsible for multidrug resistance involves the expression of the MDR-1 gene product, P-glycoprotein. However, the resistance process is multifactorial. Studies of multidrug resistance mechanisms have relied on the analysis of cancer cell lines that have been selected and present cross-reactivity to a broad range of anticancer agents. This work characterises a multidrug resistant cell line, originally selected for resistance to the Vinca alkaloid vincristine and derived from the human erythroleukaemia cell K562. This cell line, named Lucena 1, overexpresses P-glycoprotein and have its resistance reversed by the chemosensitisers verapamil, trifluoperazine and cyclosporins A, D and G. Furthermore, we demonstrated that methylene blue was capable of partially reversing the resistance in this cell line. On the contrary, the use of 5-fluorouracil increased the resistance of Lucena 1. In addition to chemotherapics, Lucena 1 cells were resistant to ultraviolet A radiation and hydrogen peroxide and failed to mobilise intracellular calcium when thapsigargin was used. Changes in the cytoskeleton of this cell line were also observed.A resistência a múltiplos fármacos é o principal obstáculo no tratamento de pacientes com câncer. O mecanismo responsável pela resistência múltipla mais bem caracterizado envolve a expressão do produto do gene MDR-1, a glicoproteína P. Entretanto, o processo de resistência tem fatores múltiplos. Estudos de mecanismos de resistência m��ltipla a fármacos têm dependido da análise de linhagens celulares tumorais que foram selecionadas e apresentam reatividade cruzada a uma ampla faixa de agentes anti-tumorais. Este trabalho caracteriza uma linhagem celular com múltipla resistência a fármacos, selecionada originalmente pela resistência ao alcalóide de Vinca vincristina e derivado

  4. Expression of the chitinase family glycoprotein YKL-40 in undifferentiated, differentiated and trans-differentiated mesenchymal stem cells.

    Directory of Open Access Journals (Sweden)

    Daniel J Hoover

    Full Text Available The glycoprotein YKL-40 (CHI3L1 is a secreted chitinase family protein that induces angiogenesis, cell survival, and cell proliferation, and plays roles in tissue remodeling and immune regulation. It is expressed primarily in cells of mesenchymal origin, is overexpressed in numerous aggressive carcinomas and sarcomas, but is rarely expressed in normal ectodermal tissues. Bone marrow-derived mesenchymal stem cells (MSCs can be induced to differentiate into various mesenchymal tissues and trans-differentiate into some non-mesenchymal cell types. Since YKL-40 has been used as a mesenchymal marker, we followed YKL-40 expression as undifferentiated MSCs were induced to differentiate into bone, cartilage, and neural phenotypes. Undifferentiated MSCs contain significant levels of YKL-40 mRNA but do not synthesize detectable levels of YKL-40 protein. MSCs induced to differentiate into chondrocytes and osteocytes soon began to express and secrete YKL-40 protein, as do ex vivo cultured chondrocytes and primary osteocytes. In contrast, MSCs induced to trans-differentiate into neurons did not synthesize YKL-40 protein, consistent with the general absence of YKL-40 protein in normal CNS parenchyma. However, these trans-differentiated neurons retained significant levels of YKL-40 mRNA, suggesting the mechanisms which prevented YKL-40 translation in undifferentiated MSCs remained in place, and that these trans-differentiated neurons differ in at least this way from neurons derived from neuronal stem cells. Utilization of a differentiation protocol containing β-mercaptoethanol resulted in cells that expressed significant amounts of intracellular YKL-40 protein that was not secreted, which is not seen in normal cells. Thus the synthesis of YKL-40 protein is a marker for MSC differentiation into mature mesenchymal phenotypes, and the presence of untranslated YKL-40 mRNA in non-mesenchymal cells derived from MSCs reflects differences between differentiated and

  5. Recombinant Outer Capsid Glycoprotein (VP7 of Rotavirus Expressed in Insect Cells Induces Neutralizing Antibodies in Rabbits

    Directory of Open Access Journals (Sweden)

    H Keyvani

    2012-04-01

    Full Text Available Background:Rotaviruses cause diarrhea in infants and young children worldwide. Rotavirus outer capsid protein, VP7 is major neutralizing antigen that is important component of subunit vaccine to prevent rotavirus infection.Many efforts have been done to produce recombinant VP7 that maintain native characteristics.We used baculovirus expression system to produce rotavirus VP7 protein and to study its immunogenicity. Methods: Simian rotavirus SA11 full-length VP7 ORF was cloned into a cloning plasmid and then the cloned gene was inserted into the linear DNA of baculovirus Autographa californica Nuclear Polyhedrosis Virus (AcNPV downstream of the polyhedrin promoter by in vitro recombination reactions. The expressed VP7 in the insect cells was recognized by rabbit hyperimmune serum raised against SA11 rotavirus by Immunofluorescence and western blotting assays. Rabbits were immunized subcutaneously by cell extracts expressing VP7 protein. Results: Reactivity with anti-rotavirus antibody suggested that expressed VP7 protein had native antigenic determinants.Injection of recombinant VP7 in rabbits elicited the production of serum antibodies,which were able to recognize VP7 protein from SA11 rotavirus by Western blotting test and neutralized SA11 rotavirus in cell culture.Conclusion: Recombinant outer capsid glycoprotein (VP7 of rotavirus expressed in insect cells induces neutralizing antibodies in rabbits and may be a candidate of rotavirus vaccine.

  6. A Molecular Switch Abrogates Glycoprotein 100 (gp100) T-cell Receptor (TCR) Targeting of a Human Melanoma Antigen*

    Science.gov (United States)

    Bianchi, Valentina; Bulek, Anna; Fuller, Anna; Lloyd, Angharad; Attaf, Meriem; Rizkallah, Pierre J.; Dolton, Garry; Sewell, Andrew K.; Cole, David K.

    2016-01-01

    Human CD8+ cytotoxic T lymphocytes can mediate tumor regression in melanoma through the specific recognition of HLA-restricted peptides. Because of the relatively weak affinity of most anti-cancer T-cell receptors (TCRs), there is growing emphasis on immunizing melanoma patients with altered peptide ligands in order to induce strong anti-tumor immunity capable of breaking tolerance toward these self-antigens. However, previous studies have shown that these immunogenic designer peptides are not always effective. The melanocyte differentiation protein, glycoprotein 100 (gp100), encodes a naturally processed epitope that is an attractive target for melanoma immunotherapies, in particular peptide-based vaccines. Previous studies have shown that substitutions at peptide residue Glu3 have a broad negative impact on polyclonal T-cell responses. Here, we describe the first atomic structure of a natural cognate TCR in complex with this gp100 epitope and highlight the relatively high affinity of the interaction. Alanine scan mutagenesis performed across the gp100280–288 peptide showed that Glu3 was critically important for TCR binding. Unexpectedly, structural analysis demonstrated that the Glu3 → Ala substitution resulted in a molecular switch that was transmitted to adjacent residues, abrogating TCR binding and T-cell recognition. These findings help to clarify the mechanism of T-cell recognition of gp100 during melanoma responses and could direct the development of altered peptides for vaccination. PMID:26917722

  7. Distribution features of the rats’ major salivary glands cells glycoproteins during early postnatal period after antenatal antigen action

    Directory of Open Access Journals (Sweden)

    Syrtsov V.K.

    2014-06-01

    animals group. Citation: Syrtsov VK, Maslova IN. Distribution features of the rats’ major salivary glands cells glycoproteins during early postnatal period after antenatal antigen action. Morphologia. 2014;8(2:56-60.

  8. Using 99mTc-MIBI to Evaluate the Effects of Chemosensitizer on P-glycoprotein in Multidrug-resistant Carcinoma Cells

    Institute of Scientific and Technical Information of China (English)

    ZHANGZhenwei; ZHANGXuemei; WUHua; ZHAOMing; XIANYUZhiqun; ZHOUJian; LAIShiying

    2005-01-01

    Objective: To establish a method to evaluate the effects of chemosensitizer on P-glycoprotein using 99mTc-MIBI, and observe the changes of 99mTc-MIBI uptake kinetics and P-glycoprotein levels after using verapamil in MDR human breast cells MCF-7/Adr. Methods: MDR breast carcinoma cells, MCF-7/Adr, were incubated and different protocols were performed. Protocol I: a chemosensitizer, verapamil (10μmol/L), was added into cell culture medium, while in control group, the same volume of DMEM was given. Cells were harvested after 2 h incubation with 99mTc-MIBI. Protocol Ⅱ: Verapamil (10μmol/L) was added into cell culture medium and incubated for 20 min, 40 min, 60 rain, 80 min, 8 h, 24 h, 48 h and 72 h respectively. Cells were harvested after 2 h incubation with 99mTc-MIBI. The radioactivity of the cells was measured and P-glycoprotein expression levels were determined with immunohistochemical stain. Results: Protocol I: After 2h incubation with verapamil the cellular uptake of 99mTc-MIBI was remarkably higher than control group (t=2.33, P0.05). Protocol

  9. The Neurospora crassa dfg5 and dcw1 Genes Encode α-1,6-Mannanases That Function in the Incorporation of Glycoproteins into the Cell Wall

    OpenAIRE

    Abhiram Maddi; Ci Fu; Free, Stephen J.

    2012-01-01

    The covalent cross-linking of cell wall proteins into the cell wall glucan/chitin matrix is an important step in the biogenesis of the fungal cell wall. We demonstrate that the Neurospora crassa DFG5 (NCU03770) and DCW1 (NCU08127) enzymes function in vivo to cross-link glycoproteins into the cell wall. Mutants lacking DFG5 or DCW1 release slightly elevated levels of cell wall proteins into their growth medium. Mutants lacking both DFG5 and DCW1 have substantially reduced levels of cell wall p...

  10. Enhancement of cellular uptake and cytotoxicity of curcumin-loaded PLGA nanoparticles by conjugation with anti-P-glycoprotein in drug resistance cancer cells

    Institute of Scientific and Technical Information of China (English)

    Wanisa PUNFA; Supachai YODKEEREE; Pornsiri PITCHAKARN; Chadarat AMPASAVATE; Pornngarm LIMTRAKUL

    2012-01-01

    Aim:To compare the anti-cancer activity and cellular uptake of curcumin (Cur) delivered by targeted and non-targeted drug delivery systems in multidrug-resistant cervical cancer cells.Methods:Cur was entrapped into poly (DL-lactide-co-glycolide) (PLGA) nanoparticles (Cur-NPs) in the presence of modified-pluronic F127 stabilizer using nano-precipitation technique.On the surface of Cur-NPs,the carboxy-terminal of modified pluronic F127 was conjugated to the amino-terminal of anti-P-glycoprotein (P-gp) (Cur-NPs-APgp).The physical properties of the Cur-NPs,including particle size,zeta potential,particle morphology and Cur release kinetics,were investigated.Cellular uptake and specificity of the Cur-NPs and Cur-NPs-APgp were detected in cervical cancer cell lines KB-V1 (higher expression of P-gp) and KB-3-1 (lower expression of P-gp) using fluorescence microscope and flow cytometry,respectively.Cytotoxicity of the Cur-NPs and Cur-NPs-APgp was determined using MTT assay.Results:The particle size of Cur-NPs and Cur-NPs-APgp was 127 and 132 nm,respectively.The entrapment efficiency and actual loading of Cur-NPs-APgp (60% and 5μg Cur/mg NP) were lower than those of Cur-NPs (99% and 7 μg Cur/mg NP).The specific binding of Cur-NPs-APgp to KB-V1 cells was significantly higher than that to KB-3-1 cells.Cellular uptake of Cur-NPs-APgp into KB-V1 cells was higher,as compared to KB-3-1 cells.However,the cellular uptake of Cur-NPs and Cur-NPs-lgG did not differ between the two types of cells.Besides,the cytotoxicity of Cur-NPs-APgp in KB-V1 cells was higher than those of Cur and Cur-NPs.Conclusion:The results demonstrate that Cur-NPs-APgp targeted to P-gp on the cell surface membrane of KB-V1 cells,thus enhancing the cellular uptake and cytotoxicity of Cur.

  11. Influence of Disulfide-Stabilized Structure on the Specificity of Helper T-Cell and Antibody Responses to HIV Envelope Glycoprotein gp120▿ †

    OpenAIRE

    Mirano-Bascos, Denise; Steede, N. Kalaya; Robinson, James E.; Landry, Samuel J.

    2010-01-01

    CD4+ helper T cells specific for human immunodeficiency virus type 1 (HIV-1) are associated with control of viremia. Nevertheless, vaccines have had limited effectiveness thus far, in part because sequence variability and other structural features of the HIV envelope glycoprotein deflect the immune response. Previous studies indicated that CD4+ T-cell epitope dominance is controlled by antigen three-dimensional structure through its influence on antigen processing and presentation. In this wo...

  12. Modulation of P-glycoprotein-mediated multidrug resistance in K562 leukemic cells by indole-3-carbinol

    International Nuclear Information System (INIS)

    Resistance to chemotherapeutic drugs is one of the major problems in the treatment of cancer. P-glycoprotein (P-gp) encoded by the mdr gene is a highly conserved protein, acts as a multidrug transporter, and has a major role in multiple drug resistance (MDR). Targeting of P-gp by naturally occurring compounds is an effective strategy to overcome MDR. Indole-3-carbinol (I3C), a glucosinolates present in cruciferous vegetables, is a promising chemopreventive agent as it is reported to possess antimutagenic, antitumorigenic, and antiestrogenic properties in experimental studies. In the present investigation, the potential of I3C to modulate P-gp expression was evaluated in vinblastine (VBL)-resistant K562 human leukemic cells. The resistant K562 cells (K562/R10) were found to be cross-resistant to vincristine (VCR), doxorubicin (DXR), and other antineoplastic agents. I3C at a nontoxic dose (10 x 10-3 M) enhanced the cytotoxic effects of VBL time dependently in VBL-resistant human leukemia (K562/R10) cells but had no effect on parent-sensitive cells (K562/S). The Western blot analysis of K 562/R 10 cells showed that I3C downregulates the induced levels of P-gp in resistant cells near to normal levels. The quantitation of immunocytochemically stained K562/R10 cells showed 24%, 48%, and 80% decrease in the levels of P-gp by I3C for 24, 48, and 72 h of incubation. The above features thus indicate that I3C could be used as a novel modulator of P-gp-mediated multidrug resistance in vitro and may be effective as a dietary adjuvant in the treatment of MDR cancers

  13. Huh-7 cell line as an alternative cultural model for the production of human like erythropoietin (EPO

    Directory of Open Access Journals (Sweden)

    Kausar Humera

    2011-11-01

    Full Text Available Abstract Background and Aims Erythropoietin (EPO is a glycoprotein hormone which is required to regulate the production of red blood cells. Deficiency of EPO is known to cause anemia in chronically infected renal patients and they require regular blood transfusion. Availability of recombinant EPO has eliminated the need for blood transfusion and now it is extensively used for the treatment of anemia. Glycosylation of erythropoietin is essential for its secretion, stability, protein conformation and biological activity. However, maintenance of human like glycosylation pattern during manufacturing of EPO is a major challenge in biotechnology. Currently, Chinese hamster ovary (CHO cell line is used for the commercial production of erythropoietin but this cell line does not maintain glycosylation resembling human system. With the trend to eliminate non-human constituent from biopharmaceutical products, as a preliminary approach, we have investigated the potential of human hepatoma cell line (Huh-7 to produce recombinant EPO. Materials and methods Initially, the secretory signal and Kozak sequences was added before the EPO mature protein sequence using overlap extension PCR technique. PCR-amplified cDNA fragments of EPO was inserted into mammalian expression vector under the control of the cytomegalovirus (CMV promoter and transiently expressed in CHO and Huh-7 cell lines. After RT-PCR analysis, ELISA and Western blotting was performed to verify the immunochemical properties of secreted EPO. Results Addition of secretory signal and Kozak sequence facilitated the extra-cellular secretion and enhanced the expression of EPO protein. Significant expression (P Conclusion Huh-7 cell line has a great potential to produce glycosylated EPO, suggesting the use of this cell line to produce glycoproteins of the therapeutic importance resembling to the natural human system.

  14. Regulatory networks define phenotypic classes of human stem cell lines

    OpenAIRE

    Müller, Franz-Josef; Louise C. Laurent; Kostka, Dennis; Ulitsky, Igor; Williams, Roy; Lu, Christina; Park, In-Hyun; Rao, Mahendra S.; Shamir, Ron; Philip H. Schwartz; Schmidt, Nils O.; Loring, Jeanne F.

    2008-01-01

    Stem cells are defined as self-renewing cell populations that can differentiate into multiple distinct cell types. However, hundreds of different human cell lines from embryonic, fetal, and adult sources have been called stem cells, even though they range from pluripotent cells, typified by embryonic stem cells, which are capable of virtually unlimited proliferation and differentiation, to adult stem cell lines, which can generate a far more limited repertory of differentiated cell types. The...

  15. Investigation of the selenium metabolism in cancer cell lines

    DEFF Research Database (Denmark)

    Lunøe, Kristoffer; Gabel-Jensen, Charlotte; Stürup, Stefan; Andresen, Lars; Skov, Søren; Gammelgaard, Bente

    2011-01-01

    The aim of this work was to compare different selenium species for their ability to induce cell death in different cancer cell lines, while investigating the underlying chemistry by speciation analysis. A prostate cancer cell line (PC-3), a colon cancer cell line (HT-29) and a leukaemia cell line...... incubated with cells for 24 h and the induction of cell death was measured using flow cytometry. The amounts of total selenium in cell medium, cell lysate and the insoluble fractions was determined by ICP-MS. Speciation analysis of cellular fractions was performed by reversed phase, anion exchange and size...... exclusion chromatography and ICP-MS detection. The selenium compounds exhibited large differences in their ability to induce cell death in the three cell lines and the susceptibilities of the cell lines were different. Full recovery of selenium in the cellular fractions was observed for all Se compounds...

  16. Development of Novel Rifampicin-Derived P-Glycoprotein Activators/Inducers. Synthesis, In Silico Analysis and Application in the RBE4 Cell Model, Using Paraquat as Substrate

    Science.gov (United States)

    Vilas-Boas, Vânia; Silva, Renata; Palmeira, Andreia; Sousa, Emília; Ferreira, Luísa Maria; Branco, Paula Sério; Carvalho, Félix; Bastos, Maria de Lourdes; Remião, Fernando

    2013-01-01

    P-glycoprotein (P-gp) is a 170 kDa transmembrane protein involved in the outward transport of many structurally unrelated substrates. P-gp activation/induction may function as an antidotal pathway to prevent the cytotoxicity of these substrates. In the present study we aimed at testing rifampicin (Rif) and three newly synthesized Rif derivatives (a mono-methoxylated derivative, MeORif, a peracetylated derivative, PerAcRif, and a reduced derivative, RedRif) to establish their ability to modulate P-gp expression and activity in a cellular model of the rat’s blood–brain barrier, the RBE4 cell line P-gp expression was assessed by western blot using C219 anti-P-gp antibody. P-gp function was evaluated by flow cytometry measuring the accumulation of rhodamine123. Whenever P-gp activation/induction ability was detected in a tested compound, its antidotal effect was further tested using paraquat as cytotoxicity model. Interactions between Rif or its derivatives and P-gp were also investigated by computational analysis. Rif led to a significant increase in P-gp expression at 72 h and RedRif significantly increased both P-gp expression and activity. No significant differences were observed for the other derivatives. Pre- or simultaneous treatment with RedRif protected cells against paraquat-induced cytotoxicity, an effect reverted by GF120918, a P-gp inhibitor, corroborating the observed P-gp activation ability. Interaction of RedRif with P-gp drug-binding pocket was consistent with an activation mechanism of action, which was confirmed with docking studies. Therefore, RedRif protection against paraquat-induced cytotoxicity in RBE4 cells, through P-gp activation/induction, suggests that it may be useful as an antidote for cytotoxic substrates of P-gp. PMID:23991219

  17. High prevalence of side population in human cancer cell lines

    OpenAIRE

    Boesch, Maximilian; Zeimet, Alain G; Fiegl, Heidi; Wolf, Barbara; Huber, Julia; Klocker, Helmut; Gastl, Guenther; Sopper, Sieghart; Wolf, Dominik

    2016-01-01

    Cancer cell lines are essential platforms for performing cancer research on human cells. We here demonstrate that, across tumor entities, human cancer cell lines harbor minority populations of putative stem-like cells, molecularly defined by dye extrusion resulting in the side population phenotype. These findings establish a heterogeneous nature of human cancer cell lines and argue for their stem cell origin. This should be considered when interpreting research involving these model systems.

  18. pH-Responsive therapeutic solid lipid nanoparticles for reducing P-glycoprotein-mediated drug efflux of multidrug resistant cancer cells

    Directory of Open Access Journals (Sweden)

    Chen HH

    2015-08-01

    enhanced passive tumor accumulation due to the loose intercellular junctions of endothelial cells lining inside blood vessels at tumor site, and the lack of lymphatic drainage. The growth of MCF-7/MDR xenografted tumor on Balb/c nude mice was inhibited to ~400 mm3 in volume as compared with the free DOX treatment group, 1,140 mm3, and the group treated with 1,2 distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol] solid lipid nanoparticles, 820 mm3. Analysis of the body weight of nude mice and the histology of organs and tumor after the administration of DOX-loaded SLNs show that the SLNs have no observable side effects. These results indicate that the C-PEG-SLN is a promising platform for the delivery of therapeutic agents for MDR cancer chemotherapy. Keywords: pH-responsive, solid lipid nanoparticles, multidrug resistance, permeability glycoprotein

  19. Radiosensitivity of Human Melanoma Cell Lines

    Energy Technology Data Exchange (ETDEWEB)

    Bergoc, R. M.; Medina, V.; Cricco, G.; Mohamed, N.; Martin, G.; Nunez, M.; Croci, M.; Crescenti, E. J.; Rivera, E. S.

    2004-07-01

    Cutaneous melanoma is a skin cancer resulting from the malign transformation of skin-pigment cells, the melanocytes. The radiotherapy, alone or in combination with other treatment, is an important therapy for this disease. the objective of this paper was to determine in vitro the radiosensitivity of two human melanoma cell lines with different metastatic capability: WM35 and MI/15, and to study the effect of drugs on radiobiological parameters. The Survival Curves were adjusted to the mathematical Linear-quadratic model using GrapsPad Prism software. Cells were seeded in RPMI medium (3000-3500 cells/flask), in triplicate and irradiated 24 h later. The irradiation was performed using an IBL 437C H Type equipment (189 TBq, 7.7 Gy/min) calibrated with a TLD 700 dosimeter. The range of Doses covered from 0 to 10 Gy and the colonies formed were counted at day 7th post-irradiation. Results obtained were: for WM35, {alpha}=0.37{+-}0.07 Gy''-1 and {beta}=0.06{+-}0.02 Gy''-2, for M1/15m {alpha}=0.47{+-}0.03 Gy''-1 and {beta}=0.06{+-}0.01 Gy''-2. The {alpha}/{beta} values WM35: {alpha}/{beta} values WM35: {alpha}/{beta}=6.07 Gy and M1/15: {alpha}/{beta}{sub 7}.33 Gy were similar, independently of their metastatic capabillity and indicate that both lines exhibit high radioresistance. Microscopic observation of irradiated cells showed multinuclear cells with few morphologic changes non-compatible with apoptosis. By means of specific fluorescent dyes and flow cytometry analysis we determined the intracellular levels of the radicals superoxide and hydrogen peroxide and their modulation in response to ionizing radiation. The results showed a marked decreased in H{sub 2}O{sub 2} intracellular levels with a simultaneous increase in superoxide that will be part of a mechanism responsible for induction of cell radioresistance. This response triggered by irradiated cells could not be abrogated by different treatments like histamine or the

  20. Effect of Procyanidin-rich Extract from Natural Cocoa Powder on Cellular Viability, Cell Cycle Progression, and Chemoresistance in Human Epithelial Ovarian Carcinoma Cell Lines

    Science.gov (United States)

    Taparia, Shruti; Khanna, Aparna

    2016-01-01

    Background: Over the last 400 years, cocoa and chocolate have been described as having potential medicinal value, being consumed as a beverage or eaten as food. Concentration–dependant, antiproliferation, and cytotoxic effects of some of their polyphenolic constituents have been demonstrated against various cancers. Such an effect remains to be demonstrated in ovarian cancer Objective: To investigate the effect of cocoa procyanidins against ovarian cancer in vitro using OAW42 and OVCAR3 cell lines. Materials and Methods: Cocoa procyanidins were extracted and enriched from non alkalized cocoa powder. The polyphenolic content and antioxidant activity were determined. Effect on cell viability was determined after the treatment with ≤1000 μg/mL cocoa procyanidin-rich extract on OAW42 and OVCAR3 and normal human dermal fibroblasts. Similarly, chemosensitization effect was determined by pretreating cancer cell lines with extract followed by doxorubicin hydrochloride treatment. The effect of treatment on cell cycle and P-glycoprotein (P-gp) expression was determined using flow cytometry. Results: The cocoa extract showed high polyphenolic content and antioxidant activity. Treatment with extract caused cytotoxicity and chemosensitization in OAW42 and OVCAR3 cell lines. Normal dermal fibroblasts showed an increase in cell viability post treatment with extract. Treatment with extract affected the cell cycle and an increasing percentage of cells in hypodiploid sub-G1/G0 phase was observed. Treatment of OVCAR3 with the extract caused reduction of P-gp expression. Conclusion: Cocoa procyanidins were found to be selectively cytotoxic against epithelial ovarian cancer, interfered with the normal cell cycle and sensitized cells to subsequent chemotherapeutic treatment. Chemosensitization was found to be associated with P-gp reduction in OVCAR3 cells. SUMMARY Among the naturally occurring flavonoids, procyanidins have been shown to be effective against cancersNon alkalized

  1. Inhibition of P-glycoprotein in Caco-2 cells: effects of herbal remedies frequently used by cancer patients.

    Science.gov (United States)

    Engdal, S; Nilsen, O G

    2008-06-01

    1. The herbal products Natto K2, Agaricus, mistletoe, noni juice, green tea and garlic were investigated for in vitro inhibitory potential on P-glycoprotein (P-gp)-mediated transport of digoxin (30 nM) in differentiated and polarized Caco-2 cells. 2. Satisfactory cell functionality was demonstrated through measurements of assay linearity, transepithelial electric resistance (TEER), cytotoxicity, mannitol permeability, and inclusion of the positive inhibition control verapamil. 3. The most potent inhibitors of the net digoxin flux (IC(50)) were mistletoe > Natto K2 > Agaricus > green tea (0.022, 0.62, 3.81, >4.5 mg ml(-1), respectively). Mistletoe also showed the lowest IC(25) value, close to that obtained by verapamil (1.0 and 0.5 microg ml(-1), respectively). The IC(50)/IC(25) ratio was found to be a good parameter for the determination of inhibition profiles. Garlic and noni juice were classified as non-inhibitors. 4. This study shows that mistletoe, Natto K2, Agaricus and green tea inhibit P-gp in vitro. Special attention should be paid to mistletoe due to very low IC(50) and IC(25) values and to Natto K2 due to a low IC(50) value and a low IC(50)/IC(25) ratio. PMID:18570158

  2. P-glycoprotein Mediates Ceritinib Resistance in Anaplastic Lymphoma Kinase-rearranged Non-small Cell Lung Cancer

    Science.gov (United States)

    Katayama, Ryohei; Sakashita, Takuya; Yanagitani, Noriko; Ninomiya, Hironori; Horiike, Atsushi; Friboulet, Luc; Gainor, Justin F.; Motoi, Noriko; Dobashi, Akito; Sakata, Seiji; Tambo, Yuichi; Kitazono, Satoru; Sato, Shigeo; Koike, Sumie; John Iafrate, A.; Mino-Kenudson, Mari; Ishikawa, Yuichi; Shaw, Alice T.; Engelman, Jeffrey A.; Takeuchi, Kengo; Nishio, Makoto; Fujita, Naoya

    2015-01-01

    The anaplastic lymphoma kinase (ALK) fusion oncogene is observed in 3%–5% of non-small cell lung cancer (NSCLC). Crizotinib and ceritinib, a next-generation ALK tyrosine kinase inhibitor (TKI) active against crizotinib-refractory patients, are clinically available for the treatment of ALK-rearranged NSCLC patients, and multiple next-generation ALK-TKIs are currently under clinical evaluation. These ALK-TKIs exhibit robust clinical activity in ALK-rearranged NSCLC patients; however, the emergence of ALK-TKI resistance restricts the therapeutic effect. To date, various secondary mutations or bypass pathway activation-mediated resistance have been identified, but large parts of the resistance mechanism are yet to be identified. Here, we report the discovery of p-glycoprotein (P-gp/ABCB1) overexpression as a ceritinib resistance mechanism in ALK-rearranged NSCLC patients. P-gp exported ceritinib and its overexpression conferred ceritinib and crizotinib resistance, but not to PF-06463922 or alectinib, which are next-generation ALK inhibitors. Knockdown of ABCB1 or P-gp inhibitors sensitizes the patient-derived cancer cells to ceritinib, in vitro and in vivo. P-gp overexpression was identified in three out of 11 cases with in ALK-rearranged crizotinib or ceritinib resistant NSCLC patients. Our study suggests that alectinib, PF-06463922, or P-gp inhibitor with ceritinib could overcome the ceritinib or crizotinib resistance mediated by P-gp overexpression. PMID:26870817

  3. P-glycoprotein Mediates Ceritinib Resistance in Anaplastic Lymphoma Kinase-rearranged Non-small Cell Lung Cancer

    Directory of Open Access Journals (Sweden)

    Ryohei Katayama

    2016-01-01

    Full Text Available The anaplastic lymphoma kinase (ALK fusion oncogene is observed in 3%–5% of non-small cell lung cancer (NSCLC. Crizotinib and ceritinib, a next-generation ALK tyrosine kinase inhibitor (TKI active against crizotinib-refractory patients, are clinically available for the treatment of ALK-rearranged NSCLC patients, and multiple next-generation ALK-TKIs are currently under clinical evaluation. These ALK-TKIs exhibit robust clinical activity in ALK-rearranged NSCLC patients; however, the emergence of ALK-TKI resistance restricts the therapeutic effect. To date, various secondary mutations or bypass pathway activation-mediated resistance have been identified, but large parts of the resistance mechanism are yet to be identified. Here, we report the discovery of p-glycoprotein (P-gp/ABCB1 overexpression as a ceritinib resistance mechanism in ALK-rearranged NSCLC patients. P-gp exported ceritinib and its overexpression conferred ceritinib and crizotinib resistance, but not to PF-06463922 or alectinib, which are next-generation ALK inhibitors. Knockdown of ABCB1 or P-gp inhibitors sensitizes the patient-derived cancer cells to ceritinib, in vitro and in vivo. P-gp overexpression was identified in three out of 11 cases with in ALK-rearranged crizotinib or ceritinib resistant NSCLC patients. Our study suggests that alectinib, PF-06463922, or P-gp inhibitor with ceritinib could overcome the ceritinib or crizotinib resistance mediated by P-gp overexpression.

  4. The studies of DNA metabolic dynamics in embryonic cell line and non-embryonic cell line of Onobrychis viciaefolia scop

    International Nuclear Information System (INIS)

    The hypocotyls of aseptic seedlings of Onobrychis viciaefolia Scop. were used as explants for inducing callus. The embryonic cell line (E-line) and non-embryonic cell line (NE line) were established. The DNA synthesis dynamics in both cell lines were studied by autoradiography. The results showed that: DNA synthesis in E-line was active and confined to embryonic cell or cell masses and then the cells divided quickly and formed somatic embryos at different stages. The changes of DNA metabolism took place in a certain pattern and the maximum rate of DNA synthesis occured during the formation of globular embryo. There was a clear relationship between the difference of DNA synthesis rate and the polarity of the embryo. In NE-line, the beginning of cell division and the forming of callus were also based on DNA replication but were much deoxythymidine. There was a significant difference in DNA synthesis dynamics between the two cell lines

  5. Susceptibility testing of fish cell lines for virus isolation

    DEFF Research Database (Denmark)

    Ariel, Ellen; Skall, Helle Frank; Olesen, Niels Jørgen

    2009-01-01

    Passage of cell cultures may adversely influence cell susceptibility to virus infection through selection of cell clones that thrive in vitro but may not necessarily display high sensitivity to virus infection. Susceptibility to a given virus can therefore vary not only between cell lines and......-cell-culture-adapted" virus by propagating the virus in heterologous cell lines to the one tested. A stock of test virus was produced and stored at - 80 °C and tests were conducted biannually. This procedure becomes complicated when several cell lines are in use and does not account for variation among lineages. In comparing...... cell lineages, we increased the number of isolates of each virus, propagated stocks in a given cell line and tested all lineages of that line in use in the laboratory. Testing of relative cell line susceptibility between laboratories is carried out annually via the Inter-laboratory Proficiency Test...

  6. Detection algorithm for the validation of human cell lines.

    Science.gov (United States)

    Eltonsy, Névine; Gabisi, Vivian; Li, Xuesong; Russe, K Blair; Mills, Gordon B; Stemke-Hale, Katherine

    2012-09-15

    Cell lines are an important tool in understanding all aspects of cancer growth, development, metastasis and tumor cell death. There has been a dramatic increase in the number of cell lines and diversity of the cancers they represent; however, misidentification and cross-contamination of cell lines can lead to erroneous conclusions. One method that has gained favor for authenticating cell lines is the use of short tandem repeats (STR) to generate a unique DNA profile. The challenge in validating cell lines is the requirement to compare the large number of existing STR profiles against cell lines of interest, particularly when considering that the profiles of many cell lines have drifted over time and original samples are not available. We report here methods that analyze the variations and the proportional changes extracted from tetra-nucleotide repeat regions in the STR analysis. This technique allows a paired match between a target cell line and a reference database of cell lines to find cell lines that match within a user designated percentage cut-off quality matrix. Our method accounts for DNA instability and can suggest whether the target cell lines are misidentified or unstable. PMID:22419365

  7. Protective effect of Porphyra yezoensis glycoprotein on D-galactosamine‑induced cytotoxicity in Hepa 1c1c7 cells.

    Science.gov (United States)

    Choi, Jeong-Wook; Kim, Young-Min; Park, Su-Jin; Kim, In-Hye; Nam, Taek-Jeong

    2015-05-01

    The present study aimed to examine the signaling pathways and enzyme activity associated with the protective effect of Porphyra yezoensis glycoprotein (PYGP) on D‑galactosamine (D‑GaIN)‑induced cytotoxicity in Hepa 1c1c7 cells. D‑GaIN is commonly used to induce hepatic injury models in vivo as well as in vitro. PYGP was extracted from Porphyra yezoensis, a red algae distributed along the coasts of Republic of Korea, China and Japan. In the present study, Hepa 1c1c7 cells were pre‑treated with PYGP (20 and 40 µg/ml) for 24 h and then the media was replaced with D‑GaIN (20 mM) and PYGP (20 and 40 µg/ml). The results demonstrated that D‑GaIN induced Hepa 1c1c7 cell death and pretreatment with PYGP was found to attenuate D‑GaIN toxicity. In addition, D‑GaIN decreased the antioxidant activity and increased lipid peroxidation processes; however, pre‑treatment with PYGP reduced the generation of lipid peroxidation products, such as thiobarbituric acid reactive substances, as well as increased the activity of antioxidant enzymes, including superoxide dismutase, catalase and glutathione‑s‑transferase (GST). PYGP was shown to suppress the overexpression of extracellular signal‑regulated kinase, c‑jun N‑terminal kinase and p38 mitogen‑activated protein kinase (MAPK) phosphorylation induced by D‑GaIN. Furthermore, PYGP increased the protein expression of nuclear factor erythroid 2‑related factor 2 (Nrf2), quinine oxidoreductase 1, GST and heme oxygenase 1 protein expression. These results suggested that PYGP had cytoprotective effects against D‑GaIN‑induced cell damage, which may be associated with MAPKs and the Nrf2 signaling pathway. PMID:25626067

  8. Protein kinase C-mediated phosphorylation of the human multidrug resistance P-glycoprotein regulates cell volume-activated chloride channels.

    OpenAIRE

    Hardy, S P; Goodfellow, H R; Valverde, M. A. (Miguel ??ngel), 1963-; Gill, D. R.; Sepúlveda, V; Higgins, C F

    1995-01-01

    The multidrug resistance P-glycoprotein (P-gp), which transports hydrophobic drugs out of cells, is also associated with volume-activated chloride currents. It is not yet clear whether P-gp is a channel itself, or whether it is a channel regulator. Activation of chloride currents by hypotonicity in cells expressing P-gp was shown to be regulated by protein kinase C (PKC). HeLa cells exhibited volume-activated chloride currents indistinguishable from those obtained in P-gp-expressing cells exc...

  9. Chloride transport in a glioma cell line

    Energy Technology Data Exchange (ETDEWEB)

    Wolpaw, E.W.

    1984-01-01

    Maintenance of the extracellular environment is a major function of central nervous system astroglia. The transport of Cl/sup -/ across the cell membrane may be an integral part of this function, since Cl/sup -/ transport has been implicated in homeostasis of cell volume, pH, and extracellular K/sup +/ concentration. The work presented here investigated Cl/sup -/ transport in the glioma cell line LRM55. Results indicate that LRM55 cells are a good model for astroglia and that these cells contain three Cl/sup -/ transporters; a Cl/sup -//HCO/sub 3//sup -/ exchanger, a K/sup +//Cl/sup -/ cotransporter, and a Cl/sup -//SO/sub 4//sup 2 -/ exchanger. Ion transport studies measured the fluxes of Cl/sup -/ (as /sup 36/Cl/sup -/), K/sup +/ (as /sup 86/Rb/sup +/), and SO/sub 4//sup 2 -/ (as /sup 35/SO/sub 4//sup 2 -/). Cl/sup -/ flux was trans-simulated by Cl/sup -/ or HCO/sub 3//sup -/ and was inhibited by SITS or furosemide. External K/sup +/ stimulated Cl/sup -/ influx and external Cl/sup -/ stimulated Rb/sup +/ influx. Furosemide, but not SITS, inhibited the K/sup +//Cl/sup -/ cotransporter. High K/sup +/ medium increased cell volume and Cl/sup -/ content. Steady-state Cl/sup -/ concentration was at least twice that predicted from passive equilibration according to the Nernst equation. SO/sub 4//sup 2 -/ flux was trans-stimulated by SO/sub 4//sup 2 -/ or by Cl/sup -/. Cl/sup -/ was a competitive inhibitor of SO/sub 4//sup 2 -/ influx, but SO/sub 4//sup 2 -/ had no detectable effect on Cl/sup -/ influx or efflux. SO/sub 4//sup 2 -/ flux was inhibited by SITS or furosemide.

  10. Effect of curcumin on multidrug resistance in resistant human gastric carcinoma cell line SGC7901/VCR

    Institute of Scientific and Technical Information of China (English)

    Xiao-qing TANG; Hu BI; Jian-qiang FENG; Jian-guo CAO

    2005-01-01

    Aim: To investigate the reversal effects of curcumin on multidrug resistance (MDR)in a resistant human gastric carcinoma cell line. Methods: The cytotoxic effect of vincristine (VCR) was evaluated by MTT assay. The cell apoptosis induced by VCR was determined by propidium iodide (PI)-stained flow cytometry (FCM) and a morphological assay using acridine orange (AO)/ethidium bromide (EB) dual staining. P-glycoprotein (P-gp) function was demonstrated by the accumulation and efflux of rhodamine123 (Rh123) using FCM. The expression of P-gp and the activation of caspase-3 were measured by FCM using fluorescein isothiocyanate (FITC)-conjugated anti-P-gp and anti-cleaved caspase-3 antibodies, respectively.Results: Curcumin, at concentrations of 5 μmol/L, 10 μmol/L, or 20 μmol/L, had no cytotoxic effect on a parent human gastric carcinoma cell line (SGC7901) or its VCR-resistant variant cell line (SGC7901/VCR). The VCR-IC50 value of the SGC7901/VCR cells was 45 times more than that of the SGC7901cells and the SGC7901/VCR cells showed apoptotic resistance to VCR. SGC7901/VCR cells treated with 5μmol/L, 10 μmol/L, or 20 μmol/L curcumin decreased the IC50 value of VCR and promoted VCR-mediated apoptosis in a dose-dependent manner. Curcumin (10μmol/L) increased Rh 123 accumulation and inhibited the efflux of Rh 123 in S GC7901/VCR cells, but did not change the accumulation and efflux of Rh123 in SGC7901cells. P-gp was overexpressed in SGC7901/VCR cells, whereas it was downregulated after a 24-h treatment with curcumin (10 μmol/L). Resistant cells treated with 1μmol/L VCR alone showed 77% lower levels of caspase-3 activation relative to SGC7901 cells, but the activation of caspase-3 in the resistant cell line increased by 44% when cells were treated with VCR in combination with curcumin.Conclusion: Curcumin can reverse the MDR of the human gastric carcinoma SGC7901/VCR cell line. This might be associated with decreased P-gp function and expression, and the promotion of

  11. CD133 expression is not selective for tumor initiating or radioresistant cell populations in the CRC line HCT-116

    International Nuclear Information System (INIS)

    The hypothesis of certain subpopulations of cancer cells with stem-cell like characteristics that might be responsible for treatment resistance and recurrence of disease is still challenging and under quite controversial discussion. In most studies, surrogate cell surface antigens such as the 92-110 kDa transmembrane glycoprotein CD133 (human Prominin-1) were labeled to isolate particular small cancer cell populations for studying their tumorigenic potential. In colorectal carcinomas (CRC) for example, a small CD133 positive (CD133+) cell population has recently been described to be enriched for tumor-initiating/cancer stem cells (TIC/CSC) as compared to the CD133 negative (CD133) population. Furthermore, it was documented that the CD133+ subpopulation could exclusively be maintained in culture as spheres under serum-free conditions. Addition of serum resulted in cell differentiation, growth in 2-D and downregulation of CD133 expression. This would imply that established colorectal cancer (CRC) cell lines that have been grown under adherent, serum-supplemented conditions for years should be devoid of CD133+ cells and TIC/CSC, respectively, which seems contradictory to the finding that many CRC lines produce tumors in nude mice models. In order to gain insight into this paradox, we studied the expression of CD133 in numerous established CRC lines under standard culture conditions and chose one particular cell line based on its expression pattern to study the behavior of CD133+ / CD133- subpopulations

  12. Elevation of Serum KL-6 Glycoprotein or Surfactant Protein-D in Adult T-cell Leukemia with Distinct Pulmonary Complications

    OpenAIRE

    Osaka, Akemi; Yanagihara, Katsunori; Yamada, Yasuaki; Hasegawa, Hiroo; Inokuchi, Naoko; Hayashi, Tomayoshi; Komoda, Minori; Nakamura, Shigeki; Aoyama, Muneo; Sawada, Takashi; Kamihira, Shimeru

    2009-01-01

    Patients with hematological malignancies frequently suffer from lung diseases as a complication. However, it is difficult to discriminate leukemic invasion into the lung from infectious pulmonary complications. The serum level of Krebs von den Lungen-6 (KL-6), which is a mucin-like glycoprotein, is increased in more than 70% of patients with interstitial pneumonia. Surfactant protein-D (SP-D) is produced mainly in the lung by alveolar type II and bronchiolar epithelial cells and is a useful s...

