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Sample records for a431 cells identification

  1. Identification of specific gravity sensitive signal transduction pathways in human A431 carcinoma cells

    NASA Astrophysics Data System (ADS)

    Rijken, P. J.; de Groot, R. P.; Kruijer, W.; de Laat, S. W.; Verkleij, A. J.; Boonstra, J.

    Epidermal growth factor (EGF) activates a well characterized signal transduction cascade in human A431 epidermoid carcinoma cells. The influence of gravity on EGF-induced EGF-receptor clustering and early gene expression as well as on actin polymerization and actin organization have been investigated. Different signalling pathways induced by the agents TPA, forskolin and A23187 that activate gene expression were tested for sensitivity to gravity. EGF-induced c-fos and c-jun expression were decreased in microgravity. However, constitutive β-2 microglobulin expression remained unaltered. Under simulated weightlessness conditions EGF- and TPA-induced c-fos expression was decreased, while forskolin- and A23187-induced c-fos expression was independent of the gravity conditions. These results suggest that gravity affects specific signalling pathways. Preliminary results indicate that EGF-induced EGF-receptor clustering remained unaltered irrespective of the gravity conditions. Furthermore, the relative filamentous actin content of steady state A431 cells was enhanced under microgravity conditions and actin filament organization was altered. Under simulated weightlessness actin filament organization in steady state cells as well as in EGF-treated cells was altered as compared to the 1 G reference experiment. Interestingly the microtubule and keratin organization in untreated cells showed no difference with the normal gravity samples. This indicates that gravity may affect specific components of the signal transduction circuitry.

  2. Inhibitory effect of berberine on human skin squamous cell carcinoma A431 cells.

    PubMed

    Li, D X; Zhang, J; Zhang, Y; Zhao, P W; Yang, L M

    2015-09-08

    Berberine (BBR) is a natural alkaloid with significant anti-tumor activity against many types of cancer cells. In this study, we investigated the molecular mechanisms employed by BBR to repress the proliferation and growth of skin squamous cell carcinoma A431 cells. Berberine was reported to inhibit the proliferation of A431 cells in a dose- and time-dependent manner and was observed to induce a series of biochemical events, including the loss of mitochondrial membrane potential, release of cytochrome-c to cytosol, induction of proteins of the Bcl-2 family and caspases, and the cleavage of poly(ADP)-ribose polymerase. This suggested its ability to induce apoptosis. The results of a wound healing test revealed that berberine inhibited the migration of A431 cells. Ezrin was transfected into A431 cells by RNA interference. The level of expression of Ezrin in the transfected A431 cells was observed to decrease with berberine treatment, which suggested that berberine might inhibit the invasion of A431 cells through Ezrin. The results of this study demonstrated that berberine could potentially inhibit proliferation, induce apoptosis, and inhibit the invasion of A431 cells.

  3. Baicalein mediates inhibition of migration and invasiveness of skin carcinoma through Ezrin in A431 cells

    PubMed Central

    2011-01-01

    Background Ezrin is highly expressed in skin cancer and promotes tumor metastasis. Ezrin serves as a promising target for anti-metastasis therapy. The aim of this study is to determine if the flavonoid bacailein inhibits the metastasis of skin cancer cells through Ezrin. Methods Cells from a cutaneous squamous carcinoma cell line, A431, were treated with baicalein at 0-60 μM to establish the non-cytotoxic concentration (NCC) range for baicalein. Following treatment with baicalein within this range, total Ezrin protein (both phosphorylated and unphosphorylated forms) and phosphorylated-Ezrin (phos-Ezrin) were detected by western blotting, and Ezrin RNA was detected in A431 cells using reverse transcription-polymerase chain reaction (RT-PCR). Thereafter, the motility and invasiveness of A431 cells following baicalein treatment were determined using wound-healing and Boyden chamber invasion assays. Short-interfering RNA (si-RNA) specifically targeting Ezrin was transfected into A431 cells, and a si-RNA Ezrin-A431 cell line was established by G418 selection. This stable cell line was transiently transfected with Ezrin and mutant Ezrin plasmids, and its motilityand invasiveness was subsequently determined to clarify whether bacailein inhibits these processes through Ezrin. Results We determined the range of NCCs for baicalein to be 2.5-40 μM in A431 cells. Baicalein displayed a dose- and time-dependent inhibition of expressions of total Ezrin and phos-Ezrin within this range NCCs. In addition, it exerted this inhibitory effect through the reduction of Ezrin RNA transcript. Baicalein also inhibited the motility and invasiveness of A431 skin carcinoma cells within the range of NCCs, in a dose- and time-dependent manner. A431 cell motility and invasiveness were inhibited by 73% and 80% respectively when cells were treated with 20 μM baicalein. However, the motility and invasiveness of A431 cells containing the Ezrin mutant were not effectively inhibited by baicalein

  4. GPR87 mediates lysophosphatidic acid-induced colony dispersal in A431 cells.

    PubMed

    Ochiai, Shoichi; Furuta, Daisuke; Sugita, Kazuya; Taniura, Hideo; Fujita, Norihisa

    2013-09-05

    We have previously reported that an orphan G protein-coupled receptor GPR87 was activated by lysophosphatidic acid (LPA) and that it induced an increase in the intracellular Ca(2+) levels in the CHO cells genetically engineered to express GPR87-Gα16 fusion protein. Because the Ca(2+) response was blocked by the LPA receptor antagonist Ki16425, GPR87 was suggested to be a putative LPA receptor. However, further studies are required to confirm whether GPR87 is an LPA receptor. A previous study showed that colonies of A431 cells treated with LPA showed rapid and synchronized dissociation. Because A431 cells have been shown to express GPR87, we used these cells to examine whether GPR87 acted as an LPA receptor. When A431 cells were treated with gpr87-specific siRNA, the expression of GPR87 was decreased and LPA-induced colony dispersal was significantly reduced. Treatment of the cells with lpa1 siRNA had an additive effect in decrease in the colony dispersal. Studies on the LPA-mediated signaling pathway in A431 cells indicated that transactivation of the epidermal growth factor receptor (EGFR) by LPA led to cell scattering. PD153035, an inhibitor of tyrosine-kinase of EGFR, and BB94, an inhibitor of metalloprotease which produces a ligand for EGFR, significantly prevented the LPA-induced scattering of A431 cells pretreated with lpa1 or gpr87-siRNA. These results strongly suggested that GPR87 acts as an LPA receptor and induces colony dispersal via the transactivation of EGFR in A431 cells. © 2013 Elsevier B.V. All rights reserved.

  5. Identification of potential glycan cancer markers with sialic acid attached to sialic acid and up-regulated fucosylated galactose structures in epidermal growth factor receptor secreted from A431 cell line.

    PubMed

    Wu, Shiaw-Lin; Taylor, Allen D; Lu, Qiaozhen; Hanash, Samir M; Im, Hogune; Snyder, Michael; Hancock, William S

    2013-05-01

    We have used powerful HPLC-mass spectrometric approaches to characterize the secreted form of epidermal growth factor receptor (sEGFR). We demonstrated that the amino acid sequence lacked the cytoplasmic domain and was consistent with the primary sequence reported for EGFR purified from a human plasma pool. One of the sEGFR forms, attributed to the alternative RNA splicing, was also confirmed by transcriptional analysis (RNA sequencing). Two unusual types of glycan structures were observed in sEGFR as compared with membrane-bound EGFR from the A431 cell line. The unusual glycan structures were di-sialylated glycans (sialic acid attached to sialic acid) at Asn-151 and N-acetylhexosamine attached to a branched fucosylated galactose with N-acetylglucosamine moieties (HexNAc-(Fuc)Gal-GlcNAc) at Asn-420. These unusual glycans at specific sites were either present at a much lower level or were not observable in membrane-bound EGFR present in the A431 cell lysate. The observation of these di-sialylated glycan structures was consistent with the observed expression of the corresponding α-N-acetylneuraminide α-2,8-sialyltransferase 2 (ST8SiA2) and α-N-acetylneuraminide α-2,8-sialyltransferase 4 (ST8SiA4), by quantitative real time RT-PCR. The connectivity present at the branched fucosylated galactose was also confirmed by methylation of the glycans followed by analysis with sequential fragmentation in mass spectrometry. We hypothesize that the presence of such glycan structures could promote secretion via anionic or steric repulsion mechanisms and thus facilitate the observation of these glycan forms in the secreted fractions. We plan to use this model system to facilitate the search for novel glycan structures present at specific sites in sEGFR as well as other secreted oncoproteins such as Erbb2 as markers of disease progression in blood samples from cancer patients.

  6. Pheophorbide a mediated photodynamic therapy against human epidermoid carcinoma cells (A431)

    NASA Astrophysics Data System (ADS)

    Chen, Yi-Chun; Li, Wen-Tyng

    2011-02-01

    The objective of this study was to characterize the death mechanism of human epidermoid carcinoma cells (A431) triggered by photodynamic therapy (PDT) with pheophorbide a. First of all, significant inhibition on the survival of A431 cells (< 20 %) was observed when an irradiation dose of 5.1 J/cm2 combined with 125 ng/ml of pheophorbide a was applied. Survival rate of human keratinocyte cells was over 70 % under the same PDT parameters, suggesting that pheophorbide a killed cancer cells selectively. Mitochondria were the main target sites where pheophorbide a accumulated. Formation of reactive oxygen species (ROS) was detected after PDT. Addition of antioxidant N-Acetyl cysteine prevented ROS production and increased cell survival thereafter. The decrease in cellular ATP level was also observed at 6 hrs after PDT. Typical apoptotic cellular morphology and a collapse of mitochondrial membrane potential occurred after PDT. The loss of mitochondrial membrane potential led to the release of cytochrome c from the mitochondria to the cytosol, followed by activation of caspase-9 and caspase-3. The activation of caspase-3 resulted in poly(ADP-ribose) polymerase (PARP) cleavage in A431 cells, followed by DNA fragmentation. In conclusion, the results demonstrated that pheophorbide a possessed photodynamic action against A431 cells, mainly through apoptosis mediated by mitochondrial intrinsic pathway triggered by ROS.

  7. Fisetin inhibits growth, induces G₂ /M arrest and apoptosis of human epidermoid carcinoma A431 cells: role of mitochondrial membrane potential disruption and consequent caspases activation.

    PubMed

    Pal, Harish C; Sharma, Samriti; Elmets, Craig A; Athar, Mohammad; Afaq, Farrukh

    2013-07-01

    Non-melanoma skin cancers (NMSCs), one of the most common neoplasms, cause serious morbidity and mortality. Therefore, identification of non-toxic phytochemicals for prevention/treatment of NMSCs is highly desirable. Fisetin (3,3',4',7-tetrahydroxyflavone), a dietary flavonoid, present in fruits and vegetables possesses anti-oxidant and antiproliferative properties. The aim of this study was to investigate the chemotherapeutic potential of fisetin in cultured human epidermoid carcinoma A431 cells. Treatment of A431 cells with fisetin (5-80 μm) resulted in a significant decrease in cell viability in a dose- and time-dependent manner. Employing clonogenic assay, we found that fisetin treatment significantly reduced colony formation in A431 cells. Fisetin treatment of A431 cells resulted in G₂ /M arrest and induction of apoptosis. Furthermore, treatment of A431 cells with fisetin resulted in (i) decreased expression of anti-apoptotic proteins (Bcl2; Bcl-xL and Mcl-1); (ii) increased expression of pro-apoptotic proteins (Bax, Bak and Bad); (iii) disruption of mitochondrial potential; (iv) release of cytochrome c and Smac/DIABLO from mitochondria; (v) activation of caspases; and (vi) cleavage of Poly(ADP-ribose) polymerase (PARP) protein. Pretreatment of A431 cells with the pan-caspase inhibitor (Z-VAD-FMK) blocked fisetin-induced cleavage of caspases and PARP. Taken together, these data provide evidence that fisetin possesses chemotherapeutic potential against human epidermoid carcinoma A431 cells. Overall, these results suggest that fisetin could be developed as a novel therapeutic agent for the management of NMSCs. © 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  8. Fisetin inhibits growth, induces G2/M arrest and apoptosis of human epidermoid carcinoma A431 cells: Role of mitochondrial membrane potential disruption and consequent caspases activation

    PubMed Central

    Pal, Harish C.; Sharma, Samriti; Elmets, Craig A.; Athar, Mohammad; Afaq, Farrukh

    2013-01-01

    Non-melanoma skin cancers (NMSCs) one of the most common neoplasms causes serious morbidity and mortality. Therefore, identification of non-toxic phytochemicals for prevention/treatment of NMSCs is highly desirable. Fisetin (3,3′,4′,7-tetrahydroxyflavone), a dietary flavonoid, present in fruits and vegetables possesses anti-oxidant and anti-proliferative properties. The aim of this study was to investigate the chemotherapeutic potential of fisetin in cultured human epidermoid carcinoma A431 cells. Treatment of A431 cells with fistein (5-80 μM) resulted in a significant decrease in cell viability in a dose- and time-dependent manner. Employing clonogenic assay, we found that fisetin treatment significantly reduced colony formation in A431 cells. Fisetin treatment of A431 cells resulted in G2/M arrest and induction of apoptosis. Furthermore, treatment of A431 cells with fisetin resulted in (i) decreased expression of anti-apoptotic proteins (Bcl2, Bcl-xL and Mcl-1), (ii) increased expression of pro-apoptotic proteins (Bax, Bak and Bad), (iii) disruption of mitochondrial potential, (iv) release of cytchrome c and Smac/DIABLO from mitochondria, (v) activation of caspases, and (vi) cleavage of PARP protein. Pretreatment of A431 cells with the pan-caspase inhibitor (Z-VAD-FMK) blocked fisetin-induced cleavage of caspases and PARP. Taken together, these data provide evidence that fisetin possesses chemotherapeutic potential against human epidermoid carcinoma A431 cells. Overall, these results suggest that fisetin could be developed as a novel therapeutic agent for the management of NMSCs. PMID:23800058

  9. Epidermal growth factor (EGF) sensitive phosphorylation of calmodulin (CAM) in A431 cell membrane

    SciTech Connect

    Lin, P.H.; Selinfreund, R.; Wharton, W.

    1986-05-01

    A431, a transformed cell line, is known to contain a high concentration of EGF receptors (EGFR). Exogenous CAM, when combined with purified membrane from A431 is strongly phosphorylated in the presence of EGF. The EGF-dependent phosphorylation of CAM did not alter the normal profile of the A431 EGFR autophosphorylation, as demonstrated by SDS-PAGE and autoradiography. In addition to its EGF dependency, the presence of divalent cations is also critical for CAM phosphorylation. The presence of a divalent cation chelator, such as EGTA, caused a complete inhibition of CAM phosphorylation, which can be reversed with cations in the following order of effectiveness: Mg/sup + +/ > Mn/sup + +/ > Ca/sup + +/. Divalent cations also break up CAM into four co-migrating bands as indicated by Coomassie Blue stained gels and the corresponding autoradiograms. Double antibody precipitation followed by phospho-amino acid analysis revealed that the EGF-sensitive CAM phosphorylation occurs exclusively on the serine residue. Using radioimmunoassay, purified A431 membrane was shown to contain a significant amount of endogenous CAM. The implications of the EGF-sensitive CAM phosphorylation are currently under investigation.

  10. [Two types of store-operated channels in A431 cells].

    PubMed

    Gusev, K O; Zubov, A N; Kaznacheeva, E V; Mozhaeva, G N

    2004-01-01

    Activation of phospholipase C-coupled receptors leads to the release of Ca2+ from Ca2+ stores, and subsequent activation of store-operated cation (SOC) channels, promoting sustained Ca2+ influx. The most studied SOC channels are CRAC ("calcium-release activated calcium") channels exhibiting a very high selectivity for Ca2+. However, there are many SOC channels permeable for Ca2+ but having a lower selectivity. And while Ca2+ influx is important for many biological processes, little is known about the types of SOC channels and mechanisms of SOC channel activation. Previously, we described store-operated Imin channels in A431 cells. Here, by whole-cell recordings, we demonstrated that the store depletion activates two types of current in A431 cells--highly selective for divalent cations (presumably, ICRAC), and moderately selective (ISOC supported by Imin channels). These currents can be registered separately and have different developing time and amplitude. Coexisting of two different types of SOC channels in A431 cells seems to facilitate the control of intracellular Ca(2+)-dependent processes.

  11. Density-dependent induction of 92-kd type IV collagenase activity in cultures of A431 human epidermoid carcinoma cells.

    PubMed Central

    Xie, B.; Bucana, C. D.; Fidler, I. J.

    1994-01-01

    We examined the in vitro regulation of the production of two type IV collagenases, MMP-2 and MMP-9, by A431 human epidermoid carcinoma cells. The A431 cells were cultured under sparse or confluent conditions. The addition of transforming growth factor-beta (TGF-beta) or phorbolester-TPA to sparse cultures induced low levels of MMP-9 secretion, whereas in confluent cultures only TGF-beta produced this effect. Neither treatment altered the level of constitutive secretion of MMP-2. Treatment of sparse, actively growing cultures but not confluent stationary cultures with both TGF-beta and TPA produced synergistic induction of MMP-9 but did not affect MMP-2. A431 cells were grown as discrete large monolayer colonies. Radiolabeling with [3H]leucine or [3H]thymidine followed by autoradiography revealed that all the A431 cells in the colonies were metabolically active and only those on the periphery were dividing. Only these dividing A431 cells stained positive by anti-MMP-9 antibodies. Our results demonstrate that the synergistic induction of MMP-9 secretion in A431 cells occurs subsequent to stimulation by external signals in only noncontact-inhibited dividing tumor cells. These regulatory mechanisms may account for the in vivo finding that many proteinases are localized at the invasion front of a neoplasm where tumor cells are dividing and accessible to various environmental signals. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 PMID:8178929

  12. Cytotoxicity and apoptotic cell death induced by Vitis vinifera peel and seed extracts in A431 skin cancer cells.

    PubMed

    Grace Nirmala, J; Evangeline Celsia, S; Swaminathan, Akila; Narendhirakannan, R T; Chatterjee, Suvro

    2017-10-05

    Vitis vinifera. L is one of the most widely consumed fruits in the world and are rich in antioxidant abundant polyphenols. The present study was carried out to assess the antiproliferative and apoptotic effects of Vitis vinifera peel and seed extracts in an in vitro model using human epidermoid carcinoma A431 cell lines. Vitis vinifera peel and seed extracts were incubated with A431 cells to evaluate the antiproliferative, apoptotic effects and the morphological apoptotic changes induced by the extracts. Mitochondrial membrane potential was also measured after incubating the cells with extracts. At the inhibitory concentration (IC50), grape seed extract (111.11 µg/mL) and grape peel extract (319.14 µg/mL) were incubated for 24 h with A431 cells. Vitis vinifera peel and seed extracts were able to impart cytotoxic effects, induced apoptosis and apoptotic morphological changes in A431 cells significantly (p < 0.01) and this effect is associated with the interference with mitochondrial membrane potential. This reduction in mitochondrial membrane potential probably initiated the apoptotic cascade in the extracts treated cells. Vitis vinifera peel and seed phytochemicals can selectively target cancer cells and the phytochemicals that are occluded can serve as potential anticancer agents providing better efficacy in killing cancer cells.

  13. Effects of epidermal growth factor on glycolysis in A431 cells.

    PubMed

    Baulida, J; Onetti, R; Bassols, A

    1992-03-31

    A431 cells were treated with epidermal growth factor (EGF) to study the mechanism by which this factor accelerates the glycolytic flux. After EGF treatment, fructose-2,6-bisphosphate (Fru-2,6-P2) levels rose up to 2-fold. This change correlated with an increase in phosphofructokinase-2 activity, which was not due to a change in the transcription or translation of the enzyme, neither in the amount of enzyme. PK-C does not appear to be involved in the signalling mechanism since EGF was equally potent in PK-C depleted cells than in control cells. The increase in Fru-2,6-P2 levels was lower and more transient in cells treated with EGF in a calcium-free medium than in the presence of the cation, and it was restored by the addition of calcium to the medium. These results suggest a possible role for calcium-mediated pathways in the control of Fru-2,6-P2 levels in A431 cells.

  14. Formation of coated vesicles from coated pits in broken A431 cells

    PubMed Central

    1989-01-01

    Biochemical and morphological techniques were used to demonstrate the early steps in the endocytosis of transferrin in broken A431 cells. After binding 125I-transferrin, the cells were broken by scraping and then warmed. 125I-transferrin became inaccessible to exogenous anti- transferrin antibody providing a measure of the internalization process. Parallel morphological experiments using transferrin coupled to horseradish peroxidase confirmed internalization in broken cells. The process was characterized and compared with endocytosis in intact cells and showed many similar features. The system was used to show that both the appearance of new coated pits and the scission of coated pits to form coated vesicles were dependent on the addition of cytosol and ATP whereas invagination of pits was dependent on neither. PMID:2564003

  15. Honokiol, a chemopreventive agent against skin cancer, induces cell cycle arrest and apoptosis in human epidermoid A431 cells.

    PubMed

    Chilampalli, Chandeshwari; Guillermo, Ruth; Kaushik, Radhey S; Young, Alan; Chandrasekher, Gudiseva; Fahmy, Hesham; Dwivedi, Chandradhar

    2011-11-01

    Honokiol is a plant lignan isolated from bark and seed cones of Magnolia officinalis. Recent studies from our laboratory indicated that honokiol pretreatment decreased ultraviolet B-induced skin cancer development in SKH-1 mice. The aim of the present investigation was to study the effects of honokiol on human epidermoid squamous carcinoma A431 cells and to elucidate possible mechanisms involved in preventing skin cancer. A431 cells were pretreated with different concentrations of honokiol for a specific time period and investigated for effects on apoptosis and cell cycle analysis. Treatment with honokiol significantly decreased cell viability and cell proliferation in a concentration- and time-dependent manner. Honokiol pretreatment at 50 μmol/L concentration induced G0/G1 cell cycle arrest significantly (P < 0.05) and decreased the percentage of cells in the S and G2/M phase. Honokiol down-regulated the expression of cyclin D1, cyclin D2, Cdk2, Cdk4 and Cdk6 proteins and up-regulated the expression of Cdk's inhibitor proteins p21 and p27. Pretreatment of A431 cells with honokiol leads to induction of apoptosis and DNA fragmentation. These findings indicate that honokiol provides its effects in squamous carcinoma cells by inducing cell cycle arrest at G0/G1 phase and apoptosis.

  16. Several types of sodium-conducting channel in human carcinoma A-431 cells.

    PubMed

    Negulyaev YuA; Vedernikova, E A; Mozhayeva, G N

    1994-08-24

    Patch clamp method in outside-out configuration was used to search for cation channels which possibly mediate sodium influx through plasma membrane in A-431 carcinoma cells. We found four types of nonvoltage-gated Na-conducting channel. The first of 9-10 pS conductance (145 mM Na+, 30 degrees C) seems to be Na-selective; three others were characterized with conductance values of 24, 35 and 65 pS and lower selectivity among cations. Na-selective channels (9-10 pS) were not blocked by tetrodotoxin (1 microM). External application of amiloride (0.1-2 mM) resulted in a reversible inhibition of single currents through Na-selective channels.