  13. Identification of human linear B-cell epitope sites on the envelope glycoproteins of Crimean-Congo haemorrhagic fever virus.

    Science.gov (United States)

    Goedhals, D; Paweska, J T; Burt, F J

    2015-05-01

    A peptide library was used to screen for regions containing potential linear B-cell epitope sites in the glycoproteins and nucleoprotein of Crimean-Congo haemorrhagic fever virus (CCHFV) in an enzyme-linked immunosorbent assay (ELISA). The library consisted of 156 peptides, spanning the nucleoprotein and mature GN and GC proteins in a 19-mer with 9-mer overlap format. Using pooled serum samples from convalescent patients to screen the library, six peptides were identified as potential epitope sites. Further testing of these six peptides with individual patient sera identified two of these peptides as probable epitope sites, with peptide G1451-1469 reacting to 13/15 and peptide G1613-1631 to 14/15 human sera. These peptides are situated on the GC protein at amino acid positions 1451-1469 (relative to CCHFV isolate SPU103/97) (TCTGCYACSSGISCKVRIH) and 1613-1631 (FMFGWRILFCFKCCRRTRG). Identified peptides may have application in ELISA for diagnostic or serosurveillance purposes. PMID:25185583

  14. Lysosome-associated membrane glycoprotein 3 is involved in influenza A virus replication in human lung epithelial (A549 cells

    Directory of Open Access Journals (Sweden)

    Wang Jianwei

    2011-08-01

    Full Text Available Abstract Background Influenza A virus mutates rapidly, rendering antiviral therapies and vaccines directed against virus-encoded targets ineffective. Knowledge of the host factors and molecular pathways exploited by influenza virus will provide further targets for novel antiviral strategies. However, the critical host factors involved in influenza virus infection have not been fully defined. Results We demonstrated that LAMP3, a member of lysosome-associated membrane glycoprotein (LAMP family, was significantly induced in human lung epithelial (A549 cells upon influenza A virus infection. Knockdown of LAMP3 expression by RNA interference attenuated production of viral nucleoprotein (NP as well as virus titers. Confocal microscopy results demonstrated that viral NP is colocalized within LAMP3 positive vesicles at early stages of virus infection. Furthermore, knockdown of LAMP3 expression led to a reduction in nuclear accumulation of viral NP and impeded virus replication. Conclusions LAMP3 is an influenza A virus inducible gene, and plays an important role in viral post-entry steps. Our observations may provide insights into the mechanism of influenza virus replication and potential targets for novel anti-influenza therapeutics.

  15. Regulatory and Helper Follicular T Cells and Antibody Avidity to Simian Immunodeficiency Virus Glycoprotein 120.

    Science.gov (United States)

    Blackburn, Matthew J; Zhong-Min, Ma; Caccuri, Francesca; McKinnon, Katherine; Schifanella, Luca; Guan, Yongjun; Gorini, Giacomo; Venzon, David; Fenizia, Claudio; Binello, Nicolò; Gordon, Shari N; Miller, Christopher J; Franchini, Genoveffa; Vaccari, Monica

    2015-10-01

    T follicular regulatory cells (TFR) are a suppressive CD4(+) T cell subset that migrates to germinal centers (GC) during Ag presentation by upregulating the chemokine receptor CXCR5. In the GC, TFR control T follicular helper cell (TFH) expansion and modulate the development of high-affinity Ag-specific responses. In this study, we identified and characterized TFR as CXCR5(+)CCR7(-) "follicular" T regulatory cells in lymphoid tissues of healthy rhesus macaques, and we studied their dynamics throughout infection in a well-defined animal model of HIV pathogenesis. TFR were infected by SIVmac251 and had comparable levels of SIV DNA to CXCR5(-)CCR7(+) "T zone" T regulatory cells and TFH. Contrary to the SIV-associated TFH expansion in the chronic phase of infection, we observed an apparent reduction of TFR frequency in cell suspension, as well as a decrease of CD3(+)Foxp3(+) cells in the GC of intact lymph nodes. TFR frequency was inversely associated with the percentage of TFH and, interestingly, with the avidity of the Abs that recognize the SIV gp120 envelope protein. Our findings show changes in the TFH/TFR ratio during chronic infection and suggest possible mechanisms for the unchecked expansion of TFH cells in HIV/SIV infection. PMID:26297759

  16. DNA profiling and characterization of animal cell lines.

    Science.gov (United States)

    Stacey, Glyn N; Byrne, Ed; Hawkins, J Ross

    2014-01-01

    The history of the culture of animal cell lines is littered with published and much unpublished experience with cell lines that have become switched, mislabelled, or cross-contaminated during laboratory handling. To deliver valid and good quality research and to avoid waste of time and resources on such rogue lines, it is vital to perform some kind of qualification for the provenance of cell lines used in research and particularly in the development of biomedical products. DNA profiling provides a valuable tool to compare different sources of the same cells and, where original material or tissue is available, to confirm the correct identity of a cell line. This chapter provides a review of some of the most useful techniques to test the identity of cells in the cell culture laboratory and gives methods which have been used in the authentication of cell lines. PMID:24297409

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

    Science.gov (United States)

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

    2015-09-01

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

  18. An Envelope Glycoprotein of the Human Endogenous Retrovirus HERV-W Is Expressed in the Human Placenta and Fuses Cells Expressing the Type D Mammalian Retrovirus Receptor

    OpenAIRE

    Blond, Jean-Luc; Lavillette, Dimitri; Cheynet, Valérie; Bouton, Olivier; Oriol, Guy; Chapel-Fernandes, Sylvie; Mandrand, Bernard; Mallet, François; Cosset, François-Loïc

    2000-01-01

    A new human endogenous retrovirus (HERV) family, termed HERV-W, was recently described (J.-L. Blond, F. Besème, L. Duret, O. Bouton, F. Bedin, H. Perron, B. Mandrand, and F. Mallet, J. Virol. 73:1175–1185, 1999). HERV-W mRNAs were found to be specifically expressed in placenta cells, and an env cDNA containing a complete open reading frame was recovered. In cell-cell fusion assays, we demonstrate here that the product of the HERV-W env gene is a highly fusogenic membrane glycoprotein. Transfe...

  19. Drug-induced trafficking of p-glycoprotein in human brain capillary endothelial cells as demonstrated by exposure to mitomycin C.

    OpenAIRE

    Noack, Andreas; Noack, Sandra; Hoffmann, Andrea; Maalouf, Katia; Buettner, Manuela; Couraud, Pierre-Olivier; Romero, Ignacio A.; Weksler, Babette; Alms, Dana; Römermann, Kerstin; Naim, Hassan Y; Löscher, Wolfgang

    2014-01-01

    P-glycoprotein (Pgp; ABCB1/MDR1) is a major efflux transporter at the blood-brain barrier (BBB), restricting the penetration of various compounds. In other tissues, trafficking of Pgp from subcellular stores to the cell surface has been demonstrated and may constitute a rapid way of the cell to respond to toxic compounds by functional membrane insertion of the transporter. It is not known whether drug-induced Pgp trafficking also occurs in brain capillary endothelial cells that form the BBB. ...

  20. MDR1 P-glycoprotein is expressed by endothelial cells of newly formed capillaries in human gliomas but is not expressed in the neovasculature of other primary tumors.

    OpenAIRE

    Tóth, K.; Vaughan, M. M.; Peress, N. S.; Slocum, H. K.; Rustum, Y M

    1996-01-01

    The expression of human MDR1 P-glycoprotein (Pgp) in the capillary endothelial cells of the central nervous system has been demonstrated. The brain capillary endothelial cells maintain the structure and function of the blood-brain barrier. Recently, the human MDR1 Pgp (and its mouse homologue MDR1a Pgp) has been shown to function as an important part of this barrier, pumping out xenobiotics from endothelial cells into the lumen of capillaries resulting in the protection of the brain parenchym...

  1. Demonstration of immunochemical identity between the nerve growth factor-inducible large external (NILE) glycoprotein and the cell adhesion molecule L1

    DEFF Research Database (Denmark)

    Bock, E; Richter-Landsberg, C; Faissner, A;

    1985-01-01

    The nerve growth factor-inducible large external (NILE) glycoprotein and the neural cell adhesion molecule L1 were shown to be immunochemically identical. Immunoprecipitation with L1 and NILE antibodies of [3H]fucose-labeled material from culture supernatants and detergent extracts of NGF......]methionine-labeled early post-natal cerebellar cell cultures or [3H]fucose-labeled NGF-treated PC12 cells, all immunoreactivity for NILE antibody could be removed by pre-clearing with L1 antibody and vice versa....

  2. Stoichiometry of monoclonal antibody neutralization of T-cell line-adapted human immunodeficiency virus type 1

    DEFF Research Database (Denmark)

    Schønning, Kristian; Lund, O; Lund, O S; Hansen, J E

    1999-01-01

    In order to study the stoichiometry of monoclonal antibody (MAb) neutralization of T-cell line-adapted human immunodeficiency virus type 1 (HIV-1) in antibody excess and under equilibrium conditions, we exploited the ability of HIV-1 to generate mixed oligomers when different env genes are...... coexpressed. By the coexpression of Env glycoproteins that either can or cannot bind a neutralizing MAb in an env transcomplementation assay, virions were generated in which the proportion of MAb binding sites could be regulated. As the proportion of MAb binding sites in Env chimeric virus increased, MAb...

  3. Direct interaction between verapamil and doxorubicin causes the lack of reversal effect of verapamil on P-glycoprotein mediated resistance to doxorubicin in vitro using L1210/VCR cells

    International Nuclear Information System (INIS)

    Mouse leukemic cell sub-line L 1210/VCR exerts expressive multidrug resistance (MDR) that is mediated by P-glycoprotein. Cells originally adapted to vincristine are also extremely resistant to doxorubicin. Resistance to both vincristine and doxorubicin is connected with depression of drug uptake. While resistance of L 121 O cells to vincristine could be reversed by verapamil as chemo-sensitizer, resistance of cells to doxorubicin was insensitive to verapamil. Action of verapamil (well-known inhibitor of PGP activity) on multidrug resistance was often used as evidence that MDR is mediated by PGP. From this point it may be possible that the resistance of L1210/VCR cells to vincristine is mediated by PGP and the resistance to doxorubicin is mediated by other PGP-independent system. Another and more probable explanation of different effect of verapamil on resistance of L1210/VCR cells to vincristine and doxorubicin may be deduced from the following fact: Using UV spectroscopy we found that doxorubicin dissolved in water buffered medium interacts effectively with verapamil. This interaction may be responsible for the decrease of concentration of both drugs in free effective form and consequently for higher survival of cells. In contrast to doxorubicin vincristine does not give any interaction with verapamil that is measurable by UV spectroscopy and resistance of L1210/VCR cells to vincristine may be fully reversed by verapamil. (authors)

  4. NMR detection and characterization of sialylated glycoproteins and cell surface polysaccharides

    Energy Technology Data Exchange (ETDEWEB)

    Barb, Adam W. [University of Georgia, Complex Carbohydrate Research Center (United States); Freedberg, Daron I.; Battistel, Marcos D. [Center for Biologics Evaluation and Research, Food and Drug Administration, Laboratory of Bacterial Polysaccharides (United States); Prestegard, James H., E-mail: jpresteg@ccrc.uga.edu [University of Georgia, Complex Carbohydrate Research Center (United States)

    2011-09-15

    Few solution NMR pulse sequences exist that are explicitly designed to characterize carbohydrates (glycans). This is despite the essential role carbohydrate motifs play in cell-cell communication, microbial pathogenesis, autoimmune disease progression and cancer metastasis, and despite that fact that glycans, often shed to extra-cellular fluids, can be diagnostic of disease. Here we present a suite of two dimensional coherence experiments to measure three different correlations (H3-C2, H3-C1, and C1-C2) on sialic acids, a group of nine-carbon carbohydrates found on eukaryotic cell surfaces that often play a key role in disease processes. The chemical shifts of the H3, C2, and C1 nuclei of sialic acids are sensitive to carbohydrate linkage, linkage conformation, and ionization state of the C1 carboxylate. The experiments reported include rigorous filter elements to enable detection and characterization of isotopically labeled sialic acids with high sensitivity in living cells and crude isolates with minimal interference from unwanted signals arising from the {approx}1% {sup 13}C-natural abundance of cellular metabolites. Application is illustrated with detection of sialic acids on living cells, in unpurified mixtures, and at the terminus of the N-glycan on the 55 kDa immunoglobulin G Fc.

  5. Pregnancy associated glycoprotein-1, -6, -7, and -17 are major products of bovine binucleate trophoblast giant cells at midpregnancy.

    Science.gov (United States)

    Klisch, Karl; De Sousa, Noelita Melo; Beckers, Jean-François; Leiser, Rudolf; Pich, Andreas

    2005-08-01

    Pregnancy associated glycoproteins (PAGs) are extensively glycosylated secretory proteins of ruminant trophoblast cells. In cattle placenta several PAG cDNAs are expressed, but the variety of correspondent proteins and their degree of glycosylation are not well characterized. Thus, we purified PAGs by using a protocol which included a lectin (Vicia villosa agglutinin) affinity chromatography. Due to their specific glycosylation pattern, PAGs derived from binucleate trophoblast giant cells were highly enriched by this protocol. PAGs were purified from cotyledons of 2 day 100 placentas and from a single placenta at day 155 and 180. In all samples three major bands (75; 66; 56 kDa) were detected by one-dimensional SDS-PAGE. Mass-spectrometric analysis identified the 75 kDa band as a mixture of PAG-7 and PAG-6, the 66 kDa band as PAG-1 and the 56 kDa band as PAG-17. N-terminal sequencing of the day 100 sample confirmed the mass spectrometric identifications. Enzymatic release of N-glycans with peptide-N-glycanase-F from PAGs reduced the molecular weight to approximately 37 kDa which corresponds to the theoretical molecular mass of PAGs. Limited peptide-N-glycanase-F treatment revealed that all four N-glycosylation sites are quantitatively occupied in PAG-1. Compared to PAG-1 the number of potential N-glycosylation sites is lower in PAG-17 (three sites) and higher in PAG-6 and -7 (five and six sites, respectively). This suggests that the number of attached N-glycans is the main determinant of molecular mass of bovine PAGs. The degree of glycosylation may be a major factor regulating the plasma half life of PAGs. PMID:15822115

  6. Identification of continuous human B-cell epitopes in the VP35, VP40, nucleoprotein and glycoprotein of Ebola virus.

    Directory of Open Access Journals (Sweden)

    Pierre Becquart

    Full Text Available Ebola virus (EBOV is a highly virulent human pathogen. Recovery of infected patients is associated with efficient EBOV-specific immunoglobulin G (IgG responses, whereas fatal outcome is associated with defective humoral immunity. As B-cell epitopes on EBOV are poorly defined, we sought to identify specific epitopes in four EBOV proteins (Glycoprotein (GP, Nucleoprotein (NP, and matrix Viral Protein (VP40 and VP35. For the first time, we tested EBOV IgG+ sera from asymptomatic individuals and symptomatic Gabonese survivors, collected during the early humoral response (seven days after the end of symptoms and the late memory phase (7-12 years post-infection. We also tested sera from EBOV-seropositive patients who had never had clinical signs of hemorrhagic fever or who lived in non-epidemic areas (asymptomatic subjects. We found that serum from asymptomatic individuals was more strongly reactive to VP40 peptides than to GP, NP or VP35. Interestingly, anti-EBOV IgG from asymptomatic patients targeted three immunodominant regions of VP40 reported to play a crucial role in virus assembly and budding. In contrast, serum from most survivors of the three outbreaks, collected a few days after the end of symptoms, reacted mainly with GP peptides. However, in asymptomatic subjects the longest immunodominant domains were identified in GP, and analysis of the GP crystal structure revealed that these domains covered a larger surface area of the chalice bowl formed by three GP1 subunits. The B-cell epitopes we identified in the EBOV VP35, VP40, NP and GP proteins may represent important tools for understanding the humoral response to this virus and for developing new antibody-based therapeutics or detection methods.

  7. Influence of group A streptococcal acid glycoprotein on expression of major virulence factors and internalization by epithelial cells.

    Science.gov (United States)

    Marouni, Mehran J; Ziomek, Edmund; Sela, Shlomo

    2003-08-01

    A single transposon insertion upstream to the open-reading-frame identified as the streptococcal acid glycoprotein (sagp) gene rendered a Tn916 isolate of Streptococcus pyogenes with elevated susceptibility to internalization by the epithelial cells. The role of SAGP in S. pyogenes internalization was further studied using isogenic mutant containing an in-frame deletion within the sagp gene. The sagp mutant displayed slower growth-rate and showed 5-fold higher internalization efficiency than the parent strain. Transcription of sagp at the logarithmic phase, but not at the stationary phase of the growth was repressed by csrR, the global regulator gene. At the same time, mutation of the sagp gene partially decreased the transcription of hasA, a gene that is required for capsule synthesis. The mutation had no effect on transcription of the emm3 gene, encoding for the M protein. The most striking effect of the sagp mutation was a down-regulation of the streptococcal pyrogenic exotoxin B (SpeB) at both translational and transcriptional level. Treatment of the SAGP mutant cells with the exogenous mSpeB (mature protease) only partially reduced their susceptibility to internalization. The exogenous mSpeB was more effective in reducing the internalization efficiency of a speB mutant and brought it to the level observed for the parent strain. In overall, results show that CsrR, directly or indirectly, affects the expression of SAGP, and that the SAGP modulates expression of not only SpeB, but also other genes that facilitate S. pyogenes internalization. PMID:12901845

  8. P-glycoprotein-mediated resistance to chemotherapy in cancer cells: using recombinant cytosolic domains to establish structure-function relationships

    Directory of Open Access Journals (Sweden)

    Di Pietro A.

    1999-01-01

    Full Text Available Resistance to chemotherapy in cancer cells is mainly mediated by overexpression of P-glycoprotein (Pgp, a plasma membrane ATP-binding cassette (ABC transporter which extrudes cytotoxic drugs at the expense of ATP hydrolysis. Pgp consists of two homologous halves each containing a transmembrane domain and a cytosolic nucleotide-binding domain (NBD which contains two consensus Walker motifs, A and B, involved in ATP binding and hydrolysis. The protein also contains an S signature characteristic of ABC transporters. The molecular mechanism of Pgp-mediated drug transport is not known. Since the transporter has an extraordinarily broad substrate specificity, its cellular function has been described as a "hydrophobic vacuum cleaner". The limited knowledge about the mechanism of Pgp, partly due to the lack of a high-resolution structure, is well reflected in the failure to efficiently inhibit its activity in cancer cells and thus to reverse multidrug resistance (MDR. In contrast to the difficulties encountered when studying the full-length Pgp, the recombinant NBDs can be obtained in large amounts as soluble proteins. The biochemical and biophysical characterization of recombinant NBDs is shown here to provide a suitable alternative route to establish structure-function relationships. NBDs were shown to bind ATP and analogues as well as potent modulators of MDR, such as hydrophobic steroids, at a region close to the ATP site. Interestingly, flavonoids also bind to NBDs with high affinity. Their binding site partly overlaps both the ATP-binding site and the steroid-interacting region. Therefore flavonoids constitute a new promising class of bifunctional modulators of Pgp.

  9. A functional glycoprotein competitive recognition and signal amplification strategy for carbohydrate-protein interaction profiling and cell surface carbohydrate expression evaluation

    Science.gov (United States)

    Wang, Yangzhong; Chen, Zhuhai; Liu, Yang; Li, Jinghong

    2013-07-01

    A simple and sensitive carbohydrate biosensor has been suggested as a potential tool for accurate analysis of cell surface carbohydrate expression as well as carbohydrate-based therapeutics for a variety of diseases and infections. In this work, a sensitive biosensor for carbohydrate-lectin profiling and in situ cell surface carbohydrate expression was designed by taking advantage of a functional glycoprotein of glucose oxidase acting as both a multivalent recognition unit and a signal amplification probe. Combining the gold nanoparticle catalyzed luminol electrogenerated chemiluminescence and nanocarrier for active biomolecules, the number of cell surface carbohydrate groups could be conveniently read out. The apparent dissociation constant between GOx@Au probes and Con A was detected to be 1.64 nM and was approximately 5 orders of magnitude smaller than that of mannose and Con A, which would arise from the multivalent effect between the probe and Con A. Both glycoproteins and gold nanoparticles contribute to the high affinity between carbohydrates and lectin. The as-proposed biosensor exhibits excellent analytical performance towards the cytosensing of K562 cells with a detection limit of 18 cells, and the mannose moieties on a single K562 cell were determined to be 1.8 × 1010. The biosensor can also act as a useful tool for antibacterial drug screening and mechanism investigation. This strategy integrates the excellent biocompatibility and multivalent recognition of glycoproteins as well as the significant enzymatic catalysis and gold nanoparticle signal amplification, and avoids the cell pretreatment and labelling process. This would contribute to the glycomic analysis and the understanding of complex native glycan-related biological processes.A simple and sensitive carbohydrate biosensor has been suggested as a potential tool for accurate analysis of cell surface carbohydrate expression as well as carbohydrate-based therapeutics for a variety of diseases and

  10. Loss of a novel mucin-like epithelial glycoprotein in oral and cervical squamous cell carcinomas

    DEFF Research Database (Denmark)

    Nielsen, P A; Mandel, U; Therkildsen, M H;

    1997-01-01

    layers of buccal epithelium and was also found in larynx, esophagus, vagina, and exocervix, but not in epidermis. Data showed that gp230 was distinct from MUC1 or CD44. It is interesting that in most cases gp230 was not expressed in squamous cell carcinomas of buccal and cervical mucosa. A few moderately...

  11. Polyphenols of Mangifera indica modulate arsenite-induced cytotoxicity in a human proximal tubule cell line

    Directory of Open Access Journals (Sweden)

    Gabino Garrido

    2012-04-01

    Full Text Available Inorganic arsenic is an ubiquitous environmental contaminant able to cause severe pathologies in humans, including kidney disorders. The possible protective effects of Mangifera indica L., Anacardiaceae, stem bark extract (MSBE and some mango phenols on the cytotoxicity of arsenite (AsIII in the proximal tubule cell line HK-2 was investigated. In cells cultured for 24 h in presence of AsIII, a dose-dependent loss of cell viability occurred that was significantly alleviated by MSBE, followed by gallic acid, catechin and mangiferin. Mangiferin complexed with Fe+++ proved more efficacious than mangiferin alone. MSBE and pure phenols increased significantly the cell surviving fraction in clonogenic assays. In cells pretreated with MSBE or phenols for 72 h the protection afforded by MSBE resulted decreased in comparison with the shorter experiments. Cells pretreated with a subcytotoxic amount of AsIII or cultured in continuous presence of low concentration of mangiferin proved to be more resistant to AsIII, while cells cultured in presence of albumin resulted more sensitive. Because all the above conditions share changes in expression/activity of P-glycoprotein (P-gp, a transporter potentially involved in arsenic resistance, the capability of M. indica phenols in modulating AsIII-induced cytotoxicity would be at least in part dependent on their interactions with P-gp.

  12. Transport of radiolabelled glycoprotein to cell surface and lysosome-like bodies of absorptive cells in cultured small-intestinal tissue from normal subjects and patients with a lysosomal storage disease

    International Nuclear Information System (INIS)

    The transport of 3H-fucose and 3H-glucosamine-labelled glycoproteins in the absorptive cells of cultured human small-intestinal tissue was investigated with light- and electron-microscopical autoradiography. The findings showed that these glycoproteins were completed in the Golgi apparatus and transported in small vesicular structures to the apical cytoplasm of these cells. Since this material arrived in the cell coat on the microvilli and in the lysosome-like bodies simultaneously, a crinophagic function of these organelles in the regulation of the transport or secretion of cell-coat material was supported. In the absorptive cells of patients with fucosidosis or Hunter's type of lysosomal storage disease, a similar transport of cell-coat material to the lysosome-like bodies and a congenital defect of a lysosomal hydrolase normally involved in the degradation of cell-coat material, can explain the accumulation of this material in the dense bodies. (orig.)

  13. Development and characterization of a new human hepatic cell line

    Science.gov (United States)

    Ramboer, Eva; De Craene, Bram; De Kock, Joey; Berx, Geert; Rogiers, Vera; Vanhaecke, Tamara; Vinken, Mathieu

    2015-01-01

    The increasing demand and hampered use of primary human hepatocytes for research purposes have urged scientists to search for alternative cell sources, such as immortalized hepatic cell lines. The aim of this study was to develop a human hepatic cell line using the combined overexpression of TERT and the cell cycle regulators cyclin D1 and mutant isoform CDK4R24C. Following transduction of adult human primary hepatocytes with the selected immortalization genes, cell growth was triggered and a cell line was established. When cultured under appropriate conditions, the cell line expressed several hepatocytic markers and liver-enriched transcription factors at the transcriptional and/or translational level, secreted liver-specific proteins and showed glycogen deposition. These results suggest that the immortalization strategy applied to primary human hepatocytes could generate a novel hepatic cell line that seems to retain some key hepatic characteristics. PMID:26869867

  14. The calcium-dependent myoblast adhesion that precedes cell fusion is mediated by glycoproteins

    OpenAIRE

    1985-01-01

    Presumptive myoblasts from explants of chick embryo pectoral muscle proliferate, differentiate, and fuse to form multinucleate myotubes. One event critical to multinucleate cell formation is the specific adhesion of myoblasts before union of their membranes. In the studies reported here five known inhibitors of myotube formation-- trifluoperazine, sodium butyrate, chloroquine, 1,10 phenanthroline, and tunicamycin--were tested for their effect on the Ca++-dependent myoblast adhesion step. The ...

  15. [Hydroxyproline: Rich glycoproteins of the plant and cell wall]. Annual technical progress report, 1993

    Energy Technology Data Exchange (ETDEWEB)

    Varner, J.E.

    1993-06-01

    Since xylem tissue includes the main cell types which are lignified, we are interested in gene expression of glycine-rich proteins and proline-rich proteins, and other proteins which are involved in secondary cell wall thickening during xylogenesis. Since the main feature of xylogenesis is the deposition of additional wall components, study of the mechanism of xylogenesis will greatly advance our knowledge of the synthesis and assembly of wall macromolecules. We are using the in vitro xylogenesis system from isolated Zinnia mesophyll cells to isolate genes which are specifically expressed during xylogenesis. We have used subtractive hybridization methods to isolate a number of cDNA clones for differentially regulated genes from the cells after hormonal induction. So far, we have partially characterized 18 different cDNA clones from 239 positive clones. These differentially regulated genes can be divided into three sets according to the characteristics of gene expression in the induction medium and the control medium. The first set is induced in both the induction medium and the control medium without hormones. The second set is induced mainly in the induction medium and in the control medium with the addition of NAA alone. Two of thesegenes are exclusively induced by auxin. The third set of genes is induced mainly in the induction medium. Since these genes are not induced by either auxin or cytokinin alone, they may be directly involved in the process of xylogenesis. Our experiments on the localization of H{sub 2}O{sub 2} production reinforce the earlier ideas of others that H{sub 2}O{sub 2} is involved in normal lignification.

  16. Varicella-zoster virus glycoprotein expression differentially induces the unfolded protein response in infected cells

    OpenAIRE

    Carpenter, John E.; Grose, Charles

    2014-01-01

    Varicella-zoster virus (VZV) is a human herpesvirus that spreads to children as varicella or chicken pox. The virus then establishes latency in the nervous system and re-emerges, typically decades later, as zoster or shingles. We have reported previously that VZV induces autophagy in infected cells as well as exhibiting evidence of the Unfolded Protein Response (UPR): XBP1 splicing, a greatly expanded Endoplasmic Reticulum (ER) and CHOP expression. Herein we report the results of a UPR specif...

  17. Varicella-Zoster Virus glycoprotein expression differentially induces the unfolded protein response in infected cells.

    OpenAIRE

    John Earl Carpenter; Charles eGrose

    2014-01-01

    Varicella-zoster virus (VZV) is a human herpesvirus that spreads to children as varicella or chicken pox. The virus then establishes latency in the nervous system and re-emerges, typically decades later, as zoster or shingles. We have reported previously that VZV induces autophagy in infected cells as well as exhibiting evidence of the Unfolded Protein Response (UPR): XBP1 splicing, a greatly expanded Endoplasmic Reticulum (ER) and CHOP expression. Herein we report the results of a UPR specif...

  18. Distinct differentiation characteristics of individual human embryonic stem cell lines

    Directory of Open Access Journals (Sweden)

    Knuutila Sakari

    2006-08-01

    Full Text Available Abstract Background Individual differences between human embryonic stem cell (hESC lines are poorly understood. Here, we describe the derivation of five hESC lines (called FES 21, 22, 29, 30 and 61 from frozen-thawed human embryos and compare their individual differentiation characteristic. Results The cell lines were cultured either on human or mouse feeder cells. The cells grew significantly faster and could be passaged enzymatically only on mouse feeders. However, this was found to lead to chromosomal instability after prolonged culture. All hESC lines expressed the established markers of pluripotent cells as well as several primordial germ cell (PGC marker genes in a uniform manner. However, the cell lines showed distinct features in their spontaneous differentiation patterns. The embryoid body (EB formation frequency of FES 30 cell line was significantly lower than that of other lines and cells within the EBs differentiated less readily. Likewise, teratomas derived from FES 30 cells were constantly cystic and showed only minor solid tissue formation with a monotonous differentiation pattern as compared with the other lines. Conclusion hESC lines may differ substantially in their differentiation properties although they appear similar in the undifferentiated state.

  19. Analysis of three marine fish cell lines by rapd assay.

    Science.gov (United States)

    Guo, H R; Zhang, S C; Tong, S L; Xiang, J H

    2001-01-01

    We tested the applicability of the random amplified polymorphic deoxyribonucleic acid (RAPD) analysis for identification of three marine fish cell lines FG, SPH, and RSBF, and as a possible tool to detect cross-contamination. Sixty commercial 10-mer RAPD primers were tested on the cell lines and on samples collected from individual fish. The results obtained showed that the cell lines could be identified to the correspondent species on the basis of identical patterns produced by 35-48% of the primers tested; the total mean similarity indices for cell lines versus correspondent species of individual fish ranged from 0.825 to 0.851, indicating the existence of genetic variation in these cell lines in relation to the species of their origin. Also, four primers, which gave a monomorphic band pattern within species/line, but different among the species/line, were obtained. These primers can be useful for identification of these cell lines and for characterization of the genetic variation of these cell lines in relation to the species of their origin. This supported the use of RAPD analysis as an effective tool in species identification and cross-contamination test among different cell lines. PMID:11573817

  20. The Changes of P-glycoprotein Activity by Interferon-γ and Tumor Necrosis Factor-α in Primary and Immortalized Human Brain Microvascular Endothelial Cells

    OpenAIRE

    Lee, Na-Young; Rieckmann, Peter; Kang, Young-Sook

    2012-01-01

    The purpose of this study was to investigate the modification of expression and functionality of the drug transporter P-glycoprotein (P-gp) by tumor necrosis factor-alpha (TNF-α) and interferon-gamma (IFN-γ) at the blood-brain barrier (BBB). We used immortalized human brain microvessel endothelial cells (iHBMEC) and primary human brain microvessel endothelial cells (pHBMEC) as in vitro BBB model. To investigate the change of p-gp expression, we carried out real time PCR analysis and Western b...

  1. Oocyte glycoproteins regulate the form and function of the follicle basal lamina and theca cells.

    Science.gov (United States)

    Christensen, Alice P; Patel, Saloni H; Grasa, Patricia; Christian, Helen C; Williams, Suzannah A

    2015-05-15

    Maintaining follicle integrity during development, whereby each follicle is a functional unit containing a single oocyte, is essential for the generation of healthy oocytes. However, the mechanisms that regulate this critical function have not been determined. In this paper we investigate the role of the oocyte in maintaining follicle development. To investigate this role, we use a mouse model with oocyte-specific deletion of C1galt1 which is required for the generation of core 1-derived O-glycans. The loss of oocyte-generated O-glycans results in the joining of follicles and the generation of Multiple-Oocyte Follicles (MOFs). The aim was to determine how Mutant follicle development is modified thus enabling follicles to join. Extracellular matrix and follicle permeability were studied using histology, immunohistochemistry and electron microscopy (EM). In ovaries containing Mutant Oocytes, the Follicle basal lamina (FBL) is altered both functionally and structurally from the primary stage onwards with Mutant follicles possessing unexpectedly thicker FBL. In Mutant ovaries, the theca cell layer is also modified with intermingling of theca between adjacent follicles. MOF function was analysed but despite increased numbers of preantral MOFs in Mutants, these do not reach the preovulatory stage after gonadotrophin stimulation. We propose a model describing how oocyte initiated changes in FBL and theca cells result in follicles joining. These data reveal new and important roles for the oocyte in follicle development and follicle integrity. PMID:25557622

  2. Pseudorabies virus glycoprotein L is necessary for virus infectivity but dispensable for virion localization of glycoprotein H.

    OpenAIRE

    Klupp, B G; Fuchs, W; Weiland, E; Mettenleiter, T.C.

    1997-01-01

    Herpesviruses contain a number of envelope glycoproteins which play important roles in the interaction between virions and target cells. Although several glycoproteins are not present in all herpesviruses, others, including glycoproteins H and L (gH and gL), are conserved throughout the Herpesviridae. To elucidate common properties and differences in herpesvirus glycoprotein function, corresponding virus mutants must be constructed and analyzed in different herpesvirus backgrounds. Analysis o...

  3. POTENTIAL CELL LINE TOXICITY OF ENVIRONMENTAL NANOPARTICLES

    Directory of Open Access Journals (Sweden)

    Mohan Durga

    2012-01-01

    Full Text Available In India, the unprecedented growth rate and urbanization along with the rapid increase in motor vehicle activity and industrialization are contributing to high levels of urban air pollution. The population is mainly exposed to high air pollution concentrations, where motor vehicle emissions constitute the main source of fine and ultrafine particles. Motor exhaust emissions is a mixture of gases and Particulate Matter (PM. Diesel and petrol fuels in vehicles produce combustion-derived particles as a result of combustion. Vehicle exhaust particles are the main constituents of environmental nanoparticles. In the present investigation, environmental nanoparticles such as Diesel Exhaust Particles (DEP and Petrol Exhaust Particles (PEP were collected from on-road vehicles using a specially designed collection chamber. The surface morphology of the collected particles was analyzed through Transmission Electron Microscope (TEM, and the elemental mapping was performed through EDAX analysis. Results indicated the presence of nanometer-size particles in both the categories of vehicle exhaust. These small-size particles of respirable range can enter the respiratory tract of humans and get deposited in the lungs and cause various effects inside the human body. The aim of this study is to assess the cytotoxicity of the collected Diesel Exhaust Nanoparticles (DENPs and Petrol Exhaust Nanoparticles (PENPs. Cytotoxicity endpoint, such as IC50 (50% Inhibitory Concentration, was determined after a 24-h exposure. Results of this study indicated that all five cell lines were sensitive to these vehicle exhaust nanoparticles at varying levels.

  4. Investigation of the selenium metabolism in cancer cell lines

    DEFF Research Database (Denmark)

    Lunøe, Kristoffer; Gabel-Jensen, Charlotte; Stürup, Stefan;

    2011-01-01

    The aim of this work was to compare different selenium species for their ability to induce cell death in different cancer cell lines, while investigating the underlying chemistry by speciation analysis. A prostate cancer cell line (PC-3), a colon cancer cell line (HT-29) and a leukaemia cell line...... (Jurkat E6-1) were incubated with five selenium compounds representing inorganic as well as organic Se compounds in different oxidation states. Selenomethionine (SeMet), Se-methylselenocysteine (MeSeCys), methylseleninic acid (MeSeA), selenite and selenate in the concentration range 5-100 mu M were...... incubated with cells for 24 h and the induction of cell death was measured using flow cytometry. The amounts of total selenium in cell medium, cell lysate and the insoluble fractions was determined by ICP-MS. Speciation analysis of cellular fractions was performed by reversed phase, anion exchange and size...

  5. EMA: a developmentally regulated cell-surface glycoprotein of CNS neurons that is concentrated at the leading edge of growth cones.

    Science.gov (United States)

    Baumrind, N L; Parkinson, D; Wayne, D B; Heuser, J E; Pearlman, A L

    1992-08-01

    To identify cell-surface molecules that mediate interactions between neurons and their environment during neural development, we used monoclonal antibody techniques to define a developmentally regulated antigen in the central nervous system of the mouse. The antibody we produced (2A1) immunolabels cells throughout the central nervous system; we analyzed its distribution in the developing cerebral cortex, where it is expressed on cells very soon after they complete mitosis and leave the periventricular proliferative zone. Expression continues into adult life. The antibody also labels the epithelium of the choroid plexus and the renal proximal tubules, but does not label neurons of the peripheral nervous system in the dorsal root ganglia. In dissociated cell culture of embryonic cerebral cortex, 2A1 labels the surface of neurons but not glia. Immunolabeling of neurons in tissue culture is particularly prominent on the edge of growth cones, including filopodia and the leading edge of lamellipodia, when observed with either immunofluorescence or freeze-etch immunoelectron microscopy. Immunopurification with 2A1 of a CHAPS-extracted membrane preparation from brains of neonatal mice produces a broad (32-36 kD) electrophoretic band and a less prominent 70 kD band that are sensitive to N-glycosidase but not endoglycosidase H. Thus the 2A1 antibody recognizes a developmentally regulated, neuronal cell surface glycoprotein (or glycoproteins) with complex N-linked oligosaccharide side chains. We have termed the glycoprotein antigen EMA because of its prominence on the edge membrane of growth cones. EMA is similar to the M6 antigen (Lagenaur et al: J. Neurobiol. 23:71-88, 1992) in apparent molecular weight, distribution in tissue sections, and immunoreactivity on Western blots, suggesting that the two antigens are similar or identical. Expression of EMA is a very early manifestation of neuronal differentiation; its distribution on growth cones suggests a role in mediating the

  6. Expression of peanut agglutinin-binding mucin-type glycoprotein in human esophageal squamous cell carcinoma as a marker

    Directory of Open Access Journals (Sweden)

    Balakrishnan Ramathilakam

    2003-11-01

    Full Text Available Abstract Background The TF (Thomson – Friedenreich blood group antigen behaves as an onco-foetal carcinoma-associated antigen, showing increased expression in malignancies and its detection and quantification can be used in serologic diagnosis mainly in adenocarcinomas. This study was undertaken to analyze the sera and tissue level detectable mucin-type glycoprotein (TF-antigen by Peanut agglutinin (PNA and its diagnostic index in serum as well tissues of human esophageal squamous cell carcinoma as marker. Results We examined 100 patients for serological analysis by Enzyme Linked Lectin Assay (ELISA and demonstrated a sensitivity of 87.5%, specificity of 90% and a positive predictive value of 95%. The immuno-histochemical localization of TF antigen by Fluorescence Antigen Technique (FAT in 25 specimens of normal esophageal squamous epithelium specimens and 92 specimens with different grades of, allowed a quicker and more precise identification of its increased expression and this did not correlate with gender and tumor size. There was a positive correlation between membrane bound TF antigen expression with different histological progression, from well differentiated to poorly differentiated, determined by PNA binding. Specimens showed morphological changes and a pronounced increase in PNA binding in Golgi apparatus, secretory granules of the cytosol of well differentiated and an increased cell membrane labeling in moderately and poorly differentiated, when compared with ESCC and normal tissues. Conclusion The authors propose that the expression of TF-antigen in human may play an important role during tumorigenesis establishing it as a chemically well-defined carcinoma-associated antigen. Identification of the circulating TF-antigen as a reactive form and as a cryptic form in the healthy individuals, using PNA-ELLA and Immunohistochemical analysis of TF antigen by FAT is positively correlated with the different histological grades as a simple

  7. Modulation of ganglioside expression in human melanoma cell lines

    International Nuclear Information System (INIS)

    Cell surface gangliosides in human melanoma cell lines were modulated by pretreatment and adaptation to 6-thioguanine and 5-bromo-deoxyuridine. Chemo- and radiation sensitivities were compared in original cell lines and modulated cells by the human tumor colony-forming assay. Modulated cells showed decreased expression of cell surface GM2 and GD2 gangliosides. This reduction was correlated with increased resistance to bleomycin, vincristine, cisplatin and radiation treatment. These results suggest that cell surface GM2 and GD2 ganglioside expression in human melanoma cells is intimately associated with several cellular biological properties, such as drug or radiation sensitivity and cellular differentiation. (author)

  8. Mechanisms of confluence-dependent expression of CD26 in colon cancer cell lines

    Directory of Open Access Journals (Sweden)

    Morimoto Chikao

    2011-02-01

    Full Text Available Abstract Background CD26 (dipeptidyl peptidase IV, DPPIV is a 110 kDa surface glycoprotein expressed in most normal tissues, and is a potential novel therapeutic target for selected cancers. Our work evaluates the mechanism involved in confluence-dependent CD26 expression in colon cancer. Methods Colon adenocarcinoma cells were grown to confluence, and expression of CD26 and transcription factors implicated in its regulation was confirmed by immunofluorescence and Western blotting. Real-time PCR was also performed to evaluate CD26 upregulation at the transcriptional level. The influence of c-Myc on CD26 expression during different growth conditions was further evaluated following transient transfection of a c-Myc-expressing plasmid and a c-Myc specific siRNA. Results We found that the colon cancer cell lines HCT-116 and HCT-15 exhibited a confluence-dependent increase in CD26 mRNA and protein, associated with decreased expression of c-Myc, increased USF-1 and Cdx 2 levels, and unchanged HNF-1α expression. Meanwhile, ectopic expression of c-Myc in both cell lines led to decreased CD26 expression. In contrast, transfection of a siRNA targeted to Cdx2 resulted in decreased CD26 level. Importantly, culturing of cells in serum-depleted media, but not acidic conditions, upregulated CD26. While HIF-1α level also increased when cells were cultured in serum-depleted media, its expression was required but not sufficient for CD26 upregulation. Conclusions CD26 mRNA and protein levels increase in a confluence-dependent manner in colon carcinoma cell lines, with c-Myc acting as a repressor and Cdx2 acting as an enhancer of CD26 expression. The enhanced expression of CD26 in serum-depleted media and a requirement for HIF-1α suggest a role for nutrients or growth factors in the regulation of CD26 protein expression.

  9. Mechanisms of confluence-dependent expression of CD26 in colon cancer cell lines

    International Nuclear Information System (INIS)

    CD26 (dipeptidyl peptidase IV, DPPIV) is a 110 kDa surface glycoprotein expressed in most normal tissues, and is a potential novel therapeutic target for selected cancers. Our work evaluates the mechanism involved in confluence-dependent CD26 expression in colon cancer. Colon adenocarcinoma cells were grown to confluence, and expression of CD26 and transcription factors implicated in its regulation was confirmed by immunofluorescence and Western blotting. Real-time PCR was also performed to evaluate CD26 upregulation at the transcriptional level. The influence of c-Myc on CD26 expression during different growth conditions was further evaluated following transient transfection of a c-Myc-expressing plasmid and a c-Myc specific siRNA. We found that the colon cancer cell lines HCT-116 and HCT-15 exhibited a confluence-dependent increase in CD26 mRNA and protein, associated with decreased expression of c-Myc, increased USF-1 and Cdx 2 levels, and unchanged HNF-1α expression. Meanwhile, ectopic expression of c-Myc in both cell lines led to decreased CD26 expression. In contrast, transfection of a siRNA targeted to Cdx2 resulted in decreased CD26 level. Importantly, culturing of cells in serum-depleted media, but not acidic conditions, upregulated CD26. While HIF-1α level also increased when cells were cultured in serum-depleted media, its expression was required but not sufficient for CD26 upregulation. CD26 mRNA and protein levels increase in a confluence-dependent manner in colon carcinoma cell lines, with c-Myc acting as a repressor and Cdx2 acting as an enhancer of CD26 expression. The enhanced expression of CD26 in serum-depleted media and a requirement for HIF-1α suggest a role for nutrients or growth factors in the regulation of CD26 protein expression

  10. DNA content analysis of insect cell lines by flow cytometry

    OpenAIRE

    Léry, Xavier; Charpentier, Guy; Belloncik, Serge

    1999-01-01

    The DNA content of insect cell lines (6 lepidoptera, 1 coleoptera and 1 diptera) was determined by flow cytometry. The DNA profiles of the 8 cell lines tested were different. They were characterized by the presence of several peaks (2 to 7) corresponding to different ploidy levels, by differences in the fluorescence intensity of each peak and by the proportion of cells in each peak. Two cell lines (Cf124 and BmN) were constituted of 2 distinct populations of cells. The DNA profiles of the cel...

  11. Routine enterovirus diagnosis in a human rhabdomyosarcoma cell line

    OpenAIRE

    Bell, Eleanor J.; Cosgrove, Bonnie P.

    1980-01-01

    For many years a substitute cell line has been sought to replace monkey kidney cell cultures for the diagnosis of enterovirus infections. Reports by various workers have shown that the RD cell line, derived from a human rhabdomyosarcoma, will support the replication of most of the prototype strains of enterovirus. The present study shows that, with the exception of the group B coxsackieviruses, RD cells are more sensitive than cynomolgus monkey kidney cultures for the isolation of a wide vari...

  12. Pseudorabies Virus Glycoprotein M Inhibits Membrane Fusion

    OpenAIRE

    Klupp, Barbara G.; Nixdorf, Ralf; Mettenleiter, Thomas C.

    2000-01-01

    A transient transfection-fusion assay was established to investigate membrane fusion mediated by pseudorabies virus (PrV) glycoproteins. Plasmids expressing PrV glycoproteins under control of the immediate-early 1 promoter-enhancer of human cytomegalovirus were transfected into rabbit kidney cells, and the extent of cell fusion was quantitated 27 to 42 h after transfection. Cotransfection of plasmids encoding PrV glycoproteins B (gB), gD, gH, and gL resulted in formation of polykaryocytes, as...

  13. Characterization of xenoantiserum produced against B cell acute lymphoblastic leukemia cell line

    Directory of Open Access Journals (Sweden)

    Akagi,Tadaatsu

    1982-10-01

    Full Text Available Antiserum was produced in white rabbit by intravenously injecting living cells of a B cell acute lymphoblastic leukemia (ALL line (BALL-1. The reactivity of the antiserum against various lymphoid cell lines was examined by membrane immunofluorescence after appropriate absorption. Serum absorbed with non-T, non-B (NALL-1 and T-ALL (TALL-1 cells recognized B cell antigens distinct from Ia-like antigens on both normal and neoplastic B cells. After further absorption with tonsillar cells or normal B cell line (KO-HL-3, it reacted only with BALL-1 cells and did not react with other leukemia/lymphoma and normal B cell lines. The serum absorbed with tonsillar cells reacted only with BALL-1 and some B cell lines. Thus we were able to obtain antisera with specificity to B cell antigen, B-ALL antigen, and B cell line antigen.

  14. Formation of infectious hybrid virions with gibbon ape leukemia virus and human T-cell leukemia virus retroviral envelope glycoproteins and the gag and pol proteins of Moloney murine leukemia virus.

    OpenAIRE

    C. Wilson; Reitz, M S; Okayama, H; Eiden, M V

    1989-01-01

    The gibbon ape leukemia virus, SEATO strain, and human T-cell leukemia virus type I envelope glycoproteins can be functionally assembled with a Moloney murine leukemia virus core into infectious particles. The envelope-host cell receptor interaction is the major determinant of the host cell specificity for these hybrid virions.

  15. Permissiveness of human hepatoma cell lines for HCV infection

    Directory of Open Access Journals (Sweden)

    Sainz Bruno

    2012-01-01

    Full Text Available Abstract Background Although primary and established human hepatoma cell lines have been evaluated for hepatitis C virus (HCV infection in vitro, thus far only Huh7 cells have been found to be highly permissive for infectious HCV. Since our understanding of the HCV lifecycle would benefit from the identification of additional permissive cell lines, we assembled a panel of hepatic and non-hepatic cell lines and assessed their ability to support HCV infection. Here we show infection of the human hepatoma cell lines PLC/PRF/5 and Hep3B with cell culture-derived HCV (HCVcc, albeit to lower levels than that achieved in Huh7 cells. To better understand the reduced permissiveness of PLC and Hep3B cells for HCVcc infection, we performed studies to evaluate the ability of each cell line to support specific steps of the viral lifecycle (i.e. entry, replication, egress and spread. Results We found that while the early events in HCV infection (i.e. entry plus replication initiation are cumulatively equivalent or only marginally reduced in PLC and Hep3B cells, later steps of the viral life cycle such as steady-state replication, de novo virus production and/or spread are impaired to different degrees in PLC and Hep3B cultures compared to Huh7 cell cultures. Interestingly, we also observed that interferon stimulated gene (i.e. ISG56 expression was significantly and differentially up-regulated in PLC and Hep3B cells following viral infection. Conclusions We conclude that the restrictions observed later during HCV infection in these cell lines could in part be attributed to HCV-induced innate signaling. Nevertheless, the identification of two new cell lines capable of supporting authentic HCVcc infection, even at reduced levels, expands the current repertoire of cell lines amendable for the study of HCV in vitro and should aid in further elucidating HCV biology and the cellular determinants that modulate HCV infection.

  16. 77 FR 5489 - Identification of Human Cell Lines Project

    Science.gov (United States)

    2012-02-03

    ... Human Subjects. Paperwork Reduction Act: This notice contains collection of information requirements... National Institute of Standards and Technology Identification of Human Cell Lines Project AGENCY: National... tandem repeat (STR) profiling up to 1500 human cell line samples as part of the Identification of...

  17. Derivation of the human embryonic stem cell line RCM1

    Directory of Open Access Journals (Sweden)

    P.A. De Sousa

    2016-03-01

    Full Text Available The human embryonic stem cell line RCM-1 was derived from a failed to fertilise egg undergoing parthenogenetic stimulation. The cell line shows normal pluripotency marker expression and differentiation to three germ layers in vitro and in vivo. It has a normal 46XX female karyotype and microsatellite PCR identity, HLA and blood group typing data is available.

  18. Trichloroethylene toxicity in a human hepatoma cell line

    Energy Technology Data Exchange (ETDEWEB)

    Thevenin, E.; McMillian, J. [Medical Univ. of Charleston South Carolina, SC (United States)

    1994-12-31

    The experiments conducted in this study were designed to determine the usefullness of hepatocyte cultures and a human hepatoma cell line as model systems for assessing human susceptibility to hepatocellular carcinoma due to exposure to trichloroethylene. The results from these studies will then be analyzed to determine if human cell lines can be used to conduct future experiments of this nature.

  19. GREG cells, a dysferlin-deficient myogenic mouse cell line

    International Nuclear Information System (INIS)

    The dysferlinopathies (e.g. LGMD2b, Myoshi myopathy) are progressive, adult-onset muscle wasting syndromes caused by mutations in the gene coding for dysferlin. Dysferlin is a large (∼ 200 kDa) membrane-anchored protein, required for maintenance of plasmalemmal integrity in muscle fibers. To facilitate analysis of dysferlin function in muscle cells, we have established a dysferlin-deficient myogenic cell line (GREG cells) from the A/J mouse, a genetic model for dysferlinopathy. GREG cells have no detectable dysferlin expression, but proliferate normally in growth medium and fuse into functional myotubes in differentiation medium. GREG myotubes exhibit deficiencies in plasma membrane repair, as measured by laser wounding in the presence of FM1–43 dye. Under the wounding conditions used, the majority (∼ 66%) of GREG myotubes lack membrane repair capacity, while no membrane repair deficiency was observed in dysferlin-normal C2C12 myotubes, assayed under the same conditions. We discuss the possibility that the observed heterogeneity in membrane resealing represents genetic compensation for dysferlin deficiency.

  20. GREG cells, a dysferlin-deficient myogenic mouse cell line

    Energy Technology Data Exchange (ETDEWEB)

    Humphrey, Glen W.; Mekhedov, Elena; Blank, Paul S. [Program in Physical Biology, Eunice Kennedy Schriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892 (United States); Morree, Antoine de [Center for Human Genetics, Leiden University Medical Center, Leiden (Netherlands); Pekkurnaz, Gulcin [Program in Physical Biology, Eunice Kennedy Schriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892 (United States); Nagaraju, Kanneboyina [Research Center for Genetic Medicine, Children' s National Medical Center, Washington, DC 20010 (United States); Zimmerberg, Joshua, E-mail: zimmerbj@mail.nih.gov [Program in Physical Biology, Eunice Kennedy Schriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892 (United States)

    2012-01-15

    The dysferlinopathies (e.g. LGMD2b, Myoshi myopathy) are progressive, adult-onset muscle wasting syndromes caused by mutations in the gene coding for dysferlin. Dysferlin is a large ({approx} 200 kDa) membrane-anchored protein, required for maintenance of plasmalemmal integrity in muscle fibers. To facilitate analysis of dysferlin function in muscle cells, we have established a dysferlin-deficient myogenic cell line (GREG cells) from the A/J mouse, a genetic model for dysferlinopathy. GREG cells have no detectable dysferlin expression, but proliferate normally in growth medium and fuse into functional myotubes in differentiation medium. GREG myotubes exhibit deficiencies in plasma membrane repair, as measured by laser wounding in the presence of FM1-43 dye. Under the wounding conditions used, the majority ({approx} 66%) of GREG myotubes lack membrane repair capacity, while no membrane repair deficiency was observed in dysferlin-normal C2C12 myotubes, assayed under the same conditions. We discuss the possibility that the observed heterogeneity in membrane resealing represents genetic compensation for dysferlin deficiency.

  1. Human embryonic stem cell lines derived from the Chinese population

    Institute of Scientific and Technical Information of China (English)

    Zhen Fu FANG; Fan JIN; Hui GAI; Ying CHEN; Li WU; Ai Lian LIU; Bin CHEN; Hui Zhen SHENG

    2005-01-01

    Six human embryonic stem cell lines were established from surplus blastocysts. The cell lines expressed alkaline phosphatase and molecules typical of primate embryonic stem cells, including Oct-4, Nanog, TDGF1, Sox2, EBAF,Thy-1, FGF4, Rex-1, SSEA-3, SSEA-4, TRA-1-60 and TRA-1-81. Five of the six lines formed embryoid bodies that expressed markers of a variety of cell types; four of them formed teratomas with tissue types representative of all three embryonic germ layers. These human embryonic stem cells are capable of producing clones of undifferentiated morphology, and one of them was propagated to become a subline. Human embryonic stem cell lines from the Chinese population should facilitate stem cell research and may be valuable in studies of population genetics and ecology.

  2. The transcriptional diversity of 25 Drosophila cell lines

    Energy Technology Data Exchange (ETDEWEB)

    Cherbas, L.; Willingham, A.; Zhang, D.; Yang, L.; Zou, Y.; Eads, B. D.; Carlson, J. W.; Landolin, J. M.; Kapranov, P.; Dumais, J.; Samsonova, A.; Choi, J. -H.; Roberts, J.; Davis, C. A.; Tang, H.; van Baren, M. J.; Ghosh, S.; Dobin, A.; Bell, K.; Lin, W.; Langton, L.; Duff, M. O.; Tenney, A. E.; Zaleski, C.; Brent, M. R.; Hoskins, R. A.; Kaufman, T. C.; Andrews, J.; Graveley, B. R.; Perrimon, N.; Celniker, S. E.; Gingeras, T. R.; Cherbas, P.

    2010-12-22

    Drosophila melanogaster cell lines are important resources for cell biologists. Here, we catalog the expression of exons, genes, and unannotated transcriptional signals for 25 lines. Unannotated transcription is substantial (typically 19% of euchromatic signal). Conservatively, we identify 1405 novel transcribed regions; 684 of these appear to be new exons of neighboring, often distant, genes. Sixty-four percent of genes are expressed detectably in at least one line, but only 21% are detected in all lines. Each cell line expresses, on average, 5885 genes, including a common set of 3109. Expression levels vary over several orders of magnitude. Major signaling pathways are well represented: most differentiation pathways are ‘‘off’’ and survival/growth pathways ‘‘on.’’ Roughly 50% of the genes expressed by each line are not part of the common set, and these show considerable individuality. Thirty-one percent are expressed at a higher level in at least one cell line than in any single developmental stage, suggesting that each line is enriched for genes characteristic of small sets of cells. Most remarkable is that imaginal discderived lines can generally be assigned, on the basis of expression, to small territories within developing discs. These mappings reveal unexpected stability of even fine-grained spatial determination. No two cell lines show identical transcription factor expression. We conclude that each line has retained features of an individual founder cell superimposed on a common ‘‘cell line‘‘ gene expression pattern. Wereport the transcriptional profiles of 25 Drosophila melanogaster cell lines, principally by whole-genome tiling microarray analysis of total RNA, carried out as part of the modENCODE project. The data produced in this study add to our knowledge of the cell lines and of the Drosophila transcriptome in several ways. We summarize the expression of previously annotated genes in each of the 25 lines with emphasis on what

  3. HIF-1α inhibition reverses multidrug resistance in colon cancer cells via downregulation of MDR1/P-glycoprotein.

    Directory of Open Access Journals (Sweden)

    Jianfang Chen

    Full Text Available BACKGROUND: Multidrug resistance (MDR is one of the major reasons chemotherapy-based treatments fail. Hypoxia is generally associated with tumor chemoresistance. However, the correlation between the heterodimeric hypoxia-inducible factor-1 (HIF-1 and the multidrug resistance (MDR1 gene/transporter P-glycoprotein (P-gp remains unclear. This study aims to explore the molecular mechanisms of reversing colon cancer MDR by focusing on the target gene HIF-1α. METHODS: A chemotherapeutic sensitivity assay was used to observe the efficiency of MDR reversal in LoVo multicellular spheroids (MCS. The apoptotic level induced by different drugs was examined by flow cytometry (FCM. Binding of HIF-1α to the MDR1 gene promoter was evaluated by Chromatin immunoprecipitation (ChIP. The relationship between HIF-1α/P-gp expression and sensitivity to chemotherapy was analyzed. RESULTS: The sensitivity of LoVo MCS to all four chemotherapy drugs was decreased to varying degrees under hypoxic conditions. After silencing the HIF-1α gene, the sensitivities of LoVo MCS to all four chemotherapy drugs were restored. The apoptotic levels that all the drugs induced were all decreased to various extents in the hypoxic group. After silencing HIF-1α, the apoptosis level induced by all four chemotherapy drugs increased. The expression of HIF-1α and P-gp was significantly enhanced in LoVo MCS after treatment with hypoxia. Inhibiting HIF-1α significantly decreased the expression of MDR1/P-gp mRNA or protein in both the LoVo monolayers and LoVo MCS. The ChIP assay showed that HIF-1α was bound to the MDR1 gene promoter. Advanced colon carcinoma patients with expression of both HIF-1α and P-gp were more resistant to chemotherapy than that with non expression. CONCLUSIONS: HIF-1α inhibition reverses multidrug resistance in colon cancer cells via downregulation of MDR1/P-gp. The expression of HIF-1α and MDR1/P-gp can be used as a predictive marker for chemotherapy resistance

  4. Derivation of Genea052 human embryonic stem cell line

    Directory of Open Access Journals (Sweden)

    Biljana Dumevska

    2016-03-01

    Full Text Available The Genea052 human embryonic stem cell line was derived from a donated, fully commercially consented ART blastocyst, through ICM outgrowth on inactivated human feeders. The line showed pluripotent cell morphology and genomic analysis verified a 46, XY karyotype and male allele pattern through CGH and STR analysis. Pluripotency of Genea052 was demonstrated with 85% of cells expressing Nanog, 87% Oct4, 60% Tra1-60 and 97% SSEA4, a PluriTest Pluripotency score of 27.21, Novelty score of 1.2 and tri-lineage teratoma formation. The cell line was negative for Mycoplasma and any visible contamination.

  5. Derivation of Genea047 human embryonic stem cell line

    Directory of Open Access Journals (Sweden)

    Biljana Dumevska

    2016-03-01

    Full Text Available The Genea047 human embryonic stem cell line was derived from a donated, fully commercially consented ART blastocyst, through ICM outgrowth on inactivated human feeders. The line showed pluripotent cell morphology and genomic analysis verified a 46, XX karyotype and female allele pattern through traditional karyotyping, CGH and STR analysis. Pluripotency of Genea047 was demonstrated with 88% of cells expressing Nanog, 95% Oct4, 59% Tra1-60 and 99% SSEA4, a PluriTest Pluripotency score of 30.86, Novelty score of 1.23 and tri-lineage teratoma formation. The cell line was negative for Mycoplasma and any visible contamination.

  6. Derivation of Genea015 human embryonic stem cell line

    Directory of Open Access Journals (Sweden)

    Biljana Dumevska

    2016-03-01

    Full Text Available The Genea015 human embryonic stem cell line was derived from a donated, fully commercially consented ART blastocyst, through ICM outgrowth on inactivated human feeders. The line showed pluripotent cell morphology and genomic analysis verified a 46, XY karyotype and male Allele pattern through traditional karyotyping, CGH and STR analysis. Pluripotency of Genea015 was demonstrated with 80% of cells expressing Nanog, 97% Oct4, 75% Tra1-60 and 98% SSEA4, a PluriTest Pluripotency score of 29.52, Novelty score of 1.3 and Alkaline Phosphatase activity. The cell line was negative for Mycoplasma and any visible contamination.

  7. Derivation of human embryonic stem cell line Genea023

    Directory of Open Access Journals (Sweden)

    Biljana Dumevska

    2016-03-01

    Full Text Available The Genea023 human embryonic stem cell line was derived from a donated, fully commercially consented ART blastocyst, through ICM outgrowth on inactivated feeders. The line showed pluripotent cell morphology and genomic analysis verified a 46, XY karyotype and male allele pattern through CGH and STR analysis. Pluripotency of Genea023 was demonstrated with 85% of cells expressed Nanog, 98% Oct4, 55% Tra1-60 and 98% SSEA4, gave a Pluritest Pluripotency score of 42.76, Novelty of 1.23, demonstrated Alkaline Phosphatase activity and tri-lineage teratoma formation. The cell line was negative for Mycoplasma and visible contamination.

  8. Derivation of human embryonic stem cell line Genea022

    Directory of Open Access Journals (Sweden)

    Biljana Dumevska

    2016-03-01

    Full Text Available The Genea022 human embryonic stem cell line was derived from a donated, fully commercially consented ART blastocyst, through ICM outgrowth on inactivated feeders. The line showed pluripotent cell morphology and genomic analysis verified a 46, XY karyotype and male allele pattern through CGH and STR analysis. Pluripotency of Genea022 was demonstrated with 84% of cells expressed Nanog, 98% Oct4, 55% Tra1–60 and 97% SSEA4, gave a Pluritest Pluripotency score of 42.95, Novelty of 1.23, demonstrated Alkaline Phosphatase activity and tri-lineage teratoma formation. The cell line was negative for Mycoplasma and visible contamination.

  9. Derivation of Genea042 human embryonic stem cell line

    Directory of Open Access Journals (Sweden)

    Biljana Dumevska

    2016-03-01

    Full Text Available The Genea042 human embryonic stem cell line was derived from a donated, fully commercially consented ART blastocyst, through ICM outgrowth on inactivated human feeders. The line showed pluripotent cell morphology and genomic analysis verified a 46, XX karyotype and female allele pattern through traditional karyotyping, CGH and STR analysis. Pluripotency of Genea042 was demonstrated with 81% of cells expressing Nanog, 95% Oct4, 53% Tra1-60 and 97% SSEA4, a PluriTest Pluripotency score of 30.06, Novelty score of 1.24 and Alkaline Phosphatase activity. The cell line was negative for Mycoplasma and any visible contamination.

  10. Non-alkaloids extract from Stemona sessilifolia enhances the activity of chemotherapeutic agents through P-glycoprotein-mediated multidrug-resistant cancer cells.

    Science.gov (United States)

    Han, Lu; Ma, Yang-Mei; An, Li; Zhang, Qiao; Wang, Chang-Li; Zhao, Qing-Chun

    2016-01-01

    One of the major impediments to the successful treatment of cancer is the development of resistant cancer cells, which could cause multidrug resistance (MDR), and overexpression of ABCB1/P-glycoprotein (P-gp) is one of the most common causes of MDR in cancer cells. Recently, natural products or plant-derived chemicals have been investigated more and more widely as potential multidrug-resistant (MDR) reversing agents. The current study demonstrated for the first time that non-alkaloids extract from Stemona sessilifolia significantly reversed the resistance of chemotherapeutic agents, adriamycin, paclitaxel and vincristine to MCF-7/ADR cells compared with MCF-7/S cells in a dose-dependent manner. The results obtained from these studies indicated that the non-alkaloids extract from S. sessilifolia plays an important role in reversing MDR of cancer as a P-gp modulator in vitro and may be effective in the treatment of multidrug-resistant cancers. PMID:26190165

  11. Cytotoxinic Mechanism of Hydroxyapatite Nanoparticles on Human Hepatoma Cell Lines

    Institute of Scientific and Technical Information of China (English)

    CAO Xian-ying; QI Zhi-tao; DAI Hong-lian; YAN Yu-hua; LI Shi-pu

    2003-01-01

    Stable and single-dispersed HAP nanoparticles were synthesized with chemical method assisted by ultrasonic treatment.HAP nanoparticles were surveyed by AFM and Zataplus.The effect on the Bel-7402 human hepatoma cell lines treated with HAP nanoparticles was investigated by the MTT methods and observation of morphology,and the mechanism was studied in changes of cell cycle and ultrastructure.The result shows that inhibition of HAP nanoparticles on the Bel-7402 human hepatoma cell lines is obviously in vitro.HAP nanoparticles the entered cancer cytoplasm,and cell proliferation is stopped at G1 phase of cell cycle,thus,cancer cells die directly.

  12. Pregnancy-specific glycoprotein 1a activates dendritic cells to provide signals for Th17-, Th2-, and Treg-cell polarization.

    Science.gov (United States)

    Martínez, Fernando F; Knubel, Carolina P; Sánchez, Maria C; Cervi, Laura; Motrán, Claudia C

    2012-06-01

    Because of their plasticity and central role in orchestrating immunity and tolerance, DCs can respond to pregnancy-specific signals, thus promoting the appropriate immune response in order to support pregnancy. Here, we show that pregnancy-specific glycoprotein (PSG1a), the major variant of PSG released into the circulation during pregnancy, targets DCs to differentiate into a subset with a unique phenotype and function. This semi-mature phenotype is able to secrete IL-6 and TGF-β. PSG1a also affected the maturation of DCs, preventing the up-regulation of some costimulatory molecules, and inducing the secretion of TGF-β or IL-10 and the expression of programmed death ligand 1 (PD-L1) in response to TLR-9 or CD40 ligation. In addition, PSG1a-treated DCs promoted the enrichment of Th2-type cytokines, IL-17-producing cells, and Treg cells from CD4(+) T cells from DO11.10 Tg mice. Moreover, in vivo expression of PSG1a promoted the expansion of Ag-specific CD4(+) CD25(+) Foxp3(+) Treg cells and IL-17-, IL-4-, IL-5-, and IL-10-secreting cells able to protect against Listeria monocytogenes infection. Taken together, our data indicate that DCs can be targeted by PSG1a to generate the signals necessary to mount an appropriate, well-balanced, and effective immune response able to protect against invading pathogens while at the same time being compatible with a successful pregnancy. PMID:22678910

  13. Establishment of human embryonic stem cell line from gamete donors

    Institute of Scientific and Technical Information of China (English)

    LI Tao; ZHOU Can-quan; MAI Qing-yun; ZHUANG Guang-lun

    2005-01-01

    Background Human embryonic stem (HES) cell derived from human blastocyst can be propagated indefinitely in the primitive undifferentiated state while remaining pluripotent. It has exciting potential in human developmental biology, drug discovery, and transplantation medicine. But there are insufficient HES cell lines for further study. Methods Three oocyte donors were studied, and 3 in vitro fertilization (IVF) cycles were carried out to get blastocysts for the establishment of HES cell line. Isolated from blastocysts immunosurgically, inner cell mass (ICM) was cultured and propagated on mouse embryonic fibroblasts (MEFs). Once established, morphology, cell surface markers, karyotype and differentiating ability of the cell line were thoroughly analyzed.Results Four ICMs from 7 blastocysts were cultured on MEFs. After culture, one cell line (cHES-1) was established and met the criteria for defining human pluripotent stem cells including a series of markers used to identify pluripotent stem cells, morphological similarity to primate embryonic stem cells and HES reported else where. Normal and stable karyotype maintained over 60 passages, and demonstrated ability to differentiate into a wide variety of cell types.Conclusions HES cell lines can be established from gamete donors at a relatively highly efficient rate. The establishment will exert a widespread impact on biomedical research.

  14. Susceptibility of various cell lines to Neospora caninum tachyzoites cultivation

    Directory of Open Access Journals (Sweden)

    Khordadmehr, M.,

    2014-05-01

    Full Text Available Neospora caninum is a coccidian protozoan parasite which is a major cause of bovine abortions and neonatal mortality in cattle, sheep, goat and horse. Occasionally, cultured cells are used for isolation and multiplication of the agent in vitro with several purposes. In this study the tachyzoite yields of N. caninum were compared in various cell cultures as the host cell lines. Among the cell cultures tested, two presented good susceptibility to the agent: cell lines Vero and MA-104. SW742 and TLI (in vitro suspension culture of lymphoid cells infected with Theileria lestoquardi showed moderate sensitivity. No viable tachyzoite were detected in the culture of MDCK and McCoy cell lines. These results demonstrate that MA-104 and SW742 cells present adequate susceptibility to N. caninum compared to Vero cells, which have been largely used to multiply the parasite in vitro. Moreover, these have easy manipulation, fast multiplication and relatively low nutritional requirements. In addition, the result of this study showed that TLI cell line as a suspension cell culture is susceptible to Nc-1 tachyzoites infection and could be used as an alternative host cell line for tachyzoites culture in vitro studies.

  15. Identification of an additional class of C3-binding membrane proteins of human peripheral blood leukocytes and cell lines.

    Science.gov (United States)

    Cole, J L; Housley, G A; Dykman, T R; MacDermott, R P; Atkinson, J P

    1985-02-01

    Proteins binding the third component of complement (C3) were isolated by affinity chromatography from surface-labeled solubilized membranes of human peripheral blood cells and cell lines. The isolated molecules were subjected to NaDodSO4/PAGE, and autoradiographs of these gels indicated that C3-binding proteins could be divided into three groups based on Mr: (i) gp200, an approximately 200,000 Mr molecule previously identified as the C3b/C4b receptor or CR1; (ii) gp140, an approximately 140,000 Mr molecule previously identified as the C3d receptor or CR2; and (iii) gp45-70, a heretofore unrecognized group of 45,000-70,000 Mr C3-binding molecules. The cell distribution, Mr, antigenic cross-reactivity, and specificity of gp45-70 were examined. Erythrocytes have no detectable gp45-70, but all leukocyte populations examined possess this group of molecules. On neutrophils and mononuclear phagocytes, CR1 is the predominant C3-binding glycoprotein, but gp45-70 is present on both cell populations and on macrophage and neutrophil cell lines. B plus null cells, chronic lymphocytic leukemia cells, and an Epstein-Barr virus-transformed B-cell line possess CR1, CR2, and gp45-70. On T cells and T-cell lines gp45-70 is the predominant or, in some cases, the only C3-binding protein isolated. gp45-70 is structurally characterized as a broad band or doublet with a mean Mr that is slightly different for each cell population. gp45-70 binds iC3, C3b, and C4b, but not C3d, indicating that the binding region is probably within the C3c portion of C3b. A polyclonal antibody to CR1 and monoclonal antibodies to CR1 and CR2 do not immunoprecipitate gp45-70. While gp45-70 has not been previously characterized on human cells, a C3b-binding glycoprotein of similar Mr is present on rabbit alveolar macrophages. We conclude that gp45-70 is an additional group of membrane proteins present on human leukocytes that possess ligand-binding activity for C3b. PMID:3871945

  16. Generation and characterization of human insulin-releasing cell lines

    OpenAIRE

    Joffé Elisa; Machado Marcel CC; Buchanan Cecilia; Terra Letícia F; Stigliano Iván; Krogh Karin; Peters Maria G; Labriola Leticia; Puricelli Lydia; Sogayar Mari C

    2009-01-01

    Abstract Background The in vitro culture of insulinomas provides an attractive tool to study cell proliferation and insulin synthesis and secretion. However, only a few human beta cell lines have been described, with long-term passage resulting in loss of insulin secretion. Therefore, we set out to establish and characterize human insulin-releasing cell lines. Results We generated ex-vivo primary cultures from two independent human insulinomas and from a human nesidioblastosis, all of which w...

  17. Off-line hyphenation of boronate affinity monolith-based extraction with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry for efficient analysis of glycoproteins/glycopeptides.

    Science.gov (United States)

    Bie, Zijun; Chen, Yang; Li, Hengye; Wu, Ronghu; Liu, Zhen

    2014-06-27

    Boronate affinity materials have attracted increasing attentions as sample enrichment platforms for glycoproteomic analysis in recent years. However, most of the boronate affinity materials that have already employed for proteomic analysis are suffering from apparent disadvantages, such as alkaline pH for binding, weak affinity, and relatively poor selectivity. Benzoboroxoles are a unique class of boronic acids which have showed excellent binding properties for the recognition of cis-diol-containing compounds. Recently, a 3-carboxy-benzoboroxole-functionalized monolithic column had been reported and it had exhibited the best selectivity and affinity as well as the lowest binding pH among all reported boronate affinity monolithic columns. In this study, an off-line hyphenation of this boronate affinity monolithic column-based extraction with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) was developed and the powerfulness of this hyphenated approach in the analysis of glycoproteins and glycopeptides in complex samples was investigated. The approach was first applied to the analysis of glycopeptides in the tryptic digest of horseradish peroxidase (HRP). Totally 22 glycopeptides were identified. To the best of our knowledge, this is the best performance among all the boronic acid-functionalized materials. We further employed this approach to the analysis of intact proteins in human saliva. Totally 6 intact glycoproteins were successfully identified. As comparison, when the samples were analyzed without extraction, only a few glycopeptides were identified from the tryptic digest of HRP while no glycoproteins were found from the saliva samples. PMID:24928239

  18. Early Expression of Pregnancy-Specific Glycoprotein 22 (PSG22) by Trophoblast Cells Modulates Angiogenesis in Mice1

    OpenAIRE

    Blois, Sandra M.; Tirado-González, Irene; Wu, Julie; Barrientos, Gabriela; Johnson, Briana; Warren, James; Freitag, Nancy; Klapp, Burghard F.; Irmak, Ster; Ergun, Suleyman; Dveskler, Gabriela S.

    2012-01-01

    Mouse and human pregnancy-specific glycoproteins (PSG) are known to exert immunomodulatory functions during pregnancy by inducing maternal leukocytes to secrete anti-inflammatory cytokines that promote a tolerogenic decidual microenvironment. Many such anti-inflammatory mediators also function as proangiogenic factors, which, along with the reported association of murine PSG with the uterine vasculature, suggest that PSG may contribute to the vascular adaptations necessary for successful impl...

  19. P-Glycoprotein/MDR1 Regulates Pokemon Gene Transcription Through p53 Expression in Human Breast Cancer Cells

    OpenAIRE

    Wei Xu; Yuyang Jiang; Xuyu Zu; Shengnan He; Zhenhua Xie; Feng Liu

    2010-01-01

    P-glycoprotein (Pgp), encoded by the multidrug resistance 1 (MDR1) gene, is an efflux transporter and plays an important role in pharmacokinetics. In this study, we demonstrated that the pokemon promoter activity, the pokemon mRNA and protein expression can be significantly inhibited by Pgp. Chromatin immunoprecipitation assay showed that Pgp can bind the pokemon prompter to repress pokemon transcription activity. Furthermore, Pgp regulated pokemon transcription activity through expression of...

  20. Generation and characterization of human insulin-releasing cell lines

    Directory of Open Access Journals (Sweden)

    Joffé Elisa

    2009-06-01

    Full Text Available Abstract Background The in vitro culture of insulinomas provides an attractive tool to study cell proliferation and insulin synthesis and secretion. However, only a few human beta cell lines have been described, with long-term passage resulting in loss of insulin secretion. Therefore, we set out to establish and characterize human insulin-releasing cell lines. Results We generated ex-vivo primary cultures from two independent human insulinomas and from a human nesidioblastosis, all of which were cultured up to passage number 20. All cell lines secreted human insulin and C-peptide. These cell lines expressed neuroendocrine and islets markers, confirming the expression profile found in the biopsies. Although all beta cell lineages survived an anchorage independent culture, none of them were able to invade an extracellular matrix substrate. Conclusion We have established three human insulin-releasing cell lines which maintain antigenic characteristics and insulin secretion profiles of the original tumors. These cell lines represent valuable tools for the study of molecular events underlying beta cell function and dysfunction.

  1. Generation and characterization of human insulin-releasing cell lines

    Science.gov (United States)

    Labriola, Leticia; Peters, Maria G; Krogh, Karin; Stigliano, Iván; Terra, Letícia F; Buchanan, Cecilia; Machado, Marcel CC; Joffé, Elisa Bal de Kier; Puricelli, Lydia; Sogayar, Mari C

    2009-01-01

    Background The in vitro culture of insulinomas provides an attractive tool to study cell proliferation and insulin synthesis and secretion. However, only a few human beta cell lines have been described, with long-term passage resulting in loss of insulin secretion. Therefore, we set out to establish and characterize human insulin-releasing cell lines. Results We generated ex-vivo primary cultures from two independent human insulinomas and from a human nesidioblastosis, all of which were cultured up to passage number 20. All cell lines secreted human insulin and C-peptide. These cell lines expressed neuroendocrine and islets markers, confirming the expression profile found in the biopsies. Although all beta cell lineages survived an anchorage independent culture, none of them were able to invade an extracellular matrix substrate. Conclusion We have established three human insulin-releasing cell lines which maintain antigenic characteristics and insulin secretion profiles of the original tumors. These cell lines represent valuable tools for the study of molecular events underlying beta cell function and dysfunction. PMID:19545371

  2. Proteolysis of the Ebola Virus Glycoproteins Enhances Virus Binding and Infectivity▿

    OpenAIRE

    Kaletsky, Rachel L.; Simmons, Graham; Bates, Paul

    2007-01-01

    Cellular cathepsins are required for Ebola virus infection and are believed to proteolytically process the Ebola virus glycoprotein (GP) during entry. However, the significance of cathepsin cleavage during infection remains unclear. Here we demonstrate a role for cathepsin L (CatL) cleavage of Ebola virus GP in the generation of a stable 18-kDa GP1 viral intermediate that exhibits increased binding to and infectivity for susceptible cell targets. Cell binding to a lymphocyte line was increase...

  3. In Situ Biochemical Demonstration That P-Glycoprotein Is a Drug Efflux Pump with Broad Specificity

    OpenAIRE

    Chen, Yu; Simon, Sanford M.

    2000-01-01

    While P-glycoprotein (Pgp) is the most studied protein involved in resistance to anti-cancer drugs, its mechanism of action is still under debate. Studies of Pgp have used cell lines selected with chemotherapeutics which may have developed many mechanisms of resistance. To eliminate the confounding effects of drug selection on understanding the action of Pgp, we studied cells transiently transfected with a Pgp-green fluorescent protein (GFP) fusion protein. This method generated a mixed popul...

  4. Efficient transduction of neurons using Ross River glycoprotein-pseudotyped lentiviral vectors

    DEFF Research Database (Denmark)

    Jakobsson, J; Nielsen, T Tolstrup; Staflin, K;

    2006-01-01

    , including the possibility to establish stable producer cell lines. After injection of RRV-LV expressing green fluorescent protein into different structures in the rat brain we found efficient transduction of both neurons and glial cells. By using two cell-type-specific promoters, neuron-specific enolase and...... human glial fibrillary acidic protein, we demonstrated cell-specific transgene expression in the desired cell type. Ross River virus glycoprotein-pseudotyped lentiviral vectors also transduced human neural progenitor cells in vitro, showing that receptors for the RRV-G are present on human neural cells....

  5. Expression of the Thy-1 glycoprotein gene by DNA-mediated gene transfer.

    OpenAIRE

    Evans, G A; Ingraham, H A; Lewis, K; Cunningham, K; Seki, T.; Moriuchi, T; Chang, H. C.; Silver, J; Hyman, R

    1984-01-01

    We isolated a gene encoding the Thy-1.2 glycoprotein from a recombinant library constructed from BALB/c mouse DNA. To evaluate the expression of this cloned gene in different genomic environments, we introduced it into cell lines derived from fibroblast, lymphoid, and neuronal tissues by DNA-mediated gene transfer. When integrated into the genome of mouse L cells, cell-surface Thy-1 can be detected with anti-Thy-1 monoclonal antibodies. These L-cell lines contain between two and four copies o...

  6. Effects of Kaempferia parviflora extracts and their flavone constituents on P-glycoprotein function

    OpenAIRE

    Patanasethanont, Denpong; Nagai, Junya; Yumoto, Ryoko; Murakami, Teruo; Sutthanut, Khaetthareeya; Sripanidkulchai, Bung-Orn; Yenjai, Chavi; Takano, Mikihisa

    2006-01-01

    The purpose of this study was to examine the effects of extracts and flavone derivatives from the rhizome of Kaempferia parviflora on P-glycoprotein (P-gp)-mediated transport in LLC-GA5-COL150, a transfectant cell line of a porcine kidney epithelial cell line LLC-PK1 with human MDR1 cDNA. Ethanol extract obtained from Kaempferia parviflora rhizome significantly increased the accumulation of rhodamine 123 and daunorubicin, P-gp substrates, in LLC-GA5-COL150 cells, but not in LLC-PK1 cells. The...