  17. Lysosomal Signaling Enhances Mitochondria-Mediated Photodynamic Therapy in A431 Cancer Cells: Role of Iron

    PubMed Central

    Saggu, Shalini; Hung, Hsin-I; Quiogue, Geraldine; Lemasters, John J.; Nieminen, Anna-Liisa

    2015-01-01

    In photodynamic therapy (PDT), light activates a photosensitizer added to a tissue, resulting in singlet oxygen formation and cell death. The photosensitizer phthalocyanine 4 (Pc 4) localizes primarily to mitochondrial membranes in cancer cells, resulting in mitochondria-mediated cell death. The aim of this study was to determine how lysosomes contribute to PDT-induced cell killing by mitochondria-targeted photosensitizers such as Pc 4. We monitored cell killing of A431 cells after Pc 4-PDT in the presence and absence of bafilomycin, an inhibitor of the vacuolar proton pump of lysosomes and endosomes. Bafilomycin was not toxic by itself, but greatly enhanced Pc 4-PDT-induced cell killing. To investigate whether iron loading of lysosomes affects bafilomycin-induced killing, cells were incubated with ammonium ferric citrate (30 μm) for 30 h prior to PDT. Ammonium ferric citrate enhanced Pc 4 plus bafilomycin-induced cell killing without having toxicity by itself. Iron chelators (desferrioxamine and starch-desferrioxamine) and the inhibitor of the mitochondrial calcium (and ferrous iron) uniporter, Ru360, protected against Pc 4 plus bafilomycin toxicity. These results support the conclusion that chelatable iron stored in the lysosomes enhances the efficacy of bafilomycin-mediated PDT and that lysosomal disruption augments PDT with Pc 4. PMID:22220628

  18. A431 cell variants lacking the blood group A antigen display increased high affinity epidermal growth factor-receptor number, protein-tyrosine kinase activity, and receptor turnover

    PubMed Central

    1988-01-01

    The epidermal growth factor receptor (EGF-R) of human A431 cells bears an antigenic determinant that is closely related to the human blood group A carbohydrate structure. Labeling studies with blood group A reactive anti-EGF-R monoclonal antibodies and various lectins revealed that A431 cultures are heterogeneous with respect to blood group A expression. We have isolated clonal variants of these cells that either express (A431A+ cells) or completely lack (A431A- cells) the blood group A specific N-acetyl-D-galactosamine (GalNAc) residue. We show that this difference is due to the absence of a UDP-GalNAc:Gal transferase activity in A431A- cells. Subsequently, we have compared EGF-R functioning in these cell lines. Scatchard analysis of EGF- binding shows that in A431A- cells 6.3% of the EGF-R belongs to a high affinity subclass (Kd = 0.4 nM) while in A431A+ this subclass represents only 3.2% of the total receptor pool. The elevated level of high affinity receptors in A431A- cells is accompanied by a parallel increase in receptor protein- tyrosine kinase activity. In membrane preparations of A431A- cells, receptor autophosphorylation as well as phosphorylation of a tyrosine-containing peptide substrate is 2-3-fold higher as compared with A431A+ cells. In intact A431A-cells, the difference in receptor activity is measured as a 2-3-fold elevated level of receptor phosphorylation and a 2-3-fold higher abundance of phosphotyrosine in total cellular protein in A431A- cells. In addition, [35S]methionine pulse-chase experiments showed a ligand-independent increase in turnover of EGF-R in A431A- cells: the receptor's half life in these cells is 10 h as compared with 17 h in A431A+ cells. Our results suggest a possible involvement of GalNAc residue(s) in determining EGF-R affinity, protein-tyrosine kinase activity and turnover in A431 cells. Furthermore, our results indicate that high affinity EGF-R are the biologically active species with respect to protein-tyrosine kinase

  19. Resveratrol enhances ultraviolet B-induced cell death through nuclear factor-{kappa}B pathway in human epidermoid carcinoma A431 cells

    SciTech Connect

    Roy, Preeti; Kalra, Neetu; Nigam, Nidhi; George, Jasmine; Ray, Ratan Singh; Hans, Rajendra K.; Prasad, Sahdeo; Shukla, Yogeshwer

    2009-06-26

    Resveratrol has been reported to suppress cancer progression in several in vivo and in vitro models, whereas ultraviolet B (UVB), a major risk for skin cancer, is known to induce cell death in cancerous cells. Here, we investigated whether resveratrol can sensitize A431 human epidermoid carcinoma cells to UVB-induced cell death. We examined the combined effect of UVB (30 mJ/cm{sup 2}) and resveratrol (60 {mu}M) on A431 cells. Exposure of A431 carcinoma cells to UVB radiation or resveratrol can inhibit cell proliferation and induce apoptosis. However, the combination of resveratrol and UVB exposure was associated with increased proliferation inhibition of A431 cells compared with either agent alone. Furthermore, results showed that resveratrol and UVB treatment of A431 cells disrupted the nuclear factor-kappaB (NF-{kappa}B) pathway by blocking phosphorylation of serine 536 and inactivating NF-{kappa}B and subsequent degradation of I{kappa}B{alpha}, which regulates the expression of survivin. Resveratrol and UVB treatment also decreased the phosphorylation of tyrosine 701 of the important transcription factor signal transducer activator of transcription (STAT1), which in turn inhibited translocation of phospho-STAT1 to the nucleus. Moreover, resveratrol/UVB also inhibited the metastatic protein LIMK1, which reduced the motility of A431 cells. In conclusion, our study demonstrates that the combination of resveratrol and UVB act synergistically against skin cancer cells. Thus, resveratrol is a potential chemotherapeutic agent against skin carcinogenesis.

  20. Direct visualization of the phosphorylated epidermal growth factor receptor during its internalization in A-431 cells

    PubMed Central

    1987-01-01

    Epidermal growth factor (EGF) rapidly stimulates receptor autophosphorylation in A-431 cells. After 1 min the phosphorylated receptor can be identified at the plasma membrane using an anti- phosphotyrosine antibody. With further incubation at 37 degrees C, approximately 50% of the phosphorylated EGF receptor was internalized (t1/2 = 5 min) and associated with the tubulovesicular system and later with multivesicular bodies, but not the nucleus. During this period, there was no change in the extent or sites of phosphorylation. At all times the phosphotyrosine remained on the cytoplasmic side of the membrane, opposite to the EGF ligand identified by anti-EGF antibody. These data indicate that (a) the tyrosine-phosphorylated EGF receptor is internalized in its activated form providing a mechanism for translocation of the receptor kinase to substrates in the cell interior; (b) the internalized receptor remains intact for at least 60 min, does not associate with the nucleus, and does not generate any tyrosine-phosphorylated fragments; and (c) tyrosine phosphorylation alone is not the signal for receptor internalization. PMID:2447100

  1. Prolonged induction of p21Cip1/WAF1/CDK2/PCNA complex by epidermal growth factor receptor activation mediates ligand-induced A431 cell growth inhibition

    PubMed Central

    1995-01-01

    Proliferation of some cultured human tumor cell lines bearing high numbers of epidermal growth factor (EGF) receptors is paradoxically inhibited by EGF in nanomolar concentrations. In the present study, we have investigated the biochemical mechanism of growth inhibition in A431 human squamous carcinoma cells exposed to exogenous EGF. In parallel, we studied a selected subpopulation, A431-F, which is resistant to EGF-mediated growth inhibition. We observed a marked reduction in cyclin-dependent kinase-2 (CDK2) activity when A431 and A431-F cells were cultured with 20 nM EGF for 4 h. After further continuous exposure of A431 cells to EGF, the CDK2 activity remained at a low level and was accompanied by persistent G1 arrest. In contrast, the early reduced CDK2 activity and G1 accumulation in A431-F cells was only transient. We found that, at early time points (4-8 h), EGF induces p21Cip1/WAF1 mRNA and protein expression in both EGF-sensitive A431 cells and EGF-resistant A431-F cells. But only in A431 cells, was p21Cip1/WAF1 expression sustained at a significantly increased level for up to 5 d after addition of EGF. Induction of p21Cip1/WAF1 by EGF could be inhibited by a specific EGF receptor tyrosine kinase inhibitor, tyrphostin AG1478, suggesting that p21Cip1/WAF1 induction was a consequence of receptor tyrosine kinase activation by EGF. We also demonstrated that the increased p21Cip1/WAF1 was associated with both CDK2 and proliferating cell nuclear antigen (PCNA). Taken together, our results demonstrate that p21Cip1/WAF1 is an important mediator of EGF-induced G1 arrest and growth inhibition in A431 cells. PMID:7559780

  2. Control of cell motility by interaction of gangliosides, tetraspanins, and epidermal growth factor receptor in A431 versus KB epidermoid tumor cells.

    PubMed

    Park, Seung-Yeol; Yoon, Seon-Joo; Freire-de-Lima, Leonardo; Kim, Jung-Hoe; Hakomori, Sen-itiroh

    2009-08-17

    Growth of epidermoid carcinoma cell lines, A431 and KB, has been known to be controlled by the interaction of epidermal growth factor (EGF) and its receptor (EGFR) with tyrosine kinase. Ganglioside GM3 was previously found to interact with EGFR and to inhibit EGFR tyrosine kinase. However, motility of these cells, controlled by EGFR and ganglioside, was not studied. The present study is focused on the control mechanism of the motility of these cells through interaction of ganglioside, tetraspanin (TSP), and EGFR. Key results are as follows: (i) The level of EGFR expressed in A431 cells is approximately 6 times higher than that expressed in KB cells, and motility of A431 cells is also much higher than that of KB cells, yet growth of A431 cells is either not affected or is inhibited by EGF. In contrast, growth of KB cells is enhanced by EGF. (ii) Levels of TSPs (CD9, CD82, and CD81) expressed in A431 cells are much higher than those expressed in KB cells, and TSPs expressed in A431 cells are reduced by treatment of cells with EtDO-P4, which inhibits the synthesis of glycosphingolipids (GSLs) and gangliosides. (iii) These TSPs are co-immunoprecipitated with EGFR in both A431 and KB cells, indicating that TSPs are closely associated with EGFR. (iv) High motility of A431 cells is greatly reduced, while low motility of KB cells is not affected, by treatment of cells with EtDO-P4. These results, taken together, suggest that there is a close correlation between high motility of A431 cells and high expression of EGFR and TSPs, and between ganglioside GM3/GM2 and TSP. A similar correlation was suggested between the low motility of KB cells and low levels of EGFR and TSP. The correlation between high motility and high level of EGFR with the ganglioside-TSP complex in A431 cells is unique. This is in contrast to our previous studies that indicate that motility of many types of tumor cells is inhibited by a high level of CD9 or CD82, together with growth factor receptors and

  3. Fumonisin B1 does not prevent apoptosis in A431 human epidermoid carcinoma cells after photosensitization with a silicon phthalocyanine.

    PubMed

    Nagy, B; Chiu, S M; Separovic, D

    2000-09-01

    Photodynamic therapy with the phthalocyanine photosensitizer Pc 4 (Pc 4-PDT), an apoptosis inducer, is associated with accumulation of ceramide in various cell lines. The role of ceramide in Pc 4-PDT-induced apoptosis was investigated in A431 cells. Caspase-3 (casp-3) was activated and TUNEL positive cells began to appear 30 and 60 min post-Pc 4-PDT, respectively. A rapid increase (10 min) in cellular ceramide levels was observed after Pc 4-PDT. Induced ceramide accumulation was maintained over 60 min, Acid sphingomyelinase, a ceramide-generating enzyme, was inhibited after photosensitization with Pc 4, suggesting that the enzyme was not required for stimulated ceramide accumulation. Co-treatment of A431 cells with fumonisin B1, a ceramide synthase inhibitor, and Pc 4-PDT led to a decrease in ceramide levels without any effect on induced casp-3 activity or apoptosis. In the presence of zVAD, a pan-caspase inhibitor, apoptosis was abolished, while ceramide levels remained elevated after Pc 4-PDT. Exposure of A431 cells to exogenous C6-ceramide for 22 h, led to induction of apoptosis, and the process was abrogated by zVAD. In conclusion, C6-ceramide-, like Pc 4-PDT-induced apoptosis, is zVAD-sensitive. Furthermore, Pc 4 photosensitization can lead to apoptosis without FB-sensitive elevation in ceramide levels upstream of caspases.

  4. Combined gene expression and proteomic analysis of EGF induced apoptosis in A431 cells suggests multiple pathways trigger apoptosis.

    PubMed

    Alanazi, Ibrahim; Ebrahimie, Esmaeil; Hoffmann, Peter; Adelson, David L

    2013-11-01

    A431 cells, derived from epidermoid carcinoma, overexpress the epidermal growth factor receptor (EGFR) and when treated with a high dose of EGF will undergo apoptosis. We exploited microarray and proteomics techniques and network prediction to study the regulatory mechanisms of EGF-induced apoptosis in A431 cells. We observed significant changes in gene expression in 162 genes, approximately evenly split between pro-apoptotic and anti-apoptotic genes and identified 30 proteins from the proteomic data that had either pro or anti-apoptotic annotation. Our correlation analysis of gene expression and proteome modeled a number of distinct sub-networks that are associated with the onset of apoptosis, allowing us to identify specific pathways and components. These include components of the interferon signalling pathway, and down stream components, including cytokines and suppressors of cytokine signalling. A central component of almost all gene expression sub-networks identified was TP53, which is mutated in A431 cells, and was down regulated. This down regulation of TP53 appeared to be correlated with proteomic sub-networks of cytoskeletal or cell adhesion components that might induce apoptosis by triggering cytochrome C release. Of the only three genes also differentially expressed as proteins, only serpinb1 had a known association with apoptosis. We confirmed that up regulation and cleavage of serpinb1 into L-DNAaseII was correlated with the induction of apoptosis. It is unlikely that a single pathway, but more likely a combination of pathways is needed to trigger EGF induced apoptosis in A431cells.

  5. Regulation of apoptosis by resveratrol through JAK/STAT and mitochondria mediated pathway in human epidermoid carcinoma A431 cells

    SciTech Connect

    Madan, Esha; Prasad, Sahdeo; Roy, Preeti; George, Jasmine; Shukla, Yogeshwer

    2008-12-26

    Resveratrol (trans-3,4',5-trihydroxystilbene), a polyphenolic phytoalexin present mainly in grapes, red wine and berries, is known to possess strong chemopreventive and anticancer properties. Here, we demonstrated the anti-proliferative and apoptosis-inducing activities of resveratrol in human epidermoid carcinoma A431 cells. Resveratrol has cytotoxic effects through inhibiting cellular proliferation of A431 cells, which leads to the induction of apoptosis, as evident by an increase in the fraction of cells in the sub-G{sub 1} phase of the cell cycle and Annexin-V binding of externalized phosphatidylserine. Results revealed that inhibition of proliferation is associated with regulation of the JAK/STAT pathway, where resveratrol prevents phosphorylation of JAK, thereby inhibiting STAT1 phosphorylation. Furthermore, resveratrol treatment actively stimulated reactive oxygen species (ROS) and mitochondrial membrane depolarization. Consequently, an imbalance in the Bax/Bcl-2 ratio triggered the caspase cascade and subsequent cleavage of PARP, thereby shifting the balance in favor of apoptosis. These observations indicate that resveratrol treatment inhibits JAK/STAT-mediated gene transcription and induce the mitochondrial cell death pathway.

  6. Chelidonium majus L. extract induces apoptosis through caspase activity via MAPK-independent NF-κB signaling in human epidermoid carcinoma A431 cells.

    PubMed

    Park, Seung-Won; Kim, Seong Ryul; Kim, Youngchul; Lee, Jang-Hoon; Woo, Hong-Jung; Yoon, Yeo-Kwang; Kim, Young Il

    2015-01-01

    Chelidonium majus L. (C. majus L.) is known to possess certain biological properties such as anti-inflammatory, antimicrobial, antiviral and antitumor activities. We investigated the effects of C. majus L. extract on human epidermoid carcinoma A431 cells through multiple mechanisms, including induction of cell cycle arrest, activation of the caspase-dependent pathway, blocking of nuclear factor-κB (NF-κB) activation and involvement in the mitogen-activated protein kinase (MAPK) pathway. C. majus L. inhibited the proliferation of A431 cells in a dose- and time-dependent manner, increased the percentage of apoptotic cells, significantly decreased the mRNA levels of cyclin D1, Bcl-2, Mcl-1 and survivin and increased p21 and Bax expression. Exposure of A431 cells to C. majus L. extract enhanced the activities of caspase-3 and caspase-9, while co-treatment with C. majus L., the pan-caspase inhibitor Z-VAD-FMK and the caspase-3 inhibitor Z-DEVE-FMK increased the proliferation of A431 cells. C. majus L. extract not only inhibited NF-κB activation, but it also activated p38 MAPK and MEK/ERK signaling. Taken together, these results demonstrate that C. majus L. extract inhibits the proliferation of human epidermoid carcinoma A431 cells by inducing apoptosis through caspase activation and NF-κB inhibition via MAPK-independent pathway.

  7. Hydroxyl radical (·OH) played a pivotal role in oridonin-induced apoptosis and autophagy in human epidermoid carcinoma A431 cells.

    PubMed

    Yu, Yang; Fan, Si Miao; Song, Jun Ke; Tashiro, Shin-ichi; Onodera, Satoshi; Ikejima, Takashi

    2012-01-01

    Oridonin, a diterpenoid compound extracted and purified from Rabdosia rubescen, has been reported to induce tumor cell apoptosis through tyrosine kinase pathway. To further examine the mechanism of oridonin, we selected human epidermoid carcinoma A431 cell as a test object. Besides apoptosis, oridonin also induced A431 cell autophagy, and this autophagy antagonized apoptosis and played a protective role for A431 cells. Reactive oxygen species (ROS) played a pivotal role in induction of cytotoxicity. Therefore, a ROS scavenger, N-acetylcysteine (NAC) combined with oridonin was appiled. Results of morphologic observation, flow cytometric analysis and Western blot analysis showed that NAC could significantly reverse both ROS generation and down-regulation of mitochondrial membrane potential in oridonin treated cells. NAC inhibited oridonin induced apoptosis through both the intrinsic and extrinsic apoptotic pathways. NAC effectively inhibited both oridonin-induced apoptosis and autophagy by reducing intracellular oxidative stress. To further examine the mechanism of ROS, exogenous enzyme antioxidants (superoxide dismutase (SOD), catalase (CAT)) and non-enzyme antioxidants (glutathione (GSH)) were applied to detect the effect of oridonin on ROS generation. Only GSH exerted a similar role with NAC, suggesting that hydroxyl radical (·OH) played the major role in oridonin-induced cell death. Oridonin could decrease the GSH level in A431 cells in a dose-dependent manner. In addition, after treatment with ·OH donor, Fenton reagent, the changes in A431cells were similar to the results of oridonin treatment. All the results proved that ·OH played the pivotal role in oridonin induced apoptosis and autophagy in A431 cells.

  8. Effects of luteolin and quercetin, inhibitors of tyrosine kinase, on cell growth and metastasis-associated properties in A431 cells overexpressing epidermal growth factor receptor

    PubMed Central

    Huang, Y -T; Hwang, J -J; Lee, P -P; Ke, F -C; Huang, J -H; Huang, C -J; Kandaswami, C; Middleton, E; Lee, M -T

    1999-01-01

    Flavonoids display a wide range of pharmacological properties including anti-inflammatory. Anti-mutagenic, anti-carcinogenic and anti-cancer effects. Here, we evaluated the effects of eight flavonoids on the tumour cell proliferation, cellular protein phosphorylation, and matrix metalloproteinase (MMPs) secretion.Of the flavonoids examined, luteolin (Lu) and quercetin (Qu) were the two most potent agents, and significantly inhibited A431 cell proliferation with IC50 values of 19 and 21 μM, respectively.The epidermal growth factor (EGF) (10 nM) promoted growth of A431 cells (+25±4.6%) and mediated epidermal growth factor receptor (EGFR) tyrosine kinase activity and autophosphorylation of EGFR were inhibited by Lu and Qu. At concentration of 20 μM, both Lu and Qu markedly decreased the levels of phosphorylation of A431 cellular proteins, including EGFR.A431 cells treated with Lu or Qu exhibited protuberant cytoplasmic blebs and progressive shrinkage morphology. Lu and Qu also time-dependently induced the appearance of a ladder pattern of DNA fragmentation, and this effect was abolished by EGF treatment.The addition of EGF only marginally diminished the inhibitory effect of luteolin and quercetin on the growth rate of A431 cells, treatment of cellular proteins with EGF and luteolin or quercetin greatly reduced protein phosphorylation, indicating Lu and Qu may act effectively to inhibit a wide range of protein kinases, including EGFR tyrosine kinase.EGF increased the levels of matrix metalloproteinase-2 (MMP-2) and matrix metalloproteinase-9 (MMP-9), while Lu and Qu appeared to suppress the secretion of these two MMPs in A431 cells.Examination of the relationship between the chemical structure and inhibitory effects of eight flavonoids reveal that the double bond between C2 and C3 in ring C and the OH groups on C3′ and C4′ in ring B are critical for the biological activities.This study demonstrates that the inhibitory effects of Lu and Qu, and the

  9. Interference of silibinin with IGF-1R signalling pathways protects human epidermoid carcinoma A431 cells from UVB-induced apoptosis

    SciTech Connect

    Liu, Weiwei; Otkur, Wuxiyar; Li, Lingzhi; Wang, Qiong; He, Hao; Zang, Linghe; Hayashi, Toshihiko; Tashiro, Shin-ichi; Onodera, Satoshi; Xia, Mingyu; Ikejima, Takashi

    2013-03-08

    Highlights: ► Silibinin protects A431 cells from UVB irradiation-induced apoptosis. ► Up-regulation of the IGF-1R-JNK/ERK pathways by UVB induces cell apoptosis. ► Silibinin inhibits IGF-1R pathways to repress caspase-8-mediated apoptosis. -- Abstract: Ultraviolet B (UVB) from sunlight is a major cause of cutaneous lesion. Silibinin, a traditional hepatic protectant, elicits protective effects against UVB-induced cellular damage. In A431 cells, the insulin-like growth factor-1 receptor (IGF-1R) was markedly up-regulated by UVB irradiation. The activation of the IGF-1R signalling pathways contributed to apoptosis of the cells rather than rescuing the cells from death. Up-regulated IGF-1R stimulated downstream mitogen-activated protein kinases (MAPKs), such as c-Jun N-terminal kinases (JNK) and extracellular signal-regulated protein kinases 1/2 (ERK1/2). The subsequent activation of caspase-8 and caspase-3 led to apoptosis. The activation of IGF-1R signalling pathways is the cause of A431 cell death. The pharmacological inhibitors and the small interfering RNA (siRNA) targeting IGF-1R suppressed the downstream activation of JNK/ERK-caspases to help the survival of the UVB-irradiated A431 cells. Indeed, silibinin treatment suppressed the IGF-1R-JNK/ERK pathways and thus protected the cells from UVB-induced apoptosis.

  10. S100A7 induction is repressed by YAP via the Hippo pathway in A431 cells

    PubMed Central

    Wang, Junhao; Hu, Enze; Wang, Rui; Liu, Jin; Xiao, Qianqian; Zhang, Weiqing; He, Dacheng; Xiao, Xueyuan

    2016-01-01

    YAP is an oncogenic transcriptional co-activator and is inhibited by the Hippo pathway. Recent studies have revealed that YAP is also a sensor of cell morphology and cell density and can be phosphorylated by cytoskeleton reorganization. Our previous study demonstrated that S100A7 was upregulated in several squamous cell carcinoma (SCC) specimens and was dramatically induced in SCC cells by suspension and dense culture as well as in xenografts. However, little is known about how S100A7 induction occurs in cancer cells. Here, we identify that S100A7 induction is accompanied by YAP phosphorylation in both suspended and dense A431 cells. This correlation reverses after recovery of cell attachment or relief from dense culture. Further examination finds that S100A7 induction is repressed by nuclear YAP, which is further validated by activation or inhibition of the Hippo pathway via loss- and/or gain-of- LATS1 and MST1 function. Strikingly, disruption of the F-actin promotes S100A7 expression via YAP by activation of the Hippo pathway. Furthermore, we demonstrate that repression of S100A7 by YAP required TEAD1 transcriptional factor. Taken together, our findings demonstrate for the first time that S100A7 is repressed by YAP via the Hippo pathway. PMID:27203549

  11. Involvement of retinoblastoma (Rb) and E2F transcription factors during photodynamic therapy of human epidermoid carcinoma cells A431.