  7. Phenotypic and cytogenetic characteristics of a new Epstein-Barr virus negative cell line (SKW 4) derived from a B-cell lymphoma.

    Science.gov (United States)

    Nilsson, K; Klareskog, L; Ralph, P; Sundström, C; Zech, L

    1983-01-01

    A new Epstein-Barr virus nuclear antigen (EBNA) negative cell line SKW 4 has been established in vitro from a patient with diffuse histiocytic lymphoma. The SKW 4 seems to be an authentic human tumour cell line as evidenced by its EBV negativity, monoclonality and aneuploidy tested during early in vitro passage. The cell line expresses surface mu and kappa-chains, HLA-DR antigen, C3 and Fc receptors and B-cell lineage antigens. The karyotypic analyses demonstrated many numerical and structural aberrations. No Burkitt lymphoma associated translocations (t8;14, t2;8, t;22) were detected, but most of the markers found are those commonly associated with various types of human cancer. The SKW 4 thus represents the most common type of 'histiocytic lymphoma', that with a B-lymphoid cell phenotype, but is unique among HL derived lymphoma lines in its strong expression of a Helix pomatia A agglutinin binding surface glycoprotein of an apparent molecular weight of 75 000 daltons. PMID:6329938

  8. McCoy cell line as a possible model containing CD4+ receptors for the study of HIV-1 replication

    Directory of Open Access Journals (Sweden)

    Nogueira Yeda L.

    2003-01-01

    Full Text Available Several studies have recently shown the use of recombinant rabies virus as potential vector-viral vaccine for HIV-1. The sequence homology between gp 120 and rabies virus glycoprotein has been reported. The McCoy cell line has therefore been used to show CD4+ or CD4+ like receptors. Samples of HIV-1 were isolated, when plasma of HIV-1 positive patients was inoculated in the McCoy cell line. The virus infection was then studied during successive virus passages. The proteins released in the extra cellular medium were checked for protein activity, by exposure to SDS Electrophoresis and blotting to nitro-cellulose filter, then reacting with sera of HIV positive and negative patients. Successive passages were performed, and showed viral replication, membrane permeabilization, the syncytium formation, and the cellular lysis (cytopathic effect. Flow cytometry analysis shows clear evidence that CD4+ receptors are present in this cell line, which enhances the likelihood of easy isolation and replication of HIV. The results observed allow the use of this cell line as a possible model for isolating HIV, as well as for carrying out studies of the dynamics of viral infection in several situations, including exposure to drugs in pharmacological studies, and possibly studies and analyses of the immune response in vaccine therapies.

  9. Molecular cloning of complementary DNAs encoding the heavy chain of the human 4F2 cell-surface antigen: a type II membrane glycoprotein involved in normal and neoplastic cell growth

    International Nuclear Information System (INIS)

    Complementary DNA (cDNA) clones encoding the heavy chain of the heterodimeric human membrane glycoprotein 4F2 have been isolated by immunoscreening of a λgt11 expression library. The identity of these clones has been confirmed by hybridization to RNA and DNA prepared from mouse L-cell transfectants, which were produced by whole cell gene transfer and selected for cell-surface expression of the human 4F2 heavy chain. DNA sequence analysis suggest that the 4F2 heavy-chain cDNAs encode an approximately 526-amino acid type II membrane glycoprotein, which is composed of a large C-terminal extracellular domain, a single potential transmembrane region, and a 50-81 amino acid N-terminal intracytoplasmic domain. Southern blotting experiments have shown that the 4F2 heavy-chain cDNAs are derived from a single-copy gene that has been highly conserved during mammalian evolution

  10. Radiobiological characteristics of cancer stem cells from esophageal cancer cell lines

    OpenAIRE

    Wang, Jian-Lin; Yu, Jing-Ping; Zhi-qiang SUN; Sun, Su-Ping

    2014-01-01

    AIM: To study the cancer stem cell population in esophageal cancer cell lines KYSE-150 and TE-1 and identify whether the resulting stem-like spheroid cells display cancer stem cells and radiation resistance characteristics.

  11. Differential heat shock response of primary human cell cultures and established cell lines

    DEFF Research Database (Denmark)

    Richter, W W; Issinger, O G

    1986-01-01

    degrees C treatment, whereas in immortalized cell lines usually 90% of the cells were found in suspension. Enhanced expression of the major heat shock protein (hsp 70) was found in all heat-treated cells. In contrast to the primary cell cultures, established and transformed cell lines synthesized a...

  12. Establishment of Jurkat tet-on cell line

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    Tet-control system is developed to tightly control target gene expression in mammalian cells by using the regulatory elements of tetracycline-repressor of the transposor Tn10 from E.Coli.We have transfected reverse tetracycline-controlled transactivator gene (rtTA) into genome of Jurkat cells and established two Jurkat tet-on cell lines.Induction of luciferase reporter activity with doxycycline,a tetracycline derivative,is dose-dependent with a peak value of 32-fold increment.Establishment of Jurkat tet-on cell lines greatly facilitates quantitative studies on target gene functions in the cells.

  13. Induction of Cell-Cell Fusion by Ebola Virus Glycoprotein: Low pH Is Not a Trigger.

    Science.gov (United States)

    Markosyan, Ruben M; Miao, Chunhui; Zheng, Yi-Min; Melikyan, Gregory B; Liu, Shan-Lu; Cohen, Fredric S

    2016-01-01

    Ebola virus (EBOV) is a highly pathogenic filovirus that causes hemorrhagic fever in humans and animals. Currently, how EBOV fuses its envelope membrane within an endosomal membrane to cause infection is poorly understood. We successfully measure cell-cell fusion mediated by the EBOV fusion protein, GP, assayed by the transfer of both cytoplasmic and membrane dyes. A small molecule fusion inhibitor, a neutralizing antibody, as well as mutations in EBOV GP known to reduce viral infection, all greatly reduce fusion. By monitoring redistribution of small aqueous dyes between cells and by electrical capacitance measurements, we discovered that EBOV GP-mediated fusion pores do not readily enlarge-a marked difference from the behavior of other viral fusion proteins. EBOV GP must be cleaved by late endosome-resident cathepsins B or L in order to become fusion-competent. Cleavage of cell surface-expressed GP appears to occur in endosomes, as evidenced by the fusion block imposed by cathepsin inhibitors, agents that raise endosomal pH, or an inhibitor of anterograde trafficking. Treating effector cells with a recombinant soluble cathepsin B or thermolysin, which cleaves GP into an active form, increases the extent of fusion, suggesting that a fraction of surface-expressed GP is not cleaved. Whereas the rate of fusion is increased by a brief exposure to acidic pH, fusion does occur at neutral pH. Importantly, the extent of fusion is independent of external pH in experiments in which cathepsin activity is blocked and EBOV GP is cleaved by thermolysin. These results imply that low pH promotes fusion through the well-known pH-dependent activity of cathepsins; fusion induced by cleaved EBOV GP is a process that is fundamentally independent of pH. The cell-cell fusion system has revealed some previously unappreciated features of EBOV entry, which could not be readily elucidated in the context of endosomal entry. PMID:26730950

  14. Expression of P-glycoprotein-mediated drug resistance in CHO cells surviving a single X-ray dose of 30 Gy

    International Nuclear Information System (INIS)

    The authors reported previously that Chinese hamster ovary (CHO) cells surviving exposure to repeated doses of 9 Gy of X-irradiation in vitro expressed a multiple drug resistance phenotype characterized by cross-resistance to epipodophyllotoxins and to Vinca alkaloids, and by P-glycoprotein (Pgp) overexpression. They now show that exposure of these CHO cells to a single 30-Gy X-ray dose similarly resulted in the survivors expressing resistance to vincristine and to etoposide and overexpressing Pgp. In agreement with data obtained on cells which received repeated X-ray exposures, this Pgp overexpression occurred in the absence of any significant elevation of Pgp mRNA. However, the reduced ability to accumulate rhodamine 123 identified in these sublines, and the ability of verapamil to reverse this accumulation defect, implies that the Pgp which was overexpressed was functional. (author)

  15. Influence of Disulfide-Stabilized Structure on the Specificity of Helper T-Cell and Antibody Responses to HIV Envelope Glycoprotein gp120▿ †

    Science.gov (United States)

    Mirano-Bascos, Denise; Steede, N. Kalaya; Robinson, James E.; Landry, Samuel J.

    2010-01-01

    CD4+ helper T cells specific for human immunodeficiency virus type 1 (HIV-1) are associated with control of viremia. Nevertheless, vaccines have had limited effectiveness thus far, in part because sequence variability and other structural features of the HIV envelope glycoprotein deflect the immune response. Previous studies indicated that CD4+ T-cell epitope dominance is controlled by antigen three-dimensional structure through its influence on antigen processing and presentation. In this work, three disulfide bonds in the outer domain of gp120 were individually deleted in order to destabilize the local three-dimensional structure and enhance the presentation of nearby weakly immunogenic epitopes. However, upon immunization of groups of BALB/c mice, the CD4+ T-cell response was broadly reduced for all three variants, and distinct epitope profiles emerged. For one variant, antibody titers were sharply increased, and the antibody exhibited significant CD4-blocking activity. PMID:20089653

  16. Antiproliferative effect of isopentenylated coumarins on several cancer cell lines.

    Science.gov (United States)

    Kawaii, S; Tomono, Y; Ogawa, K; Sugiura, M; Yano, M; Yoshizawa, Y; Ito, C; Furukawa, H

    2001-01-01

    33 coumarins, mainly the simple isopentenylated coumarins and derived pyrano- and furanocoumarins, were examined for their antiproliferative activity towards several cancer and normal human cell lines. The pyrano- and furanocoumarins showed strong activity against the cancer cell lines, whereas they had weak antiproliferative activity against the normal human cell lines. The decreasing rank order of potency was osthenone (10), clausarin (25), clausenidin (26), dentatin (24), nordentatin (23), imperatorin (29), seselin (27), xanthyletin (21), suberosin (17), phebalosin (8) and osthol (12). The structure-activity relationship established from the results revealed that the 1,1-dimethylallyl and isopentenyl groups have an important role for antiproliferative activity. PMID:11497276

  17. Isolation and Characterization of a Novel Gammaherpesvirus from a Microbat Cell Line.

    Science.gov (United States)

    Shabman, Reed S; Shrivastava, Susmita; Tsibane, Tshidi; Attie, Oliver; Jayaprakash, Anitha; Mire, Chad E; Dilley, Kari E; Puri, Vinita; Stockwell, Timothy B; Geisbert, Thomas W; Sachidanandam, Ravi; Basler, Christopher F

    2016-01-01

    While employing deep sequencing and de novo assembly to characterize the mRNA transcript profile of a cell line derived from the microbat Myotis velifer incautus, we serendipitously identified mRNAs encoding proteins with a high level of identity to herpesviruses. A majority were closely related to proteins of equine herpesvirus 2 (EHV-2), a horse gammaherpesvirus. We demonstrated by electron microscopy the presence of herpesvirus-like particles in the microbat cells. Passage of supernatants from microbat cells to Vero cells resulted in syncytium formation, and expression of viral genes and amplification of viral DNA were demonstrated by quantitative PCR. Susceptibility of human cell lines to productive infection was also demonstrated. Next-generation sequencing and de novo assembly of the viral genome from supernatants from Vero cells yielded a single contig of approximately 130 kb with at least 77 open reading frames (ORFs), predicted microRNAs (miRNAs), and a gammaherpesvirus genomic organization. Phylogenic analysis of the envelope glycoprotein (gB) and DNA polymerase (POLD1) revealed similarity to multiple gammaherpesviruses, including those from as-yet-uncultured viruses of the Rhadinovirus genus that were obtained by deep sequencing of bat tissues. Moreover, the assembled genome revealed ORFs that share little or no homology to known ORFs in EHV-2 but are similar to accessory proteins of other gammaherpesviruses. Some also have striking homology to predicted Myotis bat proteins. Cumulatively, this study provides the first isolation and characterization of a replication-competent bat gammaherpesvirus. IMPORTANCE Bats are of significant interest as reservoirs for zoonotic viral pathogens; however, tools to dissect bat-virus interactions are limited in availability. This study serendipitously identified, in an established bat cell line, a fully replication-competent gammaherpesvirus; determined the complete genome sequence of the virus; and generated a viral

  18. Thunbergia laurifolia extract minimizes the adverse effects of toxicants by regulating P-glycoprotein activity, CYP450, and lipid metabolism gene expression in HepG2 cells.

    Science.gov (United States)

    Rocejanasaroj, A; Tencomnao, T; Sangkitikomol, W

    2014-01-01

    Thunbergia laurifolia (TL) is widely used as an antidote in Thai traditional medicine against toxic substances such as alcohol, pesticides, arsenic, and strychnine. We found that the lyophilized form of TL in 80% ethanol possessed the antioxidant levels within the range 23,163.9 ± 1457.4 Trolox equivalents mM/kg dry mass and 899.8 ± 14.5 gallic acid equivalents mM/kg dry mass using the oxygen radical absorbance capacity assay and the Folin Ciocalteu phenol assay, respectively. TL extract (TLE) at a high dose (3000 mg/L) induced cytotoxicity according to the neutral red assay and the MTT assay. However, TLE doses of 800-3000 mg/L could reduce intracellular oxidative stress in a dose-dependent manner (P CYP3A4, and PPARg, but it significantly inhibited the mRNA expression of CYP3A7, CYP2D6, and CYP2E1 (P < 0.05) by reverse transcription-polymerase chain reaction. Moreover, TLE could increase the activity of a multidrug transporter, P-glycoprotein, which accelerated the excretion of toxic substances from HepG2 cells. It is suggested that TLE may be beneficial for detoxification by reducing oxidative stress, minimizing toxicity by regulating the expression CYP450 mRNAs for suitable production of CYP450 isoenzymes, and increasing PPARγ mRNA expression and P-glycoprotein activity in HepG2 cells, thereby maintaining xenobiotic biotransformation balance. PMID:24446304

  19. Characterization of an epithelial cell line from bovine mammary gland.

    Science.gov (United States)

    German, Tania; Barash, Itamar

    2002-05-01

    Elucidation of the bovine mammary gland's unique characteristics depends on obtaining an authentic cell line that will reproduce its function in vitro. Representative clones from bovine mammary cell populations, differing in their attachment capabilities, were cultured. L-1 cells showed strong attachment to the plate, whereas H-7 cells detached easily. Cultures established from these clones were nontumorigenic upon transplantation to an immunodeficient host; they exhibited the epithelial cell characteristics of positive cytokeratin but not smooth muscle actin staining. Both cell lines depended on fetal calf serum for proliferation. They exhibited distinct levels of differentiation on Matrigel in serum-free, insulin-supplemented medium on the basis of their organization and beta-lactoglobulin (BLG) secretion. H-7 cells organized into mammospheres, whereas L-1 cells arrested in a duct-like morphology. In both cell lines, prolactin activated phosphorylation of the signal transducer and activator of transcription, Stat5-a regulator of milk protein gene transcription, and of PHAS-I-an inhibitor of translation initiation in its nonphosphorylated form. De novo synthesis and secretion of BLG were detected in differentiated cultures: in L-1 cells, BLG was dependent on lactogenic hormones for maximal induction but was less stringently controlled than was beta-casein in the mouse CID-9 cell line. L-1 cells also encompassed a near-diploid chromosomal karyotype and may serve as a tool for studying functional characteristics of the bovine mammary gland. PMID:12418925

  20. Molecular profiling reveals primary mesothelioma cell lines recapitulate human disease.

    Science.gov (United States)

    Chernova, T; Sun, X M; Powley, I R; Galavotti, S; Grosso, S; Murphy, F A; Miles, G J; Cresswell, L; Antonov, A V; Bennett, J; Nakas, A; Dinsdale, D; Cain, K; Bushell, M; Willis, A E; MacFarlane, M

    2016-07-01

    Malignant mesothelioma (MM) is an aggressive, fatal tumor strongly associated with asbestos exposure. There is an urgent need to improve MM patient outcomes and this requires functionally validated pre-clinical models. Mesothelioma-derived cell lines provide an essential and relatively robust tool and remain among the most widely used systems for candidate drug evaluation. Although a number of cell lines are commercially available, a detailed comparison of these commercial lines with freshly derived primary tumor cells to validate their suitability as pre-clinical models is lacking. To address this, patient-derived primary mesothelioma cell lines were established and characterized using complementary multidisciplinary approaches and bioinformatic analysis. Clinical markers of mesothelioma, transcriptional and metabolic profiles, as well as the status of p53 and the tumor suppressor genes CDKN2A and NF2, were examined in primary cell lines and in two widely used commercial lines. Expression of MM-associated markers, as well as the status of CDKN2A, NF2, the 'gatekeeper' in MM development, and their products demonstrated that primary cell lines are more representative of the tumor close to its native state and show a degree of molecular diversity, thus capturing the disease heterogeneity in a patient cohort. Molecular profiling revealed a significantly different transcriptome and marked metabolic shift towards a greater glycolytic phenotype in commercial compared with primary cell lines. Our results highlight that multiple, appropriately characterised, patient-derived tumor cell lines are required to enable concurrent evaluation of molecular profiles versus drug response. Furthermore, application of this approach to other difficult-to-treat tumors would generate improved cellular models for pre-clinical evaluation of novel targeted therapies. PMID:26891694

  1. Impact of host cell line adaptation on quasispecies composition and glycosylation of influenza A virus hemagglutinin.

    Directory of Open Access Journals (Sweden)

    Jana Verena Roedig

    Full Text Available The genome of influenza A viruses is constantly changing (genetic drift resulting in small, gradual changes in viral proteins. Alterations within antibody recognition sites of the viral membrane glycoproteins hemagglutinin (HA and neuraminidase (NA result in an antigenetic drift, which requires the seasonal update of human influenza virus vaccines. Generally, virus adaptation is necessary to obtain sufficiently high virus yields in cell culture-derived vaccine manufacturing. In this study detailed HA N-glycosylation pattern analysis was combined with in-depth pyrosequencing analysis of the virus genomic RNA. Forward and backward adaptation from Madin-Darby Canine Kidney (MDCK cells to African green monkey kidney (Vero cells was investigated for two closely related influenza A virus PR/8/34 (H1N1 strains: from the National Institute for Biological Standards and Control (NIBSC or the Robert Koch Institute (RKI. Furthermore, stability of HA N-glycosylation patterns over ten consecutive passages and different harvest time points is demonstrated. Adaptation to Vero cells finally allowed efficient influenza A virus replication in Vero cells. In contrast, during back-adaptation the virus replicated well from the very beginning. HA N-glycosylation patterns were cell line dependent and stabilized fast within one (NIBSC-derived virus or two (RKI-derived virus successive passages during adaptation processes. However, during adaptation new virus variants were detected. These variants carried "rescue" mutations on the genomic level within the HA stem region, which result in amino acid substitutions. These substitutions finally allowed sufficient virus replication in the new host system. According to adaptation pressure the composition of the virus populations varied. In Vero cells a selection for "rescue" variants was characteristic. After back-adaptation to MDCK cells some variants persisted at indifferent frequencies, others slowly diminished and even

  2. Pathway-specific differences between tumor cell lines and normal and tumor tissue cells

    Directory of Open Access Journals (Sweden)

    Tozeren Aydin

    2006-11-01

    Full Text Available Abstract Background Cell lines are used in experimental investigation of cancer but their capacity to represent tumor cells has yet to be quantified. The aim of the study was to identify significant alterations in pathway usage in cell lines in comparison with normal and tumor tissue. Methods This study utilized a pathway-specific enrichment analysis of publicly accessible microarray data and quantified the gene expression differences between cell lines, tumor, and normal tissue cells for six different tissue types. KEGG pathways that are significantly different between cell lines and tumors, cell lines and normal tissues and tumor and normal tissue were identified through enrichment tests on gene lists obtained using Significance Analysis of Microarrays (SAM. Results Cellular pathways that were significantly upregulated in cell lines compared to tumor cells and normal cells of the same tissue type included ATP synthesis, cell communication, cell cycle, oxidative phosphorylation, purine, pyrimidine and pyruvate metabolism, and proteasome. Results on metabolic pathways suggested an increase in the velocity nucleotide metabolism and RNA production. Pathways that were downregulated in cell lines compared to tumor and normal tissue included cell communication, cell adhesion molecules (CAMs, and ECM-receptor interaction. Only a fraction of the significantly altered genes in tumor-to-normal comparison had similar expressions in cancer cell lines and tumor cells. These genes were tissue-specific and were distributed sparsely among multiple pathways. Conclusion Significantly altered genes in tumors compared to normal tissue were largely tissue specific. Among these genes downregulation was a major trend. In contrast, cell lines contained large sets of significantly upregulated genes that were common to multiple tissue types. Pathway upregulation in cell lines was most pronounced over metabolic pathways including cell nucleotide metabolism and oxidative

  3. Strategies for selecting recombinant CHO cell lines for cGMP manufacturing: improving the efficiency of cell line generation.

    Science.gov (United States)

    Porter, Alison J; Racher, Andrew J; Preziosi, Richard; Dickson, Alan J

    2010-01-01

    Transfectants with a wide range of cellular phenotypes are obtained during the process of cell line generation. For the successful manufacture of a therapeutic protein, a means is required to identify a cell line with desirable growth and productivity characteristics from this phenotypically wide-ranging transfectant population. This identification process is on the critical path for first-in-human studies. We have stringently examined a typical selection strategy used to isolate cell lines suitable for cGMP manufacturing. One-hundred and seventy-five transfectants were evaluated as they progressed through the different assessment stages of the selection strategy. High producing cell lines, suitable for cGMP manufacturing, were identified. However, our analyses showed that the frequency of isolation of the highest producing cell lines was low and that ranking positions were not consistent between each assessment stage, suggesting that there is potential to improve upon the strategy. Attempts to increase the frequency of isolation of the 10 highest producing cell lines, by in silico analysis of alternative selection strategies, were unsuccessful. We identified alternative strategies with similar predictive capabilities to the typical selection strategy. One alternate strategy required fewer cell lines to be progressed at the assessment stages but the stochastic nature of the models means that cell line numbers are likely to change between programs. In summary, our studies illuminate the potential for improvement to this and future selection strategies, based around use of assessments that are more informative or that reduce variance, paving the way to improved efficiency of generation of manufacturing cell lines. PMID:20623584

  4. Phenotypes and karyotypes of human malignant mesothelioma cell lines.

    Directory of Open Access Journals (Sweden)

    Vandana Relan

    Full Text Available BACKGROUND: Malignant mesothelioma is an aggressive tumour of serosal surfaces most commonly pleura. Characterised cell lines represent a valuable tool to study the biology of mesothelioma. The aim of this study was to develop and biologically characterise six malignant mesothelioma cell lines to evaluate their potential as models of human malignant mesothelioma. METHODS: Five lines were initiated from pleural biopsies, and one from pleural effusion of patients with histologically proven malignant mesothelioma. Mesothelial origin was assessed by standard morphology, Transmission Electron Microscopy (TEM and immunocytochemistry. Growth characteristics were assayed using population doubling times. Spectral karyotyping was performed to assess chromosomal abnormalities. Authentication of donor specific derivation was undertaken by DNA fingerprinting using a panel of SNPs. RESULTS: Most of cell lines exhibited spindle cell shape, with some retaining stellate shapes. At passage 2 to 6 all lines stained positively for calretinin and cytokeratin 19, and demonstrated capacity for anchorage-independent growth. At passage 4 to 16, doubling times ranged from 30-72 hours, and on spectral karyotyping all lines exhibited numerical chromosomal abnormalities ranging from 41 to 113. Monosomy of chromosomes 8, 14, 22 or 17 was observed in three lines. One line displayed four different karyotypes at passage 8, but only one karyotype at passage 42, and another displayed polyploidy at passage 40 which was not present at early passages. At passages 5-17, TEM showed characteristic features of mesothelioma ultrastructure in all lines including microvilli and tight intercellular junctions. CONCLUSION: These six cell lines exhibit varying cell morphology, a range of doubling times, and show diverse passage-dependent structural chromosomal changes observed in malignant tumours. However they retain characteristic immunocytochemical protein expression profiles of

  5. Surface charge characteristics of cells from malignant cell lines and normal cell lines of the human hematopoietic system.

    Science.gov (United States)

    Marikovsky, Y; Ben-Bassat, H; Leibovich, S J; Cividalli, L; Fischler, H; Danon, D

    1979-02-01

    Cells from malignant and normal lines of human hematopoietic origin were studied for their surface charge characteristics with the use of the following criteria: 1) the electron microscopic appearance of cell membranes after labeling with cationized ferritin (CF) either before or after glutaraldehyde fixation, 2) electrophoretic mobility, 3) total sialic acid content, and 4) agglutinability with poly-L-lysine (PLL). CF induced a time-dependent redistribution of surface receptors in unfixed malignant cells but not in unfixed normal cells. After 10 seconds of labeling with CF, both normal and malignant unfixed cells showed a uniform and even labeling pattern. After 5 minutes of labeling, malignant cells exhibited a highly pronounced pattern of clusters and patches, as distinct from a random and even pattern exhibited by normal cells. Both normal and malignant cells after fixation exhibited an equivalent random and even labeling pattern with CF, independent of the duration of labeling. The malignant cells studied possessed less sialic acid, had a lower electric mobility, and were agglutinated more readily with PLL than were the normal cells. PMID:310907

  6. MORPHOMETRIC SUBTYPING FOR A PANEL OF BREAST CANCER CELL LINES

    Energy Technology Data Exchange (ETDEWEB)

    Han, Ju; Chang, Hang; Fontenay, Gerald; Wang, Nicholas J.; Gray, Joe W.; Parvin, Bahram

    2009-05-08

    A panel of cell lines of diverse molecular background offers an improved model system for high-content screening, comparative analysis, and cell systems biology. A computational pipeline has been developed to collect images from cell-based assays, segment individual cells and colonies, represent segmented objects in a multidimensional space, and cluster them for identifying distinct subpopulations. While each segmentation strategy can vary for different imaging assays, representation and subpopulation analysis share a common thread. Application of this pipeline to a library of 41 breast cancer cell lines is demonstrated. These cell lines are grown in 2D and imaged through immunofluorescence microscopy. Subpopulations in this panel are identified and shown to correlate with previous subtyping literature that was derived from transcript data.

  7. Biobanking human embryonic stem cell lines

    DEFF Research Database (Denmark)

    Holm, Søren

    2016-01-01

    Stem cell banks curating and distributing human embryonic stem cells have been established in a number of countries and by a number of private institutions. This paper identifies and critically discusses a number of arguments that are used to justify the importance of such banks in policy...... curiously absent from the particular stem cell banking policy discourse. This to some extent artificially isolates this discourse from the broader discussions about the flows of reproductive materials and tissues in modern society, and such isolation may lead to the interests of important actors being...

  8. Thrombin promotes platelet-mediated melanoma cell adhesion to endothelial cells under flow conditions: role of platelet glycoproteins P-selectin and GPIIb-IIIA.

    OpenAIRE

    Dardik, R.; Savion, N; Kaufmann, Y; D. Varon

    1998-01-01

    We investigated the role of platelets in human melanoma cell (line 397) interaction with vascular endothelial cells (ECs) under flow conditions. The ability of the tumour cells to adhere to the EC monolayer was significantly reduced by application of flow at a shear rate of 250 s(-1). A 2.2-fold increase in tumour cell adhesion to ECs under flow was observed upon addition of thrombin receptor agonist peptide (TRAP)-activated platelets but not resting platelets. A similar increase (2.5-fold) i...

  9. Statistical analysis of anionic detergent-induced changes in the goblet mucous cells of opercular epidermis and gill epithelium of Rita rita (Ham.) (Bagridae: Pisces).

    Science.gov (United States)

    Roy, D

    1988-06-01

    Rita rita exposed to 96-hr LC50 (6.9 mg/liter) of an anionic detergent, dodecylbenzene sodium sulfonate, show significant changes in the number and size of goblet mucous cells in the opercular epidermis as well as in the lining epithelium of the gill arch and the gill filament at different time intervals of treatment. A shift in the staining nature of these cells from acidic glycoprotein to neutral glycoprotein, acidic glycosaminoglycans in the opercular epidermis and acidic glycoprotein to neutral glycoprotein and then again to acidic glycoprotein + acidic glycosaminoglycans in the gill filament epithelium reflects a change in the physiological status of fish. PMID:3168874

  10. Cold storage and cryopreservation of tick cell lines

    Directory of Open Access Journals (Sweden)

    Lallinger Gertrud

    2010-04-01

    Full Text Available Abstract Background Tick cell lines are now available from fifteen ixodid and argasid species of medical and veterinary importance. However, some tick cell lines can be difficult to cryopreserve, and improved protocols for short- and long-term low temperature storage will greatly enhance their use as tools in tick and tick-borne pathogen research. In the present study, different protocols were evaluated for cold storage and cryopreservation of tick cell lines derived from Rhipicephalus (Boophilus decoloratus, Rhipicephalus (Boophilus microplus, Ixodes ricinus and Ixodes scapularis. For short-term cold storage, cells were kept under refrigeration at 6°C for 15, 30 and 45 days. For cryopreservation in liquid nitrogen, use of a sucrose-phosphate-glutamate freezing buffer (SPG as cryoprotectant was compared with dimethylsulfoxide (DMSO supplemented with sucrose. Cell viability was determined by the trypan blue exclusion test and cell morphology was evaluated in Giemsa-stained cytocentrifuge smears. Results Cold storage at 6°C for up to 30 days was successful in preserving R. (B. microplus, R. (B. decoloratus, I. ricinus and I. scapularis cell lines; lines from the latter three species could be easily re-cultivated after 45 days under refrigeration. While cell lines from all four tick species cryopreserved with 6% DMSO were successfully resuscitated, the R. (B. decoloratus cells did not survive freezing in SPG and of the other three species, only the R. (B. microplus cells resumed growth during the observation period. Conclusions This constitutes the first report on successful short-term refrigeration of cells derived from R. (B. decoloratus, R. (B. microplus, and I. ricinus, and use of SPG as an alternative to DMSO for cryopreservation, thus making an important contribution to more reliable and convenient tick cell culture maintenance.

  11. Magnetic resonance spectroscopy in tumor cell lines research

    International Nuclear Information System (INIS)

    MRS can be used non-invasively to study the several trace metabolites and energy metabolism in vivo. By quantitatively analyzing the compounds changes we could detect abnormal metabolism in tumor and its surrounding tissue, and estimate tumor infiltration in vivo and vitro. In recent years, MRS has been applied in cell line research and is becoming a promising method. In this article we summarized the applications of MRS in cell lines in studying diagnosis, treatment, and tumor mechanisms. (authors)

  12. Characterization of xenoantiserum produced against B cell acute lymphoblastic leukemia cell line

    OpenAIRE

    Akagi,Tadaatsu; Sonobe, Hiroshi; Miyoshi, Isao; Yoshimoto,Shizuo

    1982-01-01

    Antiserum was produced in white rabbit by intravenously injecting living cells of a B cell acute lymphoblastic leukemia (ALL) line (BALL-1). The reactivity of the antiserum against various lymphoid cell lines was examined by membrane immunofluorescence after appropriate absorption. Serum absorbed with non-T, non-B (NALL-1) and T-ALL (TALL-1) cells recognized B cell antigens distinct from Ia-like antigens on both normal and neoplastic B cells. After further absorption with tonsillar cells or n...

  13. Reliable in vitro studies require appropriate ovarian cancer cell lines.

    Science.gov (United States)

    Jacob, Francis; Nixdorf, Sheri; Hacker, Neville F; Heinzelmann-Schwarz, Viola A

    2014-01-01

    Ovarian cancer is the fifth most common cause of cancer death in women and the leading cause of death from gynaecological malignancies. Of the 75% women diagnosed with locally advanced or disseminated disease, only 30% will survive five years following treatment. This poor prognosis is due to the following reasons: limited understanding of the tumor origin, unclear initiating events and early developmental stages of ovarian cancer, lack of reliable ovarian cancer-specific biomarkers, and drug resistance in advanced cases. In the past, in vitro studies using cell line models have been an invaluable tool for basic, discovery-driven cancer research. However, numerous issues including misidentification and cross-contamination of cell lines have hindered research efforts. In this study we examined all ovarian cancer cell lines available from cell banks. Hereby, we identified inconsistencies in the reporting, difficulties in the identification of cell origin or clinical data of the donor patients, restricted ethnic and histological type representation, and a lack of tubal and peritoneal cancer cell lines. We recommend that all cell lines should be distributed via official cell banks only with strict guidelines regarding the minimal available information required to improve the quality of ovarian cancer research in future. PMID:24936210

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

    International Nuclear Information System (INIS)

    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. Homodimers of the Antiviral Abacavir as Modulators of P-glycoprotein Transport in Cell Culture: Probing Tether Length

    OpenAIRE

    Namanja, Hilda A.; Emmert, Dana; Hrycyna, Christine A.; Chmielewski, Jean

    2013-01-01

    A major hurdle in permanently eliminating HIV from the body is the persistence of viral reservoirs, including those of the brain. One potential strategy towards eradicating HIV reservoirs of the brain is to block efflux transporters, such as P-glycoprotein (P-gp), that contribute to the limited penetration of antiviral agents across the blood-brain barrier (BBB). Herein, we described a series of dimeric inhibitors of P-gp based on the nucleoside reverse transcriptase inhibitor and P-gp substr...

  16. Mutational analysis of the hepatitis C virus E1 glycoprotein in retroviral pseudoparticles and cell-culture-derived H77/JFH1 chimeric infectious virus particles

    DEFF Research Database (Denmark)

    Russell, R S; Kawaguchi, K; Meunier, J-C; Takikawa, S; Faulk, K; Bukh, J; Purcell, R H; Emerson, S U

    2009-01-01

    Cell entry by enveloped viruses is mediated by viral glycoproteins, and generally involves a short hydrophobic peptide (fusion peptide) that inserts into the cellular membrane. An internal hydrophobic domain within E1 (aa262-290) of hepatitis C virus (HCV) may function as a fusion peptide....... Retrovirus-based HCV-pseudotyped viruses (HCVpp; genotype 1a) containing Ala or Pro substitutions at conserved amino acid positions within this putative fusion peptide were generated. Mutation of conserved residues significantly reduced efficiency of HCVpp entry into Huh-7 cells. The majority of amino acid....... However, the S283P mutation had a different effect in the two systems as it did not increase production of infectious HCVcc. This comprehensive mutational analysis of the putative HCV fusion peptide provides insight into the role of E1 in its interaction with E2 and in HCV entry....

  17. Neurohypophysial Receptor Gene Expression by Thymic T Cell Subsets and Thymic T Cell Lymphoma Cell Lines

    Directory of Open Access Journals (Sweden)

    I. Hansenne

    2004-01-01

    transcribed in thymic epithelium, while immature T lymphocytes express functional neurohypophysial receptors. Neurohypophysial receptors belong to the G protein-linked seven-transmembrane receptor superfamily and are encoded by four distinct genes, OTR, V1R, V2R and V3R. The objective of this study was to identify the nature of neurohypophysial receptor in thymic T cell subsets purified by immunomagnetic selection, as well as in murine thymic lymphoma cell lines RL12-NP and BW5147. OTR is transcribed in all thymic T cell subsets and T cell lines, while V3R transcription is restricted to CD4+ CD8+ and CD8+ thymic cells. Neither V1R nor V2R transcripts are detected in any kind of T cells. The OTR protein was identified by immunocytochemistry on thymocytes freshly isolated from C57BL/6 mice. In murine fetal thymic organ cultures, a specific OTR antagonist does not modify the percentage of T cell subsets, but increases late T cell apoptosis further evidencing the involvement of OT/OTR signaling in the control of T cell proliferation and survival. According to these data, OTR and V3R are differentially expressed during T cell ontogeny. Moreover, the restriction of OTR transcription to T cell lines derived from thymic lymphomas may be important in the context of T cell leukemia pathogenesis and treatment.

  18. Effect of P-glycoprotein on flavopiridol sensitivity

    OpenAIRE

    Boerner, S. A.; Tourne, M E; Kaufmann, S.H.; Bible, K C

    2001-01-01

    Flavopiridol is the first potent inhibitor of cyclin-dependent kinases (CDKs) to enter clinical trials. Little is known about mechanisms of resistance to this agent. In order to determine whether P-glycoprotein (Pgp) might play a role in flavopiridol resistance, we examined flavopiridol sensitivity in a pair of Chinese hamster ovary cell lines differing with respect to level of Pgp expression. The IC 50 s of flavopiridol in parental AuxB1 (lower Pgp) and colchicine-selected CHRC5 (higher Pgp)...

  19. Xenotropic retrovirus Bxv1 in human pancreatic β cell lines.

    Science.gov (United States)

    Kirkegaard, Jeannette S; Ravassard, Philippe; Ingvarsen, Signe; Diedisheim, Marc; Bricout-Neveu, Emilie; Grønborg, Mads; Frogne, Thomas; Scharfmann, Raphael; Madsen, Ole D; Rescan, Claude; Albagli, Olivier

    2016-03-01

    It has been reported that endogenous retroviruses can contaminate human cell lines that have been passaged as xenotransplants in immunocompromised mice. We previously developed and described 2 human pancreatic β cell lines (EndoC-βH1 and EndoC-βH2) that were generated in this way. Here, we have shown that B10 xenotropic virus 1 (Bxv1), a xenotropic endogenous murine leukemia virus (MuLV), is present in these 2 recently described cell lines. We determined that Bxv1 was also present in SCID mice that were used for in vivo propagation of EndoC-βH1/2 cells, suggesting that contamination occurred during xenotransplantation. EndoC-βH1/2 cells released Bxv1 particles that propagated to human 293T and Mus dunni cells. Mobilization assays demonstrated that Bxv1 transcomplements defective MuLV-based retrovectors. In contrast, common rodent β cell lines, rat INS-1E and RIN-5F cells and mouse MIN6 and βTC3 cells, displayed either no or extremely weak xenotropic helper activity toward MuLV-based retrovectors, although xenotropic retrovirus sequences and transcripts were detected in both mouse cell lines. Bxv1 propagation from EndoC-βH1/2 to 293T cells occurred only under optimized conditions and was overall poorly efficient. Thus, although our data imply that MuLV-based retrovectors should be cautiously used in EndoC-βH1/2 cells, our results indicate that an involuntary propagation of Bxv1 from these cells can be easily avoided with good laboratory practices. PMID:26901817

  20. In vitro radiosensitivity of human leukemia cell lines

    International Nuclear Information System (INIS)

    The in vitro radiobiologic survival values (n, D0) of four tumor lines derived from human hematopoietic tumors were studied. These cell lines were HL50 (n . 1.3, D0 . 117 rad[1.17 Gy]), promyelocytic leukemia; K562 (n . 1.4, D0 . 165 rad[1.65 Gy]), erythroleukemia; 45 (n . 1.1, D0 . 147 rad[1.47 Gy]), acute lymphocyte leukemia; and 176 (n . 4.0, D0 . 76 rad[0.76 Gy]), acute monomyelogenous leukemia. More cell lines must be examined before the exact relationship between in vitro radiosensitivity and clinical radiocurability is firmly established

  1. Study of tea polyphenol as a reversal agent for carcinoma cell lines' multidrug resistance (study of TP as a MDR reversal agent)

    International Nuclear Information System (INIS)

    The aim of this study was to examine MDR1 expression product P-glycoprotein (Pgp) and study the effect and mechanism of tea polyphenol (TP) in reversion of multidrug resistance (MDR) in carcinoma cell lines. Immunocytochemical method was used for qualitative detection of Pgp. A comparative study of cytotoxicity and multidrug resistance reversion effect was made by MTT assay for tea polyphenol and quinidine in MCF-7 and MCF-7/Adr cell lines. The multidrug resistance reversion effect and mechanism were studied by measuring the uptake of 99mTc-tetrofosmin in the carcinoma cell lines. (1) The Pgp overexpression in MCF-7/Adr cells was found to be strong positive, while the Pgp expression of MCF-7 was negative. (2) Although both tea polyphenol and quinidine could not remarkably change the toxicity of adriamycin to MCF-7, they could improve the sensitivity of MCF-7/Adr to adriamycin. The reversion index of tea polyphenol and quinidine was 3 and 10 respectively. (3) The cellular uptake of 99mTc-tetrofosmin was remarkably lower in MCF-7/Adr than in MCF-7. The uptake of 99mTc-tetrofosmin in MCF-7/Adr exhibited a 4, 13, 16 fold increase in the presence of 200, 400 and 500 μg/ml of tea polyphenol respectively. The uptake of 99mTc-tetrofosmin in MCF-7/Adr exhibited only a 4-fold increase in the presence of 200 μM of quinidine. Immunocytochemistry can detect P-glycoprotein expression level qualitatively. Tea polyphenol is not only an anti-tumor agent, but also a multidrug resistant modulator similar to quinidine. The multidrug resistance reversion mechanism of tea polyphenol seems to be its inhibition of the activity of P-glycoprotein. Tea polyphenol has the advantage of very low toxicity in tumor treatment

  2. Study of tea polyphenol as a reversal agent for carcinoma cell lines' multidrug resistance (study of TP as a MDR reversal agent)

    Energy Technology Data Exchange (ETDEWEB)

    Zhu Aizhi E-mail: zhuaizhi@263.net; Wang Xiangyun; Guo Zhenquan

    2001-08-01

    The aim of this study was to examine MDR1 expression product P-glycoprotein (Pgp) and study the effect and mechanism of tea polyphenol (TP) in reversion of multidrug resistance (MDR) in carcinoma cell lines. Immunocytochemical method was used for qualitative detection of Pgp. A comparative study of cytotoxicity and multidrug resistance reversion effect was made by MTT assay for tea polyphenol and quinidine in MCF-7 and MCF-7/Adr cell lines. The multidrug resistance reversion effect and mechanism were studied by measuring the uptake of {sup 99m}Tc-tetrofosmin in the carcinoma cell lines. (1) The Pgp overexpression in MCF-7/Adr cells was found to be strong positive, while the Pgp expression of MCF-7 was negative. (2) Although both tea polyphenol and quinidine could not remarkably change the toxicity of adriamycin to MCF-7, they could improve the sensitivity of MCF-7/Adr to adriamycin. The reversion index of tea polyphenol and quinidine was 3 and 10 respectively. (3) The cellular uptake of {sup 99m}Tc-tetrofosmin was remarkably lower in MCF-7/Adr than in MCF-7. The uptake of {sup 99m}Tc-tetrofosmin in MCF-7/Adr exhibited a 4, 13, 16 fold increase in the presence of 200, 400 and 500 {mu}g/ml of tea polyphenol respectively. The uptake of {sup 99m}Tc-tetrofosmin in MCF-7/Adr exhibited only a 4-fold increase in the presence of 200 {mu}M of quinidine. Immunocytochemistry can detect P-glycoprotein expression level qualitatively. Tea polyphenol is not only an anti-tumor agent, but also a multidrug resistant modulator similar to quinidine. The multidrug resistance reversion mechanism of tea polyphenol seems to be its inhibition of the activity of P-glycoprotein. Tea polyphenol has the advantage of very low toxicity in tumor treatment.

  3. Effect of dehydrodidemnin B on human colon carcinoma cell lines.

    Science.gov (United States)

    Lobo, C; García-Pozo, S G; Núñez de Castro, I; Alonso, F J

    1997-01-01

    Didemnins are cytotoxic agents belonging to a depsipeptide family isolated from marine tunicates. In the present study, a new member, dehydrodidemnin B (DDB), isolated from the mediterranean tunicate Aplidium albicans, was used. The effect of the drug on human colon cultured cell lines was tested using multiple approaches: proliferation studies, long term survival after three hours of exposure to DDB by means of a clonogenic assay and the decrease of the protooncogen, ornithine decarboxylase, activity. A dehydrodidemnin B concentration of 10(-8) M completely inhibited cell growth. The IC50 obtained using the MTT proliferation test, indicated that the most proliferative cell line (CT-2) was the most sensitive to the drug. Using a clonogenic assay a clear dose-response was obtained for the three cell lines used; HT-29 cell line showed the minimum survival after 3 hours of dehydrodidemnin B treatment. A dose-dependent decrease in ornithine decarboxylase activity was also observed in three cell lines assayed. The data presented indicate that the dehydrodidemnin B is a potent cytotoxic agent on rapidly dividing human colon cancer cells. PMID:9066673

  4. Transcription profiles of non-immortalized breast cancer cell lines

    International Nuclear Information System (INIS)

    Searches for differentially expressed genes in tumours have made extensive use of array technology. Most samples have been obtained from tumour biopsies or from established tumour-derived cell lines. Here we compare cultures of non-immortalized breast cancer cells, normal non-immortalized breast cells and immortalized normal and breast cancer cells to identify which elements of a defined set of well-known cancer-related genes are differentially expressed. Cultures of cells from pleural effusions or ascitic fluids from breast cancer patients (MSSMs) were used in addition to commercially-available normal breast epithelial cells (HMECs), established breast cancer cell lines (T-est) and established normal breast cells (N-est). The Atlas Human Cancer 1.2 cDNA expression array was employed. The data obtained were analysed using widely-available statistical and clustering software and further validated through real-time PCR. According to Significance Analysis of Microarray (SAM) and AtlasImage software, 48 genes differed at least 2-fold in adjusted intensities between HMECs and MSSMs (p < 0.01). Some of these genes have already been directly linked with breast cancer, metastasis and malignant progression, whilst others encode receptors linked to signal transduction pathways or are otherwise related to cell proliferation. Fifty genes showed at least a 2.5-fold difference between MSSMs and T-est cells according to AtlasImage, 2-fold according to SAM. Most of these classified as genes related to metabolism and cell communication. The expression profiles of 1176 genes were determined in finite life-span cultures of metastatic breast cancer cells and of normal breast cells. Significant differences were detected between the finite life-span breast cancer cell cultures and the established breast cancer cell lines. These data suggest caution in extrapolating information from established lines for application to clinical cancer research

  5. Establishment of a new cell line (MTT-95 showing basophilic differentiation from the bone marrow of a patient with acute myelogenous leukemia (M7.

    Directory of Open Access Journals (Sweden)

    Mizobuchi N

    1999-04-01

    Full Text Available A new myeloid cell line, MTT-95, was established from the bone marrow of a patient with acute myelogenous leukemia (AML, M7. MTT-95 cells differentiate into mature basophilic cells in culture medium with no chemical component or cytokine. Surface phenotypes were as follows: CD11b 79.3%, CD13 92.4%, CD33 99.8%, CD34 87.9%, CD41a 77.6% and HLA-DR 0.3%. MTT-95 cells were strongly positive for glycoprotein IIb/IIIa by immunohistochemical staining and revealed metachromatic granules. MTT-95 cells seem to possess characteristics of both megakaryocytes and basophils. These findings suggest that MTT-95 cells are basophil progenitors. MTT-95 cells might be useful in the study not only of the biological aspects of basophils, but also of the diversities of AML (M7.

  6. Technetium-99m sestamibi uptake in human breast carcinoma cell lines displaying glutathione-associated drug-resistance

    Energy Technology Data Exchange (ETDEWEB)

    Kabasakal, L. [Dept. of Nuclear Medicine and Biochemistry, Medical College of Wisconsin, Milwaukee, WI (United States); Oezker, K. [Dept. of Nuclear Medicine and Biochemistry, Medical College of Wisconsin, Milwaukee, WI (United States); Hayward, M. [Dept. of Nuclear Medicine and Biochemistry, Medical College of Wisconsin, Milwaukee, WI (United States); Akansel, G. [Dept. of Nuclear Medicine and Biochemistry, Medical College of Wisconsin, Milwaukee, WI (United States); Griffith, O. [Dept. of Nuclear Medicine and Biochemistry, Medical College of Wisconsin, Milwaukee, WI (United States); Isitman, A.T. [Dept. of Nuclear Medicine and Biochemistry, Medical College of Wisconsin, Milwaukee, WI (United States); Hellman, R. [Dept. of Nuclear Medicine and Biochemistry, Medical College of Wisconsin, Milwaukee, WI (United States); Collier, D. [Dept. of Nuclear Medicine and Biochemistry, Medical College of Wisconsin, Milwaukee, WI (United States)

    1996-05-01

    An in vitro study was designed to evaluate the uptake of sestamibi (MIBI) in P-glycoprotein (Pgp) and glutathione-associated (GSH) multidrug-resistant (MDR) cell lines. MIBI uptake was studied in various human breast carcinoma cell lines, i.e. in wild-type (MCF7/wt) cells, in adriamycin-resistant (MCF7/adr) cells which express Pgp and in melphalan-resistant (MCF7/mph) cells with increased levels of GSH. The effects of buthiomine sulphoximine (BSO) and verapamil on MIBI uptake were also studied in the MCF7/mph and MCF7/adr cells respectively. The cells were incubated for 1 h with a dose of 0.1 MBq thallium-201 and technetium-99m MIBI. Both BIBI and {sup 201}Tl uptakes were higher for MCF7/mph cells than for the other cells studied. The mean MIBI uptake in MCF7/adr cells was significantly lower than that in MCF7/wt cells (1.9%{+-}0.5% vs 3.1%.0.6%; P<0.01). Verapamil treatment increased the MIBI uptake in MCF7/adr cells (to 2.6%.0.3%; P<0.05). Treatment of MCF7/mph cells with BSO resulted in a significant reduction in GSH content (from 243.2{+-}81.1 nmol/mg protein to 17.6{+-}4.4 nmol/mg protein; P<0.001). However, MIBI uptake in BSO-treated and untreated MCF7/mph cells was similar (4.43%{+-}0.5% and 5.93%{+-}1.7%, respectively; P>0.1). This study suggests that the uptake of MIBI is not diminished by glutathione-associated drug resistance and that MIBI uptake in a tumour sample does not necessarly indicate that a cancer is sensitive to drugs. (orig.)

  7. Differences between accumulation of 99mTc-MIBI and 201Tl-Thallous Chloride in tumour cells: Role of P-glycoprotein

    International Nuclear Information System (INIS)

    Both 99mTc-MIBI and 201Tl have been used for tumour imaging. It has recently been reported that 99mTc-MIBI is a substrate for P-glycoprotein (Pgp), a membrane pump which mediates multidrug resistance. It has been evaluated the role of Pgp in the cellular accumulation of 201TI by using sensitive and resistant strains of Chinese hamster ovary (CHO) fibroblasts (AuxB1 and CHRC5, respectively) grown in suspension culture. 201Tl accumulation was the same in sensitive and resistant cells, whereas 99mTc-MIBI accumulation was much lower in resistant cells than in sensitive ones. Down-modulation of Pgp with 100 μM verapamil did not alter cellular accumulation of 201Tl while it significantly increased 99mTc-MIBI accumulation in both types of cell. Similarly, 10 μM verapamil did not affect the rate of washout of 201Tl from preloaded cells, while 99mTc-MIBI washout was greatly reduced in the presence of verapamil. These results suggest that 201Tl will accumulate in both sensitive and resistant tumour cells, whereas 99mTc-MIBI will be extruded from resistant cells and therefore may be less useful for tumour detection when the tumour cells express high Pgp levels

  8. Membrane lipidome of an epithelial cell line

    DEFF Research Database (Denmark)

    Sampaio, Julio L; Gerl, Mathias J; Klose, Christian;

    2011-01-01

    Tissue differentiation is an important process that involves major cellular membrane remodeling. We used Madin-Darby canine kidney cells as a model for epithelium formation and investigated the remodeling of the total cell membrane lipidome during the transition from a nonpolarized morphology to an...... epithelial morphology and vice versa. To achieve this, we developed a shotgun-based lipidomics workflow that enabled the absolute quantification of mammalian membrane lipidomes with minimal sample processing from low sample amounts. Epithelial morphogenesis was accompanied by a major shift from sphingomyelin...... to glycosphingolipid, together with an increase in plasmalogen, phosphatidylethanolamine, and cholesterol content, whereas the opposite changes took place during an epithelial-to-mesenchymal transition. Moreover, during polarization, the sphingolipids became longer, more saturated, and more...

  9. Cell Line Modeling to Study Biomarker Panel in Prostate Cancer

    Science.gov (United States)

    NickKholgh, Bita; Fang, Xiaolan; Winters, Shira M.; Raina, Anvi; Pandya, Komal S.; Gyabaah, Kenneth; Fino, Nora; Balaji, K.C.

    2016-01-01

    BACKGROUND African–American men with prostate cancer (PCa) present with higher-grade and -stage tumors compared to Caucasians. While the disparity may result from multiple factors, a biological basis is often strongly suspected. Currently, few well-characterized experimental model systems are available to study the biological basis of racial disparity in PCa. We report a validated in vitro cell line model system that could be used for the purpose. METHODS We assembled a PCa cell line model that included currently available African–American PCa cell lines and LNCaP (androgen-dependent) and C4-2 (castration-resistant) Caucasian PCa cells. The utility of the cell lines in studying the biological basis of variance in a malignant phenotype was explored using a multiplex biomarker panel consisting of proteins that have been proven to play a role in the progression of PCa. The panel expression was evaluated by Western blot and RT-PCR in cell lines and validated in human PCa tissues by RT-PCR. As proof-of-principle to demonstrate the utility of our model in functional studies, we performed MTS viability assays and molecular studies. RESULTS The dysregulation of the multiplex biomarker panel in primary African–American cell line (E006AA) was similar to metastatic Caucasian cell lines, which would suggest that the cell line model could be used to study an inherent aggressive phenotype in African–American men with PCa. We had previously demonstrated that Protein kinase D1 (PKD1) is a novel kinase that is down regulated in advanced prostate cancer. We established the functional relevance by over expressing PKD1, which resulted in decreased proliferation and epithelial mesenchymal transition (EMT) in PCa cells. Moreover, we established the feasibility of studying the expression of the multiplex biomarker panel in archived human PCa tissue from African–Americans and Caucasians as a prelude to future translational studies. CONCLUSION We have characterized a novel in

  10. High-efficiency secretory expression of human neutrophil gelatinase-associated lipocalin from mammalian cell lines with human serum albumin signal peptide.

    Science.gov (United States)

    Chen, Wei; Zhao, Xiaozhi; Zhang, Mingxin; Yuan, Yimin; Ge, Liyuan; Tang, Bo; Xu, Xiaoyu; Cao, Lin; Guo, Hongqian

    2016-02-01

    Human neutrophil gelatinase associated lipocalin (NGAL) is a secretory glycoprotein initially isolated from neutrophils. It is thought to be involved in the incidence and development of immunological diseases and cancers. Urinary and serum levels of NGAL have been investigated as a new biomarker of acute kidney injury (AKI), for an earlier and more accurate detection method than with creatinine level. However, expressing high-quality recombinant NGAL is difficult both in Escherichia coli and mammalian cells for the low yield. Here, we cloned and fused NGAL to the C-terminus of signal peptides of human NGAL, human interleukin-2 (IL2), gaussia luciferase (Gluc), human serum albumin preproprotein (HSA) or an hidden Markov model-generated signal sequence (HMM38) respectively for transient expression in Expi293F suspension cells to screen for their ability to improve the secretory expression of recombinant NGAL. The best results were obtained with signal peptide derived from HSA. The secretory recombinant protein could react specifically with NGAL antibody. For scaled production, we used HSA signal peptide to establish stable Chinese hamster ovary cell lines. Then we developed a convenient colony-selection system to select high-expression, stable cell lines. Moreover, we purified the NGAL with Ni-Sepharose column. The recombinant human NGAL displayed full biological activity. We provide a method to enhance the secretory expression of recombinant human NGAL by using the HSA signal peptide and produce the glycoprotein in mammalian cells. PMID:26518367

  11. Solid Oxide Fuel Cell Systems PVL Line

    Energy Technology Data Exchange (ETDEWEB)

    Susan Shearer - Stark State College; Gregory Rush - Rolls-Royce Fuel Cell Systems

    2012-05-01

    In July 2010, Stark State College (SSC), received Grant DE-EE0003229 from the U.S. Department of Energy (DOE), Golden Field Office, for the development of the electrical and control systems, and mechanical commissioning of a unique 20kW scale high-pressure, high temperature, natural gas fueled Stack Block Test System (SBTS). SSC worked closely with subcontractor, Rolls-Royce Fuel Cell Systems (US) Inc. (RRFCS) over a 13 month period to successfully complete the project activities. This system will be utilized by RRFCS for pre-commercial technology development and training of SSC student interns. In the longer term, when RRFCS is producing commercial products, SSC will utilize the equipment for workforce training. In addition to DOE Hydrogen, Fuel Cells, and Infrastructure Technologies program funding, RRFCS internal funds, funds from the state of Ohio, and funding from the DOE Solid State Energy Conversion Alliance (SECA) program have been utilized to design, develop and commission this equipment. Construction of the SBTS (mechanical components) was performed under a Grant from the State of Ohio through Ohio's Third Frontier program (Grant TECH 08-053). This Ohio program supported development of a system that uses natural gas as a fuel. Funding was provided under the Department of Energy (DOE) Solid-state Energy Conversion Alliance (SECA) program for modifications required to test on coal synthesis gas. The subject DOE program provided funding for the electrical build, control system development and mechanical commissioning. Performance testing, which includes electrical commissioning, was subsequently performed under the DOE SECA program. Rolls-Royce Fuel Cell Systems is developing a megawatt-scale solid oxide fuel cell (SOFC) stationary power generation system. This system, based on RRFCS proprietary technology, is fueled with natural gas, and operates at elevated pressure. A critical success factor for development of the full scale system is the capability

  12. Steroid hormone secretion in inflammatory breast cancer cell lines.

    Science.gov (United States)

    Illera, Juan Carlos; Caceres, Sara; Peña, Laura; de Andres, Paloma J; Monsalve, Beatriz; Illera, Maria J; Woodward, Wendy A; Reuben, James M; Silvan, Gema

    2015-12-01

    Inflammatory breast carcinoma (IBC) is a special type of breast cancer with a poor survival rate. Though several IBC cell lines have been established, recently a first IMC cell line was established. The aims of this study were: (1) to validate a highly sensitive, reliable, accurate and direct amplified enzyme immunoassay (EIA) to measure several cell-secreted steroid hormones: progesterone (P4), androstenedione (A4), testosterone (T), 17β-estradiol (E2) and estrone sulfate (SO4E1) in the culture medium. (2) To assess whether hormone production profile by IPC-366 cells validates the IMC model for human IBC. We validated a non-competitive amplified EIA for inflammatory breast cancer cell lines based on the results of accuracy, precision, sensitivity and parallelism. The low detection limits of the technique were: P4=13.2 pg/well, A4=2.3 pg/well, T=11.4 pg/well, E2=1.9 pg/well and SO4E1=4.5 pg/well. Intra- and inter-assay coefficient of variation percentages were 90%. In all hormones studied SUM149 have higher levels (1.4 times, but not significant) than IPC-366, and the correlation index between SUM149 and IPC-366 concentrations were >97%. We can coclude that cells of both cell lines, IPC-366 and SUM149, are capable to produce steroid hormone in culture media. The presented EIA methodology is very valuable for the detection of steroid production in culture media and could be used in hormone regulation studies and therapeutic agents in cell lines of inflammatory and non-inflammatory mammary carcinoma or other cancer cell lines in preclinical studies. PMID:26495931

  13. MOLECULAR AND CYTOGENETIC ANALYSIS OF LUNG TUMOR CELL LINES

    Science.gov (United States)

    We have measured the levels of amplification of oncogenes and tumor marker genes or other genes of interest in nine human lung tumor cell lines in comparison to normal human bronchial epithelial cells or normal blood lymphocytes to test the hypothesis that aberrant amplification ...

  14. Novel human multiple myeloma cell line UHKT-893

    Czech Academy of Sciences Publication Activity Database

    Uherková, L.; Vančurová, I.; Vyhlídalová, I.; Pleschnerová, M.; Špička, I.; Mihalová, R.; Březinová, J.; Hodný, Zdeněk; Čermáková, K.; Polanská, V.; Marinov, I.; Jedelský, P.L.; Kuželová, K.; Stöckbauer, P.

    2013-01-01

    Roč. 37, č. 3 (2013), s. 320-326. ISSN 0145-2126 Institutional support: RVO:68378050 Keywords : human myeloma cell line * human multiple myeloma * plasma cell * IL-6 dependence * immunoglobulin * free light chain Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 2.692, year: 2013

  15. Frequency and distribution of Notch mutations in tumor cell lines

    International Nuclear Information System (INIS)

    Deregulated Notch signaling is linked to a variety of tumors and it is therefore important to learn more about the frequency and distribution of Notch mutations in a tumor context. In this report, we use data from the recently developed Cancer Cell Line Encyclopedia to assess the frequency and distribution of Notch mutations in a large panel of cancer cell lines in silico. Our results show that the mutation frequency of Notch receptor and ligand genes is at par with that for established oncogenes and higher than for a set of house-keeping genes. Mutations were found across all four Notch receptor genes, but with notable differences between protein domains, mutations were for example more prevalent in the regions encoding the LNR and PEST domains in the Notch intracellular domain. Furthermore, an in silico estimation of functional impact showed that deleterious mutations cluster to the ligand-binding and the intracellular domains of NOTCH1. For most cell line groups, the mutation frequency of Notch genes is higher than in associated primary tumors. Our results shed new light on the spectrum of Notch mutations after in vitro culturing of tumor cells. The higher mutation frequency in tumor cell lines indicates that Notch mutations are associated with a growth advantage in vitro, and thus may be considered to be driver mutations in a tumor cell line context. The online version of this article (doi:10.1186/s12885-015-1278-x) contains supplementary material, which is available to authorized users

  16. Continuous production of erythropoietin by an established human renal carcinoma cell line: development of the cell line

    International Nuclear Information System (INIS)

    Establishment of a stable, transformed human renal carcinoma cell line that produces erythropoietin in vitro and has maintained this function continuously since 1981 and for > 150 passages in monolayer culture was accomplished by transplantation of human renal clear cell carcinoma tissue from a patient with erythrocytosis into an immunosuppressed athymic mouse. In addition to its immunocrossreactivity with native human urinary erythropoietin, the tumor erythropoietin demonstrates biological activity in the in vitro mouse erythroid colony-forming unit assay and in tumor-bearing nude mice. The cloned renal carcinoma cell line has an abnormal human karyotype and has ultrastructural features characteristic of human renal clear cell carcinoma. This cell line provides a reproducible model system for the production of an erythropoietin-like material and for the study of its synthesis and secretion

  17. Glycoengineering of Human Cell Lines Using Zinc Finger Nuclease Gene Targeting

    DEFF Research Database (Denmark)

    Steentoft, Catharina; Bennett, Eric Paul; Clausen, Henrik

    Lectin affinity chromatography is a powerful technique for isolation of glycoproteins carrying a specific glycan structure of interest. However, the enormous diversity of glycans present on the cell surface, as well as on individual proteins, makes it difficult to isolate an entire glycoproteome ...

  18. Derivation of human embryonic stem cell line Genea019

    Directory of Open Access Journals (Sweden)

    Biljana Dumevska

    2016-03-01

    Full Text Available The Genea019 human embryonic stem cell line was derived from a donated, fully commercially consented ART blastocyst, through ICM outgrowth on inactivated feeders. The line showed pluripotent cell morphology and genomic analysis verified a 46, XX karyotype, female Allele pattern and unaffected Htt CAG repeat length, compared to HD affected sibling Genea020. Pluripotency of Genea019 was demonstrated with 75% of cells expressing Nanog, 89% Oct4, 48% Tra1-60 and 85% SSEA4, a Pluritest Pluripotency score of 22.97, Novelty score of 1.42, tri-lineage teratoma formation and Alkaline Phosphatase activity. The cell line was negative for Mycoplasma and any visible contamination.

  19. Female sex bias in human embryonic stem cell lines.

    Science.gov (United States)

    Ben-Yosef, Dalit; Amit, Ami; Malcov, Mira; Frumkin, Tsvia; Ben-Yehudah, Ahmi; Eldar, Ido; Mey-Raz, Nava; Azem, Foad; Altarescu, Gheona; Renbaum, Paul; Beeri, Rachel; Varshaver, Irit; Eldar-Geva, Talia; Epsztejn-Litman, Silvina; Levy-Lahad, Ephrat; Eiges, Rachel

    2012-02-10

    The factors limiting the rather inefficient derivation of human embryonic stem cells (HESCs) are not fully understood. The aim of this study was to analyze the sex ratio in our 42 preimplantation genetic diagnosis (PGD)-HESC lines, in an attempt to verify its affect on the establishment of HESC lines. The ratio between male and female PGD-derived cell lines was compared. We found a significant increase in female cell lines (76%). This finding was further confirmed by a meta-analysis for combining the results of all PGD-derived HESC lines published to date (148) and all normal karyotyped HESC lines derived from spare in vitro fertilization embryos worldwide (397). Further, gender determination of embryos demonstrated that this difference originates from the actual derivation process rather than from unequal representation of male and female embryos. It can therefore be concluded that the clear-cut tendency for female preponderance is attributed to suboptimal culture conditions rather than from a true gender imbalance in embryos used for derivation of HESC lines. We propose a mechanism in which aberrant X chromosome inactivation and/or overexpression of critical metabolic X-linked genes might explain this sex dimorphism. PMID:21585244

  20. The cyclin-dependent kinase inhibitor roscovitine and the nucleoside analog sangivamycin induce apoptosis in caspase-3 deficient breast cancer cells independent of caspase mediated P-glycoprotein cleavage: Implications for therapy of drug resistant breast cancers

    OpenAIRE

    Cappellini, Alessandra; Chiarini, Francesca; Ognibene, Andrea; McCubrey, James A; Martelli, Alberto M.

    2009-01-01

    Resistance to multiple chemotherapeutic agents is a common clinical problem which can arise during cancer treatment. Drug resistance often involves overexpression of the multidrug resistance MDR1 gene, encoding P-glycoprotein (P-gp), a 170-kDa glycoprotein belonging to the ATP-binding cassette superfamily of membrane transporters. We have recently demonstrated apoptosis-induced, caspase-3-dependent P-gp cleavage in human T-lymphoblastoid CEM-R VBL100 cells. However, P-gp contain many aspartat...

  1. Establishment, immortalisation and characterisation of pteropid bat cell lines.

    Directory of Open Access Journals (Sweden)

    Gary Crameri

    Full Text Available BACKGROUND: Bats are the suspected natural reservoir hosts for a number of new and emerging zoonotic viruses including Nipah virus, Hendra virus, severe acute respiratory syndrome coronavirus and Ebola virus. Since the discovery of SARS-like coronaviruses in Chinese horseshoe bats, attempts to isolate a SL-CoV from bats have failed and attempts to isolate other bat-borne viruses in various mammalian cell lines have been similarly unsuccessful. New stable bat cell lines are needed to help with these investigations and as tools to assist in the study of bat immunology and virus-host interactions. METHODOLOGY/FINDINGS: Black flying foxes (Pteropus alecto were captured from the wild and transported live to the laboratory for primary cell culture preparation using a variety of different methods and culture media. Primary cells were successfully cultured from 20 different organs. Cell immortalisation can occur spontaneously, however we used a retroviral system to immortalise cells via the transfer and stable production of the Simian virus 40 Large T antigen and the human telomerase reverse transcriptase protein. Initial infection experiments with both cloned and uncloned cell lines using Hendra and Nipah viruses demonstrated varying degrees of infection efficiency between the different cell lines, although it was possible to infect cells in all tissue types. CONCLUSIONS/SIGNIFICANCE: The approaches developed and optimised in this study should be applicable to bats of other species. We are in the process of generating further cell lines from a number of different bat species using the methodology established in this study.

  2. Multidrug resistance P-glycoprotein dampens SR-BI cholesteryl ester uptake from high density lipoproteins in human leukemia cells.

    Science.gov (United States)

    Spolitu, Stefano; Uda, Sabrina; Deligia, Stefania; Frau, Alessandra; Collu, Maria; Angius, Fabrizio; Batetta, Barbara

    2016-01-01

    Tumor cells are characterised by a high content of cholesterol esters (CEs), while tumor-bearing patients show low levels of high-density lipoproteins (HDLs). The origin and significance of high CE levels in cancer cell biology has not been completely clarified. Recent evidence that lymphoblastic cells selectively acquire exogenous CE from HDL via the scavenger receptor SR-BI has drawn attention to the additional membrane proteins involved in this pathway. P-glycopotein-MDR1 (P-gp) is a product of the MDR1 gene and confers resistance to antitumor drugs. Its possible role in plasma membrane cholesterol trafficking and CE metabolism has been suggested. In the present study this aspect was investigated in a lymphoblastic cell line selected for MDR1 resistance. CEM were made resistant by stepwise exposure to low (LR) and high (HR) doses of vincristine (VCR). P-gp activity ((3)H-vinblastine), CE content, CE and triglycerides (TG) synthesis ((14)C-oleate), neutral lipids and Dil-HDL uptake (fluorescence), SR-BI, ABCA1 and P-gp protein expression (western blotting) were determined. To better evaluate the relationship between CE metabolism and P-gp activity, the ACAT inhibitor Sandoz-58035 and the P-gp inhibitors progesterone, cyclosporine and verapamil were used. CE content and synthesis were similar in the parental and resistant cells. However, in the latter population, SR-BI protein expression increased, whereas CE-HDL uptake decreased. These changes correlated with the degree of VCR-resistance. As well as reverting MDR1-resistance, the inhibitors of P-gp activity induced the CE-HDL/SR-BI pathway by reactivating membrane cholesterol trafficking. Indeed, CE-HDL uptake, SRBI expression and CE content increased, whereas there was a decrease in cholesterol esterification. These results demonstrated that P-gp overexpression impairs anticancer drug uptake as well as the SR-BI mediated selective CE-HDL uptake. This suggests that these membrane proteins act in an opposite

  3. Nestin expression in the cell lines derived from glioblastoma multiforme

    International Nuclear Information System (INIS)

    Nestin is a protein belonging to class VI of intermediate filaments that is produced in stem/progenitor cells in the mammalian CNS during development and is consecutively replaced by other intermediate filament proteins (neurofilaments, GFAP). Down-regulated nestin may be re-expressed in the adult organism under certain pathological conditions (brain injury, ischemia, inflammation, neoplastic transformation). Our work focused on a detailed study of the nestin cytoskeleton in cell lines derived from glioblastoma multiforme, because re-expression of nestin together with down-regulation of GFAP has been previously reported in this type of brain tumor. Two cell lines were derived from the tumor tissue of patients treated for glioblastoma multiforme. Nestin and other cytoskeletal proteins were visualized using imunocytochemical methods: indirect immunofluorescence and immunogold-labelling. Using epifluorescence and confocal microscopy, we described the morphology of nestin-positive intermediate filaments in glioblastoma cells of both primary cultures and the derived cell lines, as well as the reorganization of nestin during mitosis. Our most important result came through transmission electron microscopy and provided clear evidence that nestin is present in the cell nucleus. Detailed information concerning the pattern of the nestin cytoskeleton in glioblastoma cell lines and especially the demonstration of nestin in the nucleus represent an important background for further studies of nestin re-expression in relationship to tumor malignancy and invasive potential

  4. Complex formation of platelet thrombospondin with histidine-rich glycoprotein.

    OpenAIRE

    Leung, L L; Nachman, R L; Harpel, P C

    1984-01-01

    Thrombospondin and histidine-rich glycoprotein are two proteins with diverse biological activities which have been associated with human platelets and other cell systems. Using an enzyme-linked immunosorbent assay, we have demonstrated that purified human platelet thrombospondin formed a complex with purified human plasma histidine-rich glycoprotein. The formation of the thrombospondin-histidine-rich glycoprotein complex was specific, concentration dependent, and saturable. Significant bindin...

  5. An Open Receptor-Binding Cavity of Hemagglutinin-Esterase-Fusion Glycoprotein from Newly-Identified Influenza D Virus: Basis for Its Broad Cell Tropism.

    Science.gov (United States)

    Song, Hao; Qi, Jianxun; Khedri, Zahra; Diaz, Sandra; Yu, Hai; Chen, Xi; Varki, Ajit; Shi, Yi; Gao, George F

    2016-01-01

    Influenza viruses cause seasonal flu each year and pandemics or epidemic sporadically, posing a major threat to public health. Recently, a new influenza D virus (IDV) was isolated from pigs and cattle. Here, we reveal that the IDV utilizes 9-O-acetylated sialic acids as its receptor for virus entry. Then, we determined the crystal structures of hemagglutinin-esterase-fusion glycoprotein (HEF) of IDV both in its free form and in complex with the receptor and enzymatic substrate analogs. The IDV HEF shows an extremely similar structural fold as the human-infecting influenza C virus (ICV) HEF. However, IDV HEF has an open receptor-binding cavity to accommodate diverse extended glycan moieties. This structural difference provides an explanation for the phenomenon that the IDV has a broad cell tropism. As IDV HEF is structurally and functionally similar to ICV HEF, our findings highlight the potential threat of the virus to public health. PMID:26816272

  6. An Open Receptor-Binding Cavity of Hemagglutinin-Esterase-Fusion Glycoprotein from Newly-Identified Influenza D Virus: Basis for Its Broad Cell Tropism.

    Directory of Open Access Journals (Sweden)

    Hao Song

    2016-01-01

    Full Text Available Influenza viruses cause seasonal flu each year and pandemics or epidemic sporadically, posing a major threat to public health. Recently, a new influenza D virus (IDV was isolated from pigs and cattle. Here, we reveal that the IDV utilizes 9-O-acetylated sialic acids as its receptor for virus entry. Then, we determined the crystal structures of hemagglutinin-esterase-fusion glycoprotein (HEF of IDV both in its free form and in complex with the receptor and enzymatic substrate analogs. The IDV HEF shows an extremely similar structural fold as the human-infecting influenza C virus (ICV HEF. However, IDV HEF has an open receptor-binding cavity to accommodate diverse extended glycan moieties. This structural difference provides an explanation for the phenomenon that the IDV has a broad cell tropism. As IDV HEF is structurally and functionally similar to ICV HEF, our findings highlight the potential threat of the virus to public health.

  7. Characterization and endocytic internalization of Epith-2 cell surface glycoprotein during the epithelial-to-mesenchymal transition in sea urchin embryos

    Directory of Open Access Journals (Sweden)

    HidekiKatow

    2013-08-01

    Full Text Available The epithelial cells of the sea urchin Hemicentrotus pulcherrimus embryo express an Epith-2, uncharacterized glycoprotein, on the lateral surface. Here, we describe internalization of Epith-2 during mesenchyme formation through the epithelial-to-mesenchymal transition (EMT. Epith-2 was first expressed on the entire egg surface soon after fertilization and on the blastomeres until the 4-cell stage, but was localized to the lateral surface of epithelial cells at and after the 16-cell stage throughout the later developmental period. However, primary (PMC and secondary mesenchyme cells (SMC that ingress by EMT lost Epith-2 from their cell surface by endocytosis during dissociation from the epithelium, which was associated with the appearance of cytoplasmic Epith-2 dots. The cytoplasmic Epith-2 retained a similar relative molecular mass to that of the cell surface immediately after ingression through the early period of the spreading to single cells. Then, Epith-2 was completely lost from the cytoplasm. Tyrosine residues of Epith-2 were phosphorylated. The endocytic retraction of Epith-2 was inhibited by herbimycin A (HA, a protein tyrosine kinase (PTK inhibitor, and suramin, a growth factor receptor (GFR inhibitor, suggesting the involvement of the GFR/PTK (GP signaling pathway. These two GP inhibitors also inhibited PMC and SMC spreading to individual cells after ingression, but the dissociation of PMC and SMC from the epithelium was not inhibited. In suramin-treated embryos, dissociated mesenchyme cells migrated partially by retaining their epithelial morphology. In HA-treated embryos, no mesenchyme cells migrated. Thus, the EMT occurs in relation to internalization of Epith-2 from presumptive PMC and SMC.

  8. Molecular cloning and mammalian expression of human beta 2-glycoprotein I cDNA

    DEFF Research Database (Denmark)

    Kristensen, Torsten; Schousboe, Inger; Boel, Espen;

    1991-01-01

    Human β2-glycoprotein (β2gpI) cDNA was isolated from a liver cDNA library and sequenced. The cDNA encoded a 19-residue hydrophobic signal peptide followed by the mature β2gpI of 326 amino acid residues. In liver and in the hepatoma cell line HepG2 there are two mRNA species of about 1.4 and 4.3 k...

  9. The involvement of a LINE-1 element in a DNA rearrangement upstream of the mdr1a gene in a taxol multidrug-resistant murine cell line.

    Science.gov (United States)

    Cohen, D; Higman, S M; Hsu, S I; Horwitz, S B

    1992-10-01

    Two closely related but functionally distinct P-glycoprotein isoforms are encoded by the murine multidrug-resistance genes mdr1a and mdr1b. In a series of independently selected multidrug-resistant (MDR) J774.2 cell lines, mdr gene amplification and/or overexpression and overproduction of either the mdr1a or mdr1b products, or both gene products, correlates with the MDR phenotype. To investigate the possibility that mutations in the promoter regions of the mdr1a or mdr1b genes could influence their differential expression, mdr promoter-specific probes were used to detect and map potential structural alterations. An unusual structural rearrangement was found in the 5'-region of the amplified mdr1a allele in J7.T1, a cell line selected with taxol. To characterize this rearrangement, the regulatory regions of the mdr1a and mdr1b genes were analyzed. Whereas no gross structural alterations were detected by Southern blot hybridization using the mdr1b promoter probe, a novel amplified EcoRI fragment was detected by the mdr1a promoter probe. To determine the precise nature of this mutation, an mdr1a 5'-genomic clone was isolated from J7.T1 cells. Sequence analysis revealed an unusual DNA rearrangement consisting of the mdr1b gene, from its fourth intron toward its 3'-end, upstream of an intact mdr1a promoter on the amplified allele. We propose that this event occurred by an unequal sister chromatid exchange that was mediated by LINE-1 repetitive elements. PMID:1356977

  10. Human cell lines: A promising alternative for recombinant FIX production.

    Science.gov (United States)

    de Sousa Bomfim, Aline; Cristina Corrêa de Freitas, Marcela; Picanço-Castro, Virgínia; de Abreu Soares Neto, Mário; Swiech, Kamilla; Tadeu Covas, Dimas; Maria de Sousa Russo, Elisa

    2016-05-01

    Factor IX (FIX) is a vitamin K-dependent protein, and it has become a valuable pharmaceutical in the Hemophilia B treatment. We evaluated the potential of recombinant human FIX (rhFIX) expression in 293T and SK-Hep-1 human cell lines. SK-Hep-1-FIX cells produced higher levels of biologically active protein. The growth profile of 293T-FIX cells was not influenced by lentiviral integration number into the cellular genome. SK-Hep-1-FIX cells showed a significantly lower growth rate than SK-Hep-1 cells. γ-carboxylation process is significant to FIX biological activity, thus we performed a expression analysis of genes involved in this process. The 293T gene expression suggests that this cell line could efficiently carboxylate FIX, however only 28% of the total secreted protein is active. SK-Hep-1 cells did not express high amounts of VKORC1 and carboxylase, but this cell line secreted large amounts of active protein. Enrichment of culture medium with Ca(+2) and Mg(+2) ions did not affect positively rhFIX expression in SK-Hep-1 cells. In 293T cells, the addition of 0.5 mM Ca(+2) and 1 mM Mg(+2) resulted in higher rhFIX concentration. SK-Hep-1 cell line proved to be very effective in rhFIX production, and it can be used as a novel biotechnological platform for the production of recombinant proteins. PMID:26802680

  11. Mutagenesis of the fusion peptide-like domain of hepatitis C virus E1 glycoprotein: involvement in cell fusion and virus entry

    Directory of Open Access Journals (Sweden)

    Chuang Chin-Kai

    2009-09-01

    Full Text Available Abstract Background Envelope (E glycoprotein E2 of the hepatitis C virus (HCV mediates binding of the virus to target cell receptors. Nevertheless, the precise role of E1 in viral entry remains elusive. Methods To understand the involvement of the fusion peptide-like domain positioned at residues 264 to 290 within envelope glycoprotein E1 in HCV infection, mutants with Ala and Asn substitutions for residues conserved between HCV and E proteins of flaviviruses or the fusion proteins of paramyxoviruses were constructed by site-directed mutagenesis and their effects on membrane fusion and viral infectivity were examined. Results None of these mutations affected the synthesis or cell surface expression of envelope proteins, nor did they alter the formation of a non-covalent E1-E2 heterodimer or E2 binding to the large extracellular loop of CD81. The Cys residues located at positions 272 and 281 were unlikely involved in intra- or intermolecular disulfide bond formation. With the exception of the G267A mutant, which showed increased cell fusion, other mutants displayed reduced or marginally inhibited cell fusion capacities compared to the wild-type (WT E1E2. The G267A mutant was also an exception in human immunodeficiency virus type 1 (HIV-1/HCV E1E2 pseudotyping analyses, in that it showed higher one-cycle infectivity; all other mutants exhibited greatly or partially reduced viral entry versus the WT pseudotype. All but the G278A and D279N mutants showed a WT-like profile of E1E2 incorporation into HIV-1 particles. Since C272A, C281A, G282A, and G288A pseudotypes bound to Huh7 cells as effectively as did the WT pseudotype, the reduced infectivity of these pseudotypes was due to their ability to inhibit cell fusion. Conclusion Our results indicate that specific residues, but not the structure, of this fusion peptide-like domain are required for mediating cell fusion and viral entry.

  12. P-selectin glycoprotein ligand-1 forms dimeric interactions with E-selectin but monomeric interactions with L-selectin on cell surfaces.

    Directory of Open Access Journals (Sweden)

    Yan Zhang

    Full Text Available Interactions of selectins with cell surface glycoconjugates mediate the first step of the adhesion and signaling cascade that recruits circulating leukocytes to sites of infection or injury. P-selectin dimerizes on the surface of endothelial cells and forms dimeric bonds with P-selectin glycoprotein ligand-1 (PSGL-1, a homodimeric sialomucin on leukocytes. It is not known whether leukocyte L-selectin or endothelial cell E-selectin are monomeric or oligomeric. Here we used the micropipette technique to analyze two-dimensional binding of monomeric or dimeric L- and E-selectin with monomeric or dimeric PSGL-1. Adhesion frequency analysis demonstrated that E-selectin on human aortic endothelial cells supported dimeric interactions with dimeric PSGL-1 and monomeric interactions with monomeric PSGL-1. In contrast, L-selectin on human neutrophils supported monomeric interactions with dimeric or monomeric PSGL-1. Our work provides a new method to analyze oligomeric cross-junctional molecular binding at the interface of two interacting cells.

  13. Characterization of cloned cells from an immortalized fetal pulmonary type II cell line

    Energy Technology Data Exchange (ETDEWEB)

    Henderson, R.F.; Waide, J.J.; Lechner, J.F.