    PubMed

    Ahmad, N; Gupta, S; Mukhtar, H

    1999-03-11

    Photodynamic therapy (PDT), a promising new therapeutic modality for the management of a variety of solid malignancies and many non-malignant diseases, is a bimodal therapy using a porphyrin based photosensitizing chemical and visible light. The proper understanding of the mechanism of PDT-mediated cancer cell-kill may result in improving the efficacy of this treatment modality. Earlier we have shown (Proc. Natl. Acad. Sci. USA; 95: 6977-6982, 1998) that silicon phthalocyanine (Pc4)-PDT results in an induction of the cyclin kinase inhibitor WAF1/CIP1/p21 which, by inhibiting cyclins (E and D1) and cyclin dependent kinases (cdk2 and cdk6), results in a G0/G1-phase arrest followed by apoptosis in human epidermoid carcinoma cells A431. We have also demonstrated the generation of nitric oxide during PDT-mediated apoptosis (Cancer Res.; 58: 1785-1788, 1998). Retinoblastoma (pRb) and E2F family transcription factors are important proteins, which regulate the G1-->S transition in the cell cycle. Here, we provide evidence for the involvement of pRb-E2F/DP machinery as an important contributor of PDT-mediated cell cycle arrest and apoptosis. Western blot analysis demonstrated a decrease in the hyper-phosphorylated form of pRb at 3, 6 and 12 h post-PDT with a relative increase in hypo-phosphorylated pRb. Western blot analysis also revealed that PDT-caused decrease in phosphorylation of pRb occurs at serine-780. The ELISA data demonstrated a time dependent accumulation of hypo-phosphorylated pRb by PDT. This response was accompanied with down-regulation in the protein expression of all five E2F (1-5) family transcription factors, and their heterodimeric partners DP1 and DP2. These results suggest that Pc4-PDT of A431 cells results in a down regulation of hyper-phosphorylated pRb protein with a relative increase in hypo-phosphorylated pRb that, in turn, compromises with the availability of free E2F. We suggest that these events result in a stoppage of the cell cycle

  12. EGF raises cytosolic Ca sup 2+ in A431 and Swiss 3T3 cells by a dual mechanism

    SciTech Connect

    Pandiella, A.; Malgaroli, A.; Meldolesi, J.; Vicentini, L.M. )

    1987-05-01

    The changes in Ca{sup 2+} homeostasis and phosphoinositide hydrolysis induced by EGF were studied in human epidermoid carcinoma A431 cells both when attached to a substratum and after detachment and suspension. The cytosolic Ca{sup 2+} concentration was measured by the conventional fluorimetric technique, using the specific probe, quin2, as well as by a new microscopic technique in which single cells are investigated after loading with another probe, fura-2. EGF applied in the complete, Ca{sup 2+}-containing medium caused a rapid rise in the cytosolic {sup 45}Ca{sup 2+} concentration, that remained elevated for several minutes. In Ca{sup 2+}-free, EGTA-containing medium, part of this response persisted, as revealed by quin2 results in suspended cells and microscopic results with fura-2. These results, as well as additional microscopic fura-2 results in Swiss 3T3 fibroblasts, demonstrate that the Ca{sup 2+} signal elicited by EGF is due to two components: redistribution from an intracellular store and stimulated influx across the plasmalemma. This latter process was not detected in 3T3 cells treated with either PDGF or bombesin. It is therefore suggested that the {sup 45}Ca{sup 2+} influx effect of EGF is under the control of a separate, as yet unidentified mechanism.

  13. Inhibition of microRNA-21 upregulates the expression of programmed cell death 4 and phosphatase tensin homologue in the A431 squamous cell carcinoma cell line

    PubMed Central

    LI, XIAOHONG; HUANG, KAI; YU, JIANBIN

    2014-01-01

    microRNA-21 (miRNA/miR-21) is a well-known oncogenic miRNA that is overexpressed in various carcinomas. The tumor suppressor genes, programmed cell death 4 (PDCD4) and phosphatase tensin homologue (PTEN), which target miR-21, are underexpressed in several types of cancer. However, the expression of miR-21 and its target genes, PDCD4 and PTEN, has not yet been reported in skin squamous cell carcinoma (SCC). In the present study, anti-miR-21 was transfected into the A431 cell line, and the expression of miR-21, PDCD4 and PTEN and the level of cell apoptosis were detected by quantitative polymerase chain reaction, immunocytochemistry and western blotting, and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling, respectively. The expression levels of PDCD4 and PTEN in the A431 cell line transfected with anti-miR-21 were significantly increased (P<0.05) and the apoptotic ratio was significantly increased (P<0.05). The data indicate that miR-21 may play an oncogenic role in the cellular processes of SCC and represent a novel target for effective therapies. PMID:24959246

  14. The Transcription Factor AP-1 Is Required for EGF-induced Activation of Rho-like GTPases, Cytoskeletal Rearrangements, Motility, and In Vitro Invasion of A431 Cells

    PubMed Central

    Malliri, Angeliki; Symons, Marc; Hennigan, Robert F.; Hurlstone, Adam F.L.; Lamb, Richard F.; Wheeler, Tricia; Ozanne, Bradford W.

    1998-01-01

    Human squamous cell carcinomas (SCC) frequently express elevated levels of epidermal growth factor receptor (EGFR). EGFR overexpression in SCC-derived cell lines correlates with their ability to invade in an in vitro invasion assay in response to EGF, whereas benign epidermal cells, which express low levels of EGFR, do not invade. EGF-induced invasion of SCC-derived A431 cells is inhibited by sustained expression of the dominant negative mutant of c-Jun, TAM67, suggesting a role for the transcription factor AP-1 (activator protein-1) in regulating invasion. Significantly, we establish that sustained TAM67 expression inhibits growth factor–induced cell motility and the reorganization of the cytoskeleton and cell-shape changes essential for this process: TAM67 expression inhibits EGF-induced membrane ruffling, lamellipodia formation, cortical actin polymerization and cell rounding. Introduction of a dominant negative mutant of Rac and of the Rho inhibitor C3 transferase into A431 cells indicates that EGF-induced membrane ruffling and lamellipodia formation are regulated by Rac, whereas EGF-induced cortical actin polymerization and cell rounding are controlled by Rho. Constitutively activated mutants of Rac or Rho introduced into A431 or A431 cells expressing TAM67 (TA cells) induce equivalent actin cytoskeletal rearrangements, suggesting that the effector pathways downstream of Rac and Rho required for these responses are unimpaired by sustained TAM67 expression. However, EGF-induced translocation of Rac to the cell membrane, which is associated with its activation, is defective in TA cells. Our data establish a novel link between AP-1 activity and EGFR activation of Rac and Rho, which in turn mediate the actin cytoskeletal rearrangements required for cell motility and invasion. PMID:9817764

  15. Heat Induces Intracellular Acidification in Human A-431 Cells: Role of Na+ -H+ Exchange and Metabolism

    DTIC Science & Technology

    1990-01-01

    measured according to methods of for comparison of groups (40). Boron (2) and Ng and Dudley (31). pHi of cells (1 × 106 Chemicals . BCECF/AM, nigericin , and...its role in regulating pH, has been partially charac- for these cells: [Ki = 137 ± 13 mMI. Nigericin (3 uM) terized 137, 38). Finally, the effect of...valinomycin cells/ml) was set by exposing the cells to nigericin (2 were purchased from Molecular Probes (Eugene, OR). mM) for 5 min in K Hanks

  16. Ebselen reduces the toxicity of mechlorethamine in A-431 cells via inhibition of apoptosis.

    PubMed

    Lulla, Anju; Pino, Maria A; Piętka-Ottlik, Magdalena; Młochowski, Jacek; Sparavalo, Oleksiy; Billack, Blase

    2013-06-01

    A series of test compounds were evaluated for an ability to reduce the toxicity of the nitrogen mustard mechlorethamine (HN2) in vitro. The test compounds included resveratrol, pterostilbene, vitamin C, ebselen, ebselen diselenide, and ebselen-sulfur. Among them, ebselen demonstrated the highest degree of protection against HN2 toxicity. To this end, pretreatment of the cells with ebselen offered protection against the toxicant whereas no protection was observed when cells were first incubated with HN2 and then treated with ebselen. Significant increases in caspase 3 and caspase 9 activities were observed in response to HN2, and ebselen was found to reduce these effects. Taken together, the data presented here indicate that ebselen is an effective countermeasure to nitrogen mustard in vitro, which is worthy of future investigation in vivo. © 2013 Wiley Periodicals, Inc.

  17. Enhanced antitumor activity in A431 cells via encapsulation of 20(R)-ginsenoside Rg3 in PLGA nanoparticles.

    PubMed

    Zhang, Shaozhi; Liu, Jiwei; Ge, Baojian; Du, Meiling; Fu, Li; Fu, Yushan; Yan, Qiu

    2017-10-01

    The objective of this study is to investigate the encapsulation of 20(R)-ginsenoside Rg3 (20(R)-Rg3) using polylactic-co-glycolic acid (PLGA) and promotion for its antitumor activity. Preparation and evaluation of the antitumor efficacy of 20(R)-Rg3-loaded PLGA nanoparticles were the first reported. The data will be helpful to apply 20(R)-Rg3 efficiently and broadly in new drug form development and clinical cancer treatment. The nanoparticles were prepared using emulsion and solvent evaporation methods. The uniform particle size and good dispersion were further confirmed by scanning electron microscopy. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay was applied to detect cell proliferation after 20(R)-Rg3-loaded PLGA nanoparticles treatment. Western blotting and immunofluorescent staining were used for observation of key proteins related with proliferation and apoptosis. Cell cycle and apoptosis were analyzed by flow cytometer technology. The results showed that the size of 20(R)-Rg3-loaded PLGA was 97.5 nm in diameter, and zeta potential was -28 mV detected by Malvern particle size analyzer. The encapsulation efficiency was 97.5%, and drug loading was 70.2% measured by high-performance liquid chromatography. The in vitro study showed that the encapsulated 20(R)-Rg3 was consecutively released and the release ratio reached to the highest value (19.36%) at the time point of 96 h. The encapsulated 20(R)-Rg3 significantly inhibited the proliferation and induced apoptosis in A431 cancer cells compared with the unencapsulated 20(R)-Rg3, control and PLGA alone. 20(R)-Rg3-loaded PLGA nanoparticles was well prepared and characterized. The antitumor activity was increased after PLGA encapsulation. The data will be beneficial to the development of new dosage forms of 20(R)-Rg3 and extensive application.

  18. Bispecific designed ankyrin repeat proteins (DARPins) targeting epidermal growth factor receptor inhibit A431 cell proliferation and receptor recycling.

    PubMed

    Boersma, Ykelien L; Chao, Ginger; Steiner, Daniel; Wittrup, K Dane; Plückthun, Andreas

    2011-12-02

    The EGF receptor (EGFR) has been implicated in the development and progression of many tumors. Although monoclonal antibodies directed against EGFR have been approved for the treatment of cancer in combination with chemotherapy, there are limitations in their clinical efficacy, necessitating the search for robust targeting molecules that can be equipped with new effector functions or show a new mechanism of action. Designed ankyrin repeat proteins (DARPins) may provide the targeting component for such novel reagents. Previously, four DARPins were selected against EGFR with (sub)nanomolar affinity. As any targeting module should preferably be able to inhibit EGFR-mediated signaling, their effect on A431 cells overexpressing EGFR was examined: three of them were shown to inhibit proliferation by inducing G(1) arrest, as seen for the Food and Drug Administration-approved antibody cetuximab. To understand this inhibitory mechanism, we mapped the epitopes of the DARPins using yeast surface display. The epitopes for the biologically active DARPins overlapped with the EGF-binding site, whereas the fourth DARPin bound to a different domain, explaining the lack of a biological effect. To optimize the biological activity of the DARPins, we combined two DARPins binding to different epitopes with a flexible linker or with a leucine zipper, leading to a homodimer. The latter DARPin was able to reduce surface EGFR by inhibiting receptor recycling, leading to a dramatic decrease in cell viability. These results indicate that multispecific EGFR-specific DARPins are superior to cetuximab and may form the basis of new opportunities in tumor targeting and tumor therapy.

  19. Downregulated AP-1 activity is associated with inhibition of Protein-Kinase-C-dependent CD44 and ezrin localisation and upregulation of PKC theta in A431 cells.

    PubMed

    Stapleton, Genevieve; Malliri, Angeliki; Ozanne, Bradford W

    2002-07-01

    Progression to an invasive, metastatic tumour requires the coordinated expression and function of a number of gene products, as well as their regulation in the context of invasion. The transcription factor AP-1 regulates expression of many of those genes necessary for implementation of the invasion programme. Two such gene products, CD44 and ezrin, are both upregulated in fibroblasts transformed by v-fos and are commonly implicated in cell motility and invasion. Here we report that CD44 and ezrin colocalise to membrane ruffles and microvilli of A431 cells after treatment with EGF. However, A431 cells expressing dominant-negative c-Jun (TAM67), and which as a consequence fail to invade in response to EGF, also fail to correctly localise CD44 and ezrin. CD44 and ezrin are both substrates for Protein Kinase C, and we show that their EGF-dependent colocalisation requires Protein Kinase C activity. Associated with TAM67 expression and disrupted CD44 and ezrin colocalisation is the increased expression and activation of the novel PKC theta isoform. Expression of PKC theta in A431 cells results in the inhibition of cell motility and disrupted localisation of CD44 and ezrin. We propose that AP-1 regulates the integrity of Protein Kinase C signalling and identifies PKC theta as a potential suppressor of the invasion programme.

  20. Recombinant human tumor necrosis factor alpha does not potentiate cell killing after photodynamic therapy with a silicon phthalocyanine in A431 human epidermoid carcinoma cells.

    PubMed

    Azizuddin, K; Kalka, K; Chiu, S M; Ahmad, N; Mukhtar, H; Separovic, D

    2001-02-01

    Photodynamic therapy (PDT) is a novel cancer treatment utilizing a photosensitizer, visible light and oxygen. PDT with the silicon phthalocyanine Pc 4, a new photosensitizer, is highly effective in cancer cell destruction and tumor ablation. The mechanisms underlying cancer cell killing by PDT are not fully understood. Tumor necrosis factor alpha (TNF) is a multifunctional cytokine that has been implicated in photocytotoxicity. We asked whether recombinant human TNF (rhTNF) affects Pc 4-PDT cytotoxicity in A431 human epidermoid carcinoma cells. Co-treatment of A431 cells with various doses of Pc 4-PDT and a sub-lethal rhTNF dose led to a sub-additive reduction in cell survival. In addition, in the presence of Pc 4-PDT or rhTNF, caspase-3 activity and apoptosis were induced. The combined treatment, however, did not potentiate either caspase-3 activity or apoptosis. Similar to previous findings we observed that Pc 4-PDT initiated a time-dependent extracellular TNF accumulation. The data suggest that: a) PDT and rhTNF induce cancer cell killing through different mechanisms; and b) Pc 4-PDT-induced TNF production is a stress response that may not directly affect photocytotoxicity.

  1. Acquired resistance to EGFR tyrosine kinase inhibitor in A431 squamous cell carcinoma xenografts is mediated by c-Kit pathway transduction.

    PubMed

    Zhang, Lixia; Yang, Xiaokun; Zhao, Bei; Cai, Zhen

    2015-04-01

    Epidermal growth factor inhibitors (EGFRIs), the first targeted cancer therapy, are currently an essential treatment for many advance-stage epithelial cancers. These agents have the superior ability to target cancers cells and better safety profile compared to conventional chemotherapies. However, all responding patients eventually developed acquired resistance to EGFRIs and the mechanisms of acquired resistance invariably develops. In the current study, we reported the tumor xenografts of the human A431 squamous cell carcinoma, after 25-week consecutive therapy with EGFR inhibitor (gefitinib) that developed resistance as a result of c-Kit overexpression. Moreover, combined therapeutic inhibition of EGFR and c-Kit may abrogate this acquired mechanism of drug resistance due to an enhanced apoptotic effect in gefitinib-resistant xenograft model. Taken together, the results suggest that at least in the A431 xenograft model displaying acquired resistance to gefitinib can emerge in vivo, at least in part, by mechanisms involving the c-Kit overexpression.

  2. Extracellular polymeric substance from Aphanizomenon flos-aquae induces apoptosis via the mitochondrial pathway in A431 human epidermoid carcinoma cells.

    PubMed

    Xue, Xing; Lv, Ying; Liu, Qing; Zhang, Xiaolan; Zhao, Youhong; Zhang, Lili; Xu, Shiyuan

    2015-09-01

    Extracellular polymeric substance (EPS) is a substance secreted during algal growth, which has been found to have numerous health-promoting effects. In the present study, A431 human epidermoid carcinoma cells were selected as target cells and cultivated in vitro as an experimental model to investigate the anti-cancer effect of extracellular polymeric substances from Aphanizomenon flos-aquae (EPS-A) and the possible underlying mechanism. Apoptosis- and cell cycle-associated molecules as well as the mitochondrial membrane potential of the cells were quantified using flow cytometry (FCM). FCM showed that EPS-A induced cell cycle arrest, which led to a loss of mitochondrial function of the A431 cells and an increase in necrotic and late apoptotic cells. In order to evaluate the apoptosis and cell viability, acridine orange/ethidium bromide staining was used, morphological changes were observed using fluorescence microscopy and typical apoptotic characteristics were observed. Following treatment with a high dose of EPS-A, transmission electron microscopy showed nuclear fragmentation, chromosome condensation, cell shrinkage and expansion of the endoplasmic reticulum; apoptotic bodies were also observed. In conclusion, EPS-A caused cell cycle arrest, stimulated cell apoptosis via the mitochondrial pathway and exhibited important anti-cancer activity.

  3. Extracellular polymeric substance from Aphanizomenon flos-aquae induces apoptosis via the mitochondrial pathway in A431 human epidermoid carcinoma cells

    PubMed Central

    XUE, XING; LV, YING; LIU, QING; ZHANG, XIAOLAN; ZHAO, YOUHONG; ZHANG, LILI; XU, SHIYUAN

    2015-01-01

    Extracellular polymeric substance (EPS) is a substance secreted during algal growth, which has been found to have numerous health-promoting effects. In the present study, A431 human epidermoid carcinoma cells were selected as target cells and cultivated in vitro as an experimental model to investigate the anti-cancer effect of extracellular polymeric substances from Aphanizomenon flos-aquae (EPS-A) and the possible underlying mechanism. Apoptosis- and cell cycle-associated molecules as well as the mitochondrial membrane potential of the cells were quantified using flow cytometry (FCM). FCM showed that EPS-A induced cell cycle arrest, which led to a loss of mitochondrial function of the A431 cells and an increase in necrotic and late apoptotic cells. In order to evaluate the apoptosis and cell viability, acridine orange/ethidium bromide staining was used, morphological changes were observed using fluorescence microscopy and typical apoptotic characteristics were observed. Following treatment with a high dose of EPS-A, transmission electron microscopy showed nuclear fragmentation, chromosome condensation, cell shrinkage and expansion of the endoplasmic reticulum; apoptotic bodies were also observed. In conclusion, EPS-A caused cell cycle arrest, stimulated cell apoptosis via the mitochondrial pathway and exhibited important anti-cancer activity. PMID:26622416

  4. Turmeric toxicity in A431 epidermoid cancer cells associates with autophagy degradation of anti-apoptotic and anti-autophagic p53 mutant.

    PubMed

    Thongrakard, Visa; Titone, Rossella; Follo, Carlo; Morani, Federica; Suksamrarn, Apichart; Tencomnao, Tewin; Isidoro, Ciro

    2014-12-01

    The keratinocyte-derived A431 Squamous Cell Carcinoma cells express the p53R273H mutant, which has been reported to inhibit apoptosis and autophagy. Here, we show that the crude extract of turmeric (Curcuma longa), similarly to its bioactive component Curcumin, could induce both apoptosis and autophagy in A431 cells, and these effects were concomitant with degradation of p53. Turmeric and curcumin also stimulated the activity of mTOR, which notoriously promotes cell growth and acts negatively on basal autophagy. Rapamycin-mediated inhibition of mTOR synergized with turmeric and curcumin in causing p53 degradation, increased the production of autophagosomes and exacerbated cell toxicity leading to cell necrosis. Small-interference mediated silencing of the autophagy proteins BECLIN 1 or ATG7 abrogated the induction of autophagy and largely rescued p53 stability in Turmeric-treated or Curcumin-treated cells, indicating that macroautophagy was mainly responsible for mutant p53 degradation. These data uncover a novel mechanism of turmeric and curcumin toxicity in chemoresistant cancer cells bearing mutant p53.

  5. Oligomerization of epidermal growth factor receptors on A431 cells studied by time-resolved fluorescence imaging microscopy. A stereochemical model for tyrosine kinase receptor activation

    PubMed Central

    1995-01-01

    The aggregation states of the epidermal growth factor receptor (EGFR) on single A431 human epidermoid carcinoma cells were assessed with two new techniques for determining fluorescence resonance energy transfer: donor photobleaching fluorescence resonance energy transfer (pbFRET) microscopy and fluorescence lifetime imaging microscopy (FLIM). Fluorescein-(donor) and rhodamine-(acceptor) labeled EGF were bound to the cells and the extent of oligomerization was monitored by the spatially resolved FRET efficiency as a function of the donor/acceptor ratio and treatment conditions. An average FRET efficiency of 5% was determined after a low temperature (4 degrees C) incubation with the fluorescent EGF analogs for 40 min. A subsequent elevation of the temperature for 5 min caused a substantial increase of the average FRET efficiency to 14% at 20 degrees C and 31% at 37 degrees C. In the context of a two-state (monomer/dimer) model for the EGFR, these FRET efficiencies were consistent with minimal average receptor dimerizations of 13, 36, and 69% at 4, 20, and 37 degrees C, respectively. A431 cells were pretreated with the monoclonal antibody mAb 2E9 that specifically blocks EGF binding to the predominant population of low affinity EGFR (15). The average FRET efficiency increased dramatically to 28% at 4 degrees C, indicative of a minimal receptor dimerization of 65% for the subpopulation of high affinity receptors. These results are in accordance with prior studies indicating that binding of EGF leads to a fast and temperature- dependent microclustering of EGFR, but suggest in addition that the high affinity functional subclass of receptors on quiescent A431 cells are present in a predimerized or oligomerized state. We propose that the transmission of the external ligand-binding signal to the cytoplasmic domain is effected by a concerted relative rotational rearrangement of the monomeric units comprising the dimeric receptor, thereby potentiating a mutual activation of

  6. The endocytosis of epidermal growth factor in A431 cells: A pH of microenvironment and the dynamics of receptor complex dissociation

    SciTech Connect

    Sorkin, A.D.; Teslenko, L.V.; Nikolsky, N.N. )

    1988-03-01

    The endocytosis and intracellular fate of epidermal growth factor (EGF) were studied in A431 cells. After 15-20 min of internalization at 37{degree}C, rhodomaine-labeled ({sup 125}-I) EGF (EGR-Rh) accumulated into large juxtanuclear compartment consisting of closely related vesicles. This structure was shown to be localized in the para-Golgi region. Fluorescein-labeled transferrin (Tr-FITC) was observed in the same region when added to the cell simultaneously with EGF-Rh. Using microscopy spectrofluorometer, the authors determined that the Tr-FITC-containing para-Golgi structures have a pH of 6.1{plus minus}0.3 while lysosomes containing dextran-fluorescein have a pH of 5.0{plus minus}0.2. To study the dynamics of EGF-receptor dissociation during endocytosis a mild detergent treatment of living cells was used for extraction of an intracellular receptor-unbound EGF. These results suggest that EGF remains associated with receptors during endocytosis in A431 cells until it is transferred to lysosomes where the pH of the EGF microenvironment is dropped to 5. A prolonged presence of EGF-receptor complexes in the para-Golgi region might be of importance in mitotic signaling.

  7. Evaluating the promiscuous nature of tyrosine kinase inhibitors assessed in A431 epidermoid carcinoma cells by both chemical- and phosphoproteomics.

    PubMed

    Giansanti, Piero; Preisinger, Christian; Huber, Kilian V M; Gridling, Manuela; Superti-Furga, Giulio; Bennett, Keiryn L; Heck, Albert J R

    2014-07-18

    Deregulation of protein tyrosine kinase signaling has been linked to many diseases, most notably cancer. As a consequence, small molecule inhibitors of protein tyrosine kinases may provide powerful strategies for treatment. Following the successful introduction of imatinib in the treatment of chronic myelogenous leukemia, such drugs are also now evaluated for other types of cancer. However, many developed kinase inhibitors are not very target-specific and therefore may induce side effects. The importance of such side effects is certainly cell-proteome dependent. Understanding the all-inclusive action of a tyrosine kinase inhibitor on each individual cell-type entails the identification of potential targets, combined with monitoring the downstream effects revealing the signaling networks involved. Here, we explored a multilevel quantitative mass spectrometry-based proteomic strategy to identify the direct targets and downstream signaling effect of four tyrosine kinase inhibitors (imatinib, dasatinib, bosutinib, and nilotinib) in epidermoid carcinoma cells, as a model system for skin-cancer. More than 25 tyrosine kinases showed affinity to the drugs, with imatinib and nilotinib displaying a high specificity, especially when compared to dasatinib and bosutinib. Consequently, the latter two drugs showed a larger effect on downstream phosphotyrosine signaling. Many of the proteins affected are key regulators in cell adhesion and invasion. Our data represents a multiplexed view on the promiscuous action of certain tyrosine kinase inhibitors that needs to be taking into consideration prior to the application of these drugs in the treatment of different forms of cancer.

  8. Differential responses of skin cancer-chemopreventive agents silibinin, quercetin, and epigallocatechin 3-gallate on mitogenic signaling and cell cycle regulators in human epidermoid carcinoma A431 cells.

    PubMed

    Bhatia, N; Agarwal, C; Agarwal, R

    2001-01-01

    Silibinin, quercetin, and epigallocatechin 3-gallate (EGCG) have been shown to be skin cancer-preventive agents, albeit by several different mechanisms. Here, we assessed whether these agents show their cancer-preventive potential by a differential effect on mitogenic signaling molecules and cell cycle regulators. Treatment of human epidermoid carcinoma A431 cells with these agents inhibited the activation of the epidermal growth factor receptor and the downstream adapter protein Shc, but only silibinin showed a marked inhibition of mitogen-activated protein kinase-extracellular signal-regulated kinase-1 and -2 activation. In terms of cell cycle regulators, silibinin treatment showed an induction of Cip1/p21 and Kip1/p27 together with a significant decrease in cyclin-dependent kinase (CDK)-4, CDK2, and cyclin D1. Quercetin treatment, however, resulted in a moderate increase in Cip1/p21 with no change in Kip1/p27 and a decrease in CDK4 and cyclin D1. EGCG treatment also led to an induction of Cip1/p21 but no change in Kip1/27, CDK2, and cyclin D1 and a decrease in CDK4 only at low doses. Treatment of cells with these agents resulted in a strong dose- and time-dependent cell growth inhibition. A high dose of silibinin and low and high doses of quercetin and EGCG also led to cell death by apoptosis, suggesting that a lack of their inhibitory effect on mitogen-activated protein kinase-extracellular signal-regulated kinase-1 and -2 activation possibly "turns on" an apoptotic cell death response associated with their cancer-preventive and anticarcinogenic effects. Together, these results suggest that silibinin, quercetin, and EGCG exert their cancer-preventive effects by differential responses on mitogenic signaling and cell cycle regulators.

  9. Divalent lead cations induce cyclooxygenase-2 gene expression by epidermal growth factor receptor/nuclear factor-kappa B signaling in A431carcinoma cells.

    PubMed

    Chou, Yii-Her; Woon, Peng-Yeong; Huang, Wan-Chen; Shiurba, Robert; Tsai, Yao-Ting; Wang, Yu-Shiuan; Hsieh, Tusty-Jiuan; Chang, Wen-Chang; Chuang, Hung-Yi; Chang, Wei-Chiao

    2011-06-10

    Divalent lead cations (Pb²+) are toxic metal pollutants that may contribute to inflammatory diseases in people and animals. Human vascular smooth muscle cells in culture respond to low concentrations of Pb²+ ions by activating mediators of inflammation via the plasma membrane epidermal growth factor receptor (EGFR). These include cyclooxygenase-2 (COX-2) and cytosolic phospholipase A₂ as well as the hormone-like lipid compound prostaglandin E₂. To further clarify the mechanism by which Pb²+ induces such mediators of inflammation, we tested human epidermoid carcinoma cell line A431 that expresses high levels of EGFR. Reverse transcription PCR and western blots confirmed A431 cells treated with a low concentration (1 μM) of Pb²+ in the form of lead (II) nitrate increased expression of COX-2 mRNA and its encoded protein in a time-dependent manner. Promoter deletion analysis revealed the transcription factor known as nuclear factor-kappa B (NF-κB) was a necessary component of the COX-2 gene response. NF-κB inhibitor BAY 11-7082 suppressed Pb²+-induced COX-2 mRNA expression, and EGFR inhibitors AG1478 and PD153035 as well as EGFR small interfering RNA reduced the coincident nuclear translocation of NF-κB. Our findings support the hypothesis that low concentrations of Pb²+ ions incite inflammation by inducing COX-2 gene expression via the EGFR/NF-κB signal transduction pathway. Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.

  10. Induction of apoptosis in A431 skin cancer cells by Cissus quadrangularis Linn stem extract by altering Bax-Bcl-2 ratio, release of cytochrome c from mitochondria and PARP cleavage.

    PubMed

    Bhujade, Arti; Gupta, G; Talmale, S; Das, S K; Patil, M B

    2013-02-01

    Skin is generally damaged through genetic and environmental factors such as smoking, exposure to xenobiotics, heat, hormonal changes, and ultraviolet light. These factors can cause skin diseases. Cissus quadrangularis Linn. (CQ) has been used in folk medicine for the treatment of skin diseases since ancient times. Taking in to consideration the medicinal properties exhibited by this genus, it was decided to investigate the anti-cancer activity of CQ. Extracts obtained from CQ and their phenolic contents were subjected to in vitro evaluation of anticancer activity by using A431 (skin epidermoid carcinoma, human) cell line. The A431 cells were treated with different extracts of CQ in a dose dependent manner. Out of five extracts, the acetone extract demonstrated significant anti-cancer activity in the A431 cell line. Hexane, chloroform, ethyl acetate and methanol extracts also exhibited cytotoxicity but to a comparatively lesser extent than the acetone extract. The GI(50) value of the acetone extract was found to be 8 μg mL(-1), whereas GI(50) value of purified fraction of acetone extract, termed as AFCQ (active acetone fraction of CQ) with respect to A431 cells, was found to be 4.8 μg mL(-1). Furthermore, the mechanism of anticancer activity exhibited by AFCQ was investigated by comparing its effect with the standard anticancer drug Doxorubicin (DOX) by evaluating the status of apoptotic markers after treatment of A431 cells with AFCQ and DOX. Bax-Bcl-2 ratio along with the release of cytochrome c from mitochondria to cytoplasm, which is a hallmark of apoptosis, was also evaluated. Cleavage of PARP revealed that AFCQ induces apoptosis in A431 cells with reference to DOX.

  11. Recombinant human IgG antibodies recognizing distinct extracellular domains of EGF receptor exhibit different degrees of growth inhibitory effects on human A431 cancer cells.

    PubMed

    Chang, Chialun; Takayanagi, Atsushi; Yoshida, Tetsuhiko; Shimizu, Nobuyoshi

    2013-05-01

    Recently, we isolated 4 distinct kinds of single chain antibody against human EGF receptor (EGFR) after screening the Keio phage display scFv library by using two methods of target-guided proximity labeling. In the current study, these monovalent scFv antibodies were converted to bivalent IgGs of humanized forms (hIgGs) by recombinant technology using the specially designed expression vectors followed by protein production in CHO cells. The resulting recombinant hIgGs were examined for their binding specificity using several different transformed human BJ cell lines that express deletion mutants of EGFR, each lacking one of 4 distinct extracellular domains (L1, L2, C1 and C2). Immuno-fluorescent microscopy and immuno-precipitation assay on these cells indicated that 4 distinct kinds of hIgGs bind to one of 3 different domains (L1, C1 and C2). Then, these hIgGs were further examined for biological effects on human A431 cancer cells, which overexpress EGFR. The results indicated that hIgG38 binding to L1 and hIgG45 binding to C2 substantially suppressed the EGF-induced phosphorylation of EGFR, resulting in the growth inhibition of A431 cancer cells. On the contrary, hIgG40 binding to C1 and hIgG42 binding to another site (epitope) of C2 exhibited no such inhibitory effects. Thus, the newly produced four recombinant hIgG antibodies recognize 4 different sites (epitopes) in 3 different extracellular domains of EGFR and exhibit different biological effects on cancer cells. These characteristics are somewhat different from the currently utilized therapeutic anti-EGFR antibodies. Hence, these hIgG antibodies will be invaluable as a research tool for the detailed molecular analysis of the EGFR-mediated signal transduction mechanism and more importantly a possible application as new therapeutic agents to treat certain types of cancers.

  12. MicroRNAs Are Part of the Regulatory Network that Controls EGF Induced Apoptosis, Including Elements of the JAK/STAT Pathway, in A431 Cells

    PubMed Central

    Alanazi, Ibrahim; Hoffmann, Peter; Adelson, David L.

    2015-01-01

    MiRNAs are known to regulate gene expression and in the context of cancer have been shown to regulate metastasis, cell proliferation and cell death. In this report we describe potential miRNA regulatory roles with respect to induction of cell death by pharmacologic dose of Epidermal Growth Factor (EGF). Our previous work suggested that multiple pathways are involved in the induction of apoptosis, including interferon induced genes, cytokines, cytoskeleton and cell adhesion and TP53 regulated genes. Using miRNA time course expression profiling of EGF treated A431 cells and coupling this to our previous gene expression and proteomic data, we have been able to implicate a number of additional miRNAs in the regulation of apoptosis. Specifically we have linked miR-134, miR-145, miR-146b-5p, miR-432 and miR-494 to the regulation of both apoptotic and anti-apoptotic genes expressed as a function of EGF treatment. Whilst additional miRNAs were differentially expressed, these had the largest number of apoptotic and anti-apoptotic targets. We found 5 miRNAs previously implicated in the regulation of apoptosis and our results indicate that an additional 20 miRNAs are likely to be involved based on their correlated expression with targets. Certain targets were linked to multiple miRNAs, including PEG10, BTG1, ID1, IL32 and NCF2. Some miRNAs that target the interferon pathway were found to be down regulated, consistent with a novel layer of regulation of interferon pathway components downstream of JAK/STAT. We have significantly expanded the repertoire of miRNAs that may regulate apoptosis in cancer cells as a result of this work. PMID:25781916

  13. Characterization of the interaction of 5'-p-fluorosulfonylbenzoyl adenosine with the epidermal growth factor receptor/protein kinase in A431 cell membranes.

    PubMed

    Buhrow, S A; Cohen, S; Garbers, D L; Staros, J V

    1983-06-25

    Treatment of membrane vesicles from A431 cells, a human epidermoid carcinoma line, with the affinity label 5'-p-fluorosulfonylbenzoyl [8-14C]adenosine (5'-p-FSO2Bz[14C]Ado) results in an inhibition of the epidermal growth factor (EGF)-stimulable protein kinase and in the modification of proteins having the same molecular weight (Mr = 170,000 and 150,000) as the receptor for EGF (Buhrow, S. A., Cohen, S., and Staros, J. V. (1982) J. Biol. Chem. 257, 4019-4022). Modification of the vesicles with 5'-p-FSO2BzAdo inhibits not only the EGF-stimulated phosphorylation of endogenous membrane proteins but also the EGF-stimulated phosphorylation of an exogenous synthetic tyrosine-containing peptide substrate. This indicates that the EGF-stimulable protein kinase is modified by 5'-p-FSO2BzAdo at a site affecting catalytic activity. Membrane vesicles were treated with 5'-p-FSO2Bz-[14C]Ado to affinity label the kinase, then the EGF receptor was purified by affinity chromatography on immobilized EGF. The EGF receptor thus purified contains the 5'-p-SO2Bz[14C]Ado moiety. These data strongly support our hypothesis that the EGF receptor and EGF-stimulable kinase are two parts of the same polypeptide chain.

  14. Bromelain inhibits nuclear factor kappa-B translocation, driving human epidermoid carcinoma A431 and melanoma A375 cells through G(2)/M arrest to apoptosis.

    PubMed

    Bhui, Kulpreet; Tyagi, Shilpa; Srivastava, Amit Kumar; Singh, Madhulika; Roy, Preeti; Singh, Richa; Shukla, Yogeshwer

    2012-03-01

    Bromelain, obtained from pineapple, is already in use clinically as adjunct in chemotherapy. Our objective was to test its ability to act as a sole anti-cancer agent. Therefore, we describe its anti-proliferative, anti-inflammatory and subsequent anti-cancer effects in vitro, against human epidermoid carcinoma-A431 and melanoma-A375 cells. Bromelain exhibited reduction in proliferation of both these cell-lines and suppressed their potential for anchorage-independent growth. Further, suppression of inflammatory signaling by bromelain was evident by inhibition of Akt regulated-nuclear factor-kappaB activation via suppression of inhibitory-kappaBα phosphorylation and concomitant reduction in cyclooxygenase-2. Since, the inflammatory cascade is well-known to be closely allied to cancer; we studied the effect of bromelain on events/molecules central to it. Bromelain caused depletion of intracellular glutathione and generation of reactive oxygen-species followed by mitochondrial membrane depolarization. This led to bromelain-induced cell-cycle arrest at G(2)/M phase which was mediated by modulation of cyclin B1, phospho-cdc25C, Plk1, phospho-cdc2, and myt1. This was subsequently followed by induction of apoptosis, indicated by membrane-blebbing, modulation of Bax-Bcl-2 ratio, Apaf-1, caspase-9, and caspase-3; chromatin-condensation, increase in caspase-activity and DNA-fragmentation. Bromelain afforded substantial anti-cancer potential in these settings; hence we suggest it as a potential prospect for anti-cancer agent besides only an additive in chemotherapy.

  15. Corticotropin-releasing factor induces phosphorylation of phospholipase C-gamma at tyrosine residues via its receptor 2beta in human epidermoid A-431 cells.

    PubMed

    Kiang, J G; Ding, X Z; Gist, I D; Jones, R R; Tsokos, G C

    1998-12-18

    This laboratory previously reported that corticotropin-releasing factor (CRF) increased intracellular free calcium concentrations, cellular cAMP, inositol 1,4,5-trisphosphate, protein kinase C activity, and protein phosphorylation in human A-431 cells. The increase was blocked by CRF receptor antagonist. In this study, we identified the type of CRF receptors present and investigated whether CRF induced tyrosine phosphorylation of phospholipase C-gamma via CRF receptors. Using novel primers in reverse transcriptase-polymerase chain reaction, we determined the CRF receptor type to be that of 2beta. The levels of the CRF receptor type 2beta were not altered in cells treated with activators of protein kinase C, Ca2+ ionophore, or cells overexpressing heat shock protein 70 kDa. Cells treated with CRF displayed increases in protein tyrosine phosphorylation approximately at 150 kDa as detected by immunoblotting using an antibody against phosphotyrosine. Immunoprecipitation with antibodies directed against phospholipase C-beta3, -gamma1, or -gamma2 isoforms (which have molecular weights around 150 kDa) followed by Western blotting using an anti-phosphotyrosine antibody showed that only phospholipase C-gamma1 and -gamma2 were phosphorylated. The increase in phospholipase C-gamma phosphorylation was concentration-dependent with an EC50 of 4.2+/-0.1 pM. The maximal phosphorylation by CRF at 1 nM occurred by 5 min. The CRF-induced phosphorylation was inhibited by the protein tyrosine kinase inhibitors genistein and herbimycin A, suggesting that CRF activates protein tyrosine kinases. Treatment of cells with CRF receptor antagonist, but not pertussis toxin, prior to treatment with CRF inhibited the CRF-induced phosphorylation, suggesting it is mediated by the CRF receptor type 2beta that is not coupled to pertussis toxin-sensitive G-proteins. Treatment with 1,2-bis(2iminophenoxy)ethane-N,N,N',N'-tetraacetic acid attenuated the phospholipase C-gamma phosphorylation. In summary

  16. Grape seed proanthocyanidins promote apoptosis in human epidermoid carcinoma A431 cells through alterations in Cdki-Cdk-cyclin cascade, and caspase-3 activation via loss of mitochondrial membrane potential.

    PubMed

    Meeran, Syed M; Katiyar, Santosh K

    2007-05-01

    Dietary grape seed proanthocyanidins (GSPs) prevent photocarcinogenesis in mice. Here, we report that in vitro treatment of human epidermoid carcinoma A431 cells with GSPs inhibited cellular proliferation (13-89%) and induced cell death (1-48%) in a dose (5-100 mug/ml)- and time (24, 48 and 72 h)-dependent manner. GSP-induced inhibition of cell proliferation was associated with an increase in G1-phase arrest at 24 h, which was mediated through the inhibition of cyclin-dependent kinases (Cdk) Cdk2, Cdk4, Cdk6 and cyclins D1, D2 and E and simultaneous increase in protein expression of cyclin-dependent kinase inhibitors (Cdki), Cip1/p21 and Kip1/p27, and enhanced binding of Cdki-Cdk. The treatment of A431 cells with GSPs (20-80 mug/ml) resulted in a dose-dependent increase in apoptotic cell death (26-58%), which was associated with an increased protein expression of proapoptotic Bax, decreased expression of antiapoptotic Bcl-2 and Bcl-xl, loss of mitochondrial membrane potential, and cleavage of caspase-9, caspase-3 and PARP. Pretreatment with the pan-caspase inhibitor (z-VAD-fmk) blocked the GSP-induced apoptosis in A431 cells suggesting that GSP-induced apoptosis is associated primarily with the caspase-3-dependent pathway. Together, our study suggests that GSPs possess chemotherapeutic potential against human epidermoid carcinoma cells in vitro, further in vivo mechanistic studies are required to verify the chemotherapeutic effect of GSPs in skin cancers.

  17. In contrast to their murine counterparts, normal human keratinocytes and human epidermoid cell lines A431 and HaCaT fail to express IL-10 mRNA and protein

    PubMed Central

    TEUNISSEN, M B M; KOOMEN, C W; JANSEN, J; DE WAAL MALEFYT, R; SCHMITT, E; VAN DEN WIJNGAARD, R M J G J; DAS, P K; BOS, J D

    1997-01-01

    In mice, keratinocyte-derived IL-10 is up-regulated by ultraviolet-B (UVB) radiation and plays a major role in UVB-induced immunosuppression. The present study was designed to examine whether a comparable phenomenon can be detected in man. Freshly isolated or cultured normal human keratinocytes (NHK) and keratinocyte cell lines A431 and HaCaT were stimulated with graded doses of UVB (up to 200 J/m2) or with a variety of other stimuli. RNA was extracted at various time points post-stimulation and analysed by reverse transcriptase-polymerase chain reaction (RT-PCR) using four different IL-10-specific primer pairs and RNA from monocytes or T cells as positive controls. We failed to detect IL-10 mRNA in NHK from 40 different donors (breast, abdomen, leg, scalp, foreskin) and in A431 and HaCaT cells, irrespective of the stimulation used and despite successful stimulation. Supernatants of NHK, A431 and HaCaT cultures were negative for IL-10 protein, as tested by four different ELISAs and a bioassay. Murine keratinocytes, stimulated under comparable conditions and tested by the same techniques, displayed a strong expression of IL-10 mRNA and protein. Remarkably, an IL-10 mRNA signal could be detected in NHK after a second round of PCR amplification. Because NHK suspensions are contaminated with Langerhans cells, melanocytes and possibly fibroblasts, we tested pure populations of each individual cell type to determine the origin of this IL-10 mRNA. Our results clearly indicate that NHK, Langerhans cells and fibroblasts fail to express IL-10 and that melanocytes are the principal source of IL-10 mRNA in normal human epidermis. PMID:9010278

  18. Accelerated degradation of 160 kDa epidermal growth factor (EGF) receptor precursor by the tyrosine kinase inhibitor herbimycin A in the endoplasmic reticulum of A431 human epidermoid carcinoma cells.

    PubMed Central

    Murakami, Y; Mizuno, S; Uehara, Y

    1994-01-01

    The effect of herbimycin A on the biosynthesis of epidermal growth factor (EGF) receptor was examined in human epidermoid carcinoma A431 cells. Cells were pulse-labelled with [35S]methionine, and EGF receptor biosynthesis was quantified by immunoprecipitation using a monoclonal anti-(EGF receptor) antibody. In the presence of herbimycin A, an immature 160 kDa EGF receptor precursor accumulated in 1 h and disappeared completely in 4 h. Pulse-labelled 160 kDa receptor precursor in the absence of herbimycin A, however, was converted normally into a 170 kDa one by chase with herbimycin A. Herbimycin A affected neither the synthesis of the secreted form of EGF receptor devoid of cytoplasmic domain, nor that of the transferrin receptor in A431 cells. The herbimycin A-induced degradation of 160 kDa EGF receptor precursor was not inhibited by an inhibitor of lysosomal enzymes, NH4Cl. Endoglycosidase H digestion of the 160 kDa precursor converted it into the deglycosylated 130 kDa precursor peptide. These results suggested that herbimycin A selectively acted on the EGF receptor precursor during the synthesis of the 160 kDa form, probably on the cytoplasmic domain, to form an aberrant molecule which was subjected to rapid degradation in the endoplasmic reticulum. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 PMID:8037692

  19. 1,4-Naphthoquinone activates the HSP90/HSF1 pathway through the S-arylation of HSP90 in A431 cells: Negative regulation of the redox signal transduction pathway by persulfides/polysulfides.

    PubMed

    Abiko, Yumi; Sha, Liang; Shinkai, Yasuhiro; Unoki, Takamitsu; Luong, Nho Cong; Tsuchiya, Yukihiro; Watanabe, Yasuo; Hirose, Reiko; Akaike, Takaaki; Kumagai, Yoshito

    2017-03-01

    The current consensus is that environmental electrophiles activate redox signal transduction pathways through covalent modification of sensor proteins with reactive thiol groups at low concentrations, while they cause cell damage at higher concentrations. We previously exposed human carcinoma A431 cells to the atmospheric electrophile 1,4-naphthoquinone (1,4-NQ) and found that heat shock protein 90 (HSP90), a negative regulator of heat shock factor 1 (HSF1), was a target of 1,4-NQ. In the study presented here, we determined whether 1,4-NQ activates HSF1. We also examined whether such redox signaling could be regulated by nucleophilic sulfur species. Exposure of A431 cells to 1,4-NQ covalently modified cellular HSP90, resulting in repression of the association between HSF1 with HSP90, thereby enhancing HSF1 translocation into the nuclei. Liquid chromatography-tandem mass spectrometry analysis with recombinant HSP90 revealed that the modifications site were Cys412 and Cys564. We found that HSF1 activation mediated by 1,4-NQ upregulated downstream genes, such as HSPA6. HSF1 knockdown accelerated 1,4-NQ-mediated cytotoxicity in the cells. While simultaneous treatment with reactive persulfide and polysulfide, Na2S2 and Na2S4, blocked 1,4-NQ-dependent protein modification and HSF1 activation in A431 cells, the knockdown of Cys persulfide producing enzymes cystathionine β-synthase (CBS) and/or cystathionine γ-lyase (CSE) enhanced these phenomena. 1,4-NQ-thiol adduct and 1,4-NQ-S-1,4-NQ adduct were produced during the enzymatic reaction of recombinant CSE in the presence of 1,4-NQ. The results suggest that activation of the HSP90-HSF1 signal transduction pathway mediated by 1,4-NQ protects cells against 1,4-NQ and that per/polysulfides can diminish the reactivity of 1,4-NQ by forming sulfur adducts.