    1995-12-01

    A cultured cell line that maintained expression of pulmonary type II cell markers of differentiation would be advantageous to generate a large number of homogenous cells in which to study the biochemical functions of type II cells. Type II epithelial cells are the source of pulmonary surfactant and a cell of origin for pulmonary adenomas. Last year our laboratory reported the induction of expression of two phenotypic markers of pulmonary type II cells (alkaline phosphatase activity and surfactant lipid synthesis) in cultured fetal rat lung epithelial (FRLE) cells, a spontaneously immortalized cell line of fetal rat lung type II cell origin. Subsequently, the induction of the ability to synthesize surfactant lipid became difficult to repeat. We hypothesized that the cell line was heterogenuous and some cells were more like type II cells than others. The purpose of this study was to test this hypothesis and to obtain a cultured cell line with type II cell phenotypic markers by cloning several FRLE cells and characterizing them for phenotypic markers of type II cells (alkaline phosphatase activity and presence of surfactant lipids). Thirty cloned cell lines were analyzed for induced alkaline phosphatase activity (on x-axis) and for percent of phospholipids that were disaturated (i.e., surfactant).

  14. Antiproliferative effect of Tualang honey on oral squamous cell carcinoma and osteosarcoma cell lines

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    Ismail Noorliza M

    2010-09-01

    Full Text Available Abstract Background The treatment of oral squamous cell carcinomas (OSCC and human osteosarcoma (HOS includes surgery and/or radiotherapy which often lead to reduced quality of life. This study was aimed to study the antiproliferative activity of local honey (Tualang on OSCC and HOS cell lines. Methods Several concentrations of Tualang honey (1% - 20% were applied on OSCC and HOS cell lines for 3, 6, 12, 24, 48 and 72 hours. Morphological characteristics were observed under light and fluorescent microscope. Cell viability was assessed using MTT assay and the optical density for absorbance values in each experiment was measured at 570 nm by an ELISA reader. Detection of cellular apoptosis was done using the Annexin V-FITC Apoptosis Detection Kit. Results Morphological appearance showed apoptotic cellular changes like becoming rounded, reduction in cell number, blebbed membrane and apoptotic nuclear changes like nuclear shrinkage, chromatin condensation and fragmented nucleus on OSCC and HOS cell lines. Cell viability assay showed a time and dose-dependent inhibitory effect of honey on both cell lines. The 50% inhibitory concentration (IC50 for OSCC and HOS cell lines was found to be 4% and 3.5% respectively. The maximum inhibition of cell growth of ≥80% was obtained at 15% for both cell lines. Early apoptosis was evident by flow cytometry where percentage of early apoptotic cells increased in dose and time dependent manner. Conclusion Tualang honey showed antiproliferative effect on OSCC and HOS cell lines by inducing early apoptosis.

  15. Comparison of repair capability of four human tumor cell lines

    International Nuclear Information System (INIS)

    A fast and reliable method for assessment of repair capacity of cells based on the restoration of transcription of the gene for the green fluorescent protein carried by a plasmid vector is presented. Repair capacity of the cells counted under fluorescent microscope 24 hours following transcription. This approach has been applied to compare the rates of repair of different types of DNA lesions in four human tumor cell lines. The analysis of the obtained results show that there are considerable differences in the repair capacity of the different cell lines towards the different types of lesions. It should be noted that the difference in repair capacity of the host cells are better evident when plasmids with lower rather that higher number of lesions per unit length of plasmid DNA are used. This is probably because the egfp genes with fewer lesions can be fully repaired while egfp genes with more lesions remain inactive even after some of the lesions have been repaired

  16. Simultaneous measurement of natural killer cell cytotoxicity against each of three different target cell lines.

    Science.gov (United States)

    Blomberg, K

    1994-02-10

    A time-resolved fluorometric assay for the simultaneous measurement of natural killer cell activity against three different lanthanide diethylenetriaminopentaacetate (LaDTPA) labelled target cell lines is described. The target cell line K-562 was labelled with SmDTPA, the cell line Molt with TbDTPA and the cell line Raji with EuDTPA. After co-incubation of the three target cell lines with effector cells the fluorescence of the lanthanides released from the lysed target cells was measured in an enhancer solution in which they formed highly fluorescent complexes. It was possible to differentiate the specific release from the three target cell lines because the emission lines of the europium, samarium and terbium complexes formed in the enhancer solution are well separated from each other. The autofluorescence from culture media supplemented with serum was avoided by the use of time-resolved fluorometry. The results show that applying fluorometry based on the combination of spectral and temporal resolution to natural killer cell assays, makes possible the simultaneous determination of lysis in up to three target cell lines in complex culture medium. PMID:8308301

  17. Dipeptidyl peptidase IV in two human glioma cell lines

    Directory of Open Access Journals (Sweden)

    A Sedo

    2009-12-01

    Full Text Available There is growing evidence that dipeptidyl peptidase IV [DPP-IV, EC 3.4.14.5] takes part in the metabolism of biologically active peptides participating in the regulation of growth and transformation of glial cells. However, the knowledge on the DPP-IV expression in human glial and glioma cells is still very limited. In this study, using histochemical and biochemical techniques, the DPP-IV activity was demonstrated in two commercially available human glioma cell lines of different transformation degree, as represented by U373 astrocytoma (Grade III and U87 glioblastoma multiforme (Grade IV lines. Higher total activity of the enzyme, as well as its preferential localisation in the plasma membrane, was observed in U87 cells. Compared to U373 population, U87 cells were morphologically more pleiomorphic, they were cycling at lower rate and expressing less Glial Fibrillary Acidic Protein. The data revealed positive correlation between the degree of transformation of cells and activity of DPP-IV. Great difference in expression of this enzyme, together with the phenotypic differences of cells, makes these lines a suitable standard model for further 57 studies of function of this enzyme in human glioma cells.

  18. Alloreactive cloned T cell lines. I. Interactions between cloned amplifier and cytolytic T cell lines

    OpenAIRE

    1980-01-01

    Several T cell clones have been derived by limiting dilution of secondary mixed leukocyte culture cells stimulated by H-2- and M locus (Mls)-disparate spleen cells. When examined for the expression of cytolytic activity and the ability to proliferate, these cell clones can be classified into two major categories. One type of cell is noncytolytic; when cultured with irradiated spleen cells, such clones proliferate in response to Mls determinants. Some, but not all, of these clones express Lyt-...

  19. Reversal of P-glycoprotein-mediated multidrug resistance in human hepatoma cells by hedyotiscone A, a compound isolated from Hedyotis corymbosa.

    Science.gov (United States)

    Yue, Grace Gar-Lee; Kin-Ming Lee, Julia; Cheng, Ling; Chung-Lap Chan, Ben; Jiang, Lei; Fung, Kwok-Pui; Leung, Ping-Chung; Bik-San Lau, Clara

    2012-06-01

    Multidrug resistance is a major problem in hepatocellular carcinoma. Hedyotiscone A, a compound isolated from Chinese herbal medicine Hedyotis corymbosa (HC, family Rubiaceae), was used as the chemical marker to distinguish between HC and an anticancer herb Hedyotis diffusa (HD) in our previous study. The present study aimed to investigate whether HA exhibited antiproliferative activities in multidrug-resistant hepatocellular carcinoma cells R-HepG2 and the parental cells HepG2 using MTT assay and [(3)H]-thymidine incorporation assay. Our results showed that HA could significantly inhibit cell proliferation in R-HepG2 and HepG2 (IC(50) = 43.7 and 56.3 µg/mL, respectively), but not in normal human liver cells WRL-68 (IC(50) > 100 µg/mL) cells, suggesting its selective cytotoxic effects. Besides, HA induced apoptosis in R-HepG2 cells, as confirmed by annexin-V & propidium iodide staining, and DNA fragmentation assay. The caspase cascade was activated as shown by a significant increase of cleaved caspases-3, -7 and -9 in HA-treated R-HepG2 cells. The activities and protein expression of P-glycoprotein as well as mRNA expression of MDR1 were also decreased in HA-treated R-HepG2 cells. Our study demonstrated for the first time the antiproliferative activities of hedyotiscone A in multidrug-resistant R-HepG2 cells. The findings revealed the potential of this compound in treating multidrug-resistant tumor. PMID:22352391

  20. Influence of colchicine and vinblastine on the intracellular migration of secretory and membrane glycoproteins: III. Inhibition of intracellular migration of membrane glycoproteins in rat intestinal columnar cells and hepatocytes as visualized by light and electron-microscope radioautography after 3H-fucose injection

    International Nuclear Information System (INIS)

    In the present work, the effects of these drugs on migration of membrane glycoproteins have been examined at the ultrastructural level in duodenal villous columnar cells and hepatocytes. Young (40 gm) rats were given a single intravenous injection of colchicine (4.0 mg) or vinblastine (2.0 mg). At 10 min after colchicine and 30 min after vinblastine administration, the rats were injected with 3H-fucose. Control rats received 3H-fucose only. All rats were sacrificed 90 min after 3H-fucose injection and their tissues processed for radioautography. In duodenal villous columnar cells, 3H-fucose labeling of the apical plasma membrane was reduced by 51% after colchicine and by 67% after vinblastine treatment; but there was little change in labeling of the lateral plasma membrane. Labeling of the Golgi apparatus increased. This suggests that labeled glycoproteins destined for the apical plasma membrane were inhibited from leaving the Golgi region, while migration to the lateral plasma membrane was not impaired. In hepatocytes, labeling of the sinusoidal plasma membrane was reduced by 83% after colchicine and by 85% after vinblastine treatment. Labeling of the lateral plasma membrane also decreased, although not so dramatically. Labeling of the Golgi apparatus and neighboring secretory vesicles increased. This indicates that the drugs inhibited migration of membrane glycoproteins from the Golgi region to the various portions of the plasma membrane. Accumulation of secretory vesicles at the sinusoidal front suggests that exocytosis may also have been partially inhibited. In both cell types, microtubules almost completely disappeared after drug treatment. Microtubules may, therefore, be necessary for intracellular transport of membrane glycoproteins, although the possibility of a direct action of these drugs on Golgi or plasma membranes must also be considered

  1. Changes of Tc-99m sestamibi uptake in P-glycoprotein expressing leukaemia cells treated in vivo with antisense oligodeoxynucleotide complementary to mdr1 mRNA

    International Nuclear Information System (INIS)

    We examined the feasibility of Tc-99m sestamibi to monitor changes of mRNA expression of MDRl/P-glycoprotein (Pgp) following antisense oligodeoxynucleotide (AS-ODN) treatment in vivo. Three days after the intraperitoneal inoculation of murine leukaemia P388/R cells expressing MDR1/P-gp in CDFI mice, 15-mer phosphorothioate ASODN to the initiation codon of mouse mdr1 mRNA was administered intraperitoneally at 10 mg/kg daily for 3 or 4 days. Cells collected from ascites were suspended in medium for Tc-99m sestamibi uptake studies. To know the duration of antisense effects, cells were harvested 2 days later after the 3-day treatment. AS-ODN treatment increased Tc-99m sestamibi uptake. Effects of 3-day treatment and 4-day treatment were the same. Treatment effects were not detected when uptake was observed 2 days after 3-day treatment. Based on the results it was concluded that in vivo treatment with AS-ODN specific to the coding portion of mdr1 mRNA increased Tc-99m sestamibi uptake in leukaemia cells possessing MDR function. (author)

  2. Cellular and Phenotypic Characterization of Canine Osteosarcoma Cell Lines

    Directory of Open Access Journals (Sweden)

    Marie E. Legare, Jamie Bush, Amanda K. Ashley, Taka Kato, William H. Hanneman

    2011-01-01

    Full Text Available Canine and human osteosarcoma (OSA have many similarities, with the majority of reported cases occurring in the appendicular skeleton, gender predominance noted, high rate of metastasis at the time of presentation, and a lack of known etiology for this devastating disease. Due to poor understanding of the molecular mechanisms underlying OSA, we have characterized seven different OSA canine cell lines: Abrams, D17, Grey, Hughes, Ingles, Jarques, and Marisco and compared them to U2, a human OSA cell line, for the following parameters: morphology, growth, contact inhibition, migrational tendencies, alkaline phosphatase staining, heterologous tumor growth, double-strand DNA breaks, and oxidative damage. All results demonstrated the positive characteristics of the Abrams cell line for use in future studies of OSA. Of particular interest, the robust growth of a subcutaneous tumor and rapid pulmonary metastasis of the Abrams cell line in an immunocompromised mouse shows incredible potential for the future use of Abrams as a canine OSA model. Further investigations utilizing a canine cell model of OSA, such as Abrams, will be invaluable to understanding the molecular events underlying OSA, pharmaceutical inhibition of metastasis, and eventual prevention of this devastating disease.

  3. GPC3 reduces cell proliferation in renal carcinoma cell lines

    OpenAIRE

    Valsechi, Marina Curado; Oliveira, Ana Beatriz Bortolozo; Conceição, André Luis Giacometti; Stuqui, Bruna; Candido, Natalia Maria; Provazzi, Paola Jocelan Scarin; de Araújo, Luiza Ferreira; Silva, Wilson Araújo; Calmon, Marilia Freitas; Rahal, Paula

    2014-01-01

    Background Glypican 3 (GPC3) is a member of the family of glypican heparan sulfate proteoglycans (HSPGs). The GPC3 gene may play a role in controlling cell migration, negatively regulating cell growth and inducing apoptosis. GPC3 is downregulated in several cancers, which can result in uncontrolled cell growth and can also contribute to the malignant phenotype of some tumors. The purpose of this study was to analyze the mechanism of action of the GPC3 gene in clear cell renal cell carcinoma. ...

  4. Mouse DRG Cell Line with Properties of Nociceptors.

    Directory of Open Access Journals (Sweden)

    Ciara Doran

    Full Text Available In vitro cell lines from DRG neurons aid drug discovery because they can be used for early stage, high-throughput screens for drugs targeting pain pathways, with minimal dependence on animals. We have established a conditionally immortal DRG cell line from the Immortomouse. Using immunocytochemistry, RT-PCR and calcium microfluorimetry, we demonstrate that the cell line MED17.11 expresses markers of cells committed to the sensory neuron lineage. Within a few hours under differentiating conditions, MED17.11 cells extend processes and following seven days of differentiation, express markers of more mature DRG neurons, such as NaV1.7 and Piezo2. However, at least at this time-point, the nociceptive marker NaV1.8 is not expressed, but the cells respond to compounds known to excite nociceptors, including the TRPV1 agonist capsaicin, the purinergic receptor agonist ATP and the voltage gated sodium channel agonist, veratridine. Robust calcium transients are observed in the presence of the inflammatory mediators bradykinin, histamine and norepinephrine. MED17.11 cells have the potential to replace or reduce the use of primary DRG culture in sensory, pain and developmental research by providing a simple model to study acute nociception, neurite outgrowth and the developmental specification of DRG neurons.

  5. The effect of gallic acid on Jurkat cell line

    Directory of Open Access Journals (Sweden)

    Sourani Zahra

    2015-10-01

    Full Text Available Introduction: Acute lymphoblastic leukemia (ALL is the most prevalent leukemia in children. Fruits and plants have a wide range of biological functions including anti-proliferative effect. Gallic acid (GA, is a polyhydroxyphenolic compound that is widely distributed in the natural plants. The aim of the present study was the evaluation of the effect of GA on proliferation inhibition of Jurkat cells, the lymphoblastic leukemia cell line. Methods: Jurkat cell line was cultured in blood cells culture media in a standard conditions with different concentrations of GA (0-100 μm for 24, 48 and 72 hours. The effect of GA on cell viability was measured using MTS assay. Results: Decline of cell viability to less than 50% was observed at 60, 50 and 30 μm concentrations after 24, 48 and 72 hours incubation time, respectively. Conclusion: The results demonstrated that the polyphenolic compound, GA with antioxidant capability is effective in proliferation inhibition in Jurkat lymphoblastic leukemia cell line with a time and dose dependent manner. Therefore, GA may be a potential compound for cancer prevention and treatment.

  6. Effect of selected insecticides on SF9 insect cell line

    International Nuclear Information System (INIS)

    The toxic effect of three insecticides: dimethoate (organophosphate insecticide), acetamiprid (neonicotinoid insecticide) and deltamethrin (pyrethroid insecticide) were evaluated in vitro on cultured Sf9 cell line. Cell growth inhibition was measured by the 3- (4,5- dimethylthiazol - 2-yl) - 2,5 - diphenyl tetrazolium bromide (MTT) assay. Regression Analysis was used to estimate the 20% inhibition of cells growth (IC 20). The IC 20 values obtained for deltamethrin, acetamipridand dimethoate were: 46.8, 61.6 and 68.9 μM, respectively. The proportion of phagocytic cells was positively correlated with the applied concentrations of the insecticides. (author)

  7. Proteoglycans secreted by packaging cell lines inhibit retrovirus infection.

    OpenAIRE

    Le Doux, J M; Morgan, J.R.; Snow, R G; Yarmush, M. L.

    1996-01-01

    Using a model recombinant retrovirus encoding the Escherichia coli lacZ gene, we have found that medium conditioned with NIH 3T3 cells and packaging cell lines derived from NIH 3T3 cells inhibits infection. Most of the inhibitory activity was greater than 100 kDa and was sensitive to chondroitinase ABC digestion, which is consistent with the inhibitor being a chondroitin sulfate proteoglycan. Proteoglycans secreted by NIH 3T3 cells and purified by anion-exchange chromatography inhibited ampho...

  8. Selection of Phage Display Peptides Targeting Human Pluripotent Stem Cell-Derived Progenitor Cell Lines.

    Science.gov (United States)

    Bignone, Paola A; Krupa, Rachel A; West, Michael D; Larocca, David

    2016-01-01

    The ability of human pluripotent stem cells (hPS) to both self-renew and differentiate into virtually any cell type makes them a promising source of cells for cell-based regenerative therapies. However, stem cell identity, purity, and scalability remain formidable challenges that need to be overcome for translation of pluripotent stem cell research into clinical applications. Directed differentiation from hPS cells is inefficient and residual contamination with pluripotent cells that have the potential to form tumors remains problematic. The derivation of scalable (self-renewing) embryonic progenitor stem cell lines offers a solution because they are well defined and clonally pure. Clonally pure progenitor stem cell lines also provide a means for identifying cell surface targeting reagents that are useful for identification, tracking, and repeated derivation of the corresponding progenitor stem cell types from additional hPS cell sources. Such stem cell targeting reagents can then be applied to the manufacture of genetically diverse banks of human embryonic progenitor cell lines for drug screening, disease modeling, and cell therapy. Here we present methods to identify human embryonic progenitor stem cell targeting peptides by selection of phage display libraries on clonal embryonic progenitor cell lines and demonstrate their use for targeting quantum dots (Qdots) for stem cell labeling. PMID:25410289

  9. Development and characterization of multidrug resistant human hepatocarcinoma cell line in nude mice

    Institute of Scientific and Technical Information of China (English)

    Bao-Jin Zhai; Ze-Yong Shao; Chun-Liang Zhao; Kai Hu; Feng Wu

    2006-01-01

    AIM: To establish a multidrug resistant (MDR) cell subline from the human hepatocarcinoma cell line (HepG2)in nude mice.METHODS: HepG2 cell cultures were incubated with increasing concentrations of adriamycin (ADM) to develop an ADM-resistant cell subline (HepG2/ADM) with crossresistance to other chemotherapeutic agents. Twenty male athymic BALB/c-nu/nu mice were randomized into HepG2/nude and HepG2/ADM/nude groups (10 in each group). A cell suspension (either HepG2 or HepG2/ADM)was injected subcutaneously into mice in each group.Tumor growth was recorded, and animals were sacrificed 4-5 wk after cell implantation. Tumors were prepared for histology, and viable tumor was dispersed into a single-cell suspension. The IC50 values for a number of chemotherapeutic agents were determined by 2, 3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide inner salt (MTT) assay. Rhodamine-123retention/efflux and the level of resistance-associated proteins were determined by flow cytometry. The mRNA expression of mdr1, mrp and Irp genes was detected using reverse transcriptase polymerase chain reaction (RT-PCR) in HepG2/nude and HepG2/ADM/nude groups.RESULTS: The appearances of HepG2/nude cells were slightly different from those of HepG2/ADM/nude cells.Similar tumor growth curves were determined in both groups. A cross-resistance to ADM, vincristine, cisplatin and 5-fiuorouracil was seen in HepG2/ADM/nude group.The levels of P-glycoprotein and multidrug resistanceassociated proteins were significantly increased. The mRNA expression levels of mdr1, mrp and Irp were higher in HepG2/ADM/nude cells.CONCLUSION: ADM-resistant HepG2 subline in nude mice has a cross resistance to chemotherapeutic drugs.Tt may be used as an in vivo model to investigate the mechanisms of MDR, and explore the targeted approaches to overcoming MDR.

  10. Osmotic stress affects functional properties of human melanoma cell lines

    CERN Document Server

    La Porta, Caterina A M; Pasini, Maria; Laurson, Lasse; Alava, Mikko J; Zapperi, Stefano; Amar, Martine Ben

    2015-01-01

    Understanding the role of microenvironment in cancer growth and metastasis is a key issue for cancer research. Here, we study the effect of osmotic pressure on the functional properties of primary and metastatic melanoma cell lines. In particular, we experimentally quantify individual cell motility and transmigration capability. We then perform a circular scratch assay to study how a cancer cell front invades an empty space. Our results show that primary melanoma cells are sensitive to a low osmotic pressure, while metastatic cells are less. To better understand the experimental results, we introduce and study a continuous model for the dynamics of a cell layer and a stochastic discrete model for cell proliferation and diffusion. The two models capture essential features of the experimental results and allow to make predictions for a wide range of experimentally measurable parameters.

  11. Cell membrane fatty acid composition differs between normal and malignant cell lines.

    Science.gov (United States)

    Meng, Xialong; Riordan, Neil H; Riordan, Hugh D; Mikirova, Nina; Jackson, James; González, Michael J; Miranda-Massari, Jorge R; Mora, Edna; Trinidad Castillo, Waleska

    2004-06-01

    Twenty-eight fatty acids (C8:0 to C24:l n-9) were measured by gas chromatography in four normal cell lines (C3H / 10T1 / 2, CCD-18Co, CCD-25SK and CCD-37Lu) and seven cancer cell lines (C-41, Caov-3, LS-180, PC-3, SK-MEL-28, SK-MES-1 and U-87 MG). Results show differences in the content and proportions of fatty acids when comparing cancer cell lines with their normal counterparts. Cancer cell lines showed lower C20: 4 n-6, C24:1 n-9, polyunsaturated fatty acids (PUFA's) and ratios of C20:4 n-6 to C20:5 n-3 and C16:0 to C18:1 n-9 and stearic to oleic (SA/OA) than their normal counterparts. All cancer cell lines had SA/OA ratios lower than 7.0 while normal cell lines had ratios greater than 0.7 (p<0.05). In addition, the ratios of total saturated fatty acids (SFA) to PUFA'S and the concentration of C18:1 n-9, C18:2 n-6, C20:5 n-3 were higher in cancer cell lines as compared to normal cell lines. A positive correlation was detected between C16:0 and longer SFA'S (r = +0.511, p<0.05) in normal cell lines whereas a negative correlation (r=0.608, p<0.05) was obtained for malignant cell lines. Moreover, cancerous cell lines exhibited a particular desaturation defect and an abnormal incorporation of C18:2 n-6 and C20-4 n-6 fatty acids. PMID:15377057

  12. Detection of immunotoxicity using T-cell based cytokine reporter cell lines ('Cell Chip')

    International Nuclear Information System (INIS)

    Safety assessment of chemicals and drugs is an important regulatory issue. The evaluation of potential adverse effects of compounds on the immune system depends today on animal experiments. An increasing demand, however, exists for in vitro alternatives. Cytokine measurement is a promising tool to evaluate chemical exposure effects on the immune system. Fortunately, this type of measurement can be performed in conjunction with in vitro exposure models. We have taken these considerations as the starting point to develop an in vitro method to efficiently screen compounds for potential immunotoxicity. The T-cell lymphoma cell line EL-4 was transfected with the regulatory sequences of interleukin (IL)-2, IL-4, IL-10, interferon (IFN)-γ or actin fused to the gene for enhanced green fluorescent protein (EGFP) in either a stabile or a destabilised form. Consequently, changes in fluorescence intensity represent changes in cytokine expression with one cell line per cytokine. We used this prototype 'Cell Chip' to test, by means of flow cytometry, the immunomodulatory potential of 13 substances and were able to detect changes in cytokine expression in 12 cases (successful for cyclosporine, rapamycin, pentamidine, thalidomide, bis(tri-n-butyltin)oxide, house dust mite allergen (Der p I), 1-chloro-2,4-dinitrobenzene, benzocaine, tolylene 2,4-diisocyanate, potassium tetrachloroplatinate, sodium dodecyl sulphate and mercuric chloride; unsuccessful for penicillin G). In conclusion, this approach seems promising for in vitro screening for potential immunotoxicity, especially when additional cell lines besides T-cells are included

  13. Effect of brefeldin A and castanospermine on resistant cell lines as supplements in anticancer therapy.

    Science.gov (United States)

    Wojtowicz, Karolina; Januchowski, Radosław; Sosińska, Patrycja; Nowicki, Michał; Zabel, Maciej

    2016-05-01

    In the present study, we analyzed the influence of brefeldin A (BFA) and castanospermine (CAS) on the activity, stability and localization of P-glycoprotein (Pgp) and breast cancer resistance protein (BCRP) in various resistant cell lines. The impact of BFA and CAS on cell viability was assessed using the MTT test. Western blotting (WB) was performed to assess the effect of the inhibitors on the expression of the investigated proteins. Immunofluorescence was employed to assess the effect of BFA and CAS on the cellular localization of the proteins. Flow cytometry was used to verify the functional role of inhibitors on drug uptake and efflux. The MTT test showed that BFA had a significant effect on cell viability in LoVo/Dx and W1PR cell lines. WB analysis demonstrated that BFA partially blocked Pgp N-glycosylation and induced BCRP degradation and CASP 3-dependent apoptosis in W1TR cells; however, the BFA activity was p53-independent. CAS had no effect on the stability of Pgp but increased the level of non-glycosylated BCRP. The expression of p53 protein decreased in all of the cells that were treated with CAS. Immunofluorescence revealed that BFA caused a more granular Pgp signal in W1PR and BCRP in A2780T1 cells. Furthermore, BFA caused morphological changes in LoVo/Dx and W1TR cell lines. CAS also induced a granular signal in all of the cell lines, except W1TR. The flow cytometry showed higher dye accumulation in sensitive cell lines. We observed an increase in the mean fluorescence intensity (MFI) of Rho123 in LoVo/Dx cells treated with BFA and CAS, but no differences were observed in W1PR. BFA had no effect on the MFI of W1TR, but CAS led to an increase in the level of intracellular H33342 in W1TR and A2780T1 cells. These results suggest that these compounds are likely to be useful as supplements in anticancer therapy. PMID:26985570

  14. Cysteine modified polyaniline films improve biocompatibility for two cell lines

    International Nuclear Information System (INIS)

    This work focuses on one of the most exciting application areas of conjugated conducting polymers, which is cell culture and tissue engineering. To improve the biocompatibility of conducting polymers we present an easy method that involves the modification of the polymer backbone using L-cysteine. In this publication, we show the synthesis of polyaniline (PANI) films supported onto Polyethylene terephthalate (PET) films, and modified using cysteine (PANI-Cys) in order to generate a biocompatible substrate for cell culture. The PANI-Cys films are characterized by Fourier Transform infrared and UV–visible spectroscopy. The changes in the hydrophilicity of the polymer films after and before the modification were tested using contact angle measurements. After modification the contact angle changes from 86° ± 1 to 90° ± 1, suggesting a more hydrophylic surface. The adhesion properties of LM2 and HaCaT cell lines on the surface of PANI-Cys films in comparison with tissue culture plastic (TCP) are studied. The PANI-Cys film shows better biocompatibility than PANI film for both cell lines. The cell morphologies on the TCP and PANI-Cys film were examined by florescence and Atomic Force Microscopy (AFM). Microscopic observations show normal cellular behavior when PANI-Cys is used as a substrate of both cell lines (HaCaT and LM2) as when they are cultured on TCP. The ability of these PANI-Cys films to support cell attachment and growth indicates their potential use as biocompatible surfaces and in tissue engineering. - Highlights: • A new surface PANI-Cys was produced on films of polyethylene terephthalate. • The relationship between surface characteristics and biocompatibility is analyzed. • The PANI-Cys film presents good biocompatibility for two cell lines

  15. Adhesion of Actinobacillus actinomycetemcomitans to a human oral cell line.

    OpenAIRE

    Mintz, K. P.; Fives-Taylor, P M

    1994-01-01

    Two quantitative, rapid assays were developed to study the adhesion of Actinobacillus actinomycetemcomitans, an oral bacterium associated with periodontal disease, to human epithelial cells. The human oral carcinoma cell line KB was grown in microtiter plates, and adherent bacteria were detected by an enzyme-linked immunosorbent assay with purified anti-A. actinomycetemcomitans serum and horseradish peroxidase-conjugated secondary antibody or [3H]thymidine-labeled bacteria. Adhesion was found...

  16. Cysteine modified polyaniline films improve biocompatibility for two cell lines

    Energy Technology Data Exchange (ETDEWEB)

    Yslas, Edith I., E-mail: eyslas@exa.unrc.edu.ar [Departamento de Biología Molecular, Universidad Nacional de Río Cuarto, Agencia Postal Nro3, X580BYA Río Cuarto (Argentina); Cavallo, Pablo; Acevedo, Diego F.; Barbero, César A. [Departamento de Química, Universidad Nacional de Río Cuarto, Agencia Postal Nro3, X580BYA Río Cuarto (Argentina); Rivarola, Viviana A. [Departamento de Biología Molecular, Universidad Nacional de Río Cuarto, Agencia Postal Nro3, X580BYA Río Cuarto (Argentina)

    2015-06-01

    This work focuses on one of the most exciting application areas of conjugated conducting polymers, which is cell culture and tissue engineering. To improve the biocompatibility of conducting polymers we present an easy method that involves the modification of the polymer backbone using L-cysteine. In this publication, we show the synthesis of polyaniline (PANI) films supported onto Polyethylene terephthalate (PET) films, and modified using cysteine (PANI-Cys) in order to generate a biocompatible substrate for cell culture. The PANI-Cys films are characterized by Fourier Transform infrared and UV–visible spectroscopy. The changes in the hydrophilicity of the polymer films after and before the modification were tested using contact angle measurements. After modification the contact angle changes from 86° ± 1 to 90° ± 1, suggesting a more hydrophylic surface. The adhesion properties of LM2 and HaCaT cell lines on the surface of PANI-Cys films in comparison with tissue culture plastic (TCP) are studied. The PANI-Cys film shows better biocompatibility than PANI film for both cell lines. The cell morphologies on the TCP and PANI-Cys film were examined by florescence and Atomic Force Microscopy (AFM). Microscopic observations show normal cellular behavior when PANI-Cys is used as a substrate of both cell lines (HaCaT and LM2) as when they are cultured on TCP. The ability of these PANI-Cys films to support cell attachment and growth indicates their potential use as biocompatible surfaces and in tissue engineering. - Highlights: • A new surface PANI-Cys was produced on films of polyethylene terephthalate. • The relationship between surface characteristics and biocompatibility is analyzed. • The PANI-Cys film presents good biocompatibility for two cell lines.

  17. Establishment and applications of male germ cell and Sertoli cell lines.

    Science.gov (United States)

    Wang, Hong; Wen, Liping; Yuan, Qingqing; Sun, Min; Niu, Minghui; He, Zuping

    2016-08-01

    Within the seminiferous tubules there are two major cell types, namely male germ cells and Sertoli cells. Recent studies have demonstrated that male germ cells and Sertoli cells can have significant applications in treating male infertility and other diseases. However, primary male germ cells are hard to proliferate in vitro and the number of spermatogonial stem cells is scarce. Therefore, methods that promote the expansion of these cell populations are essential for their use from the bench to the bed side. Notably, a number of cell lines for rodent spermatogonia, spermatocytes and Sertoli cells have been developed, and significantly we have successfully established a human spermatogonial stem cell line with an unlimited proliferation potential and no tumor formation. This newly developed cell line could provide an abundant source of cells for uncovering molecular mechanisms underlying human spermatogenesis and for their utilization in the field of reproductive and regenerative medicine. In this review, we discuss the methods for establishing spermatogonial, spermatocyte and Sertoli cell lines using various kinds of approaches, including spontaneity, transgenic animals with oncogenes, simian virus 40 (SV40) large T antigen, the gene coding for a temperature-sensitive mutant of p53, telomerase reverse gene (Tert), and the specific promoter-based selection strategy. We further highlight the essential applications of these cell lines in basic research and translation medicine. PMID:27069011

  18. Biological characteristics of side population cells in a self-established human ovarian cancer cell line

    Science.gov (United States)

    WEI, ZHENTONG; LV, SHUANG; WANG, YISHU; SUN, MEIYU; CHI, GUANGFAN; GUO, JUN; SONG, PEIYE; FU, XIAOYU; ZHANG, SONGLING; LI, YULIN

    2016-01-01

    The aim of the present study was to establish an ovarian cancer (OC) cell line from ascites of an ovarian serous cystadenocarcinoma patient and investigate the biological characteristics of its side population (SP) cells. The OC cell line was established by isolating, purifying and subculturing primary cells from ascites of an ovarian serous cystadenocarcinoma patient (stage IIIc; grade 3). SP and non-SP (NSP) cells were isolated by fluorescence-activated cell sorting and cultured in serum-free medium and soft agar to compare the tumorsphere and colony formation capacities. Furthermore, SP and NSP cell tumorigenesis was examined by subcutaneous and intraperitoneal injection of the cells to non-obese diabetic/severe combined immune deficiency (NOD/SCID) mice. Drug resistance to cisplatin was examined by cell counting kit-8. The OC cell line was successfully established from ascites of an ovarian serous cystadenocarcinoma patient, which exhibited properties similar to primary tumors subsequent to >50 passages and >2 years of culture. The SP cell ratio was 0.38% in the OC cell line, and a similar SP cell ratio (0.39%) was observed when sorted SP cells were cultured for 3 weeks. Compared with NSP cells, SP cells exhibited increased abilities in differentiation and tumorsphere and colony formation, in addition to the formation of xenografted tumors and ascites and metastasis of the tumors in NOD/SCID mice, even at low cell numbers (3.0×103 cells). The xenografted tumors demonstrated histological features similar to primary tumors and expressed the ovarian serous cystadenocarcinoma marker CA125. In addition, SP cells demonstrated a significantly stronger drug resistance to cisplatin compared with NSP and unsorted cells, while treatment with verapamil, an inhibitor of ATP-binding cassette transporters, potently abrogated SP cell drug resistance. In conclusion, the present study verified SP cells from an established OC cell line and characterized the cells with self

  19. Sphere-forming cell subpopulations with cancer stem cell properties in human hepatoma cell lines

    Directory of Open Access Journals (Sweden)

    Chen Lei

    2011-06-01

    Full Text Available Abstract Background Cancer stem cells (CSCs are regarded as the cause of tumor formation and recurrence. The isolation and identification of CSCs could help to develop novel therapeutic strategies specifically targeting CSCs. Methods Human hepatoma cell lines were plated in stem cell conditioned culture system allowed for sphere forming. To evaluate the stemness characteristics of spheres, the self-renewal, proliferation, chemoresistance, tumorigenicity of the PLC/PRF/5 sphere-forming cells, and the expression levels of stem cell related proteins in the PLC/PRF/5 sphere-forming cells were assessed, comparing with the parental cells. The stem cell RT-PCR array was performed to further explore the biological properties of liver CSCs. Results The PLC/PRF/5, MHCC97H and HepG2 cells could form clonal nonadherent 3-D spheres and be serially passaged. The PLC/PRF/5 sphere-forming cells possessed a key criteria that define CSCs: persistent self-renewal, extensive proliferation, drug resistance, overexpression of liver CSCs related proteins (Oct3/4, OV6, EpCAM, CD133 and CD44. Even 500 sphere-forming cells were able to form tumors in NOD/SCID mice, and the tumor initiating capability was not decreased when spheres were passaged. Besides, downstream proteins DTX1 and Ep300 of the CSL (CBF1 in humans, Suppressor of hairless in Drosophila and LAG1 in C. elegans -independent Notch signaling pathway were highly expressed in the spheres, and a gamma-secretase inhibitor MRK003 could significantly inhibit the sphere formation ability. Conclusions Nonadherent tumor spheres from hepatoma cell lines cultured in stem cell conditioned medium possess liver CSC properties, and the CSL-independent Notch signaling pathway may play a role in liver CSCs.

  20. Cryopreservation of specialized chicken lines using cultured primordial germ cells.

    Science.gov (United States)

    Nandi, S; Whyte, J; Taylor, L; Sherman, A; Nair, V; Kaiser, P; McGrew, M J

    2016-08-01

    Biosecurity and sustainability in poultry production requires reliable germplasm conservation. Germplasm conservation in poultry is more challenging in comparison to other livestock species. Embryo cryopreservation is not feasible for egg-laying animals, and chicken semen conservation has variable success for different chicken breeds. A potential solution is the cryopreservation of the committed diploid stem cell precursors to the gametes, the primordial germ cells ( PGCS: ). Primordial germ cells are the lineage-restricted cells found at early embryonic stages in birds and form the sperm and eggs. We demonstrate here, using flocks of partially inbred, lower-fertility, major histocompatibility complex- ( MHC-: ) restricted lines of chicken, that we can easily derive and cryopreserve a sufficient number of independent lines of male and female PGCs that would be sufficient to reconstitute a poultry breed. We demonstrate that germ-line transmission can be attained from these PGCs using a commercial layer line of chickens as a surrogate host. This research is a major step in developing and demonstrating that cryopreserved PGCs could be used for the biobanking of specialized flocks of birds used in research settings. The prospective application of this technology to poultry production will further increase sustainability to meet current and future production needs. PMID:27099306

  1. 9-β-arabinofuranosyladenine preferentially sensitizes radioresistant squamous cell carcinoma cell lines to x-rays

    International Nuclear Information System (INIS)

    The effect of 9-β-arabinofuranosyladenine (ara-A) on sensitivity to the deleterious effects of x-rays was studied in six squamous cell carcinoma cell lines. Three lines were relatively radioresistant, having D0 values of 2.31 to 2.89 Gy, and the other three lines were relatively radiosensitive, having D0 values of between 1.07 and 1.45 Gy. Ara-A (50 or 500 μM) was added to cultures 30 min prior to irradiation and removed 30 min after irradiation, and sensitivity was measured in terms of cell survival. The radiosensitizing effect of ara-A was very dependent on the inherent radiosensitivity of the tumor cell line. Fifty micromolar concentrations of ara-A sensitized only the two most radioresistant lines, SCC-12B.2 and JSQ-3. Five hundred micromolar concentrations of ara-A sensitized the more sensitive cell lines, SQ-20B and SQ-9G, but failed to have any effect on the radiation response of the two most sensitive cell lines, SQ-38 and SCC-61. Concentrations of ara-A as low as 10 μM were equally efficient in inhibiting DNA synthesis in all six cell lines. These results suggest that the target for the radiosensitizing effect of ara-A is probably related to the factor controlling the inherent radiosensitivity of human tumor cells. Therefore, ara-A might be useful in overcoming radiation resistance in vivo

  2. Human Embryonic St me Cell Lines fromthe Chinese Population and Differentiation to Liver and Muscle Cell Types

    Institute of Scientific and Technical Information of China (English)

    2005-01-01

    We have established 6 hES cell lines from IVF surplus blastocysts. Characterization of these lines have shown that 4 of the 6 lines meet all of the criterion (Science) for hES cell lines and 2 of them display most characteristics of hES cells but do not form teratoma. In order to produce hES cell lines without using mouse feeders, we have produced a hES cell line using feeders derived from hES cells themselves, and showed that hES-derived feeders are capable of supporting the derivation of new hES cell line...