  20. Development of an ErbB-overexpressing A-431 Optical Reporting Tumor Xenograft Model to Assess Targeted Photodynamic Therapy Regimens

    PubMed Central

    Savellano, Mark D.; Owusu-Brackett, Nicci; Son, Ji; Callier, Thierri; Savellano, Dagmar Högemann

    2010-01-01

    To better assess the efficacy of erbB-targeted therapies, it would help to have optical reporting human tumor xenograft models that abundantly express erbB receptors. A-431 cells have frequently been used in erbB1-targeting studies, but a well-characterized optical reporting version of the cell line has not been readily available. In this study, optical reporting A-431 clones were developed that express both a fluorescent protein reporter (green, GFP; or red, RFP) and a bioluminescent reporter, firefly luciferase. Reporter genes were transduced into cells using commercial lentiviral vectors, and clonal selection was carried out using a series of procedures. A number of clones were isolated for further characterization. A GFP/luciferase clone, A-431/D4, and an RFP/luciferase clone, A-431/G4, were obtained that exhibit erbB1 expression levels and tumor growth kinetics similar to the parental cells. To demonstrate the utility of the optical reporting clones, A-431/G4 tumors were grown subcutaneously in nude mice and treated with vascular-targeted photodynamic therapy (PDT), which targets the angiogenic consequences of erbB signaling. The A-431/G4 tumor model permitted highly sensitive longitudinal monitoring of PDT treatment response using optical imaging. A-431/D4 and A-431/G4 optical reporting tumor models should also prove useful for assessing therapies that directly target the erbB1 receptor. PMID:20880229

  1. PP2B-mediated Dephosphorylation of c-Jun C Terminus Regulates Phorbol Ester-induced c-Jun/Sp1 Interaction in A431 Cells

    PubMed Central

    Chen, Ben-Kuen; Huang, Chi-Chen; Chang, Wei-Chiao; Chen, Yun-Ju; Kikkawa, Ushio; Nakahama, Ken-ichi; Morita, Ikuo

    2007-01-01

    The c-Jun/Sp1 interaction is essential for growth factor- and phorbol 12-myristate 13-acetate (PMA)-induced genes expression, including human 12(S)-lipoxygenase, keratin 16, cytosolic phospholipase A2, p21WAF1/CIP1, and neuronal nicotinic acetylcholine receptor β4. Here, we examined the mechanism underlying the PMA-induced regulation on the interaction between c-Jun and Sp1. We found that treatment of cells with PMA induced a dephosphorylation at the C terminus of c-Jun at Ser-243 and a concomitant inhibition of PP2B by using PP2B small interfering RNA, resulting in reduction of PMA-induced gene expression as well as the c-Jun/Sp1 interaction. The c-Jun mutant TAM-67-3A, which contains three substitute alanines at Thr-231, Ser-243, and Ser-249 compared with TAM-67, binds more efficaciously with Sp1 and is about twice as efficacious as TAM-67 in inhibiting the PMA-induced activation of the 12(S)-lipoxygenase promoter. Importantly, PP2B not only dephosphorylates the c-Jun at Ser-243 but also interacts with c-Jun in PMA-treated cells. PMA stimulates the association of the PP2B/c-Jun/Sp1 complex with the promoter. These findings indicate the dephosphorylation of c-Jun C terminus is required for the c-Jun/Sp1 interaction and reveal that PP2B plays an important role in regulating c-Jun/Sp1 interaction in PMA-induced gene expression. PMID:17215518

  2. Purification and properties of the alpha-3/4-L-fucosyltransferase released into the culture medium during the growth of the human A431 epidermoid carcinoma cell line.

    PubMed

    Johnson, P H; Donald, A S; Watkins, W M

    1993-04-01

    A soluble alpha-3/4-fucosyltransferase secreted into the growth medium of the human A431 epidermoid carcinoma cell line has been purified 700,000 fold by a series of steps involving chromatography on Phenyl Sepharose 4B, CM-Sephadex C-50 and GDP-hexanolamine Sepharose 4B. The untreated spent culture medium transferred almost ten times more fucose to the subterminal N-acetylglucosamine residue in the Type 1 (Gal beta 1-3GlcNAc) disaccharide than to the subterminal sugar in the Type 2 (Gal beta 1-4GlcNAc) disaccharide; the relative activity with these two substrates remained virtually unchanged throughout the purification procedure. At no stage was any alpha-3-fucosyltransferase species acting solely on N-acetylglucosamine residues in Type 2 chains separated from the bulk of the alpha-3/4-fucosyltransferase activity. The purified enzyme preparation showed insignificant activity with glycoprotein substrates having N-linked oligosaccharide chains with terminal Type 2 sequences but transferred fucose to a mucin-type glycoprotein with O-linked oligosaccharide chains with terminal Type 1 structures. Lactose was a poor substrate but the activity of the enzyme was influenced by the presence of substituents on the terminal beta-galactosyl residue and 2'-fucosyllactose was almost as good an acceptor as the Type 1 disaccharide. The properties of the purified enzyme with regard to specificity, divalent cation requirements, pH optimum, and M(r), closely resembled those of the Lewis-blood-group gene associated alpha-3/4-fucosyltransferase isolated from human milk.

  3. Red Blood Cell Antibody Identification

    MedlinePlus

    ... name: Red Blood Cell Antibody Identification Related tests: Direct Antiglobulin Test ; RBC Antibody Screen ; Blood Typing ; Type ... a positive RBC antibody screen or a positive direct antiglobulin test (DAT) . It is used to identify ...

  4. Blue light activates phase 2 response proteins and slows growth of a431 epidermoid carcinoma xenografts.

    PubMed

    Patel, Alpesh D; Rotenberg, Shaun; Messer, Regina L W; Wataha, John C; Ogbureke, Kalu U E; McCloud, Veronica V; Lockwood, Petra; Hsu, Stephen; Lewis, Jill B

    2014-11-01

    Recent studies suggest that light in the UVA range (320-400 nm) activates signaling pathways that are anti-inflammatory, antioxidative and play a critical role in protection against cancer. These effects have been attributed to NF-E2-related factor (NRF2)-mediated up-regulation of 'phase 2' genes that neutralize oxidative stress and metabolize electrophiles. We had previously shown that small doses of blue light (400-500 nm) had selective toxicity for cultured oral tumor cells and increased levels of peroxiredoxin phase 2 proteins, which led to our hypothesis that blue light activates NRF2 signaling. A431 epidermoid carcinoma cells were treated in culture and as nude mouse xenografts with doses of blue light. Cell lysates and tumor samples were tested for NRF2 activation, and for markers of proliferation and oxidative stress. Blue light activated the phase 2 response in cultured A431 cells and reduced their viability dose dependently. Light treatment of tumors reduced tumor growth, and levels of proliferating cell nuclear antigen (PCNA), and oxidized proteins. Cellular responses to these light energies are worth further study and may provide therapeutic interventions for inflammation and cancer. Copyright© 2014 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.

  5. Novel Gefitinib Formulation with Improved Oral Bioavailability in Treatment of A431 Skin Carcinoma

    PubMed Central

    Godugu, Chandraiah; Doddapaneni, Ravi; Patel, Apurva R; Singh, Rakesh; Mercer, Roger; Singh, Mandip

    2016-01-01

    Purpose Oral administration of anticancer agents presents a series of advantages for patients. However, most of the anti-cancer drugs have poor water solubility leading to low bioavailability. Methods Controlled released spray dried matrix system of Gefitinib with hydroxypropyl β-cyclodextrin, chitosan, hydroxy propyl methyl cellulose, vitamin E TPGS, succinic acid were used for the design of formulations to improve the oral absorption of Gefitinib. Spray drying with a customized spray gun which allows simultaneous/pulsatile flow of two different liquid systems through single nozzle was used to prepare Gefitinib spray dried formulations (Gef-SD). Formulation was characterized by in vitro drug release and Caco-2 permeability studies. Pharmacokinetic studies were performed in Sprague Dawley rats. Efficacy of Gef-SD was carried out in A431 xenografts models in nude mice. Results In Gef-SD group 9.14-fold increase in the AUC was observed compared to free Gef. Improved pharmacokinetic profile of Gef-SD translated into increase (1.75 fold compared to Gef free drug) in anticancer effects. Animal survival was significantly increased in Gef formulation treated groups, with superior reduction in the tumor size (1.48-fold) and volumes (1.75-fold) and also increase in the anticancer effects (TUNEL positive apoptotic cells) was observed in Gef-SD treated groups. Further, western blot, immunohistochemical and proteomics analysis demonstrated the increased pharmacodynamic effects of Gef-SD formulations in A431 xenograft tumor models. Conclusion Our studies suggested that Gefitinib can be successfully incorporated into control release microparticles based oral formulation with enhanced pharmacokinetic and pharmacodynamic activity. This study demonstrates the novel application of Gef in A431 tumor models. PMID:26286185

  6. Detergent solubilization of the EGF receptor from A431 cells

    NASA Technical Reports Server (NTRS)

    Dayanidhi, R.; Rintoul, D. A.; Spooner, B. S. (Principal Investigator)

    1993-01-01

    Functional reconstitution of purified preparations of human epidermal growth factor receptor (EGFR) requires dissociation of the protein from its plasma membrane lipid environment. Solubilization of membrane proteins in this manner requires the use of detergents, which are known to disrupt plasma membrane lipid/protein interactions. We have investigated the ability of three nonionic detergents to solubilize the human EGFR selectively, and have also analyzed the effect of these various treatments on the intrinsic tyrosyl kinase activity of the receptor. The nonionic detergent known as n-octyl glucoside (n-octyl beta-D-glucopyranoside) was found to give the best combination of selectivity, yield, and maintenance of enzymatic activity of the human EGFR.

  7. Detergent solubilization of the EGF receptor from A431 cells

    NASA Technical Reports Server (NTRS)

    Dayanidhi, R.; Rintoul, D. A.; Spooner, B. S. (Principal Investigator)

    1993-01-01

    Functional reconstitution of purified preparations of human epidermal growth factor receptor (EGFR) requires dissociation of the protein from its plasma membrane lipid environment. Solubilization of membrane proteins in this manner requires the use of detergents, which are known to disrupt plasma membrane lipid/protein interactions. We have investigated the ability of three nonionic detergents to solubilize the human EGFR selectively, and have also analyzed the effect of these various treatments on the intrinsic tyrosyl kinase activity of the receptor. The nonionic detergent known as n-octyl glucoside (n-octyl beta-D-glucopyranoside) was found to give the best combination of selectivity, yield, and maintenance of enzymatic activity of the human EGFR.

  8. Optical trapping dynamics for cell identification

    NASA Astrophysics Data System (ADS)

    Volpe, Giovanni; Singh, Gajendra P.; Buettner, Florian; Drets, Anna M.; Petrov, Dmitri V.

    2006-08-01

    Living cells show a variety of morphological traits upon which numerous identification techniques have already been developed. However most of them involve lengthy biochemical procedures and can compromise the viability of the cell. We demonstrate a method to differentiate cells only on the basis of its trapping dynamics while it is being drawn into an optical trap (Optical Trapping Dynamics). Since it relies only on the inherent properties of the optical trap, without requiring external markers or biochemically sensitive spectroscopic techniques, it can be readily combined with existing optical tweezers setups. We applied it to the study of the yeast cell-cycle stages, showing, in particular, how it can be amenable for the measurement of the budding index of a cell population.

  9. Identification of human brain tumour initiating cells.

    PubMed

    Singh, Sheila K; Hawkins, Cynthia; Clarke, Ian D; Squire, Jeremy A; Bayani, Jane; Hide, Takuichiro; Henkelman, R Mark; Cusimano, Michael D; Dirks, Peter B

    2004-11-18

    The cancer stem cell (CSC) hypothesis suggests that neoplastic clones are maintained exclusively by a rare fraction of cells with stem cell properties. Although the existence of CSCs in human leukaemia is established, little evidence exists for CSCs in solid tumours, except for breast cancer. Recently, we prospectively isolated a CD133+ cell subpopulation from human brain tumours that exhibited stem cell properties in vitro. However, the true measures of CSCs are their capacity for self renewal and exact recapitulation of the original tumour. Here we report the development of a xenograft assay that identified human brain tumour initiating cells that initiate tumours in vivo. Only the CD133+ brain tumour fraction contains cells that are capable of tumour initiation in NOD-SCID (non-obese diabetic, severe combined immunodeficient) mouse brains. Injection of as few as 100 CD133+ cells produced a tumour that could be serially transplanted and was a phenocopy of the patient's original tumour, whereas injection of 10(5) CD133- cells engrafted but did not cause a tumour. Thus, the identification of brain tumour initiating cells provides insights into human brain tumour pathogenesis, giving strong support for the CSC hypothesis as the basis for many solid tumours, and establishes a previously unidentified cellular target for more effective cancer therapies.

  10. Identification of cells initiating human melanomas

    PubMed Central

    Schatton, Tobias; Murphy, George F.; Frank, Natasha Y.; Yamaura, Kazuhiro; Waaga-Gasser, Ana Maria; Gasser, Martin; Zhan, Qian; Jordan, Stefan; Duncan, Lyn M.; Weishaupt, Carsten; Fuhlbrigge, Robert C.; Kupper, Thomas S.; Sayegh, Mohamed H.; Frank, Markus H.

    2012-01-01

    Tumour-initiating cells capable of self-renewal and differentiation, which are responsible for tumour growth, have been identified in human haematological malignancies1,2 and solid cancers3–6. If such minority populations are associated with tumour progression in human patients, specific targeting of tumour-initiating cells could be a strategy to eradicate cancers currently resistant to systemic therapy. Here we identify a subpopulation enriched for human malignant-melanoma-initiating cells (MMIC) defined by expression of the chemoresistance mediator ABCB5 (refs 7, 8) and show that specific targeting of this tumorigenic minority population inhibits tumour growth. ABCB5+ tumour cells detected in human melanoma patients show a primitive molecular phenotype and correlate with clinical melanoma progression. In serial human-to-mouse xenotransplantation experiments, ABCB5+ melanoma cells possess greater tumorigenic capacity than ABCB5− bulk populations and re-establish clinical tumour heterogeneity. In vivo genetic lineage tracking demonstrates a specific capacity of ABCB5+ sub-populations for self-renewal and differentiation, because ABCB5+ cancer cells generate both ABCB5+ and ABCB5− progeny, whereas ABCB5− tumour populations give rise, at lower rates, exclusively to ABCB5− cells. In an initial proof-of-principle analysis, designed to test the hypothesis that MMIC are also required for growth of established tumours, systemic administration of a monoclonal antibody directed at ABCB5, shown to be capable of inducing antibody-dependent cell-mediated cytotoxicity in ABCB5+ MMIC, exerted tumour-inhibitory effects. Identification of tumour-initiating cells with enhanced abundance in more advanced disease but susceptibility to specific targeting through a defining chemoresistance determinant has important implications for cancer therapy. PMID:18202660

  11. Identification of cells initiating human melanomas.

    PubMed

    Schatton, Tobias; Murphy, George F; Frank, Natasha Y; Yamaura, Kazuhiro; Waaga-Gasser, Ana Maria; Gasser, Martin; Zhan, Qian; Jordan, Stefan; Duncan, Lyn M; Weishaupt, Carsten; Fuhlbrigge, Robert C; Kupper, Thomas S; Sayegh, Mohamed H; Frank, Markus H

    2008-01-17

    Tumour-initiating cells capable of self-renewal and differentiation, which are responsible for tumour growth, have been identified in human haematological malignancies and solid cancers. If such minority populations are associated with tumour progression in human patients, specific targeting of tumour-initiating cells could be a strategy to eradicate cancers currently resistant to systemic therapy. Here we identify a subpopulation enriched for human malignant-melanoma-initiating cells (MMIC) defined by expression of the chemoresistance mediator ABCB5 (refs 7, 8) and show that specific targeting of this tumorigenic minority population inhibits tumour growth. ABCB5+ tumour cells detected in human melanoma patients show a primitive molecular phenotype and correlate with clinical melanoma progression. In serial human-to-mouse xenotransplantation experiments, ABCB5+ melanoma cells possess greater tumorigenic capacity than ABCB5- bulk populations and re-establish clinical tumour heterogeneity. In vivo genetic lineage tracking demonstrates a specific capacity of ABCB5+ subpopulations for self-renewal and differentiation, because ABCB5+ cancer cells generate both ABCB5+ and ABCB5- progeny, whereas ABCB5- tumour populations give rise, at lower rates, exclusively to ABCB5- cells. In an initial proof-of-principle analysis, designed to test the hypothesis that MMIC are also required for growth of established tumours, systemic administration of a monoclonal antibody directed at ABCB5, shown to be capable of inducing antibody-dependent cell-mediated cytotoxicity in ABCB5+ MMIC, exerted tumour-inhibitory effects. Identification of tumour-initiating cells with enhanced abundance in more advanced disease but susceptibility to specific targeting through a defining chemoresistance determinant has important implications for cancer therapy.

  12. Secretome identification of immune cell factors mediating metastatic cell homing

    PubMed Central

    Aguado, Brian A.; Wu, Jia J.; Azarin, Samira M.; Nanavati, Dhaval; Rao, Shreyas S.; Bushnell, Grace G.; Medicherla, Chaitanya B.; Shea, Lonnie D.

    2015-01-01

    Metastatic cell homing is a complex process mediated in part by diffusible factors secreted from immune cells found at a pre-metastatic niche. We report on connecting secretomics and TRanscriptional Activity CEll aRray (TRACER) data to identify functional paracrine interactions between immune cells and metastatic cells as novel mediators of homing. Metastatic breast cancer mouse models were used to generate a diseased splenocyte conditioned media (D-SCM) containing immune cell secreted factors. MDA-MB-231 metastatic cell activity including cell invasion, migration, transendothelial migration, and proliferation were increased in D-SCM relative to control media. Our D-SCM secretome analysis yielded 144 secreted factor candidates that contribute to increased metastatic cell activity. The functional mediators of homing were identified using MetaCore software to determine interactions between the immune cell secretome and the TRACER-identified active transcription factors within metastatic cells. Among the 5 candidate homing factors identified, haptoglobin was selected and validated in vitro and in vivo as a key mediator of homing. Our studies demonstrate a novel systems biology approach to identify functional signaling factors associated with a cellular phenotype, which provides an enabling tool that complements large-scale protein identification provided by proteomics. PMID:26634905

  13. Global Identification of Disease Associated Genes in Fragile X Cells

    DTIC Science & Technology

    2016-08-01

    AWARD NUMBER: W81XWH-15-1-0204 TITLE: Global Identification of Disease-Associated Genes in Fragile X Cells PRINCIPAL INVESTIGATOR: Wenyi Feng...Global Identification of Disease-Associated Genes in Fragile X Cells 5a. CONTRACT NUMBER 5b. GRANT NUMBER W81XWH-15-1-0204 GRANT1171 2389...conflict. We have performed three biological replicate experiments to rigorously test if the Fragile X cell line produces more DSBs than the normal

  14. Biologically synthesised silver nanoparticles from three diverse family of plant extracts and their anticancer activity against epidermoid A431 carcinoma.

    PubMed

    Nayak, Debasis; Pradhan, Sonali; Ashe, Sarbani; Rauta, Pradipta Ranjan; Nayak, Bismita

    2015-11-01

    Biological synthesis of silver nanoparticles is a cost effective natural process where the phytochemicals specifically phenols, flavonoids and terpenoids present in the plant extracts act as capping and reducing agent. Due to their nano size regime the silver nanoparticles may directly bind to the DNA of the pathogenic bacterial strains leading to higher antimicrobial activity. In the current study silver nanoparticles were synthesised using plant extracts from different origin Cucurbita maxima (petals), Moringa oleifera (leaves) and Acorus calamus (rhizome). The synthesised nanoparticles were characterized by UV-visible spectroscopy, dynamic light scattering (DLS), X-ray diffraction (XRD), field emission scanning electron microscopy (Fe-SEM) and Fourier transform infrared spectroscopy (FTIR). Highly crystalline, roughly spherical and cuboidal silver nanoparticles of 30-70 nm in size were synthesised. The nanoparticles provided strong antimicrobial activity against pathogenic strains. The effect of the synthesised nanoparticles against A431 skin cancer cell line was tested for their toxicity by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) dye. The IC50 values of 82.39±3.1, 83.57±3.9 and 78.58±2.7 μg/ml were calculated for silver nanoparticles synthesised by C. maxima, M. oleifera and A. calamus respectively. Copyright © 2015 Elsevier Inc. All rights reserved.

  15. Identification and isolation of synovial dendritic cells.

    PubMed

    Pettit, Allison R; Cavanagh, Lois; Boyce, Amanda; Padmanabha, Jagadish; Peng, Judy; Thomas, Ranjeny

    2007-01-01

    In rheumatoid arthritis patients, three compartments need to be considered: peripheral blood, synovial fluid, and synovial tissue. Dendritic cells characterized from each compartment have different properties. The methods given are based on cell sorting for isolation of cells, and flow cytometry and immunohistochemical staining for analysis of cells in these compartments.

  16. Raman spectroscopic identification of single bacterial cells under antibiotic influence.

    PubMed

    Münchberg, Ute; Rösch, Petra; Bauer, Michael; Popp, Jürgen

    2014-05-01

    The identification of pathogenic bacteria is a frequently required task. Current identification procedures are usually either time-consuming due to necessary cultivation steps or expensive and demanding in their application. Furthermore, previous treatment of a patient with antibiotics often renders routine analysis by culturing difficult. Since Raman microspectroscopy allows for the identification of single bacterial cells, it can be used to identify such difficult to culture bacteria. Yet until now, there have been no investigations whether antibiotic treatment of the bacteria influences the Raman spectroscopic identification. This study aims to rapidly identify bacteria that have been subjected to antibiotic treatment on single cell level with Raman microspectroscopy. Two strains of Escherichia coli and two species of Pseudomonas have been treated with four antibiotics, all targeting different sites of the bacteria. With Raman spectra from untreated bacteria, a linear discriminant analysis (LDA) model is built, which successfully identifies the species of independent untreated bacteria. Upon treatment of the bacteria with subinhibitory concentrations of ampicillin, ciprofloxacin, gentamicin, and sulfamethoxazole, the LDA model achieves species identification accuracies of 85.4, 95.3, 89.9, and 97.3 %, respectively. Increasing the antibiotic concentrations has no effect on the identification performance. An ampicillin-resistant strain of E. coli and a sample of P. aeruginosa are successfully identified as well. General representation of antibiotic stress in the training data improves species identification performance, while representation of a specific antibiotic improves strain distinction capability. In conclusion, the identification of antibiotically treated bacteria is possible with Raman microspectroscopy for diverse antibiotics on single cell level.

  17. Isolation and identification of normal killer cells from Syrian hamsters

    SciTech Connect

    Matveeva, V.A.; Klyuchareva, T.E.

    1986-09-01

    This paper gives data on isolation of normal killer cells from the blood and various tissues of Syrian hamsters in a Percoll density gradient and their identification on the basis of morphologic criteria and cytotoxic activity (CTA). CTA of the isolated cells was studied in the cytotoxic test with target cells of a human MOLT-4 thymoma cell labeled with /sup 51/Cr. Isolation of large granular lymphocytes from blood, spleen, and bone marrow of Syrian hamsters in Percoll density gradient is shown in the results of five experiments used for cells of each type.

  18. Diffraction pattern study for cell type identification.

    PubMed

    Mihailescu, M; Costescu, J

    2012-01-16

    This paper presents our study regarding diffracted intensity distribution in Fresnel and Fraunhofer approximation from different cell types. Starting from experimental information obtained through digital holographic microscopy, we modeled the cell shapes as oblate spheroids and built their phase-only transmission functions. In Fresnel approximation, the experimental and numerical diffraction patterns from mature and immature red blood cells have complementary central intensity values at different distances. The Fraunhofer diffraction patterns of deformed red blood cells were processed in the reciprocal space where, the isoamplitude curves were formed independently for each degree of cell deformation present within every sample; the values on each separate isoamplitude curve are proportional with the percentage of the respective cell type within the sample.

  19. Identification of cell density signal molecule

    DOEpatents

    Schwarz, R.I.

    1998-04-21

    Disclosed herein is a novel proteinaceous cell density signal molecule (CDS) between 25 and 35 kD, which is secreted by fibroblastic primary avian tendon cells in culture, and causes the cells to self-regulate their proliferation and the expression of differentiated function. It effects an increase of procollagen production in avian tendon cell cultures of ten fold while proliferation rates are decreased. CDS, and the antibodies which recognize them, are important for the development of diagnostics and treatments for injuries and diseases involving connective tissues, particularly tendon. Also disclosed are methods of production and use. 2 figs.

  20. Identification of cell density signal molecule

    DOEpatents

    Schwarz, Richard I.

    1998-01-01

    Disclosed herein is a novel proteinaceous cell density signal molecule (CDS) between 25 and 35 kD, which is secreted by fibroblastic primary avian tendon cells in culture, and causes the cells to self-regulate their proliferation and the expression of differentiated function. It effects an increase of procollagen production in avian tendon cell cultures of ten fold while proliferation rates are decreased. CDS, and the antibodies which recognize them, are important for the development of diagnostics and treatments for injuries and diseases involving connective tissues, particularly tendon. Also disclosed are methods of production and use.

  1. Identification of Potential Germ-Cell Mutagens

    EPA Science Inventory

    The existence of agents that can induce germ-cell mutations in experimental systems has been recognized since 1927 with the discovery of the ability of X-rays to induce such mutations in Drosophila. Various rodent-based germ-cell mutation assays have been developed, and ~50 germ...

  2. Identification of Potential Germ-Cell Mutagens

    EPA Science Inventory

    The existence of agents that can induce germ-cell mutations in experimental systems has been recognized since 1927 with the discovery of the ability of X-rays to induce such mutations in Drosophila. Various rodent-based germ-cell mutation assays have been developed, and ~50 germ...

  3. Identification of Novel Cell Wall Components

    SciTech Connect

    Michelle Momany

    2009-10-26

    Our DOE Biosciences-funded work focused on the fungal cell wall and morphogenesis. We are especially interested in how new cell wall material is targeted to appropriate areas for polar (asymmetric) growth. Polar growth is the only way that filamentous fungi explore the environment to find suitable substrates to degrade. Work funded by this grant has resulted in a total of twenty peer-reviewed publications. In work funded by this grant, we identified nine Aspergillus nidulans temperature-sensitive (ts) mutants that fail to send out a germ tube and show a swollen cell phenotype at restrictive temperature, the swo mutants. In other organisms, a swollen cell phenotype is often associated with misdirected growth or weakened cell walls. Our work shows that several of the A. nidulans swo mutants have defects in the establishment and maintenance of polarity. Cloning of several swo genes by complementation also showed that secondary modification of proteins seems is important in polarity. We also investigated cell wall biosynthesis and branching based on leads in literature from other organisms and found that branching and nuclear division are tied and that the cell wall reorganizes during development. In our most recent work we have focused on gene expression during the shift from isotropic to polar growth. Surprisingly we found that genes previously thought to be involved only in spore formation are important in early vegetative growth as well.

  4. Isolation, Identification, and Purification of Murine Thymic Epithelial Cells

    PubMed Central

    Xing, Yan; Hogquist, Kristin A.

    2014-01-01

    The thymus is a vital organ for T lymphocyte development. Of thymic stromal cells, thymic epithelial cells (TECs) are particularly crucial at multiple stages of T cell development: T cell commitment, positive selection and negative selection. However, the function of TECs in the thymus remains incompletely understood. In the article, we provide a method to isolate TEC subsets from fresh mouse thymus using a combination of mechanical disruption and enzymatic digestion. The method allows thymic stromal cells and thymocytes to be efficiently released from cell-cell and cell-extracellular matrix connections and to form a single-cell suspension. Using the isolated cells, multiparameter flow cytometry can be applied to identification and characterization of TECs and dendritic cells. Because TECs are a rare cell population in the thymus, we also describe an effective way to enrich and purify TECs by depleting thymocytes, the most abundant cell type in the thymus. Following the enrichment, cell sorting time can be decreased so that loss of cell viability can be minimized during purification of TECs. Purified cells are suitable for various downstream analyses like Real Time-PCR, Western blot and gene expression profiling. The protocol will promote research of TEC function and as well as the development of in vitro T cell reconstitution. PMID:25145384

  5. 77 FR 5489 - Identification of Human Cell Lines Project

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-02-03

    ... identification as part of this project will undergo STR profiling, a DNA profiling method that examines/screens for STRs (DNA elements 2-6 bps long repeated in tandem) in the human chromosomes, that has been shown... are expected between cell line DNA samples originating from unrelated individuals. Each unique...

  6. [Identification of a multihit model for nonhomogeneous cell population].

    PubMed

    Pavlova, L V; Khanin, L G; Iakovlev, A Iu

    1992-01-01

    A generalized multihit-multitarget model for a nonhomogeneous, with respect to radiosensitivity, population of irradiated cells is presented. The least squares and the maximum likelihood estimation of the model parameters is given. The estimates quality is evaluated by the computer-based study. The results obtained show the possibility of the parametric identification of cell radiosensitivity distribution according to the "dose-response" data.

  7. Identification of a candidate stem cell in human gallbladder

    PubMed Central

    Manohar, Rohan; Li, Yaming; Fohrer, Helene; Guzik, Lynda; Stolz, Donna Beer; Chandran, Uma R.; LaFramboise, William A.; Lagasse, Eric

    2015-01-01

    There are currently no reports of the identification of stem cells in the human gallbladder. The differences between human gallbladder and intrahepatic bile duct (IHBD) cells have also not been explored. The goals of this study were to evaluate if human fetal gallbladder contains a candidate stem cell population and if fetal gallbladder cells are distinct from fetal IHBD cells. We found that EpCAM+CD44+CD13+ cells represent the cell population most enriched for clonal self-renewal from primary gallbladder. Primary EpCAM+CD44+CD13+ cells gave rise to EpCAM+CD44+CD13+ and EpCAM+CD44+CD13− cells in vitro, and gallbladder cells expanded in vitro exhibited short-term engraftment in vivo. Last, we found that CD13, CD227, CD66, CD26 and CD49b were differentially expressed between gallbladder and IHBD cells cultured in vitro indicating clear phenotypic differences between the two cell populations. Microarray analyses of expanded cultures confirmed that both cell types have unique transcriptional profiles with predicted functional differences in lipid, carbohydrate, nucleic acid and drug metabolism. In conclusion, we have isolated a distinct clonogenic population of epithelial cells from primary human fetal gallbladder with stem cell characteristics and found it to be unique compared to IHBD cells. PMID:25765520

  8. Functional identification of islet cell types by electrophysiological fingerprinting

    PubMed Central

    Zhang, Quan; Vergari, Elisa; Kellard, Joely A.; Rodriguez, Blanca; Ashcroft, Frances M.; Rorsman, Patrik

    2017-01-01

    The α-, β- and δ-cells of the pancreatic islet exhibit different electrophysiological features. We used a large dataset of whole-cell patch-clamp recordings from cells in intact mouse islets (N = 288 recordings) to investigate whether it is possible to reliably identify cell type (α, β or δ) based on their electrophysiological characteristics. We quantified 15 electrophysiological variables in each recorded cell. Individually, none of the variables could reliably distinguish the cell types. We therefore constructed a logistic regression model that included all quantified variables, to determine whether they could together identify cell type. The model identified cell type with 94% accuracy. This model was applied to a dataset of cells recorded from hyperglycaemic βV59M mice; it correctly identified cell type in all cells and was able to distinguish cells that co-expressed insulin and glucagon. Based on this revised functional identification, we were able to improve conductance-based models of the electrical activity in α-cells and generate a model of δ-cell electrical activity. These new models could faithfully emulate α- and δ-cell electrical activity recorded experimentally. PMID:28275121

  9. Betulin as an antitumor agent tested in vitro on A431, HeLa and MCF7, and as an angiogenic inhibitor in vivo in the CAM assay.

    PubMed

    Dehelean, Cristina Adriana; Feflea, Stefana; Molnár, Judit; Zupko, Istvan; Soica, Codruta

    2012-08-01

    Betulin, an important compound found in birch tree bark, can be converted to betulinic acid, an important pharmacological substance. Betulin has recently been reported as a cytotoxic agent for several tumor cell lines and as an apoptotic inductor. Angiogenesis is a key process involved in tumor metastasis and in developing tumor resistance to cytotoxic therapy. There are little data on betulin as an anti angiogenic agent. This preliminary study aimed to evaluate the cytotoxic effect of betulin on three cancer cell lines: HeLa (cervix adenocarcinoma), MCF7 (breast adenocarcinoma) and A431 (skin epidermoid carcinoma), and the apoptotic mechanism, as well as the implication in the capillary formation of the chicken embryo chorioallantoic membrane. The analysis consisted in the interpretation of the MTT assay and fluorescence double staining with Hoechst dye 33258 and propidium iodide, while the angiogenic effect was evaluated using morphological and immunohistochemical techniques. The antitumor activity is revealed by the double fluorescence staining, indicating that at higher concentrations, the cell membrane permeability is enhanced, while at lower concentrations there is evidence for nuclear fragmentation. In what concerns its effect on the process of blood vessel formation, betulin induced the reduction of newly formed capillaries, especially in the mesenchyme, possible through targeting the normal function of endothelial cells. In vitro results proved the superior specificity of betulin on cervical cancer cells, followed by skin cancer cells.

  10. Identification of a cancer stem cell in human brain tumors.

    PubMed

    Singh, Sheila K; Clarke, Ian D; Terasaki, Mizuhiko; Bonn, Victoria E; Hawkins, Cynthia; Squire, Jeremy; Dirks, Peter B

    2003-09-15

    Most current research on human brain tumors is focused on the molecular and cellular analysis of the bulk tumor mass. However, there is overwhelming evidence in some malignancies that the tumor clone is heterogeneous with respect to proliferation and differentiation. In human leukemia, the tumor clone is organized as a hierarchy that originates from rare leukemic stem cells that possess extensive proliferative and self-renewal potential, and are responsible for maintaining the tumor clone. We report here the identification and purification of a cancer stem cell from human brain tumors of different phenotypes that possesses a marked capacity for proliferation, self-renewal, and differentiation. The increased self-renewal capacity of the brain tumor stem cell (BTSC) was highest from the most aggressive clinical samples of medulloblastoma compared with low-grade gliomas. The BTSC was exclusively isolated with the cell fraction expressing the neural stem cell surface marker CD133. These CD133+ cells could differentiate in culture into tumor cells that phenotypically resembled the tumor from the patient. The identification of a BTSC provides a powerful tool to investigate the tumorigenic process in the central nervous system and to develop therapies targeted to the BTSC.

  11. Identification and Quantitation of Circulating Tumor Cells.

    PubMed

    Rawal, Siddarth; Yang, Yu-Ping; Cote, Richard; Agarwal, Ashutosh

    2017-03-06

    Circulating tumor cells (CTCs) are shed from the primary tumor into the circulatory system and act as seeds that initiate cancer metastasis to distant sites. CTC enumeration has been shown to have a significant prognostic value as a surrogate marker in various cancers. The widespread clinical utility of CTC tests, however, is still limited due to the inherent rarity and heterogeneity of CTCs, which necessitate robust techniques for their efficient enrichment and detection. Significant recent advances have resulted in technologies with the ability to improve yield and purity of CTC enrichment as well as detection sensitivity. Current efforts are largely focused on the translation and standardization of assays to fully realize the clinical utility of CTCs. In this review, we aim to provide a comprehensive overview of CTC enrichment and detection techniques with an emphasis on novel approaches for rapid quantification of CTCs. Expected final online publication date for the Annual Review of Analytical Chemistry Volume 10 is June 12, 2017. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.

  12. Identification of Human Memory-Like NK Cells.

    PubMed

    Kovalenko, Elena I; Streltsova, Maria A; Kanevskiy, Leonid M; Erokhina, Sophia A; Telford, William G

    2017-01-05

    Our understanding of NK biology is increased dramatically, a product of improved flow-cytometric techniques for analyzing these cells. NK cells undergo significant changes in repertoire during differentiation. A repeating stimulus, such as a cytomegalovirus infection, may result in accumulation of certain types of highly differentiated NK cells designated as memory-like, or adaptive NK cells. Adaptive NK cells are capable of rapid expansion and effective response to the recall stimulus. These cells differ significantly from conventional NK cells both functionally and phenotypically. Here we describe an approach for identification and analysis of adaptive NK cells in human peripheral blood. CD57-positive cells with high expression of activating-receptor NKG2C, increased expression of KIR receptors, lack of co-expression with inhibitory receptor NKG2A, and decreased expression of activating receptor NCR3 (NKp30) all characterize this cell type. The flow-cytometric method described below can identify this NK cell subset on a relatively simple flow cytometer. © 2017 by John Wiley & Sons, Inc.

  13. Clostridium perfringens Iota-Toxin b Induces Rapid Cell Necrosis▿

    PubMed Central

    Nagahama, Masahiro; Umezaki, Mariko; Oda, Masataka; Kobayashi, Keiko; Tone, Shigenobu; Suda, Taiji; Ishidoh, Kazumi; Sakurai, Jun

    2011-01-01

    Clostridium perfringens iota-toxin is a binary toxin composed of an enzyme component (Ia) and a binding component (Ib). Each component alone lacks toxic activity, but together they produce cytotoxic effects. We examined the cytotoxicity of iota-toxin Ib in eight cell lines. A431 and A549 cells were susceptible to Ib, but MDCK, Vero, CHO, Caco-2, HT-29, and DLD-1 cells were not. Ib bound and formed oligomers in the membranes of A431 and MDCK cells. However, Ib entered MDCK cells but not A431 cells, suggesting that uptake is essential for cellular survival. Ib also induced cell swelling and the rapid depletion of cellular ATP in A431 and A549 cells but not the insensitive cell lines. In A431 cells, Ib binds and oligomerizes mainly in nonlipid rafts in the membranes. Disruption of lipid rafts by methyl-β-cyclodextrin did not impair ATP depletion or cell death caused by Ib. Ib induced permeabilization by propidium iodide without DNA fragmentation in A431 cells. Ultrastructural studies revealed that A431 cells undergo necrosis after treatment with Ib. Ib caused a disruption of mitochondrial permeability and the release of cytochrome c. Staining with active-form-specific antibodies showed that the proapoptotic Bcl-2-family proteins Bax and Bak were activated and colocalized with mitochondria in Ib-treated A431 cells. We demonstrate that Ib by itself produces cytotoxic activity through necrosis. PMID:21911469

  14. 76 FR 16609 - Proposed Information Collection; Comment Request; Identification of Human Cell Lines Project

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-03-24

    ...; Identification of Human Cell Lines Project AGENCY: National Institute of Standards and Technology (NIST...) profiling up to 1500 human cell line samples as part of the Identification of Human Cell Lines Project. All... for Biotechnology Information (NCBI) and will be used to differentiate among cell lines, as described...

  15. Identification of stable endogenous control genes for transcriptional profiling of photon, proton and carbon-ion irradiated cells.

    PubMed

    Sharungbam, Geeta D; Schwager, Christian; Chiblak, Sara; Brons, Stephan; Hlatky, Lynn; Haberer, Thomas; Debus, Jürgen; Abdollahi, Amir

    2012-05-17

    Quantitative analysis of transcriptional regulation of genes is a prerequisite for a better understanding of the molecular mechanisms of action of different radiation qualities such as photon, proton or carbon ion irradiation. Microarrays and real-time quantitative RT-PCR (qRT-PCR) are considered the two cornerstones of gene expression analysis. In interpreting these results it is critical to normalize the expression levels of the target genes by that of appropriately selected endogenous control genes (ECGs) or housekeeping genes. We sought to systematically investigate common ECG candidates for their stability after different radiation modalities in different human cell lines by qRT-PCR. We aimed to identify the most robust set of ECGs or housekeeping genes for transcriptional analysis in irradiation studies. We tested the expression stability of 32 ECGs in three human cancer cell lines. The epidermoid carcinoma cells (A431), the non small cell lung carcinoma cells (A549) and the pancreatic adenocarincoma cells (BxPC3) were irradiated with photon, proton and carbon ions. Expression Heat maps, clustering and statistic algorithms were employed using SUMO software package. The expression stability was evaluated by computing: mean, standard deviation, ANOVA, coefficient of variation and the stability measure (M) given by the geNorm algorithm. Expression analysis revealed significant cell type specific regulation of 18 out of 32 ECGs (p < 0.05). A549 and A431 cells shared a similar pattern of ECG expression as the function of different radiation qualities as compared to BxPC3. Of note, the ribosomal protein 18S, one of the most frequently used ECG, was differentially regulated as the function of different radiation qualities (p ≤ 0.01). A comprehensive search for the most stable ECGs using the geNorm algorithm identified 3 ECGs for A431 and BxPC3 to be sufficient for normalization. In contrast, 6 ECGs were required to properly normalize expression data in the

  16. Identification of stable endogenous control genes for transcriptional profiling of photon, proton and carbon-ion irradiated cells

    PubMed Central

    2012-01-01

    Background Quantitative analysis of transcriptional regulation of genes is a prerequisite for a better understanding of the molecular mechanisms of action of different radiation qualities such as photon, proton or carbon ion irradiation. Microarrays and real-time quantitative RT-PCR (qRT-PCR) are considered the two cornerstones of gene expression analysis. In interpreting these results it is critical to normalize the expression levels of the target genes by that of appropriately selected endogenous control genes (ECGs) or housekeeping genes. We sought to systematically investigate common ECG candidates for their stability after different radiation modalities in different human cell lines by qRT-PCR. We aimed to identify the most robust set of ECGs or housekeeping genes for transcriptional analysis in irradiation studies. Methods We tested the expression stability of 32 ECGs in three human cancer cell lines. The epidermoid carcinoma cells (A431), the non small cell lung carcinoma cells (A549) and the pancreatic adenocarincoma cells (BxPC3) were irradiated with photon, proton and carbon ions. Expression Heat maps, clustering and statistic algorithms were employed using SUMO software package. The expression stability was evaluated by computing: mean, standard deviation, ANOVA, coefficient of variation and the stability measure (M) given by the geNorm algorithm. Results Expression analysis revealed significant cell type specific regulation of 18 out of 32 ECGs (p < 0.05). A549 and A431 cells shared a similar pattern of ECG expression as the function of different radiation qualities as compared to BxPC3. Of note, the ribosomal protein 18S, one of the most frequently used ECG, was differentially regulated as the function of different radiation qualities (p ≤ 0.01). A comprehensive search for the most stable ECGs using the geNorm algorithm identified 3 ECGs for A431 and BxPC3 to be sufficient for normalization. In contrast, 6 ECGs were required to properly

  17. Prospective Identification of Glioblastoma Cells Generating Dormant Tumors

    PubMed Central

    Segal, Ehud; Ma, Lili; Dixit, Niharika; Ijaz, Ambreen; Hlatky, Lynn; Abdollahi, Amir; Almog, Nava

    2012-01-01

    Although dormant tumors are highly prevalent within the human population, the underlying mechanisms are still mostly unknown. We have previously identified the consensus gene expression pattern of dormant tumors. Here, we show that this gene expression signature could be used for the isolation and identification of clones which generate dormant tumors. We established single cell-derived clones from the aggressive tumor-generating U-87 MG human glioblastoma cell line. Based only on the expression pattern of genes which were previously shown to be associated with tumor dormancy, we identified clones which generate dormant tumors. We show that very high expression levels of thrombospondin and high expression levels of angiomotin and insulin-like growth factor binding protein 5 (IGFBP5), together with low levels of endothelial specific marker (ESM) 1 and epithelial growth factor receptor (EGFR) characterize the clone which generates dormant U-87 MG derived glioblastomas. These tumors remained indolent both in subcutaneous and orthotopic intracranial sites, in spite of a high prevalence of proliferating cells. We further show that tumor cells which form U-87 MG derived dormant tumors have an impaired angiogenesis potential both in vitro and in vivo and have a slower invasion capacity. This work demonstrates that fast-growing tumors contain tumor cells that when isolated will form dormant tumors and serves as a proof-of-concept for the use of transcriptome profiles in the identification of such cells. Isolating the tumor cells that form dormant tumors will facilitate understanding of the underlying mechanisms of dormant micro-metastases, late recurrence, and changes in rate of tumor progression. PMID:22970208

  18. A Morphological identification cell cytotoxicity assay using cytoplasm-localized fluorescent probe (CLFP) to distinguish living and dead cells.

    PubMed

    Lai, Fangfang; Shen, Zhengwei; Wen, Hui; Chen, Jialing; Zhang, Xiang; Lin, Ping; Yin, Dali; Cui, Huaqing; Chen, Xiaoguang

    2017-01-08

    Cell cytotoxicity assays include cell activity assays and morphological identification assays. Currently, all frequently used cytotoxicity assays belong to cell activity assays but suffer from detection limitations. Morphological identification of cell death remains as the gold standard, although the method is difficult to scale up. At present there is no generally accepted morphological identification based cell cytotoxicity assay. In this study, we applied previous developed cell cytoplasm-localized fluorescent probe (CLFP) to display cell morphologies. Under fluorescence microscopy, the fluorescence morphology and intensity of living cells are distinct from dead cells. Based on these characters we extracted the images of living cells from series of samples via computational analysis. Thus, a novel cell morphological identification cytotoxicity assay (CLFP assay) is developed. The performance of the CLFP assay was similar to cell activity assay (MTT assay), but the accuracy of the CLFP assay was superior when measuring the cytotoxicity of active compounds. Copyright © 2016 Elsevier Inc. All rights reserved.

  19. Identification and immunophenotypic characterization of normal and pathological mast cells.

    PubMed

    Morgado, José Mário; Sánchez-Muñoz, Laura; Teodósio, Cristina; Escribano, Luís

    2014-01-01

    Mast cells (MCs) are secretory cells that are central players in human allergic disease and immune responses. With the exception of a few pathological situations, MCs are usually present at relatively low frequencies in most tissues. Since their first description, MCs in tissues were identified mostly using their morphological characteristics and their typical coloration when stained with aniline dyes. However, increasing availability of highly specific antibodies now permits the use of fluorescence-based flow cytometry as the method of choice for the quantification, characterization, and purification of cells in suspension. This technique allows for a rapid analysis of thousands of events and for the identification of cells present at frequencies as low as one event in 10(6) unwanted cells. This method also permits for simultaneous characterization of multiple antigens at a single-cell level, which is ideal in order to study rare populations of cells like MCs. Here we describe the basis of flow cytometry-based immunophenotyping applied to the study of MC. The protocol focuses on the study of human MCs present in body fluids (mainly bone marrow) but can easily be adapted to study MCs from other tissues and species.