  3. Choosing the right chondrocyte cell line: Focus on nitric oxide.

    Science.gov (United States)

    Santoro, Anna; Conde, Javier; Scotece, Morena; Abella, Vanessa; López, Verónica; Pino, Jesús; Gómez, Rodolfo; Gómez-Reino, Juan Jesús; Gualillo, Oreste

    2015-12-01

    Nitric oxide (NO) has been considered a catabolic factor that contributes to OA pathology by inducing chondrocytes apoptosis, matrix metalloproteinases synthesis, and pro-inflammatory cytokines expression. Thus, the research on NO regulation in chondrocytes represents a relevant field which needs to be explored in depth. However, to date, only the murine ATDC-5 cell line and primary chondrocytes are well-established cells to study NO production in cartilage tissues. The goal of this study is to determine whether two commonly used human chondrocytic cell lines: SW-1353 and T/C-28a2 cell lines are good models to examine lipopolysaccharide and/or pro-inflammatory cytokine-driven NO release and iNOS expression. To this aim, we carefully examined NO production and iNOS protein expression in human T/C-28a2 and SW-1353 chondrocytes stimulated with LPS and interleukin (IL)-1 alone or in combination. We also use ATDC-5 cells as a positive control for NO production. NO accumulation has been determined by colorimetric Griess reaction, whereas NOS type II expression was determined by Western Blot analysis. Our results clearly demonstrated that neither human T/C-28a2 nor SW-1353 chondrocytes showed a detectable increase in NO production or iNOS expression after bacterial endotoxin or cytokines challenge with IL-1. Our study demonstrated that T/C-28a2 and SW-1353 human cell lines are not suitable for studying NO release and iNOS expression confirming that ATDC5 and human primary cultured chondrocytes are the best in vitro cell system to study the actions derived from this mediator. PMID:26016689

  4. Induced pluripotent stem cell lines derived from equine fibroblasts.

    Science.gov (United States)

    Nagy, Kristina; Sung, Hoon-Ki; Zhang, Puzheng; Laflamme, Simon; Vincent, Patrick; Agha-Mohammadi, Siamak; Woltjen, Knut; Monetti, Claudio; Michael, Iacovos Prodromos; Smith, Lawrence Charles; Nagy, Andras

    2011-09-01

    The domesticated horse represents substantial value for the related sports and recreational fields, and holds enormous potential as a model for a range of medical conditions commonly found in humans. Most notable of these are injuries to muscles, tendons, ligaments and joints. Induced pluripotent stem (iPS) cells have sparked tremendous hopes for future regenerative therapies of conditions that today are not possible to cure. Equine iPS (EiPS) cells, in addition to bringing promises to the veterinary field, open up the opportunity to utilize horses for the validation of stem cell based therapies before moving into the human clinical setting. In this study, we report the generation of iPS cells from equine fibroblasts using a piggyBac (PB) transposon-based method to deliver transgenes containing the reprogramming factors Oct4, Sox2, Klf4 and c-Myc, expressed in a temporally regulated fashion. The established iPS cell lines express hallmark pluripotency markers, display a stable karyotype even during long-term culture, and readily form complex teratomas containing all three embryonic germ layer derived tissues upon in vivo grafting into immunocompromised mice. Our EiPS cell lines hold the promise to enable the development of a whole new range of stem cell-based regenerative therapies in veterinary medicine, as well as aid the development of preclinical models for human applications. EiPS cell could also potentially be used to revive recently extinct or currently threatened equine species. PMID:21347602

  5. Simultaneous measurement of NK cell cytotoxicity against two target cell lines labelled with fluorescent lanthanide chelates.

    Science.gov (United States)

    Lövgren, J; Blomberg, K

    1994-07-12

    We describe a cytotoxicity assay which permits the simultaneous measurement of natural killer cell activity against two different cell lines. The target cell lines are labelled either with a fluorescent europium chelate or with a fluorescent terbium chelate and cell death is quantified by measuring the chelate release. K-562, Molt4 and Daudi cell lines have been used as targets. The release of the two chelates from the target cells can be detected with the help of time resolved fluorometry. As the measurements are made after background fluorescence has decayed no additional steps are needed to correct for the background from the medium. The assay procedure used for measurement of cytotoxicity against two target cell lines is very similar to the widely used 51Cr release assay. PMID:8034979

  6. Every Single Cell Clones from Cancer Cell Lines Growing Tumors In Vivo May Not Invalidate the Cancer Stem Cell Concept

    OpenAIRE

    Li, Fengzhi

    2009-01-01

    We present the result of our research on the tumorigenic ability of single cell clones isolated from an aggressive murine breast cancer cell line in a matched allografting mouse model. Tumor formation is basically dependent on the cell numbers injected per location. We argue that in vivo tumor formation from single cell clones, isolated in vitro from cancer cell lines, may not provide conclusive evidence to disprove the cancer stem cell (CSC) theory without additional data.

  7. The Glycoprotein and the Matrix Protein of Rabies Virus Affect Pathogenicity by Regulating Viral Replication and Facilitating Cell-to-Cell Spread▿

    OpenAIRE

    Pulmanausahakul, Rojjanaporn; Li, Jianwei; Schnell, Matthias J.; Dietzschold, Bernhard

    2007-01-01

    While the glycoprotein (G) of rabies virus (RV) is known to play a predominant role in the pathogenesis of rabies, the function of the RV matrix protein (M) in RV pathogenicity is not completely clear. To further investigate the roles of these proteins in viral pathogenicity, we constructed chimeric recombinant viruses by exchanging the G and M genes of the attenuated SN strain with those of the highly pathogenic SB strain. Infection of mice with these chimeric viruses revealed a significant ...

  8. N-alkylated isatins evade P-gp mediated efflux and retain potency in MDR cancer cell lines.

    Science.gov (United States)

    Vine, Kara L; Belfiore, Lisa; Jones, Luke; Locke, Julie M; Wade, Samantha; Minaei, Elahe; Ranson, Marie

    2016-01-01

    The search for novel anticancer therapeutics with the ability to overcome multi-drug resistance (MDR) mechanisms is of high priority. A class of molecules that show potential in overcoming MDR are the N-alkylated isatins. In particular 5,7-dibromo-N-alkylisatins are potent microtubule destabilizing agents that act to depolymerize microtubules, induce apoptosis and inhibit primary tumor growth in vivo. In this study we evaluated the ability of four dibrominated N-alkylisatin derivatives and the parent compound, 5,7-dibromoisatin, to circumvent MDR. All of the isatin-based compounds examined retained potency against the MDR cell lines; U937VbR and MES-SA/Dx5 and displayed bioequivalent dose-dependent cytotoxicity to that of the parental control cell lines. We show that one mechanism by which the isatin-based compounds overcome MDR is by circumventing P-glycoprotein (P-gp) mediated drug efflux. Thus, as the isatin-based compounds are not susceptible to extrusion from P-gp overexpressing tumor cells, they represent a promising alternative strategy as a stand-alone or combination therapy for treating MDR cancer. PMID:27441242

  9. Are there any differences in uptake of Tc-99m MIBI and tetrofosmin between cancer cells expressing P-glycoprotein and MDR-related protein?

    International Nuclear Information System (INIS)

    Uptake of Tc-99m MIBI and Tc-99m tetrofosmin(TF) in two multidrug resistance cells, human colon cancer HCT/CLO2 cells expressing P-glycoprotein(Pgp) and non-small cell lung cancer A549 cells showing multidrug-resistance related protein(MRP) in vivo. RT-PCR Western blot analysis and immunohistochemistry(IHC) were used to detect Pgp and MRP. Cellular uptake of MIBI and TF was measured with single cell suspension of HCR/CLO2 and 549 cells. Cells were inoculated in flanks of 24 nude mice. Group 1 (Gr1) and Gr3 mice were injected with only MIBI or TF, and Gr2 and Gr4 mice were treated with cyclosporin A(CsA) before injection of MIBI or TF. Mice were sacrificed at 10, 60 and 240-min after tracer injection. Radioactivities of organs and tumors were measured. 549 cells expressed MRPr1 protein detected by Western blot and IHC staining of tumor tissue at cell membrane but not Pgp, A549/CLO2 was vice versa. Cellular uptake and % increases with CsA were higher for MIBI than TF for both cells in vitro. Animal studies showed that MIBI and TF uptake were progressively increased by time in both Pgp(+) HCT/CLO2 tumor and MRP(+) A549 cells in both Gr1 and Gr3. There were no significant differences in tumoral uptake of MIBI and TF between Grl and Gr3 for both cells except higher MIBI uptake in Pgp cells at 60-min. % increases of MIBI (111% at 10 min, 220% at 60 min, 426% at 240 min) and TF uptake (186% at 10 min 579% at 60 min, 390% at 240 min) were progressively increased with time up to 240-min with CsA % increases of MIBI (114% at 10 min, 257% at 60 min 396% at 240 min) and TF uptake (110% at 10 min, 205% at 60 min, 410% at 240 min) were similar with those in HCT/CLO2 tumor. There were no differences in MIBI and TF uptake between Pgp and MRP tumors. Although MIBI showed better MDR reversal effect of modulator CsA than TF in vitro, there were no differences for both tumors in vivo. MIBI and TF are suitable tracers for imaging Pgp and MRP-mediated drug resistance in cancers

  10. Are there any differences in uptake of Tc-99m MIBI and tetrofosmin between cancer cells expressing P-glycoprotein and MDR-related protein?

    Energy Technology Data Exchange (ETDEWEB)

    Seo, J. H.; Yoo, J. A.; Seo, M. R.; Bae, J. H.; Jeong, S. Y.; Ahn, B. C.; Lee, K. B.; Lee, J [Kyungpook National University Hospital, Daegu (Korea, Republic of)

    2004-07-01

    Uptake of Tc-99m MIBI and Tc-99m tetrofosmin(TF) in two multidrug resistance cells, human colon cancer HCT/CLO2 cells expressing P-glycoprotein(Pgp) and non-small cell lung cancer A549 cells showing multidrug-resistance related protein(MRP) in vivo. RT-PCR Western blot analysis and immunohistochemistry(IHC) were used to detect Pgp and MRP. Cellular uptake of MIBI and TF was measured with single cell suspension of HCR/CLO2 and 549 cells. Cells were inoculated in flanks of 24 nude mice. Group 1 (Gr1) and Gr3 mice were injected with only MIBI or TF, and Gr2 and Gr4 mice were treated with cyclosporin A(CsA) before injection of MIBI or TF. Mice were sacrificed at 10, 60 and 240-min after tracer injection. Radioactivities of organs and tumors were measured. 549 cells expressed MRPr1 protein detected by Western blot and IHC staining of tumor tissue at cell membrane but not Pgp, A549/CLO2 was vice versa. Cellular uptake and % increases with CsA were higher for MIBI than TF for both cells in vitro. Animal studies showed that MIBI and TF uptake were progressively increased by time in both Pgp(+) HCT/CLO2 tumor and MRP(+) A549 cells in both Gr1 and Gr3. There were no significant differences in tumoral uptake of MIBI and TF between Grl and Gr3 for both cells except higher MIBI uptake in Pgp cells at 60-min. % increases of MIBI (111% at 10 min, 220% at 60 min, 426% at 240 min) and TF uptake (186% at 10 min 579% at 60 min, 390% at 240 min) were progressively increased with time up to 240-min with CsA % increases of MIBI (114% at 10 min, 257% at 60 min 396% at 240 min) and TF uptake (110% at 10 min, 205% at 60 min, 410% at 240 min) were similar with those in HCT/CLO2 tumor. There were no differences in MIBI and TF uptake between Pgp and MRP tumors. Although MIBI showed better MDR reversal effect of modulator CsA than TF in vitro, there were no differences for both tumors in vivo. MIBI and TF are suitable tracers for imaging Pgp and MRP-mediated drug resistance in cancers.

  11. The effect of vitamin A supplementation on stimulated T-cell proliferation with myelin oligodendrocyte glycoprotein in patients with multiple sclerosis

    Directory of Open Access Journals (Sweden)

    Sima Jafarirad

    2012-01-01

    Full Text Available Background: Multiple sclerosis (MS is an autoimmune disease whereby myelin sheath of the central nervous system is destroyed. Vitamin A is known to play a role in the immune system. It has been recognized that some metabolites of vitamin A can be used effectively to treat experimental autoimmune encephalomyelitis (EAE. Aims: The effect of vitamin A as retinyl palmitate on T-cell proliferation in MS patients. Setting and Design: This study is a double blind clinical trial of two test groups over a period of 6 months. Materials and Methods: Thirty five multiple sclerosis (MS patients were divided into two groups. One group received 25,000 IU/day vitamin A (as retinyl palmitate and the other group were administered a placebo. The peripheral blood mononuclear cells (PBMCs were separated and stimulated with myelin oligodendrocyte glycoprotein (MOG and phytohemagglutinin (PHA before and after the trial period. BrdU calorimetric assay was performed to measure cell proliferation. Statistical Analysis: Analysis of covariance (ANCOVA and paired t-test were used to analyze the data. Results: Observations showed statistical significant differences in the reduction of cell proliferation in the presence of MOG and fetal calf serum (FCS in the culture medium, between patients receiving vitamin A and the placebo (P = 0.046. Although, this difference was not significant between the two vitamin A and placebo groups in MOG treatment with human serum, a decrease was observed in the group of patients taking vitamin A supplements (P = 0.019. Phytohemagglutinin did not cause any change in cell proliferation between the two groups. Conclusion: The results suggest supplementation with retinyl palmitate in patients with MS reduce MOG stimulatory effects on T-cells.

  12. Continuous production of erythropoietin by an established human renal carcinoma cell line: development of the cell line.

    OpenAIRE

    Sherwood, J B; Shouval, D

    1986-01-01

    Establishment of a stable, transformed human renal carcinoma cell line that produces erythropoietin in vitro and has maintained this function continuously since 1981 and for greater than 150 passages in monolayer culture was accomplished by transplantation of human renal clear cell carcinoma tissue from a patient with erythrocytosis into an immunosuppressed athymic mouse. In addition to its immunocrossreactivity with native human urinary erythropoietin, the tumor erythropoietin demonstrates b...

  13. Expression of particulate-form of Japanese encephalitis virus envelope protein in a stably transfected Drosophila cell line

    Directory of Open Access Journals (Sweden)

    Zhang Li

    2007-02-01

    Full Text Available Abstract Background Japanese encephalitis virus (JEV, a member of the family Flaviviridae, is an important mosquito-borne human pathogen. Its envelope glycoprotein (E is the major determinant of the pathogenicity and host immune responses. In the present study, we explored the feasibility of producing recombinant JEV E protein in the virus-free Drosophila expression system. Results The coding sequence for the signal sequence of premembrane and E protein was cloned into the Drosophila expression vector pAc5.1/V5-His. A Drosophila cell line S2 was cotransfected with this construct as well as a plasmid providing hygromycin B resistance. A cell line expressing the JEV E protein was selected by immunofluoresence, confocal microscopy, and western blot analysis using three different monoclonal antibodies directed against JEV E protein. This cell line was stable in the yield of JEV E protein during two months in vitro maintenance in the presence of hygromycin B. The results showed that the recombinant E protein had an expected molecular weight of about 50 kilodalton, was immunoreactive with all three monoclonal antibodies, and found in both the cytoplasm and culture supernatant. Sucrose gradient ultracentrifugation analysis revealed that the secreted E protein product was in a particulate form. It migrated to the sucrose fraction with a density of 1.13 g/ml. Balb/c mice immunised with the sucrose fraction containing the E protein particles developed specific antibodies. These data show that functioning JEV E protein was expressed in the stable S2 cell line. Conclusion The Drosophila expression system is a more convenient, cheaper and safer approach to the production of vaccine candidates and diagnostic reagents for JEV.

  14. Evaluation Of Lenalidomide Activity On Glioblastoma Cell Lines In Vitro

    OpenAIRE

    Mut, Melike; Gregory POLAR; Carpenter, Joan E.; Gerard REDPATH; Larner, James; Schiff, David; Shaffrey, Mark E.

    2007-01-01

    Purpose: Thalidomide analog, Lenalidomide (Revlimid®) is a chemotherapeutic agent. In this study, lenalidomide was used in human glioblastoma multiforme (GBM) cell lines to determine its pro-apoptotic, anti-proliferative and radiosensitizing properties. Methods: The GBM cells were treated with lenalidomide [0, 1, 5, 30, 60 µM] before ultravioletB (UVB) [0, 50, 100 mj] or γ -irradiation [0, 5, 20 Gy], and kept in drug for 5 days. Viable cell numbers were determined by trypan blue exclusion. Th...

  15. Cytotoxic studies of paclitaxel (Taxol) in human tumour cell lines.

    OpenAIRE

    Liebmann, J. E.; Cook, J. A.; Lipschultz, C.; Teague, D.; Fisher, J; Mitchell, J B

    1993-01-01

    The cytotoxicity of paclitaxel against eight human tumour cell lines has been studied with in vitro clonogenic assays. The fraction of surviving cells fell sharply after exposure for 24 h to paclitaxel concentrations ranging from 2 to 20 nM; the paclitaxel IC50 was found to range between 2.5 and 7.5 nM. Increasing the paclitaxel concentration above 50 nM, however, resulted in no additional cytotoxicity after a 24 h drug exposure. Cells incubated in very high concentrations of paclitaxel (10,0...

  16. Growth inhibition by tyrosine kinase inhibitors in mesothelioma cell lines.

    Science.gov (United States)

    Nutt, Joyce E; O'Toole, Kieran; Gonzalez, David; Lunec, John

    2009-06-01

    Clinical outcome following chemotherapy for malignant pleural mesothelioma is poor and improvements are needed. This preclinical study investigates the effect of five tyrosine kinase inhibitors (PTK787, ZD6474, ZD1839, SU6668 and SU11248) on the growth of three mesothelioma cell lines (NCI H226, NCI H28 and MSTO 211H), the presence of growth factor receptors and inhibition of their downstream signalling pathways. GI50 values were determined: ZD6474 and SU11248, mainly VEGFR2 inhibitors, gave the lowest GI50 across all cell lines (3.5-6.9 microM) whereas ZD1839 gave a GI50 in this range only in H28 cells. All cell lines were positive for EGFR, but only H226 cells were positive for VEGFR2 by Western blotting. ZD6474 and ZD1839 inhibited EGF-induced phosphorylation of EGFR, AKT and ERK, whereas VEGF-induced phosphorylation of VEGFR2 was completely inhibited with 0.1 microM SU11248. VEGFR2 was detected in tumour samples by immunohistochemistry. VEGFR2 tyrosine kinase inhibitors warrant further investigation in mesothelioma. PMID:19318229

  17. Plasmids and packaging cell lines for use in phage display

    Science.gov (United States)

    Bradbury, Andrew M.

    2012-07-24

    The invention relates to a novel phagemid display system for packaging phagemid DNA into phagemid particles which completely avoids the use of helper phage. The system of the invention incorporates the use of bacterial packaging cell lines which have been transformed with helper plasmids containing all required phage proteins but not the packaging signals. The absence of packaging signals in these helper plasmids prevents their DNA from being packaged in the bacterial cell, which provides a number of significant advantages over the use of both standard and modified helper phage. Packaged phagemids expressing a protein or peptide of interest, in fusion with a phage coat protein such as g3p, are generated simply by transfecting phagemid into the packaging cell line.

  18. MAC-1 Glycoprotein Family mediates adherence of neutrophils to endothelial cells stimulated by leukotriene B4 and platelet activating factor

    International Nuclear Information System (INIS)

    The process of neutrophil (N) adhesion to and migration through endothelium (EC), an early event in the induction of the acute inflammatory response, has been attributed to the generation of extravascular chemoattractant peptides and lipids. Although both leukotriene B4 (LTB4) and platelet activating factor (PAF) enhance N adherence to EC, the mechanisms involved in this interaction are still not completely understood. Since the MAC-1 Glycoprotein (GP) Family has recently been shown to be required for a variety of adherence-dependent functions of stimulated N, the authors questioned whether these adherence-associated GP might be involved in N adherence to EC stimulated by LTB4 or PAF. Using a microtiter adherence assay with 111In labeled N, they assessed the ability of N from patients with MAC-1, LFA-1 Deficiency to adhere to monolayers of human omental microvascular or umbilical vein EC as well as to serum-coated plastic. Patient N exhibited markedly diminished adherence in response to LTB4 or PAF compared to normal controls. LTB4 and PAF enhanced expression of the MAC-1 GP Family on the surface of normal N as determined by flow cytofluorimetry using a monoclonal antibody (TS1/18) to the GP common beta subunit. In addition TS1/18 (20 μg/ml) completely inhibited N adherence stimulated by either LTB4 (10-8M) or PAF(10-11M). Thus, the MAC-1 GP Family appears to be important in chemotactic factor regulation of N adherence to EC

  19. Effects of irradiation on cytokine production in glioma cell lines

    International Nuclear Information System (INIS)

    The effects of irradiation on cytokine production in glioma cell lines, NP1, NP2 and NP3, were studied. Culture supernatants were collected after 6, 24, 48 or 72 hours and the concentrations of interleukin (IL)-6 and IL-8 measured by enzyme-linked immunosorbent assay. Spontaneous and IL-1β-stimulated productions were analyzed. Some cells were given a single dose of Lineac irradiation (10 or 20 Gy). Production of IL-6 (with or without IL-1β stimulation) increased gradually to a maximum after 72 hours, more in the 20 Gy-irradiated cells than 10 Gy cells (p<0.01). Production of IL-8 increased gradually to a maximum after 48 or 72 hours. Spontaneous production of IL-8 increased more in 20 Gy-irradiated cells than 10 Gy cells after 6 and 24 hours (p<0.01), but increased more in 10 Gy cells than 20 Gy cells after 48 and 72 hours (p<0.01). The production of IL-8 stimulated by IL-1β increased more in 10 Gy cells than 20 Gy cells 24 hours later (p<0.01). IL-6 and IL-8 production differed in the response to irradiation. Our data suggest that bidirectional communication between the immune system and glioma cells changes after radiotherapy. (author)

  20. Heterogeneity of a human T-lymphoblastoid cell line

    Energy Technology Data Exchange (ETDEWEB)

    Snow, K.; Judd, W.

    1987-08-01

    A human T-lymphoblastoid cell line (Jurkat) was cloned, and four resulting sublines were characterized in a variety of ways with the objective of gaining information on heterogeneity in cell lines. Within a few weeks of cloning, distinct cellular morphologies and growth patterns became apparent in the four sublines. Growth rate measurements made over 3 months did not show any significant differences between the sublines. Surface protein profiles obtained by radioimmunoprecipitation using antisera in conjunction with extracts from (/sup 35/S)Met and /sup 125/I-labeled cells revealed differences between the sublines. Analysis of total cell DNA showed that one of the sublines possessed only half the chromosome complement of the other sublines and the parental line. Karyotyping confirmed this result and, in addition, demonstrated that chromosome numbers fluctuated around a mean value for each subline. Karyotypic variability became apparent within 2 months of cloning and tended to increase with time in culture. G-banding analysis showed that the analyzed cell populations contained distinctive cytogenetic aberrations. Properties of the cloned sublines were monitored over a 9-month period. One of the sublines that had shown heterogeneous morphology even after 6 weeks maintained the heterogeneity throughout this time. Another subline underwent a marked change in morphology (round to irregular) and growth habit (single cells to large clumps) with increasing time in culture. Interestingly, several alterations to surface proteins accompanied these growth changes. A third subline had relatively stable morphology and chromosome number throughout the 9-month period. The modal chromosome number was hypotetraploid for three sublines and the parent line, but was diploid for another subline.

  1. Survey of Differentially Methylated Promoters in Prostate Cancer Cell Lines

    Directory of Open Access Journals (Sweden)

    Yipeng Wang

    2005-08-01

    Full Text Available DNA methylation, copy number in the genomes of three immortalized prostate epithelial, five cancer cell lines (LNCaP, PC3, PC3M, PC3M-Pro4, PC3MLN4 were compared using a microarray-based technique. Genomic DNA is cut with a methylation-sensitive enzyme Hpall, followed by linker ligation, polymerase chain reaction (PCR amplification, labeling, hybridization to an array of promoter sequences. Only those parts of the genomic DNA that have unmethylated restriction sites within a few hundred base pairs generate PCR products detectable on an array. Of 2732 promoter sequences on a test array, 504 (18.5% showed differential hybridization between immortalized prostate epithelial, cancer cell lines. Among candidate hypermethylated genes in cancer-derived lines, there were eight (CD44, CDKN1A, ESR1, PLAU, RARB, SFN, TNFRSF6, TSPY previously observed in prostate cancer, 13 previously known methylation targets in other cancers (ARHI, bcl-2, BRCA1, CDKN2C, GADD45A, MTAP, PGR, SLC26A4, SPARC, SYK, TJP2, UCHL1, WIT-1. The majority of genes that appear to be both differentially methylated, differentially regulated between prostate epithelial, cancer cell lines are novel methylation targets, including PAK6, RAD50, TLX3, PIR51, MAP2K5, INSR, FBN1, GG2-1, representing a rich new source of candidate genes used to study the role of DNA methylation in prostate tumors.

  2. The genomic landscape of epithelioid sarcoma cell lines and tumours.

    Science.gov (United States)

    Jamshidi, Farzad; Bashashati, Ali; Shumansky, Karey; Dickson, Brendan; Gokgoz, Nalan; Wunder, Jay S; Andrulis, Irene L; Lazar, Alexander J; Shah, Sohrab P; Huntsman, David G; Nielsen, Torsten O

    2016-01-01

    We carried out whole genome and transcriptome sequencing on four tumour/normal pairs of epithelioid sarcoma. These index cases were supplemented with whole transcriptome sequencing of three additional tumours and three cell lines. Unlike rhabdoid tumour (the other major group of SMARCB1-negative cancers), epithelioid sarcoma shows a complex genome with a higher mutational rate, comparable to that of ovarian carcinoma. Despite this mutational burden, SMARCB1 mutations remain the most frequently recurring event and are probably critical drivers of tumour formation. Several cases show heterozygous SMARCB1 mutations without inactivation of the second allele, and we explore this further in vitro. Finding CDKN2A deletions in our discovery cohort, we evaluated CDKN2A protein expression in a tissue microarray. Six out of 16 cases had lost CDKN2A in greater than or equal to 90% of cells, while the remaining cases had retained the protein. Expression analysis of epithelioid sarcoma cell lines by transcriptome sequencing shows a unique profile that does not cluster with any particular tissue type or with other SWI/SNF-aberrant lines. Evaluation of the levels of members of the SWI/SNF complex other than SMARCB1 revealed that these proteins are expressed as part of a residual complex, similarly to previously studied rhabdoid tumour lines. This residual SWI/SNF is susceptible to synthetic lethality and may therefore indicate a therapeutic opportunity. PMID:26365879

  3. Melatonin and Doxorubicin synergistically induce cell apoptosis in human hepatoma cell lines

    Institute of Scientific and Technical Information of China (English)

    2010-01-01

    AIM:To investigate whether Melatonin has synergistic effects with Doxorubicin in the growth-inhibition and apoptosis-induction of human hepatoma cell lines HepG2 and Bel-7402.METHODS:The synergism of Melatonin and Doxorubicin inhibited the cell growth and induced cell apoptosis in human hepatoma cell lines HepG2 and Bel-7402.Cell viability was analyzed by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide(MTT)assay.Cell apoptosis was evaluated using TUNEL method and flow cytometry.Apoptosis-r...

  4. Synthesis and characterization of rabies virus glycoprotein-tagged amphiphilic cyclodextrins for siRNA delivery in human glioblastoma cells: in vitro analysis.

    Science.gov (United States)

    Gooding, Matt; Malhotra, Meenakshi; McCarthy, David J; Godinho, Bruno M D C; Cryan, John F; Darcy, Raphael; O'Driscoll, Caitriona M

    2015-04-25

    In man brain cancer is an aggressive, malignant form of tumour, it is highly infiltrative in nature, is associated with cellular heterogeneity and affects cerebral hemispheres of the brain. Current drug therapies are inadequate and an unmet clinical need exists to develop new improved therapeutics. The ability to silence genes associated with disease progression by using short interfering RNA (siRNA) presents the potential to develop safe and effective therapies. In this work, in order to protect the siRNA from degradation, promote cell specific uptake and enhance gene silencing efficiency, a PEGylated cyclodextrin (CD)-based nanoparticle, tagged with a CNS-targeting peptide derived from the rabies virus glycoprotein (RVG) was formulated and characterized. The modified cyclodextrin derivatives were synthesized and co-formulated to form nanoparticles containing siRNA which were analysed for size, surface charge, stability, cellular uptake and gene-knockdown in brain cancer cells. The results identified an optimised co-formulation prototype at a molar ratio of 1:1.5:0.5 (cationic cyclodextrin:PEGylated cyclodextrin:RVG-tagged PEGylated cyclodextrin) with a size of 281 ± 39.72 nm, a surface charge of 26.73 ± 3 mV, with efficient cellular uptake and a 27% gene-knockdown ability. This CD-based formulation represents a potential nanocomplex for systemic delivery of siRNA targeting brain cancer. PMID:25703259

  5. Absence of cytotoxic antibody to human immunodeficiency virus-infected cells in humans and its induction in animals after infection or immunization with purified envelope glycoprotein gp120

    Energy Technology Data Exchange (ETDEWEB)

    Nara, P.L.; Robey, W.G.; Gonda, M.A.; Carter, S.G.; Fischinger, P.J.

    1987-06-01

    The presence of antibody-dependent complement-mediated cytotoxicity (ACC) was assessed in humans and chimpanzees, which are capable of infection with human immunodeficiency virus isolate HTLV-IIIb, and examined in the goat after immunization with the major viral glycoprotein (gp120) of HTLV-IIIb. In infected humans no antibody mediating ACC was observed regardless of the status of disease. Even healthy individuals with high-titer, broadly reactive, neutralizing antibodies has no ACC. In contrast, chimpanzees infected with HTLV-IIIb, from whom virus could be isolated, not only had neutralizing antibody but also antibodies broadly reactive in ACC, even against distantly related human immunodeficiency virus isolates, as well as against their own reisolated virus. In the goat, the gp120 of HTLV-IIIb induced a highly type-specific response as measured by both ACC and flow cytofluorometry of live infected H9 cells. Normal human cells were not subject to ACC by animal anti-HTLV-III gp120-specific sera. Induction of ACC and neutralizing antibody were closely correlated in the animal experimental models but not in humans. The presence of ACC in gp120-inoculated goats and HTLV-III-infected chimpanzees represent a qualitative difference that may be important in the quest for the elicitation of a protective immunity in humans.

  6. Absence of cytotoxic antibody to human immunodeficiency virus-infected cells in humans and its induction in animals after infection or immunization with purified envelope glycoprotein gp120

    International Nuclear Information System (INIS)

    The presence of antibody-dependent complement-mediated cytotoxicity (ACC) was assessed in humans and chimpanzees, which are capable of infection with human immunodeficiency virus isolate HTLV-IIIb, and examined in the goat after immunization with the major viral glycoprotein (gp120) of HTLV-IIIb. In infected humans no antibody mediating ACC was observed regardless of the status of disease. Even healthy individuals with high-titer, broadly reactive, neutralizing antibodies has no ACC. In contrast, chimpanzees infected with HTLV-IIIb, from whom virus could be isolated, not only had neutralizing antibody but also antibodies broadly reactive in ACC, even against distantly related human immunodeficiency virus isolates, as well as against their own reisolated virus. In the goat, the gp120 of HTLV-IIIb induced a highly type-specific response as measured by both ACC and flow cytofluorometry of live infected H9 cells. Normal human cells were not subject to ACC by animal anti-HTLV-III gp120-specific sera. Induction of ACC and neutralizing antibody were closely correlated in the animal experimental models but not in humans. The presence of ACC in gp120-inoculated goats and HTLV-III-infected chimpanzees represent a qualitative difference that may be important in the quest for the elicitation of a protective immunity in humans

  7. Feeder-independent continuous culture of the PICM-19 pig liver stem cell line

    Science.gov (United States)

    The PICM-19 pig liver stem cell line is a bipotent cell line, i.e., capable of forming either bile ductules or hepatocyte monolayers in vitro, that was derived from the primary culture of pig embryonic stem cells. The cell line has been strictly feeder-dependent in that cell replication morphology,...

  8. Absence of C-type virus production in human leukemic B cell, T cell and null cell lines.

    Directory of Open Access Journals (Sweden)

    Ogura,Hajime

    1978-06-01

    Full Text Available Electron microscope observation of cultured human leukemic B cell, T cell and null cell lines and reverse transcriptase assay of the culture supernatants were all negative for the presence of C-type virus. Bat cell line, which propagates primate C-type viruses well, was cocultivated with the human leukemic cell lines, in the hope of amplification of virus if present. Three weeks after mixed culture, the culture supernatants were again examined for reverse transcriptase activity and the cells were tested for syncytia formation by cocultivation with rat XC, human KC and RSb cell lines. All these tests, except for the positive control using a simian sarcoma virus, were negative, suggesting that no C-type was produced from these human leukemic cell lines.

  9. Cytolytic replication of echoviruses in colon cancer cell lines

    Directory of Open Access Journals (Sweden)

    Gullberg Maria

    2011-10-01

    Full Text Available Abstract Background Colorectal cancer is one of the most common cancers in the world, killing nearly 50% of patients afflicted. Though progress is being made within surgery and other complementary treatments, there is still need for new and more effective treatments. Oncolytic virotherapy, meaning that a cancer is cured by viral infection, is a promising field for finding new and improved treatments. We have investigated the oncolytic potential of several low-pathogenic echoviruses with rare clinical occurrence. Echoviruses are members of the enterovirus genus within the family Picornaviridae. Methods Six colon cancer cell lines (CaCo-2, HT29, LoVo, SW480, SW620 and T84 were infected by the human enterovirus B species echovirus 12, 15, 17, 26 and 29, and cytopathic effects as well as viral replication efficacy were investigated. Infectivity was also tested in spheroids grown from HT29 cells. Results Echovirus 12, 17, 26 and 29 replicated efficiently in almost all cell lines and were considered highly cytolytic. The infectivity of these four viruses was further evaluated in artificial tumors (spheroids, where it was found that echovirus 12, 17 and 26 easily infected the spheroids. Conclusions We have found that echovirus 12, 17 and 26 have potential as oncolytic agents against colon cancer, by comparing the cytolytic capacity of five low-pathogenic echoviruses in six colon cancer cell lines and in artificial tumors.

  10. Derivation of human embryonic stem cell lines from parthenogenetic blastocysts

    Institute of Scientific and Technical Information of China (English)

    Qingyun Mai; Yang Yu; Tao Li; Liu Wang; Mei-jue Chen; Shu-zhen Huang; Canquan Zhou; Qi Zhou

    2007-01-01

    Parthenogenesis is one of the main, and most useful, methods to derive embryonic stem cells (ESCs), which may be an important source of histocompatible cells and tissues for cell therapy. Here we describe the derivation and characterization of two ESC lines (hPES-1 and hPES-2) from in vitro developed blastocysts following parthenogenetic activation of human oocytes. Typical ESC morphology was seen, and the expression of ESC markers was as expected for alkaline phosphatase, octamer-binding transcription factor 4, stage-specific embryonic antigen 3, stage-specific embryonic antigen 4, TRA-1-60, and TRA-1-81, and there was absence of expression of negative markers such as stage-specific embryonic antigen 1. Expression of genes specific for different embryonic germ layers was detected from the embryoid bodies (EBs) of both hESC lines, suggesting their differentiation potential in vitro. However, in vivo, only hPES-1 formed teratoma consisting of all three embryonic germ layers (hPES-2 did not). Interestingly, after continuous proliferation for more than 100 passages, hPES-1 cells still maintained a normal 46 XX karyotype; hPES-2 displayed abnormalities such as chromosome translocation after long term passages. Short Tandem Repeat (STR) results demonstrated that the hPES lines were genetic matches with the egg donors, and gene imprinting data confirmed the parthenogenetic origin of these ES cells. Genome-wide SNP analysis showed a pattern typical of parthenogenesis. All of these results demonstrated the feasibility to isolate and establish human parthenogenetic ESC lines, which provides an important tool for studying epigenetic effects in ESCs as well as for future therapeutic interventions in a clinical setting.

  11. Apoptosis in Raji cell line induced by influenza A virus

    Institute of Scientific and Technical Information of China (English)

    李虹; 肖丽英; 李华林; 李婉宜; 蒋中华; 张林; 李明远

    2003-01-01

    Objective To study the apoptotic effects of influenza A virus on the Raji cell line. Methods Cultured Raji cells were infected with influenza A virus at a multiplicity of infection (m.o.i) of 20 and the effects of apoptosis were detected at different time points post infection using the following methods: electron microscope, DNA agarose gel electrophoresis, PI stained flow cytometry (FCM) and Annexin-V FITC/PI stained FCM.Results Raji cells infected with influenza A virus showed changes of morphology apoptotis, DNA agarose electrophoresis also demonstrated a ladder-like pattern of DNA fragments in a time-dependent manner. PI stained FCM showed "apoptosis peak" and FITC/PI stained FCM showed apoptotic cells. Quantitative analysis indicated that the percentage of apoptotic Raji cells increased after infection, and cycloheximide (CHX), an eukaryotic transcription inhibitor, could effectively inhibit the apoptotic effects of influenza A virus in vitro.Conclusions Influenza A virus can induce apoptosis in Raji cell line suggesting that it may lead to a potential method for tumor therapy.

  12. Regulation of glycoprotein synthesis in yeast by mating pheromones

    International Nuclear Information System (INIS)

    In Saccharomyces cerevisiae, glycosylated proteins amount to less than 2% of the cell protein. Two intensively studied examples of yeast glycoproteins are the external cell wall - associated invertase and the vacuolar carboxypeptidase Y. Recently, it was shown that the mating pheromone, alpha factor, specifically and strongly inhibits the synthesis of N-glycosylated proteins in haploid a cells, whereas O-glycosylated proteins are not affected. In this paper, the pathways of glycoprotein biosynthesis are summarized briefly, and evidence is presented that mating pheomones have a regulatory function in glycoprotein synthesis

  13. Temozolomide Resistance in Glioblastoma Cell Lines: Implication of MGMT, MMR, P-Glycoprotein and CD133 Expression

    OpenAIRE

    Gloria Perazzoli; Jose Prados; Raul Ortiz; Octavio Caba; Laura Cabeza; Maria Berdasco; Beatriz Gónzalez; Consolación Melguizo

    2015-01-01

    Background The use of temozolomide (TMZ) has improved the prognosis for glioblastoma multiforme patients. However, TMZ resistance may be one of the main reasons why treatment fails. Although this resistance has frequently been linked to the expression of O6-methylguanine-DNA methyltransferase (MGMT) it seems that this enzyme is not the only molecular mechanism that may account for the appearance of drug resistance in glioblastoma multiforme patients as the mismatch repair (MMR) complex, P-gly...

  14. Inhibition of N-linked glycosylation of P-glycoprotein by tunicamycin results in a reduced multidrug resistance phenotype.

    OpenAIRE

    Kramer, R.; Weber, T K; Arceci, R.; Ramchurren, N.; Kastrinakis, W. V.; Steele, G.; Summerhayes, I. C.

    1995-01-01

    Characterisation of altered glycosylation of P-glycoprotein (P-gp) found associated with the absence of a multidrug resistance (MDR) phenotype in cell lines prompted an investigation to assess the role of post-translational processing in establishing P-gp efflux pump functionally. The clone A cell line used in this study displays a strong MDR phenotype mediated by high constitutive levels of expression of P-gp. Incubation of clone A cells with tunicamycin for different periods resulted in a t...

  15. Establishment and characterization of primary lung cancer cell lines from Chinese population

    Institute of Scientific and Technical Information of China (English)

    Chao ZHENG; Yi-hua SUN; Xiao-lei YE; Hai-quan CHEN; Hong-bin JI

    2011-01-01

    Aim: To establish and characterize primary lung cancer cell lines from Chinese population.Methods: Lung cancer specimens or pleural effusions were collected from Chinese lung cancer patients and cultured in vitro with ACL4 medium (for non-small cell lung carcinomas (NSCLC)) or HITES medium (for small cell lung carcinomas (SCLC)) supplemented with 5%FBS. All cell lines were maintained in culture for more than 25 passages. Most of these cell lines were further analyzed for oncogenic mutations, karyotype, cell growth kinetics, and tumorigenicity in nude mice.Results: Eight primary cell lines from Chinese lung cancer patients were established and characterized, including seven NSCLC cell lines and one SCLC cell line. Five NSCLC cell lines were found to harbor epidermal growth factor receptor (EGFR) kinase domain mutations.Conclusion: These well-characterized primary lung cancer cell lines from Chinese population provide a unique platform for future studies of the ethnic differences in lung cancer biology and drug response.

  16. Designing of promiscuous inhibitors against pancreatic cancer cell lines

    Science.gov (United States)

    Kumar, Rahul; Chaudhary, Kumardeep; Singla, Deepak; Gautam, Ankur; Raghava, Gajendra P. S.

    2014-04-01

    Pancreatic cancer remains the most devastating disease with worst prognosis. There is a pressing need to accelerate the drug discovery process to identify new effective drug candidates against pancreatic cancer. We have developed QSAR models for predicting promiscuous inhibitors using the pharmacological data. Our models achieved maximum Pearson correlation coefficient of 0.86, when evaluated on 10-fold cross-validation. Our models have also successfully validated the drug-to-oncogene relationship and further we used these models to screen FDA approved drugs and tested them in vitro. We have integrated these models in a webserver named as DiPCell, which will be useful for screening and designing novel promiscuous drug molecules. We have also identified the most and least effective drugs for pancreatic cancer cell lines. On the other side, we have identified resistant pancreatic cancer cell lines, which need investigative scanner on them to put light on resistant mechanism in pancreatic cancer.

  17. RBE of neutrons for induction of cell reproductive death and chromosome aberrations in three cell lines

    International Nuclear Information System (INIS)

    The authors have compared the RBE values for induction of dicentrics and centric rings with those for cell inactivation and with the mean or effective quality factors (Q) recommended for radiation protection. The induction of cell reproductive death and chromosome aberrations has been investigated in plateau phase cultures of established lines of a rat rhabdomyosarcoma, a rat ureter carcinoma and Chinese hamster cells for single doses of 300 kV X-rays and 0.5, 4.2 and 15 MeV neutrons. The different cell lines show considerable variations in sensitivity and the RBE values obtained are presented in tabular form. The mean RBE values for the rat rhabdomyosarcoma cells are lower than those for the other two relatively resistant cell lines. Those for the Chinese hamster cells extrapolated to levels according to low doses of X-rays are in good agreement with the quoted Q values. (Auth./C.F.)