  20. Sheep poxvirus identification by PCR in cell cultures.

    PubMed

    Mangana-Vougiouka, O; Markoulatos, P; Koptopoulos, G; Nomikou, K; Bakandritsos, N; Papadopoulos, O

    1999-01-01

    A simple, rapid and specific diagnostic polymerase chain reaction (PCR) method was developed for sheep poxvirus identification. The primers used were from the sequenced genomes of the capripox viruses KS-1 and InS-1. Six different sheep pox isolates were tested against two orf (parapox) and three animal herpesviruses as controls. Material from uninfected cell cultures was also used as control. The sensitivity of the PCR was approximately equivalent with each of the two primers and for the six sheep pox isolates. All the negative control virus DNAs were negative and differed clearly from those of the sheep pox strains.

  1. Identification of multipotent stem cells from adult dog periodontal ligament.

    PubMed

    Wang, Wen-Jun; Zhao, Yu-Ming; Lin, Bi-Chen; Yang, Jie; Ge, Li-Hong

    2012-08-01

    Periodontal diseases, which are characterized by destruction of the connective tissues responsible for restraining the teeth within the jaw, are the main cause of tooth loss. Periodontal regeneration mediated by human periodontal ligament stem cells (hPDLSCs) may offer an alternative strategy for the treatment of periodontal disease. Dogs are a widely used large-animal model for the study of periodontal-disease progression, tissue regeneration, and dental implants, but little attention has been paid to the identification of the cells involved in this species. This study aimed to characterize stem cells isolated from canine periodontal ligament (cPDLSCs). The cPDLSCs, like hPDLSCs, showed clonogenic capability and expressed the mesenchymal stem cell markers STRO-1, CD146, and CD105, but not CD34. After induction of osteogenesis, cPDLSCs showed calcium accumulation in vitro. Moreover, cPDLSCs also showed both adipogenic and chondrogenic potential. Compared with cell-free controls, more cementum/periodontal ligament-like structures were observed in CB-17/SCID mice into which cPDLSCs had been transplanted. These results suggest that cPDLSCs are clonogenic, highly proliferative, and have multidifferentiation potential, and that they could be used as a new cellular therapeutic approach to facilitate successful and more predictable regeneration of periodontal tissue using a canine model of periodontal disease.

  2. Statistical mixture modeling for cell subtype identification in flow cytometry.

    PubMed

    Chan, Cliburn; Feng, Feng; Ottinger, Janet; Foster, David; West, Mike; Kepler, Thomas B

    2008-08-01

    Statistical mixture modeling provides an opportunity for automated identification and resolution of cell subtypes in flow cytometric data. The configuration of cells as represented by multiple markers simultaneously can be modeled arbitrarily well as a mixture of Gaussian distributions in the dimension of the number of markers. Cellular subtypes may be related to one or multiple components of such mixtures, and fitted mixture models can be evaluated in the full set of markers as an alternative, or adjunct, to traditional subjective gating methods that rely on choosing one or two dimensions. Four color flow data from human blood cells labeled with FITC-conjugated anti-CD3, PE-conjugated anti-CD8, PE-Cy5-conjugated anti-CD4, and APC-conjugated anti-CD19 Abs was acquired on a FACSCalibur. Cells from four murine cell lines, JAWS II, RAW 264.7, CTLL-2, and A20, were also stained with FITC-conjugated anti-CD11c, PE-conjugated anti-CD11b, PE-Cy5-conjugated anti-CD8a, and PE-Cy7-conjugated-CD45R/B220 Abs, respectively, and single color flow data were collected on an LSRII. The data were fitted with a mixture of multivariate Gaussians using standard Bayesian statistical approaches and Markov chain Monte Carlo computations. Statistical mixture models were able to identify and purify major cell subsets in human peripheral blood, using an automated process that can be generalized to an arbitrary number of markers. Validation against both traditional expert gating and synthetic mixtures of murine cell lines with known mixing proportions was also performed. This article describes the studies of statistical mixture modeling of flow cytometric data, and demonstrates their utility in examples with four-color flow data from human peripheral blood samples and synthetic mixtures of murine cell lines.

  3. Identification of Phosphorylation Sites on Extracellular Corneal Epithelial Cell Maspin

    PubMed Central

    Narayan, Malathi; Mirza, Shama P.; Twining, Sally S.

    2011-01-01

    Maspin, a 42-kDa non classical serine protease inhibitor (serpin) is expressed by epithelial cells of various tissues including the cornea. The protein localizes to the nucleus and cytosol, and is present in the extracellular space. While extracellular maspin regulates corneal stromal fibroblast adhesion and inhibits angiogenesis during wound healing in the cornea, the molecular mechanism of its extracellular functions is unclear. We hypothesized that identifying post-translational modifications of maspin, such as phosphorylation, may help decipher its mode of action. The focus of this study was on the identification of phosphorylation sites on extracellular maspin, since the extracellular form of the molecule is implicated in several functions. Multi-stage fragmentation mass spectrometry was used to identify sites of phosphorylation on extracellular corneal epithelial cell maspin. A total of eight serine and threonine phosphorylation sites (Thr50, Ser97, Thr118, Thr157, Ser240, Ser298, Thr310, Ser316) were identified on the extracellular forms of the molecule. Phosphorylation of tyrosine residues on extracellular maspin was not detected on extracellular maspin from corneal epithelial cell, in contrast to breast epithelial cells. This study provides the basis for further investigation into the functional role of phosphorylation of corneal epithelial maspin. PMID:21365746

  4. Identification to a breached fuel pin in the IEM cell

    SciTech Connect

    McGuinness, P.W.; Kalk, J.J.; Hicks, D.F.

    1987-01-01

    Novel methods were successfully employed to identify one breached fuel pin in a 217-pin fuel assembly. The assembly was an experiment that had been irradiated at the Fast Flux Test Facility (FFTF), an experimental liquid-metal reactor operated by Westinghouse Hanford Company for the US Dept. of Energy. A fuel assembly known to contain breached fuel pins was removed from the sodium-cooled FFTF reactor in November 1984. Later, this assembly was brought into the FFTF's Interim Examination and Maintenance (IEM) cell to be disassembled and, for the first time ever at FFTF, to identify a breached fuel pin. The synergistic evaluation of the four different verification techniques - visual examination, cladding swipe activity, wash water radiochemistry, and pin weight - provided rapid and positive identification. The capability to perform future detective work of this kind has been conclusively demonstrated.

  5. [Isolation, purification and identification of epithelial cells derived from fetal islet-like cell clusters].

    PubMed

    Qiao, Hai; Zhao, Ting; Wang, Yun; Yang, Chun-Rong; Xiao, Mei; Dou, Zhong-Ying

    2007-03-01

    The aim of this article is to provide methods for the isolation and identification of pancreatic stem cells and cell source for research and therapy of diabetes. ICCs were isolated by collagenase IV digesting and then cultured; epithelial cells were purified from monolayer cultured ICCs. The growth curve of the epithelial cells was measured by MTT. The expression of molecular markers in the cells was identified by immunohistochemical staining. The surface markers in the epithelial cells were analyzed by FACS. Epithelial cells were purified from isolated human fetal ICCs and passaged 40 times, and 10(6) - 10(8) cells were cryopreservated per passage. The growth curve demonstrated that the epithelial cells proliferated rapidly. The epithelial cells expressed PDX-1, PCNA, CK-7, CK-19, Nestin, Glut2, and Vimentin, but Insulin was undetected. The cells expressed CD29, CD44, and CD166, but did not express CD11a, CD14, CD34, CD45, CD90, CD105, and CD117. Taken together, these results indicate that self-renewable epithelial cells can be isolated and purified from human fetal pancreas. These also show that the epithelial cells originate from ducts and have the characteristics of pancreatic stem cells.

  6. Selection of peptidoglycan-specific aptamers for bacterial cells identification.

    PubMed

    Ferreira, Iêda Mendes; de Souza Lacerda, Camila Maria; de Faria, Lígia Santana; Corrêa, Cristiane Rodrigues; de Andrade, Antero Silva Ribeiro

    2014-12-01

    Peptidoglycan is a highly complex and essential macromolecule of bacterial outer cell wall; it is a heteropolymer made up of linear glycan strands cross-linked by peptides. Peptidoglycan has a particular composition which makes it a possible target for specific bacterial recognition. Aptamers are single-stranded DNA or RNA oligonucleotides that bind to target molecules with high affinity and specificity. Aptamers can be labeled with different radioisotopes and possess several properties that make them suitable for molecular imaging. The purpose of this study was to obtain aptamers for use as radiopharmaceutical in bacterial infection diagnosis. Two aptamers (Antibac1 and Antibac2) against peptidoglycan were selected through the Systematic Evolution of Ligands by Exponential Enrichment (SELEX) methodology. The dissociation constant (Kd) for Antibac1 was 0.415 + 0.047 μM and for Antibac2 was 1.261 + 0.280 μM. These aptamers labeled with (32)P showed high affinity for Staphylococcus aureus cells. The binding to S. aureus and Escherichia coli in vitro were significantly higher than for Candida albicans and human fibroblasts, demonstrating their specificity for bacterial cells. These results point Antibac1 and Antibac2 as promising tools for bacterial infections identification.

  7. Identification of lipid derivatives in Hep G2 cells.

    PubMed

    Gdula-Argasińska, Joanna; Garbacik, Aneta; Tyszka-Czochara, Małgorzata; Woźniakiewicz, Michał; Paśko, Paweł; Czepiel, Jacek

    2013-01-01

    Metabolism of polyunsaturated fatty acids results in biosynthesis of mediators with different physiological effects. These metabolites include prostaglandins, prostacyclins, isoprostanes and others that are important signalling molecules and regulate a variety of physiological and pathophysiological processes including inflammation. Prostaglandins and isoprostanes are produced by either non-enzymatic lipid peroxidation or by enzyme-induced peroxidation (cyclooxygenases and lipoxygenases). They are used as biomarkers of oxidative stress. The aim of our study was to assess the effect of eicosapentaenoic acid (EPA) supplementation with added benzo(a)pyrene (BaP) on HepG2 cells by using a UHPLC/MS-TOF method. This rapid and simple method was developed for the identification, separation and quantification of 8-iPGF3α, PGF3α, 8-isoPGF2α and 5-iPF2α in cultured cells. The UHPLC/MS-TOF method was validated. The calculated limit of detection was in the range of 0.16-0.50 ng/mL, precision (% RSD): 1.2-2.1% and recoveries better than 88%. This method empowered qualitative and quantitative analysis of the selected individual prostaglandins derived from arachidonic acid and eicosapentaenoic acid from cell extracts.

  8. Impact of cell dissociation on identification of breast cancer stem cells.

    PubMed

    Quan, Yi; Yan, Ying; Wang, Xiaoli; Fu, Qibin; Wang, Weikang; Wu, Jingwen; Yang, Gen; Ren, Jun; Wang, Yugang

    Fluorescence-activated cell sorting was commonly used for identification of cancer stem cells (CSCs), which relied on specific cell surface markers. And this approach makes it possible for us to study characteristics of CSCs in vitro. However, the pattern of membrane protein including surface makers might be vitally influenced during the dissociation of the adherent cells, thus it might heavily impact the quantity and quality of CSCs identified by flow cytometry. To address this question, in present study, three commonly used digestive reagents and two different temperatures were performed in MCF-7 cells to assay CD44(+)CD24(-) CSCs subpopulation. The potential of sorted CD44(+)CD24(-) cells from different digestion to form mammosphere in culture was also compared. The results showed that trypsin, a commonly used reagent in CSCs studies, most aggressively reduced antigenicity for surface markers and make part of CD44(+)CD24(-) CSCs subpopulation cleaved into CD44(+)CD24(-) non-stem cancer cells (NSCCs). And it also increased the mammosphere formation efficiency of CD44(-)CD24(-) subpopulation. This cleavage effect is especially serious when cells are digested at 37°C. While accutase, a purified collagenase/neutral protease cocktail, provides the best balance of dissociation efficiency and antigen retention. Taken together; these results indicate that enzymatic digestion process plays an important role in identification of CSCs with surface marker via flow cytometer, suggesting that researchers need to reconsider this process seriously.

  9. Identification and IVC of spermatogonial stem cells in prepubertal buffaloes.

    PubMed

    Yu, Xue; Riaz, Hasan; Dong, Ping; Chong, Zhenlu; Luo, Xuan; Liang, Aixin; Yang, Liguo

    2014-06-01

    Development of suitable selective marker for buffalo spermatogonial stem cells (SSCs), optimization of long-term IVC conditions, and their pluripotent retention capacity in buffaloes can be of prime importance in selective genetic modifications of this species. In the present study, we identified CDH1 as a specific marker for buffalo SSCs and revealed that it existed in two protein isoforms (large [135 kDa] and small [90 kDa] subunits) in the buffalo testis; furthermore, immunohistochemical analysis revealed that CDH1 expression was present in spermatogonia but absent in the somatic cells of 4-month-old buffalo testis. After 7 days of enrichment, expression of CDH1 was also detectable in IVC colonies (∼53% enrichment efficiency by Fluorescence-activated cell sorting (FACS)). For long-term culture of SSCs, proliferation studies with different factors showed that combination of 20 ng/mL GDNF, 10 ng/mL FGF2, and 1000 U/mL LIF could significantly promote number of colonies (∼two folds) and proliferation of buffalo SSCs (∼three folds) compared with those of control or single-treatment groups; furthermore, addition of these combination growth factors significantly upregulated the messenger RNA level of spermatogonial-specific and pluripotency-related markers (BCL6B, GFRA1, and POU5F1), whereas downregulated receptor tyrosine kinase (KIT). For confirmation of their stem cell potential, Dolichos biflorus agglutinin-stained cells were identified in the basal membrane of seminiferous tubules of xenotransplanted mice testis. These findings indicate the identification of a new buffalo SSCs marker; furthermore, it may help in establishing long-term culture that would assist in genetic modification of these buffaloes.

  10. Identification of human plasma cells with a lamprey monoclonal antibody

    PubMed Central

    Yu, Cuiling; Liu, Yanling; Chan, Justin Tze Ho; Tong, Jiefei; Li, Zhihua; Shi, Mengyao; Davani, Dariush; Parsons, Marion; Khan, Srijit; Zhan, Wei; Kyu, Shuya; Grunebaum, Eyal; Campisi, Paolo; Propst, Evan J.; Jaye, David L.; Trudel, Suzanne; Moran, Michael F.; Ostrowski, Mario; Herrin, Brantley R.; Lee, F. Eun-Hyung; Sanz, Ignacio; Cooper, Max D.; Ehrhardt, Götz R.A.

    2016-01-01

    Ab-producing plasma cells (PCs) serve as key participants in countering pathogenic challenges as well as being contributors to autoimmune and malignant disorders. Thus far, only a limited number of PC–specific markers have been identified. The characterization of the unique variable lymphocyte receptor (VLR) Abs that are made by evolutionarily distant jawless vertebrates prompted us to investigate whether VLR Abs could detect novel PC antigens that have not been recognized by conventional Abs. Here, we describe a monoclonal lamprey Ab, VLRB MM3, that was raised against primary multiple myeloma cells. VLRB MM3 recognizes a unique epitope of the CD38 ectoenzyme that is present on plasmablasts and PCs from healthy individuals and on most, but not all, multiple myelomas. Binding by the VLRB MM3 Ab coincides with CD38 dimerization and NAD glycohydrolase activity. Our data demonstrate that the lamprey VLRB MM3 Ab is a unique reagent for the identification of plasmablasts and PCs, with potential applications in the diagnosis and therapeutic intervention of PC or autoimmune disorders. PMID:27152361

  11. Fluorescent protein vectors for pancreatic islet cell identification in live-cell imaging.

    PubMed

    Shuai, Hongyan; Xu, Yunjian; Yu, Qian; Gylfe, Erik; Tengholm, Anders

    2016-10-01

    The islets of Langerhans contain different types of endocrine cells, which are crucial for glucose homeostasis. β- and α-cells that release insulin and glucagon, respectively, are most abundant, whereas somatostatin-producing δ-cells and particularly pancreatic polypeptide-releasing PP-cells are more scarce. Studies of islet cell function are hampered by difficulties to identify the different cell types, especially in live-cell imaging experiments when immunostaining is unsuitable. The aim of the present study was to create a set of vectors for fluorescent protein expression with cell-type-specific promoters and evaluate their applicability in functional islet imaging. We constructed six adenoviral vectors for expression of red and green fluorescent proteins controlled by the insulin, preproglucagon, somatostatin, or pancreatic polypeptide promoters. After transduction of mouse and human islets or dispersed islet cells, a majority of the fluorescent cells also immunostained for the appropriate hormone. Recordings of the sub-plasma membrane Ca(2+) and cAMP concentrations with a fluorescent indicator and a protein biosensor, respectively, showed that labeled cells respond to glucose and other modulators of secretion and revealed a striking variability in Ca(2+) signaling among α-cells. The measurements allowed comparison of the phase relationship of Ca(2+) oscillations between different types of cells within intact islets. We conclude that the fluorescent protein vectors allow easy identification of specific islet cell types and can be used in live-cell imaging together with organic dyes and genetically encoded biosensors. This approach will facilitate studies of normal islet physiology and help to clarify molecular defects and disturbed cell interactions in diabetic islets.

  12. ALA-PpIX variability quantitatively imaged in A431 epidermoid tumors using in vivo ultrasound fluorescence tomography and ex vivo assay

    NASA Astrophysics Data System (ADS)

    DSouza, Alisha V.; Flynn, Brendan P.; Gunn, Jason R.; Samkoe, Kimberley S.; Anand, Sanjay; Maytin, Edward V.; Hasan, Tayyaba; Pogue, Brian W.

    2014-03-01

    Treatment monitoring of Aminolevunilic-acid (ALA) - Photodynamic Therapy (PDT) of basal-cell carcinoma (BCC) calls for superficial and subsurface imaging techniques. While superficial imagers exist for this purpose, their ability to assess PpIX levels in thick lesions is poor; additionally few treatment centers have the capability to measure ALA-induced PpIX production. An area of active research is to improve treatments to deeper and nodular BCCs, because treatment is least effective in these. The goal of this work was to understand the logistics and technical capabilities to quantify PpIX at depths over 1mm, using a novel hybrid ultrasound-guided, fiber-based fluorescence molecular spectroscopictomography system. This system utilizes a 633nm excitation laser and detection using filtered spectrometers. Source and detection fibers are collinear so that their imaging plane matches that of ultrasound transducer. Validation with phantoms and tumor-simulating fluorescent inclusions in mice showed sensitivity to fluorophore concentrations as low as 0.025μg/ml at 4mm depth from surface, as presented in previous years. Image-guided quantification of ALA-induced PpIX production was completed in subcutaneous xenograft epidermoid cancer tumor model A431 in nude mice. A total of 32 animals were imaged in-vivo, using several time points, including pre-ALA, 4-hours post-ALA, and 24-hours post-ALA administration. On average, PpIX production in tumors increased by over 10-fold, 4-hours post-ALA. Statistical analysis of PpIX fluorescence showed significant difference among all groups; p<0.05. Results were validated by exvivo imaging of resected tumors. Details of imaging, analysis and results will be presented to illustrate variability and the potential for imaging these values at depth.

  13. Compact and field-portable 3D printed shearing digital holographic microscope for automated cell identification.

    PubMed

    Rawat, Siddharth; Komatsu, Satoru; Markman, Adam; Anand, Arun; Javidi, Bahram

    2017-03-20

    We propose a low-cost, compact, and field-portable 3D printed holographic microscope for automated cell identification based on a common path shearing interferometer setup. Once a hologram is captured from the portable setup, a 3D reconstructed height profile of the cell is created. We extract several morphological cell features from the reconstructed 3D height profiles, including mean physical cell thickness, coefficient of variation, optical volume (OV) of the cell, projected area of the cell (PA), ratio of PA to OV, cell thickness kurtosis, cell thickness skewness, and the dry mass of the cell for identification using the random forest (RF) classifier. The 3D printed prototype can serve as a low-cost alternative for the developing world, where access to laboratory facilities for disease diagnosis are limited. Additionally, a cell phone sensor is used to capture the digital holograms. This enables the user to send the acquired holograms over the internet to a computational device located remotely for cellular identification and classification (analysis). The 3D printed system presented in this paper can be used as a low-cost, stable, and field-portable digital holographic microscope as well as an automated cell identification system. To the best of our knowledge, this is the first research paper presenting automatic cell identification using a low-cost 3D printed digital holographic microscopy setup based on common path shearing interferometry.

  14. Identification of glycan structure alterations on cell membrane proteins in desoxyepothilone B resistant leukemia cells.

    PubMed

    Nakano, Miyako; Saldanha, Rohit; Göbel, Anja; Kavallaris, Maria; Packer, Nicolle H

    2011-11-01

    Resistance to tubulin-binding agents used in cancer is often multifactorial and can include changes in drug accumulation and modified expression of tubulin isotypes. Glycans on cell membrane proteins play important roles in many cellular processes such as recognition and apoptosis, and this study investigated whether changes to the glycan structures on cell membrane proteins occur when cells become resistant to drugs. Specifically, we investigated the alteration of glycan structures on the cell membrane proteins of human T-cell acute lymphoblastic leukemia (CEM) cells that were selected for resistance to desoxyepothilone B (CEM/dEpoB). The glycan profile of the cell membrane glycoproteins was obtained by sequential release of N- and O-glycans from cell membrane fraction dotted onto polyvinylidene difluoride membrane with PNGase F and β-elimination respectively. The released glycan alditols were analyzed by liquid chromatography (graphitized carbon)-electrospray ionization tandem MS. The major N-glycan on CEM cell was the core fucosylated α2-6 monosialo-biantennary structure. Resistant CEM/dEpoB cells had a significant decrease of α2-6 linked sialic acid on N-glycans. The lower α2-6 sialylation was caused by a decrease in activity of β-galactoside α2-6 sialyltransferase (ST6Gal), and decreased expression of the mRNA. It is clear that the membrane glycosylation of leukemia cells changes during acquired resistance to dEpoB drugs and that this change occurs globally on all cell membrane glycoproteins. This is the first identification of a specific glycan modification on the surface of drug resistant cells and the mechanism of this downstream effect on microtubule targeting drugs may offer a route to new interventions to overcome drug resistance.