  18. Cell line profiling to improve monoclonal antibody production.

    Science.gov (United States)

    Kang, Sohye; Ren, Da; Xiao, Gang; Daris, Kristi; Buck, Lynette; Enyenihi, Atim A; Zubarev, Roman; Bondarenko, Pavel V; Deshpande, Rohini

    2014-04-01

    Mammalian cell culture performance is influenced by both intrinsic (genetic) and extrinsic (media and process) factors. In this study, intrinsic capacity of various monoclonal antibody-producing Chinese Hamster Ovary (CHO) cell lines was compared by exposing them to the same culture condition. Microarray-based transcriptomics and LC-MS/MS shotgun proteomics technologies were utilized to obtain expression landscape of different cell lines. Specific transcripts and proteins correlating with productivity, growth rate and cell size have been identified. The proteomics analysis results showed a strong correlation between the intracellular protein expression levels of the recombinant DHFR and productivity. In contrast, neither the light chain nor the heavy chain of the recombinant monoclonal antibody showed correlation to productivity. Other top ranked proteins which demonstrated positive correlation to productivity included the adaptor protein complex subunits AP3D1and AP2B2, DNA repair protein DDB1 and the ER translocation complex component, SRPR. The subunits of molecular chaperone T-complex protein 1 and the regulator of mitochondrial one-carbon metabolism MTHFD2 showed negative correlation to productivity. The transcriptomics analysis has identified the regulators of calcium signaling, Tmem20 and Rcan1, as the top ranked genes displaying positive and negative correlation to productivity, respectively. For the second part of the study, the principal component analysis (PCA) was generated to view the underlying global structure of the expression data. A clear division and expression polarity was observed between the two distinct clusters of cell lines, independent of link to productivity or any other traits examined. The primary component of the PCA generated from either transcriptomics or proteomics data displayed a strong correlation to cell size and doubling time, while none of the main principal components showed correlation to productivity. Our findings suggest

  19. Glycoprotein 130 receptor signaling mediates α-cell dysfunction in a rodent model of type 2 diabetes

    DEFF Research Database (Denmark)

    Chow, Samuel Z; Speck, Madeleine; Yoganathan, Piriya;

    2014-01-01

    knockout (αgp130KO) mice showed no differences in glycemic control, α-cell function, or α-cell mass. However, when subjected to streptozotocin plus high-fat diet to induce islet inflammation and pathophysiology modeling type 2 diabetes, αgp130KO mice had reduced fasting glycemia, improved glucose tolerance......, reduced fasting insulin, and improved α-cell function. Hyperinsulinemic-euglycemic clamps revealed no differences in insulin sensitivity. We conclude that in a setting of islet inflammation and pathophysiology modeling type 2 diabetes, activation of α-cell gp130 receptor signaling has deleterious effects...... on α-cell function, promoting hyperglycemia. Antagonism of α-cell gp130 receptor signaling may be useful for the treatment of type 2 diabetes....

  20. UV light blocks EGFR signalling in human cancer cell lines

    DEFF Research Database (Denmark)

    Olsen, BB; Neves-Petersen, M T; Klitgaard, S;

    2007-01-01

    antibodies. There was a threshold level, below which the receptor could not be blocked. In addition, illumination caused the cells to upregulate the cyclin-dependent kinase inhibitor p21WAF1, irrespective of the p53 status. Since the EGF receptor is often overexpressed in cancers and other proliferative skin......UV light excites aromatic residues, causing these to disrupt nearby disulphide bridges. The EGF receptor is rich in aromatic residues near the disulphide bridges. Herein we show that laser-pulsed UV illumination of two different skin-derived cancer cell lines i.e. Cal-39 and A431, which both...

  1. In vitro chemosensitivity of head and neck cancer cell lines

    Directory of Open Access Journals (Sweden)

    Schuler PJ

    2010-08-01

    Full Text Available Abstract Background Systemic treatment of head and neck squamous cell carcinoma (HNSCC includes a variety of antineoplastic drugs. However, drug-resistance interferes with the effectiveness of chemotherapy. Preclinical testing models are needed in order to develop approaches to overcome chemoresistance. Methods Ten human cell lines were obtained from HNSCC, including one with experimentally-induced cisplatin resistance. Inhibition of cell growth by seven chemotherapeutic agents (cisplatin, carboplatin, 5- fluorouracil, methotrexate, bleomycin, vincristin, and paclitaxel was measured using metabolic MTT-uptake assay and correlated to clinically-achievable plasma concentrations. Results All drugs inhibited cell growth in a concentration-dependent manner with an IC50 comparable to that achievable in vivo. However, response curves for methotrexate were unsatisfactory and for paclitaxel, the solubilizer cremophor EL was toxic. Cross-resistance was observed between cisplatin and carboplatin. Conclusion Chemosensitivity of HNSCC cell lines can be determined using the MTT-uptake assay. For DNA-interfering cytostatics and vinca alkaloids this is a simple and reproducible procedure. Determined in vitro chemosensitivity serves as a baseline for further experimental approaches aiming to modulate chemoresistance in HNSCC with potential clinical significance.

  2. Molecular mechanisms of alkylation sensitivity in Indian muntjac cell lines.

    Science.gov (United States)

    Musk, S R; Hatton, D H; Bouffler, S D; Margison, G P; Johnson, R T

    1989-07-01

    The responses of two Indian muntjac cell lines to two monofunctional alkylating agents were investigated. An SV40-transformed line (SVM) had an increased sensitivity to cell killing when compared to the other, euploid line (DM) after exposure both to methyl nitrosourea (MNU) and to dimethylsulphate (DMS) and also exhibited higher frequencies of sister chromatid exchanges (SCEs) following alkylation. The hypersensitivity of SVM to DMS correlates with the defective repair of single-strand breaks that results in the generation of long-lived breaks in the DNA following exposure, leading eventually to the formation of chromosome aberrations. In contrast no difference is seen in the formation of long-lived breaks in the DNA of SVM and DM after treatment with biologically relevant doses of MNU; in this case hypersensitivity may be due to the loss of O6-alkylguanine-DNA-alkyltransferase activity. The conclusion that the hypersensitivites of SVM to MNU and to DMS have different molecular bases is supported by transfection of SVM with plasmids containing the protein coding region of the Escherichia coli ada+ gene; subsequent expression within the cell corrects its hypersensitivity to the cytotoxic and SCE-inducing effects of MNU but has very little influence upon the lethality, SCE induction or the repair of long-lived DNA strand breaks after exposure to DMS. PMID:2544312

  3. Rift Valley Fever Virus Incorporates the 78 kDa Glycoprotein into Virions Matured in Mosquito C6/36 Cells

    OpenAIRE

    Weingartl, Hana M.; Zhang, Shunzhen; Marszal, Peter; McGreevy, Alan; Burton, Lynn; Wilson, William C.

    2014-01-01

    Rift Valley fever virus (RVFV), genus Phlebovirus, family Bunyaviridae is a zoonotic arthropod-borne virus able to transition between distant host species, causing potentially severe disease in humans and ruminants. Viral proteins are encoded by three genomic segments, with the medium M segment coding for four proteins: nonstructural NSm protein, two glycoproteins Gn and Gc and large 78 kDa glycoprotein (LGp) of unknown function. Goat anti-RVFV polyclonal antibody and mouse monoclonal antibod...

  4. Murine Pregnancy-Specific Glycoprotein 23 Induces the Proangiogenic Factors Transforming-Growth Factor Beta 1 and Vascular Endothelial Growth Factor A in Cell Types Involved in Vascular Remodeling in Pregnancy1

    OpenAIRE

    Wu, Julie A.; Johnson, Briana L.; Chen, Yongqing; Ha, Cam T.; Dveksler, Gabriela S.

    2008-01-01

    Haemochorial placentation is a unique physiological process in which the fetal trophoblast cells remodel the maternal decidual spiral arteries to establish the fetoplacental blood supply. Pregnancy-specific glycoproteins (PSGs) are members of the carcinoembryonic antigen family. PSGs are produced by the placenta of rodents and primates and are secreted into the bloodstream. PSG23 is one of 17 members of the murine PSG family (designated PSG16 to PSG32). Previous studies determined that PSGs h...

  5. A novel lineage restricted, pericyte-like cell line isolated from human embryonic stem cells.

    Science.gov (United States)

    Greenwood-Goodwin, Midori; Yang, Jiwei; Hassanipour, Mohammad; Larocca, David

    2016-01-01

    Pericytes (PCs) are endothelium-associated cells that play an important role in normal vascular function and maintenance. We developed a method comparable to GMP quality protocols for deriving self-renewing perivascular progenitors from the human embryonic stem cell (hESC), line ESI-017. We identified a highly scalable, perivascular progenitor cell line that we termed PC-A, which expressed surface markers common to mesenchymal stromal cells. PC-A cells were not osteogenic or adipogenic under standard differentiation conditions and showed minimal angiogenic support function in vitro. PC-A cells were capable of further differentiation to perivascular progenitors with limited differentiation capacity, having osteogenic potential (PC-O) or angiogenic support function (PC-M), while lacking adipogenic potential. Importantly, PC-M cells expressed surface markers associated with pericytes. Moreover, PC-M cells had pericyte-like functionality being capable of co-localizing with human umbilical vein endothelial cells (HUVECs) and enhancing tube stability up to 6 days in vitro. We have thus identified a self-renewing perivascular progenitor cell line that lacks osteogenic, adipogenic and angiogenic potential but is capable of differentiation toward progenitor cell lines with either osteogenic potential or pericyte-like angiogenic function. The hESC-derived perivascular progenitors described here have potential applications in vascular research, drug development and cell therapy. PMID:27109637

  6. HIV-1 latency in actively dividing human T cell lines

    Directory of Open Access Journals (Sweden)

    Berkhout Ben

    2008-04-01

    Full Text Available Abstract Background Eradication of HIV-1 from an infected individual cannot be achieved by current drug regimens. Viral reservoirs established early during the infection remain unaffected by anti-retroviral therapy and are able to replenish systemic infection upon interruption of the treatment. Therapeutic targeting of viral latency will require a better understanding of the basic mechanisms underlying the establishment and long-term maintenance of HIV-1 in resting memory CD4 T cells, the most prominent reservoir of transcriptional silent provirus. However, the molecular mechanisms that permit long-term transcriptional control of proviral gene expression in these cells are still not well understood. Exploring the molecular details of viral latency will provide new insights for eventual future therapeutics that aim at viral eradication. Results We set out to develop a new in vitro HIV-1 latency model system using the doxycycline (dox-inducible HIV-rtTA variant. Stable cell clones were generated with a silent HIV-1 provirus, which can subsequently be activated by dox-addition. Surprisingly, only a minority of the cells was able to induce viral gene expression and a spreading infection, eventhough these experiments were performed with the actively dividing SupT1 T cell line. These latent proviruses are responsive to TNFα treatment and alteration of the DNA methylation status with 5-Azacytidine or genistein, but not responsive to the regular T cell activators PMA and IL2. Follow-up experiments in several T cell lines and with wild-type HIV-1 support these findings. Conclusion We describe the development of a new in vitro model for HIV-1 latency and discuss the advantages of this system. The data suggest that HIV-1 proviral latency is not restricted to resting T cells, but rather an intrinsic property of the virus.

  7. Responding to hypoxia: lessons from a model cell line.

    Science.gov (United States)

    Seta, K A; Spicer, Z; Yuan, Y; Lu, G; Millhorn, D E

    2002-08-20

    Mammalian cells require a constant supply of oxygen to maintain adequate energy production, which is essential for maintaining normal function and for ensuring cell survival. Sustained hypoxia can result in cell death. It is, therefore, not surprising that sophisticated mechanisms have evolved that allow cells to adapt to hypoxia. "Oxygen-sensing" is a special phenotype that functions to detect changes in oxygen tension and to transduce this signal into organ system functions that enhance the delivery of oxygen to tissue in various organisms. Oxygen-sensing cells can be segregated into two distinct cell types: those that functionally depolarize (excitable) and those that do not functionally depolarize (nonexcitable) in response to reduced oxygen. Theoretically, excitable cells have all the same signaling capabilities as the nonexcitable cells, but the nonexcitable cells cannot have all the signaling capabilities as excitable cells. A number of signaling pathways have been identified that regulate gene expression during hypoxia. These include the Ca2+-calmodulin pathway, the 3'-5' adenosine monophosphate (cAMP)-protein kinase A (PKA) pathway, the p42 and p44 mitogen-activated protein kinase [(MAPK); also known as the extracellular signal-related kinase (ERK) for ERK1 and ERK2] pathway, the stress-activated protein kinase (SAPK; also known as p38 kinase) pathway, and the phosphatidylinositol 3-kinase (PI3K)-Akt pathway. In this review, we describe hypoxia-induced signaling in the model O2-sensing rat pheochromocytoma (PC12) cell line, the current level of understanding of the major signaling events that are activated by reduced O2, and how these signaling events lead to altered gene expression in both excitable and nonexcitable oxygen-sensing cells. PMID:12189251

  8. Boldine: a potential new antiproliferative drug against glioma cell lines.

    Science.gov (United States)

    Gerhardt, Daniéli; Horn, Ana Paula; Gaelzer, Mariana Maier; Frozza, Rudimar Luiz; Delgado-Cañedo, Andrés; Pelegrini, Alessandra Luiza; Henriques, Amélia T; Lenz, Guido; Salbego, Christianne

    2009-12-01

    Malignant gliomas are the most common and devastating primary tumors of the central nervous system. Currently no efficient treatment is available. This study evaluated the effect and underlying mechanisms of boldine, an aporphine alkaloid of Peumus boldus, on glioma proliferation and cell death. Boldine decreased the cell number of U138-MG, U87-MG and C6 glioma lines at concentrations of 80, 250 and 500 muM. We observed that cell death caused by boldine was cell-type specific and dose-dependent. Exposure to boldine for 24 h did not activate key mediators of apoptosis. However, it induced alterations in the cell cycle suggesting a G(2)/M arrest in U138-MG cells. Boldine had no toxic effect on non-tumor cells when used at the same concentrations as those used on tumor cells. Based on these results, we speculate that boldine may be a promising compound for evaluation as an anti-cancer agent. PMID:19050827

  9. Immune Response Induced by an Immunodominant 60 kDa Glycoprotein of the Cell Wall of Sporothrix schenckii in Two Mice Strains with Experimental Sporotrichosis

    Science.gov (United States)

    Alba-Fierro, Carlos A.; Pérez-Torres, Armando; Toriello, Conchita; Pulido-Camarillo, Evelyn; Romo-Lozano, Yolanda; Gutiérrez-Sánchez, Gerardo

    2016-01-01

    Cell wall (CW) components of fungus Sporothrix schenckii are the major inductors antigens of immune responses. The immunodominant 60 kDa glycoprotein (gp60) has been shown to be associated with the virulence of this fungus but its role in experimental sporotrichosis is unknown. In this work, the immunological effects of CW-purified gp60 were investigated in a model of experimental subcutaneous sporotrichosis in normal and gp60-preimmunized C57BL/6 and BALB/c mice strains which were then infected with S. schenckii conidia. Results showed that both mice strains use different cytokine profiles in order to fight S. schenckii infection; C57BL/6 mice seem to use a Th17 response while BALB/c mice tend to depend on a Th1 profile. Preimmunization with gp60 showed a downregulatory effect on the immune response since cytokines levels were diminished in both strains. There were no significant differences in the magnitude of dorsoplantar inflammation between gp60-preimmunized and nonimmunized mice of both strains. However, skin lesions due to the infection in gp60-preimmunized mice were more severe in BALB/c than in C57BL/6 mice, suggesting that the antigen exerts a higher downregulatory effect on the Th1 response. PMID:27051673

  10. The Ebola virus glycoprotein mediates entry via a non-classical dynamin-dependent macropinocytic pathway

    International Nuclear Information System (INIS)

    Ebola virus (EBOV) has been reported to enter cultured cell lines via a dynamin-2-independent macropinocytic pathway or clathrin-mediated endocytosis. The route(s) of productive EBOV internalization into physiologically relevant cell types remain unexplored, and viral-host requirements for this process are incompletely understood. Here, we use electron microscopy and complementary chemical and genetic approaches to demonstrate that the viral glycoprotein, GP, induces macropinocytic uptake of viral particles into cells. GP's highly-glycosylated mucin domain is dispensable for virus-induced macropinocytosis, arguing that interactions between other sequences in GP and the host cell surface are responsible. Unexpectedly, we also found a requirement for the large GTPase dynamin-2, which is proposed to be dispensable for several types of macropinocytosis. Our results provide evidence that EBOV uses an atypical dynamin-dependent macropinocytosis-like entry pathway to enter Vero cells, adherent human peripheral blood-derived monocytes, and a mouse dendritic cell line.

  11. Digoxin and ouabain induce P-glycoprotein by activating calmodulin kinase II and hypoxia-inducible factor-1α in human colon cancer cells

    International Nuclear Information System (INIS)

    Digoxin and ouabain are cardioactive glycosides, which inhibit the Na+/K+-ATPase pump and in this way they increase the intracellular concentration of cytosolic calcium ([Ca++]i). They are also strong inducers of the P-glycoprotein (Pgp), a transmembrane transporter which extrudes several drugs, including anticancer agents like doxorubicin. An increased amount of Pgp limits the absorption of drugs through epithelial cells, thus inducing resistance to chemotherapy. The mechanism by which cardioactive glycosides increase Pgp is not known and in this work we investigated whether digoxin and ouabain elicited the expression of Pgp with a calcium-driven mechanism. In human colon cancer HT29 cells both glycosides increased the [Ca++]i and this event was dependent on the calcium influx via the Na+/Ca++ exchanger. The increased [Ca++]i enhanced the activity of the calmodulin kinase II enzyme, which in turn activated the transcription factor hypoxia-inducible factor-1α. This one was responsible for the increased expression of Pgp, which actively extruded doxorubicin from the cells and significantly reduced the pro-apoptotic effect of the drug. All the effects of glycosides were prevented by inhibiting the Na+/Ca++ exchanger or the calmodulin kinase II. This work clarified the molecular mechanisms by which digoxin and oubain induce Pgp and pointed out that the administration of cardioactive glycosides may widely affect the absorption of drugs in colon epithelia. Moreover, our results suggest that the efficacy of chemotherapeutic agent substrates of Pgp may be strongly reduced in patients taking digoxin.

  12. Lactobacillus acidophilus stimulates intestinal P-glycoprotein expression via a c-Fos/c-Jun-dependent mechanism in intestinal epithelial cells.

    Science.gov (United States)

    Priyamvada, Shubha; Anbazhagan, Arivarasu N; Kumar, Anoop; Soni, Vikas; Alrefai, Waddah A; Gill, Ravinder K; Dudeja, Pradeep K; Saksena, Seema

    2016-04-15

    Our previous studies showed that Lactobacillus acidophilus (LA) culture supernatant (CS) increased P-glycoprotein [Pgp/multidrug resistance 1 (MDR1)] function, expression, and promoter activity in Caco-2 cells. The current studies were designed to elucidate the molecular mechanisms mediating the stimulatory effects of LA CS on Pgp promoter activity. Deletion analysis indicated that the LA CS response element(s) is located in the -172/+428-bp region, and sequence analysis of this region revealed three potential binding sites for c-Fos or c-Jun: proximal activating protein (AP) 1a (-119/-98 bp), distal AP1b (-99/-78 bp), and AP1c (+175/+196 bp). LA CS (24 h) showed an approximately twofold increase in the protein expression of c-Fos and c-Jun in Caco-2 cells. Electrophoretic mobility shift assay showed that LA CS markedly increased the binding of Caco-2 nuclear proteins to AP1a and AP1b, but not AP1c. The DNA-protein complex was completely eliminated by c-Fos antibody, while c-Jun antibody partially eliminated the complex. Chromatin immunoprecipitation analysis also showed that LA CS enhanced the association of c-Fos and c-Jun (by ∼4- and 1.5-fold, respectively) with endogenous Pgp promoter in Caco-2 cells (p-172/+1). Interestingly, overexpression of c-Fos or c-Jun activated Pgp promoter by nearly twofold each. This increase was further enhanced (∼14-fold) when c-Fos and c-Jun were simultaneously overexpressed, suggesting that the presence of one of these transcription factors potentiates the effect of the other. These studies, for the first time, provide evidence for the involvement of c-Fos/c-Jun in stimulation of Pgp gene expression by LA CS in the human intestine. PMID:26867563

  13. Effects of Genistein on Cell Cycle and Apoptosis of Two Murine Melanoma Cell Lines

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    The effects of genistein on several tumor cell lines were investigated to study the effects of genistein on cell growth, cell cycle, and apoptosis of two murine melanoma cell lines, B16 and K1735M2. These two closely related murine melanoma cell lines, however, have different responses to the genistein treatment. Genistein inhibits the growth of both the B16 and K1735M2 cell lines and arrests the growth at the G2/M phase. After treatment with 60 μmol/L genistein for 72 h, apoptosis and caspase activities were detected in B16 cells, while such effects were not found in K1735M2. Further tests showed that after genistein treatment the protein content and mRNA levels of p53 increased in B16, but remained the same in K1735M2. The protein content and mRNA levels of p21WAF1/CIP1 increased in both cell lines after treatment.The results show that genistein might induce apoptosis in B16 cells by damaging the DNA, inhibiting topoisomerase Ⅱ, increasing p53 expression, releasing cytochrome c from the mitochondria, and activating the caspases which will lead to apoptosis.

  14. Fluorouracil Selectively Enriches Stem-like Leukemic Cells in a Leukemic Cell Line

    Directory of Open Access Journals (Sweden)

    Ling Zhang, Song Yang, Yu-Juan He, Hui-Yuan Shao, Li Wang, Hui Chen, Yu-Jie Gao, Feng-Xian Qing, Xian-Chun Chen, Liu-Yang Zhao, Shi Tan

    2010-01-01

    Full Text Available Recent studies have reported that cancer stem cells (CSCs could be isolated from solid cancer cell lines, in which the purity of CSCs was higher than that from tumor tissues. Separation of CSCs from leukemic cell lines was rarely reported. In this study, CD34+CD38- stem-like cell subsets in human KG-1a leukemic cell line were enriched by cytotoxic agent 5-fluorouracil (5-FU. After 4 days incubation of KG-1a cell line with 5-FU (50 μg/ml, the CD34+CD38- subpopulation of cell lines was enriched more than 10 times. The enriched cells had proliferate potential in vitro, low level of RNA transcription and Hoechst 33342 dye efflux ability, accompanied by high expression of ATP-binding cassette transporter protein ABCG2. Our findings suggest that treatment with 5-FU offers an easy method to isolate leukemic stem-like subpopulation. It can facilitate studies of leukemic stem cell biology and the development of new therapeutic strategies.

  15. Establishment of a novel human medulloblastoma cell line characterized by highly aggressive stem-like cells.

    Science.gov (United States)

    Silva, Patrícia Benites Gonçalves da; Rodini, Carolina Oliveira; Kaid, Carolini; Nakahata, Adriana Miti; Pereira, Márcia Cristina Leite; Matushita, Hamilton; Costa, Silvia Souza da; Okamoto, Oswaldo Keith

    2016-08-01

    Medulloblastoma is a highly aggressive brain tumor and one of the leading causes of morbidity and mortality related to childhood cancer. These tumors display differential ability to metastasize and respond to treatment, which reflects their high degree of heterogeneity at the genetic and molecular levels. Such heterogeneity of medulloblastoma brings an additional challenge to the understanding of its physiopathology and impacts the development of new therapeutic strategies. This translational effort has been the focus of most pre-clinical studies which invariably employ experimental models using human tumor cell lines. Nonetheless, compared to other cancers, relatively few cell lines of human medulloblastoma are available in central repositories, partly due to the rarity of these tumors and to the intrinsic difficulties in establishing continuous cell lines from pediatric brain tumors. Here, we report the establishment of a new human medulloblastoma cell line which, in comparison with the commonly used and well-established cell line Daoy, is characterized by enhanced proliferation and invasion capabilities, stem cell properties, increased chemoresistance, tumorigenicity in an orthotopic metastatic model, replication of original medulloblastoma behavior in vivo, strong chromosome structural instability and deregulation of genes involved in neural development. These features are advantageous for designing biologically relevant experimental models in clinically oriented studies, making this novel cell line, named USP-13-Med, instrumental for the study of medulloblastoma biology and treatment. PMID:26358937

  16. Evaluation of nitroimidazole hypoxic cell radiosensitizers in a human tumor cell line high in intracellular glutathione

    International Nuclear Information System (INIS)

    Five nitroimidazole hypoxic cell radiosensitizers were evaluated in a human lung adenocarcinoma cell line (A549) whose GSH level was 8-fold higher than Chinese hamster V79 cells. One millimolar concentrations of Misonidazole (MISO), SR-2508, RSU-1164, RSU-1172, and Ro-03-8799 sensitized hypoxic A549 cells to radiation, with Ro-03-8799 giving the highest sensitizer enhancement ration (SER) (2.3). However, MISO, SR-2508 and Ro-03-8799 were less effective in this cell line than in V79 cells, presumably due to higher GSH content of the A549 cells. Increased hypoxic radiosensitization was seen with 0.1 mM Ro-03-8799 after GSH depletion by BSO as compared to 0.1 mM Ro-03-8799 alone (SER-1.8 vs 1.3). The combination of GSH depletion and 0.1 mM Ro-03-8799 was considerably more toxic than 0.1 mM or 1.0 mM Ro-03-8799 alone. This sensitivity was much greater than has been observed for SR-2508. These data show that Ro-03-8799 was the most efficient hypoxic cell radiosensitizer in a human tumor cell line considerably higher in GSH than the rodent cell lines often used in hypoxic radiosensitization studies. Thus, Ro-03-8799 may be a more effective hypoxic cell sensitizer in human tumors that are high in GSH

  17. Multidrug resistance and retroviral transduction potential in human small cell lung cancer cell lines

    DEFF Research Database (Denmark)

    Theilade, M D; Gram, G J; Jensen, P B;

    1999-01-01

    Multidrug resistance (MDR) remains a major problem in the successful treatment of small cell lung cancer (SCLC). New treatment strategies are needed, such as gene therapy specifically targeting the MDR cells in the tumor. Retroviral LacZ gene-containing vectors that were either pseudotyped...... for the gibbon ape leukemia virus (GALV-1) receptor or had specificity for the amphotropic murine leukemia virus (MLV-A) receptor were used for transduction of five SCLC cell lines differing by a range of MDR mechanisms. Transduction efficiencies in these cell lines were compared by calculating the percentage...... of blue colonies after X-Gal staining of the cells grown in soft agar. All examined SCLC cell lines were transducible with either vector. Transduction efficiencies varied from 5.7% to 33.5% independent of the presence of MDR. These results indicate that MDR does not severely impair transduction of SCLC...

  18. Multidrug resistance and retroviral transduction potential in human small cell lung cancer cell lines

    DEFF Research Database (Denmark)

    Theilade, M D; Gram, G J; Jensen, P B;

    1999-01-01

    blue colonies after X-Gal staining of the cells grown in soft agar. All examined SCLC cell lines were transducible with either vector. Transduction efficiencies varied from 5.7% to 33.5% independent of the presence of MDR. These results indicate that MDR does not severely impair transduction of SCLC......Multidrug resistance (MDR) remains a major problem in the successful treatment of small cell lung cancer (SCLC). New treatment strategies are needed, such as gene therapy specifically targeting the MDR cells in the tumor. Retroviral LacZ gene-containing vectors that were either pseudotyped for the...... gibbon ape leukemia virus (GALV-1) receptor or had specificity for the amphotropic murine leukemia virus (MLV-A) receptor were used for transduction of five SCLC cell lines differing by a range of MDR mechanisms. Transduction efficiencies in these cell lines were compared by calculating the percentage of...

  19. Cloning of partial cDNA encoding differentiation and tumor-associated mucin glycoproteins expressed by human mammary epithelium

    International Nuclear Information System (INIS)

    Human mammary epithelial cells secrete and express on their cell surfaces complex mucin glycoproteins that are developmentally regulated, tumor-associated, and highly immunogenic. Studies using monoclonal antibodies directed to these glycoproteins suggest that their molecular structures can vary with differentiation stages in the normal gland and in malignancy. To analyze the molecular nature of these glycoproteins, milk mucin was affinity-purifed and deglycosylated with hydrogen fluoride, yielding bands at 68 and 72 kDa on silver-stained gels. Polyclonal and monoclonal antibodies to the stripped core protein were developed and used to screen a λgt11 expression library of cDNA made from mRNA of the mammary tumor cell line MCF-7. Seven crossreacting clones were isolated, with inserts 0.1-1.8 kilobases long. RNA blot analysis, using as a probe the 1.8-kilobase insert subcloned in plasmid pUC8 (pMUC10), revealed transcripts of 4.7 and 6.4 kilobases in MCF-7 and T47D mammary tumor cells, whereas normal mammary epithelial cells from pooled milks have additional transcripts. The expression of mRNA correlates with antigen expression as determined by binding of two previously characterized anti-mucin monoclonal antibodies (HMFG-1 and HMFG-2) to seven cell lines. Restriction enzyme analysis detected a restriction fragment length polymorphism when human genomic DNA was digested with EcoRI or HinfI

  20. The comparison of glycosphingolipids isolated from an epithelial ovarian cancer cell line and a nontumorigenic epithelial ovarian cell line using MALDI-MS and MALDI-MS/MS.

    Science.gov (United States)

    Rajanayake, Krishani K; Taylor, William R; Isailovic, Dragan

    2016-08-01

    Glycosphingolipids (GSLs) are important biomolecules, which are linked to many diseases such as GSL storage disorders and cancer. Consequently, the expression of GSLs may be altered in ovarian cancer cell lines in comparison to apparently healthy cell lines. Here, differential expressions of GSLs in an epithelial ovarian cancer cell line SKOV3 and a nontumorigenic epithelial ovarian cell line T29 were studied using matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS) and MALDI-MS/MS. The isolation of GSLs from SKOV3 and T29 cell lines was carried out using Folch partition. GSLs were successfully detected by MALDI-MS, and structurally assigned by a comparison of their MALDI-MS/MS fragmentation patterns with MS/MS data found in SimLipid database. Additionally, LIPID MAPS was used to assign GSL ion masses in MALDI-MS spectra. Seventeen neutral GSLs were identified in Folch partition lower (chloroform/methanol) phases originating from both cell lines, while five globo series neutral GSLs were identified only in the Folch partition lower phase of SKOV3 cell line. Several different sialylated GSLs were detected in Folch partition upper (water/methanol) phases of SKOV3 and T29 cell lines. Overall, this study demonstrates the alteration and increased glycosylation of GSLs in an epithelial ovarian cancer cell line in comparison to a nontumorigenic epithelial ovarian cell line. PMID:27267063

  1. Cloned goats (Gapra hircus) from foetal fibroblast cell lines

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    Mammalian cloning has been one of the most active research topics in the world.Cloning with in vitro culured foetal fibroblast cells,in comparison with embryonic cells,can be used not only to theoretically study the embryonic or cellular development and differentiation in mammals,but also to utilize the unlimited fibroblast cells to produce large numbers of clonings.The preliminary results are as follows:(i) The division and development of the cloned embryos with embryonic donor cells and goat foetal fibroblast donor cells were 55%,77% and 35%,31%,respectively.There is no significant statistical difference between them.(ii) These studies result in the birth of two cloned goats derived from two 30-day foetal fibroblast cell lines,which are the first cloned mammals from somatic cells in China.This project has established a technological data base for the furture research on adult mammalian somatic cloning and nucleocytoplasmic interactions in animal development,and a novel technique for the cloning of animals with a high-level expression of transgene(s).

  2. CD3 receptor modulation in Jurkat leukemic cell line.

    Directory of Open Access Journals (Sweden)

    Jacek M Witkowski

    2004-03-01

    Full Text Available CD3 antigen is a crucial molecule in T cell signal transduction. Although its expression on cell surface is constitutive, dynamic regulation of TCR-CD3 level is probably the most important mechanism allowing T cells to calibrate their response to different levels of stimuli. In our study we examined the role of two main T cell signal transduction pathways in controlling the surface level of CD3 antigen, one based on protein kinase C activity and the other dependent on calcineurin. As an experimental model we used three clones derived from Jurkat cell line, expressing different levels of CD3 antigen surface expression: CD3(low (217.6, CD3+(217.9 or CD3(low (217.7. The cells were stimulated with PMA or ionomycin, acting directly on PKC and calcineurin, respectively. Prior to the stimulation cells were incubated with PKC inhibitor--chelerythrine or calcineurin blocker--cyclosporine A. Changes in CD3 surface expression were measured by flow cytometry. Only PMA and chelerythrine were able to change CD3 expression suggesting important involvement of PKC in the regulation of its expression. To confirm these findings, PKC activity was estimated in Jurkat clones. Our data demonstrated that Jurkat clones with different CD3 expression showed also different PKC activities, so we conclude that PKC-dependent pathway is the main way of controlling CD3 level on Jurkat clones.

  3. Characterization of cell lines stably transfected with rubella virus replicons

    Energy Technology Data Exchange (ETDEWEB)

    Tzeng, Wen-Pin; Xu, Jie [Department of Biology, Georgia State University, P.O. Box 4010, Atlanta GA 30302-4010 (United States); Frey, Teryl K., E-mail: tfrey@gsu.edu [Department of Biology, Georgia State University, P.O. Box 4010, Atlanta GA 30302-4010 (United States)

    2012-07-20

    Rubella virus (RUBV) replicons expressing a drug resistance gene and a gene of interest were used to select cell lines uniformly harboring the replicon. Replicons expressing GFP and a virus capsid protein GFP fusion (C-GFP) were compared. Vero or BHK cells transfected with either replicon survived drug selection and grew into a monolayer. However, survival was {approx}9-fold greater following transfection with the C-GFP-replicon than with the GFP-expressing replicon and while the C-GFP-replicon cells grew similarly to non-transfected cells, the GFP-replicon cells grew slower. Neither was due to the ability of the CP to enhance RNA synthesis but survival during drug selection was correlated with the ability of CP to inhibit apoptosis. Additionally, C-GFP-replicon cells were not cured of the replicon in the absence of drug selection. Interferon-alpha suppressed replicon RNA and protein synthesis, but did not cure the cells, explaining in part the ability of RUBV to establish persistent infections.

  4. Interactions of Streptococcus iniae with phagocytic cell line.

    Science.gov (United States)

    El Aamri, Fatima; Remuzgo-Martínez, S; Acosta, Félix; Real, Fernando; Ramos-Vivas, José; Icardo, José M; Padilla, Daniel

    2015-04-01

    Streptococcus iniae has become one of the most serious aquatic pathogens in the last decade, causing large losses in wild and farmed fish worldwide. There is clear evidence that this pathogen is capable not only of causing serious disease in fish but also of being transferred to and infecting humans. In this study, we investigate the interaction of S. iniae with two murine macrophage cell lines, J774-A1 and RAW 264.7. Cytotoxicity assay demonstrated significant differences between live and UV-light killed IUSA-1 strains. The burst respiratory activity decreased to baseline after 1 and 4 h of exposure for J774-A1 and RAW 264.7, respectively. Immunofluorescent and ultrastructural study of infected cells confirmed the intracellular localization of bacteria at 1 h and 24 h post-infection. Using qRT-PCR arrays, we investigated the changes in the gene expression of immune relevant genes associated with macrophage activation. In this screening, we identified 11 of 84 genes up-regulated, we observed over-expression of pro-inflammatory response as IL-1α, IL-1β, and TNF-α, without a good anti-inflammatory response. Present findings suggest a capacity of S. iniae to modulate a mammalian macrophages cell lines to their survival and replication intracellular, which makes this cell type as a reservoir for continued infection. PMID:24956597

  5. Hypoxia induces adipogenic differentitation of myoblastic cell lines

    Energy Technology Data Exchange (ETDEWEB)

    Itoigawa, Yoshiaki [Tohoku University School of Medicine, Sendai (Japan); Juntendo University School of Medicine, Tokyo (Japan); Kishimoto, Koshi N., E-mail: kishimoto@med.tohoku.ac.jp [Tohoku University School of Medicine, Sendai (Japan); Okuno, Hiroshi; Sano, Hirotaka [Tohoku University School of Medicine, Sendai (Japan); Kaneko, Kazuo [Juntendo University School of Medicine, Tokyo (Japan); Itoi, Eiji [Tohoku University School of Medicine, Sendai (Japan)

    2010-09-03

    Research highlights: {yields} C2C12 and G8 myogenic cell lines treated by hypoxia differentiate into adipocytes. {yields} The expression of C/EBP{beta}, {alpha} and PPAR{gamma} were increased under hypoxia. {yields} Myogenic differentiation of C2C12 was inhibited under hypoxia. -- Abstract: Muscle atrophy usually accompanies fat accumulation in the muscle. In such atrophic conditions as back muscles of kyphotic spine and the rotator cuff muscles with torn tendons, blood flow might be diminished. It is known that hypoxia causes trans-differentiation of mesenchymal stem cells derived from bone marrow into adipocytes. However, it has not been elucidated yet if hypoxia turned myoblasts into adipocytes. We investigated adipogenesis in C2C12 and G8 murine myogenic cell line treated by hypoxia. Cells were also treated with the cocktail of insulin, dexamethasone and IBMX (MDI), which has been known to inhibit Wnt signaling and promote adipogenesis. Adipogenic differentiation was seen in both hypoxia and MDI. Adipogenic marker gene expression was assessed in C2C12. CCAAT/enhancer-binding protein (C/EBP) {beta}, {alpha} and peroxisome proliferator activating receptor (PPAR) {gamma} were increased by both hypoxia and MDI. The expression profile of Wnt10b was different between hypoxia and MDI. The mechanism for adipogenesis of myoblasts in hypoxia might be regulated by different mechanism than the modification of Wnt signaling.

  6. Hypoxia induces adipogenic differentitation of myoblastic cell lines

    International Nuclear Information System (INIS)

    Research highlights: → C2C12 and G8 myogenic cell lines treated by hypoxia differentiate into adipocytes. → The expression of C/EBPβ, α and PPARγ were increased under hypoxia. → Myogenic differentiation of C2C12 was inhibited under hypoxia. -- Abstract: Muscle atrophy usually accompanies fat accumulation in the muscle. In such atrophic conditions as back muscles of kyphotic spine and the rotator cuff muscles with torn tendons, blood flow might be diminished. It is known that hypoxia causes trans-differentiation of mesenchymal stem cells derived from bone marrow into adipocytes. However, it has not been elucidated yet if hypoxia turned myoblasts into adipocytes. We investigated adipogenesis in C2C12 and G8 murine myogenic cell line treated by hypoxia. Cells were also treated with the cocktail of insulin, dexamethasone and IBMX (MDI), which has been known to inhibit Wnt signaling and promote adipogenesis. Adipogenic differentiation was seen in both hypoxia and MDI. Adipogenic marker gene expression was assessed in C2C12. CCAAT/enhancer-binding protein (C/EBP) β, α and peroxisome proliferator activating receptor (PPAR) γ were increased by both hypoxia and MDI. The expression profile of Wnt10b was different between hypoxia and MDI. The mechanism for adipogenesis of myoblasts in hypoxia might be regulated by different mechanism than the modification of Wnt signaling.

  7. Proteomic identification of alpha-2-HS-glycoprotein as a plasma biomarker of hypopharyngeal squamous cell carcinoma

    OpenAIRE

    TIAN, WEN-DONG; Li, Jun-Zheng; Hu, Shui-Wang; Peng, Xiao-wei; Li, Gang; LIU, Xiong; Chen, Huai-hong; Xu, Xia; Li, Xiang-ping

    2015-01-01

    Hypopharyngeal squamous cell carcinoma (HSCC) has very poor prognosis compared with other head and neck squamous cell carcinomas. Late-stage diagnosis of HSCC increases mortality. Therefore, more effective biomarkers for early diagnosis of HSCC are necessary. Unfortunately, appropriate biomarkers for clinical diagnosis and prognosis have not been identified yet. However, recent progresses in quantitative proteomics have offered opportunities to identify plasma proteins as biomarkers for HSCC....

  8. Glycoprotein Hypersecretion Alters the Cell Wall in Trichoderma reesei Strains Expressing the Saccharomyces cerevisiae Dolichylphosphate Mannose Synthase Gene▿

    OpenAIRE

    Perlińska-Lenart, Urszula; Orłowski, Jacek; Laudy, Agnieszka E.; Zdebska, Ewa; Palamarczyk, Grażyna; Kruszewska, Joanna S.

    2006-01-01

    Expression of the Saccharomyces cerevisiae DPM1 gene (coding for dolichylphosphate mannose synthase) in Trichoderma reesei (Hypocrea jecorina) increases the intensity of protein glycosylation and secretion and causes ultrastructural changes in the fungal cell wall. In the present work, we undertook further biochemical and morphological characterization of the DPM1-expressing T. reesei strains. We established that the carbohydrate composition of the fungal cell wall was altered with an increas...

  9. Radiosensitivity of brain cancer stem cells from malignant glicoma cell line U251 in vitro

    International Nuclear Information System (INIS)

    Objective: To investigate the radiosensitivity of brain cancer stem cells of different conditions isolated from malignant glioma cell line U251 irt vitro. Methods: The brain cancer stem cells in U251 or the brain cancer stem cells isolated from U251 were irradiated by 60Co γ-rays. TUNEL and Annexin-FITC were employed to detect the apoptosis. The brain cancer stem cells were subcutaneously transplanted to nude mouse. Flow cytometry was used to detect cell cycle. Results: The brain cancer stem cells isolated from malignant glioma cell line U251 were in active cell cycle and sensitive to 60Co γ-rays. Thed apoptotic cells were increased obviously after irradiation. After subcutaneously transplanted to unde mouse, there was no tumor appear. However; the brain cancer stem cells existed in U251 were in G0-G1 and resisted to 60Co γ-rays. They differentiated into the parent glioma type after traqnsplantation. Conclusions: The brain cancer stem cells existed in the malignant glioma cell line is resisted to irradiation, and this phenomenon may explain the glioma relapse irt situ after radiation therapy. (authors)

  10. Molecular signatures in response to Isoliquiritigenin in lymphoblastoid cell lines