  15. Identification of miRNAs Involved in Reprogramming Acinar Cells into Insulin Producing Cells

    PubMed Central

    Teichenne, Joan; Morró, Meritxell; Casellas, Alba; Jimenez, Veronica; Tellez, Noelia; Leger, Adrien; Bosch, Fatima; Ayuso, Eduard

    2015-01-01

    Reprogramming acinar cells into insulin producing cells using adenoviral (Ad)-mediated delivery of Pdx1, Ngn3 and MafA (PNM) is an innovative approach for the treatment of diabetes. Here, we aimed to investigate the molecular mechanisms involved in this process and in particular, the role of microRNAs. To this end, we performed a comparative study of acinar-to-β cell reprogramming efficiency in the rat acinar cell line AR42J and its subclone B13 after transduction with Ad-PNM. B13 cells were more efficiently reprogrammed than AR42J cells, which was demonstrated by a strong activation of β cell markers (Ins1, Ins2, IAPP, NeuroD1 and Pax4). miRNome panels were used to analyze differentially expressed miRNAs in acinar cells under four experimental conditions (i) non-transduced AR42J cells, (ii) non-transduced B13 cells, (iii) B13 cells transduced with Ad-GFP vectors and (iv) B13 cells transduced with Ad-PNM vectors. A total of 59 miRNAs were found to be differentially expressed between non-transduced AR42J and B13 cells. Specifically, the miR-200 family was completely repressed in B13 cells, suggesting that these cells exist in a less differentiated state than AR42J cells and as a consequence they present a greater plasticity. Adenoviral transduction per se induced dedifferentiation of acinar cells and 11 miRNAs were putatively involved in this process, whereas 8 miRNAs were found to be associated with PNM expression. Of note, Ad-PNM reprogrammed B13 cells presented the same levels of miR-137-3p, miR-135a-5p, miR-204-5p and miR-210-3p of those detected in islets, highlighting their role in the process. In conclusion, this study led to the identification of miRNAs that might be of compelling importance to improve acinar-to-β cell conversion for the future treatment of diabetes. PMID:26690959

  16. Identification of miRNAs Involved in Reprogramming Acinar Cells into Insulin Producing Cells.

    PubMed

    Teichenne, Joan; Morró, Meritxell; Casellas, Alba; Jimenez, Veronica; Tellez, Noelia; Leger, Adrien; Bosch, Fatima; Ayuso, Eduard

    2015-01-01

    Reprogramming acinar cells into insulin producing cells using adenoviral (Ad)-mediated delivery of Pdx1, Ngn3 and MafA (PNM) is an innovative approach for the treatment of diabetes. Here, we aimed to investigate the molecular mechanisms involved in this process and in particular, the role of microRNAs. To this end, we performed a comparative study of acinar-to-β cell reprogramming efficiency in the rat acinar cell line AR42J and its subclone B13 after transduction with Ad-PNM. B13 cells were more efficiently reprogrammed than AR42J cells, which was demonstrated by a strong activation of β cell markers (Ins1, Ins2, IAPP, NeuroD1 and Pax4). miRNome panels were used to analyze differentially expressed miRNAs in acinar cells under four experimental conditions (i) non-transduced AR42J cells, (ii) non-transduced B13 cells, (iii) B13 cells transduced with Ad-GFP vectors and (iv) B13 cells transduced with Ad-PNM vectors. A total of 59 miRNAs were found to be differentially expressed between non-transduced AR42J and B13 cells. Specifically, the miR-200 family was completely repressed in B13 cells, suggesting that these cells exist in a less differentiated state than AR42J cells and as a consequence they present a greater plasticity. Adenoviral transduction per se induced dedifferentiation of acinar cells and 11 miRNAs were putatively involved in this process, whereas 8 miRNAs were found to be associated with PNM expression. Of note, Ad-PNM reprogrammed B13 cells presented the same levels of miR-137-3p, miR-135a-5p, miR-204-5p and miR-210-3p of those detected in islets, highlighting their role in the process. In conclusion, this study led to the identification of miRNAs that might be of compelling importance to improve acinar-to-β cell conversion for the future treatment of diabetes.

  17. Identification of Genes Required for the Survival of BRCA 1-/- Cells

    DTIC Science & Technology

    2010-02-01

    to double-strand breaks. Science 286, 1162-6 (1999). 36. Smogorzewska, A. et al. Identification of the FANCI protein, a monoubiquitinated FANCD2 paralog required for DNA repair. Cell 129, 289-301 (2007).

  18. Identification of cell surface molecules involved in dystroglycan-independent Lassa virus cell entry.

    PubMed

    Shimojima, Masayuki; Ströher, Ute; Ebihara, Hideki; Feldmann, Heinz; Kawaoka, Yoshihiro

    2012-02-01

    Although O-mannosylated dystroglycan is a receptor for Lassa virus, a causative agent of Lassa fever, recent findings suggest the existence of an alternative receptor(s). Here we identified four molecules as receptors for Lassa virus: Axl and Tyro3, from the TAM family, and dendritic cell-specific intercellular adhesion molecule 3-grabbing nonintegrin (DC-SIGN) and liver and lymph node sinusoidal endothelial calcium-dependent lectin (LSECtin), from the C-type lectin family. These molecules enhanced the binding of Lassa virus to cells and mediated infection independently of dystroglycan. Axl- or Tyro3-mediated infection required intracellular signaling via the tyrosine kinase activity of Axl or Tyro3, whereas DC-SIGN- or LSECtin-mediated infection and binding were dependent on a specific carbohydrate and on ions. The identification of these four molecules as Lassa virus receptors advances our understanding of Lassa virus cell entry.

  19. Single-cell bacterium identification with a SOI optical microcavity

    NASA Astrophysics Data System (ADS)

    Tardif, M.; Jager, J.-B.; Marcoux, P. R.; Uchiyamada, K.; Picard, E.; Hadji, E.; Peyrade, D.

    2016-09-01

    Photonic crystals and microcavities act as on-chip nano-optical tweezers for identification and manipulation of biological objects. Until now, optical trapping of virus and bacteria has been achieved and their presence in the vicinity of the optical resonator is deduced by the shift in the resonant wavelength. Here, we show trapping and identification of bacteria through a properly tuned silicon on insulator microcavity. Through the spatial and temporal observations of bacteria-cavity interaction, the optical identification of three different kinds of bacteria is demonstrated.

  20. Cell identification using Raman spectroscopy in combination with optical trapping and microfluidics

    NASA Astrophysics Data System (ADS)

    Krafft, Christoph; Dochow, Sebastian; Beleites, Claudia; Popp, Jürgen

    2014-03-01

    Cell identification by Raman spectroscopy has evolved to be an attractive complement to established optical techniques. Raman activated cell sorting (RACS) offers prospects to complement the widely applied fluorescence activated cell sorting. RACS can be realized by combination with optical traps and microfluidic devices. The progress of RACS is reported for a cellular model system that can be found in peripheral blood of tumor patients. Lymphocytes and erythrocytes were extracted from blood samples. Breast carcinoma derived tumor cells (MCF-7, BT-20) and acute myeloid leukemia cells (OCI-AML3) were grown in cell cultures. First, Raman images were collected from dried cells on calcium fluoride slides. Support vector machines (SVM) classified 99.7% of the spectra to the correct cell type. Second, a 785 nm laser was used for optical trapping of single cells in aqueous buffer and for excitation of the Raman spectrum. SVM distinguished 1210 spectra of tumor and normal cells with a sensitivity of >99.7% and a specificity of >99.5%. Third, a microfluidic glass chip was designed to inject single cells, modify the flow speed, accommodate fibers of an optical trap and sort single cells after Raman based identification with 514 nm for excitation. Forth, the microfluidic chip was fabricated by quartz which improved cell identification results with 785 nm excitation. Here, partial least squares discriminant analysis gave classification rates of 98%. Finally, a Raman-on-chip approach was developed that integrates fibers for trapping, Raman excitation and signal detection in a single compact unit.

  1. Identification of Prostate Cancer-Related Genes Using Inhibition of NMD in Prostate Cancer Cell Lines

    DTIC Science & Technology

    2005-01-01

    TITLE AND SUBTITLE 5. FUNDING NUMBERS Identification of Prostate Cancer -Related Genes Using W81XWH-04- 1 -0045 Inhibition of NMD in Prostate Cancer Cell...analytical filter to the prostate cancer cell lines 22RV- 1 and DU-145. Ten genes for each cell line have been selected for sequencing analysis.(Table...list of candidate genes for sequencing analysis from the LNCaP, PC3, 22RV- 1 and DU- 145 prostate cancer cell lines has been produced REPORTABLE

  2. Dendritic cells and skin sensitization: Biological roles and uses in hazard identification

    SciTech Connect

    Ryan, Cindy A.; Kimber, Ian; Basketter, David A.; Pallardy, Marc; Gildea, Lucy A.; Gerberick, G. Frank . E-mail: gerberick.gf@pg.com

    2007-06-15

    Recent advances have been made in our understanding of the roles played by cutaneous dendritic cells (DCs) in the induction of contact allergy. A number of associated changes in epidermal Langerhans cell phenotype and function required for effective skin sensitization are providing the foundations for the development of cellular assays (using DC and DC-like cells) for skin sensitization hazard identification. These alternative approaches to the identification and characterization of skin sensitizing chemicals were the focus of a Workshop entitled 'Dendritic Cells and Skin Sensitization: Biological Roles and Uses in Hazard Identification' that was given at the annual Society of Toxicology meeting held March 6-9, 2006 in San Diego, California. This paper reports information that was presented during the Workshop.

  3. Identification of various testicular cell populations in pubertal and adult cockerels

    USDA-ARS?s Scientific Manuscript database

    Precise identification of the male germinal stem cell population is important for their practical use in programs dedicated to the integration of exogenous genetic material in testicular tissues. In the present study, our aim was to identify germinal cell populations in the testes of pubertal and ad...

  4. Effect of Heat Shock, (Ca(2+))-i, and cAMP on Inositol Trisphosphate in Human Epidermoid A-431 Cells

    DTIC Science & Technology

    1993-01-01

    processes blocked the increasin InsP, resulting fro, heat exposure Both pertuasi tomi (30 ng/mL, 24 h), an inhibitor of (3 inhibitory protein, and cholen...lylimidodiphosphate stimulated that component of the InsP 3 those studies a mechanism(s) underlying the heat-in- increase due to G proteins. U-73122, an inhibitor ...heat exposure. Both pertussis toxin (30 ng/ml, 24 shock may directly stimulate PLC (23). On the other h), an inhibitor of G inhibitory protein, and

  5. Isolation, Culture and Identification of Porcine Skeletal Muscle Satellite Cells.

    PubMed

    Li, Bo-Jiang; Li, Ping-Hua; Huang, Rui-Hua; Sun, Wen-Xing; Wang, Han; Li, Qi-Fa; Chen, Jie; Wu, Wang-Jun; Liu, Hong-Lin

    2015-08-01

    The objective of this study was to establish the optimum protocol for the isolation and culture of porcine muscle satellite cells. Mononuclear muscle satellite cells are a kind of adult stem cell, which is located between the basal lamina and sarcolemma of muscle fibers and is the primary source of myogenic precursor cells in postnatal muscle. Muscle satellite cells are a useful model to investigate the mechanisms of muscle growth and development. Although the isolation and culture protocols of muscle satellite cells in some species (e.g. mouse) have been established successfully, the culture system for porcine muscle satellite cells is very limited. In this study, we optimized the isolation procedure of porcine muscle satellite cells and elaborated the isolation and culture process in detail. Furthermore, we characterized the porcine muscle satellite cells using the immunofluorecence. Our study provides a reference for the isolation of porcine muscle satellite cells and will be useful for studying the molecular mechanisms in these cells.

  6. Identification of the Molecular Determinants of Breast Epithelial Cell Polarity

    DTIC Science & Technology

    2005-10-01

    cells as well as bladder carcinoma cell migration ( Wittchen et al. 2005). In addition, RapI was activated in metastatic mammary carcinoma of Crkl...human breast cells in three-dimensional culture and in vivo by integrin blocking antibodies. J Cell Biol. 137(1):231-45. Wittchen ES, van Buul JD

  7. Isolation and identification of epithelial-like cells in culture by a collagenase-separation technique.

    PubMed

    Kanoza, R J; Brunette, D M; Purdon, A D; Sodek, J

    1978-09-01

    An operational criterion for the identification and isolation of epithelial-like (E) cells, based on their ability to cover and protect a collagen gel from the action of collagenase, has been developed. The E cells isolated by this collagenase-separation technique (CST) exhibited the ultrastructural features, including desmosomes and abundant tonofilaments, that are considered characteristic of this cell type. Unlike confluent cultures of fibroblast-like (F) cells, E cells were not found to have large external transformation-sensitive (LETS) protein on their surface membranes. The CST provides a nondestructive and efficient means of identifying and isolating E cells from mixed populations.

  8. Identification of new cell size control genes in S. cerevisiae

    PubMed Central

    2012-01-01

    Cell size homeostasis is a conserved attribute in many eukaryotic species involving a tight regulation between the processes of growth and proliferation. In budding yeast S. cerevisiae, growth to a “critical cell size” must be achieved before a cell can progress past START and commit to cell division. Numerous studies have shown that progression past START is actively regulated by cell size control genes, many of which have implications in cell cycle control and cancer. Two initial screens identified genes that strongly modulate cell size in yeast. Since a second generation yeast gene knockout collection has been generated, we screened an additional 779 yeast knockouts containing 435 new ORFs (~7% of the yeast genome) to supplement previous cell size screens. Upon completion, 10 new strong size mutants were identified: nine in log-phase cells and one in saturation-phase cells, and 97% of the yeast genome has now been screened for cell size mutations. The majority of the logarithmic phase size mutants have functions associated with translation further implicating the central role of growth control in the cell division process. Genetic analyses suggest ECM9 is directly associated with the START transition. Further, the small (whi) mutants mrpl49Δ and cbs1Δ are dependent on CLN3 for cell size effects. In depth analyses of new size mutants may facilitate a better understanding of the processes that govern cell size homeostasis. PMID:23234503

  9. Study on activity measurement of Nostoc flagelliforme cells based on color identification

    NASA Astrophysics Data System (ADS)

    Wang, Yizhong; Su, Jianyu; Liu, Tiegen; Kong, Fanzhi; Jia, Shiru

    2008-12-01

    In order to measure the activities of Nostoc flagelliforme cells, a new method based on color identification was proposed in this paper. N. flagelliforme cells were colored with fluoreseein diaeetate. Then, an image of colored N. flagelliforme cells was taken, and changed from RGB model to HIS model. Its histogram of hue H was calculated, which was used as the input of a designed BP network. The output of the BP network was the description of measured activity of N. flagelliforme cells. After training, the activity of N. flagelliforme cells was identified by the BP network according to the histogram of H of their colored image. Experiments were conducted with satisfied results to show the feasibility and usefulness of activity measurement of N. flagelliforme cells based on color identification.

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

    PubMed Central

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

    2016-01-01

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

  11. Human satellite cells: identification on human muscle fibres

    PubMed Central

    Boldrin, Luisa; Morgan, Jennifer E

    2012-01-01

    Satellite cells, normally quiescent underneath the myofibre basal lamina, are skeletal muscle stem cells responsible for postnatal muscle growth, repair and regeneration. Since their scarcity and small size have limited study on transverse muscle sections, techniques to isolate individual myofibres, bearing their attendant satellite cells, were developed. Studies on mouse myofibres have generated much information on satellite cells, but the limited availability and small size of human muscle biopsies have hampered equivalent studies of satellite cells on human myofibres. Here, we identified satellite cells on fragments of human and mouse myofibres, using a method applicable to small muscle biopsies. PMID:22333991

  12. Functional identification of neural stem cell-derived oligodendrocytes.

    PubMed

    Grade, Sofia; Agasse, Fabienne; Bernardino, Liliana; Malva, João O

    2012-01-01

    Directing neural stem cells (NSCs) differentiation towards oligodendroglial cell lineage is a crucial step in the endeavor of developing cell replacement-based therapies for demyelinating diseases. Evaluation of NSCs differentiation is mostly performed by methodologies that use fixed cells, like immunocytochemistry, or lysates, like Western blot. On the other hand, electrophysiology allows differentiation studies on living cells, but it is highly time-consuming and endowed with important limitations concerning population studies. Herein, we describe a functional method, based on single cell calcium imaging, which accurately and rapidly distinguishes cell types among NSCs progeny, in living cultures prepared from the major reservoir of NSCs in the postnatal mouse brain, the subventricular zone (SVZ). Indeed, by applying a rational sequence of three stimuli-KCl, histamine, and thrombin-to the heterogeneous SVZ cell population, one can identify each cell phenotype according to its unique calcium signature. Mature oligodendrocytes, the myelin-forming cells of the central nervous system, are the thrombin-responsive cells in SVZ cell culture and display no intracellular calcium increase upon KCl or histamine perfusion. On the other hand, KCl and histamine stimulate neurons and immature cells, respectively. The method described in this chapter is a valuable tool to identify novel pro-oligodendrogenic compounds, which may play an important role in the design of future treatments for demyelinating disorders such as multiple sclerosis.

  13. Identification and behavior of label-retaining cells in epithelia

    SciTech Connect

    Bickenbach, J.R.

    1982-01-01

    A subpopulation of stem cells has been demonstrated in several renewing tissues. Such cells have a slow cell cycle and provide differentiating cells during normal turnover and during regeneration of the tissue following damage. The presence of slowly-cycling cells in epithelia from regions of skin and oral mucosa was examined by labeling 10-day-old mice and 5-day-old hamsters with tritiated thymidine (/sup 3/H-TdR) and observing the rate at which label was diluted from the basal cells. Label was rapidly diluted by cell division in most cells but a small percentage of basal cells (label-retaining cells, LRCS) was found to retain label for up to ninety days. Electron microscopic autoradiography and ..beta..-glucuronidase histochemistry with autoradiography were used to distinguish slowly-cycling keratinocytes from Langerhans cells. Such findings of slowly-cycling keratinocytes in epithelia with the ability to proliferate in culture and with a direct relationship to patterns of tissue architecture suggest that LRCs in epithelia correspond to stem cells described in other continuously renewing tissues.

  14. Isolation, cultivation and identification of human lung adenocarcinoma stem cells

    PubMed Central

    ZHANG, DE-GENG; JIANG, AI-GUI; LU, HUI-YU; ZHANG, LI-XIN; GAO, XIAO-YAN

    2015-01-01

    Recently, an increasing number of studies have demonstrated that lung cancer is a stem cell disease. However, ideal cell surface markers for isolating stem cells in lung cancer are yet to be identified. In the present study, a cell population with a cluster of differentiation (CD)133+ phenotype was successfully isolated from a single cell suspension of lung adenocarcinoma tissue using magnetic-activated cell sorting (MACS) and enriched in a serum-free culture. In comparison to CD133− cells, the CD133+ cells exhibited an enhanced capacity for self-renewal and differentiation, and a greater potential for in vivo tumor formation, in non-obese diabetic/severe combined immunodeficient (NOD/SCID) mice. Tumors could be induced in NOD/SCID mice by the transplantation of 102 stem-like cells per mouse. The results of the present study demonstrated that CD133 may serve as a specific cell surface marker for lung adenocarcinoma stem cells, and that MACS combined with serum-free culture is an effective method for isolating and enriching lung cancer stem cells. PMID:25435932

  15. Proteomic identification of mammalian cell surface derived glycosylphosphatidylinositol-anchored proteins through selective glycan enrichment

    PubMed Central

    Cortes, Leslie K; Vainauskas, Saulius; Dai, Nan; McClung, Colleen M; Shah, Manesh; Benner, Jack S; Corrêa, Ivan R; VerBerkmoes, Nathan C; Taron, Christopher H

    2014-01-01

    Glycosylphosphatidylinositol-anchored proteins (GPI-APs) are an important class of glycoproteins that are tethered to the surface of mammalian cells via the lipid GPI. GPI-APs have been implicated in many important cellular functions including cell adhesion, cell signaling, and immune regulation. Proteomic identification of mammalian GPI-APs en masse has been limited technically by poor sensitivity for these low abundance proteins and the use of methods that destroy cell integrity. Here, we present methodology that permits identification of GPI-APs liberated directly from the surface of intact mammalian cells through exploitation of their appended glycans to enrich for these proteins ahead of LC-MS/MS analyses. We validate our approach in HeLa cells, identifying a greater number of GPI-APs from intact cells than has been previously identified from isolated HeLa membranes and a lipid raft preparation. We further apply our approach to define the cohort of endogenous GPI-APs that populate the distinct apical and basolateral membrane surfaces of polarized epithelial cell monolayers. Our approach provides a new method to achieve greater sensitivity in the identification of low abundance GPI-APs from the surface of live cells and the nondestructive nature of the method provides new opportunities for the temporal or spatial analysis of cellular GPI-AP expression and dynamics. PMID:25262930

  16. Identification of cell lines permissive for human coronavirus NL63.

    PubMed

    Schildgen, Oliver; Jebbink, Maarten F; de Vries, Michel; Pyrc, Krzysztov; Dijkman, Ronald; Simon, Arne; Müller, Andreas; Kupfer, Bernd; van der Hoek, Lia

    2006-12-01

    Six cell lines routinely used in laboratories were tested for permissiveness to the infection with the newly identified human coronavirus NL63. Two monkey epithelial cell lines, LLC-MK2 and Vero-B4, showed a cytopathic effect (CPE) and clear viral replication, whereas no CPE or replication was observed in human lung fibroblasts MRC-5s. In Rhabdomyosarcoma cells, Madin-Darby-Canine-kidney cells and in an undefined monkey kidney cell line some replication was observed but massive exponential rise in virus yield lacked The results will lead to an improved routine diagnostic algorithm for the detection of the human coronavirus NL63.

  17. Raman spectroscopic identification of normal and malignant human stomach cells

    NASA Astrophysics Data System (ADS)

    Yang, Jipeng; Guo, Jianyu; Wu, Liangping; Sun, Zhenrong; Cai, Weiying; Wang, Zugeng

    2005-12-01

    Micro-Raman spectroscopy is employed to identify the normal and malignant human stomach cells. For the cancer cell, the reduced intensity of the Raman peak at 1250 cm^(-1) indicates that the protein secondary structure transforms from ?-sheet or disordered structures to ?-helical, while the increased intensity of the symmetric PO2 stretching vibration mode at 1094 cm^(-1) shows the increased DNA content. The ratio of the intensity at 1315 cm^(-1) to that at 1340 cm^(-1) reduces from 1.8 for the normal cell to 1.1 for the cancer cell in the course of canceration, and the ratio of the intensity at 1655 cm^(-1) to that at 1450 cm^(-1) increases from 1.00 for the cancer cell to 1.26 for the normal cell which indicates that the canceration of stomach cell may induce saturation of the lipid chain.

  18. Identification of cell cycle-regulated genes in fission yeast.

    PubMed

    Peng, Xu; Karuturi, R Krishna Murthy; Miller, Lance D; Lin, Kui; Jia, Yonghui; Kondu, Pinar; Wang, Long; Wong, Lim-Soon; Liu, Edison T; Balasubramanian, Mohan K; Liu, Jianhua

    2005-03-01

    Cell cycle progression is both regulated and accompanied by periodic changes in the expression levels of a large number of genes. To investigate cell cycle-regulated transcriptional programs in the fission yeast Schizosaccharomyces pombe, we developed a whole-genome oligonucleotide-based DNA microarray. Microarray analysis of both wild-type and cdc25 mutant cell cultures was performed to identify transcripts whose levels oscillated during the cell cycle. Using an unsupervised algorithm, we identified 747 genes that met the criteria for cell cycle-regulated expression. Peaks of gene expression were found to be distributed throughout the entire cell cycle. Furthermore, we found that four promoter motifs exhibited strong association with cell cycle phase-specific expression. Examination of the regulation of MCB motif-containing genes through the perturbation of DNA synthesis control/MCB-binding factor (DSC/MBF)-mediated transcription in arrested synchronous cdc10 mutant cell cultures revealed a subset of functional targets of the DSC/MBF transcription factor complex, as well as certain gene promoter requirements. Finally, we compared our data with those for the budding yeast Saccharomyces cerevisiae and found approximately 140 genes that are cell cycle regulated in both yeasts, suggesting that these genes may play an evolutionarily conserved role in regulation of cell cycle-specific processes. Our complete data sets are available at http://giscompute.gis.a-star.edu.sg/~gisljh/CDC.

  19. Identification of peptides that bind to irradiated pancreatic tumor cells

    SciTech Connect

    Huang Canhui; Liu, Xiang Y.; Rehemtulla, Alnawaz; Lawrence, Theodore S. . E-mail: tsl@med.umich.edu

    2005-08-01

    Purpose: Peptides targeting tumor vascular cells or tumor cells themselves have the potential to be used as vectors for delivering either DNA in gene therapy or antitumor agents in chemotherapy. We wished to determine if peptides identified by phage display could be used to target irradiated pancreatic cancer cells. Methods and Materials: Irradiated Capan-2 cells were incubated with 5 x 10{sup 12} plaque-forming units of a phage display library. Internalized phage were recovered and absorbed against unirradiated cells. After five such cycles of enrichment, the recovered phage were subjected to DNA sequencing analysis and synthetic peptides made. The binding of both phage and synthetic peptides was evaluated by fluorescence staining and flow cytometry in vitro and in vivo. Results: We identified one 12-mer peptide (PA1) that binds to irradiated Capan-2 pancreatic adenocarcinoma cells but not to unirradiated cells. The binding of peptide was significant after 48 h incubation with cells. In vivo experiments with Capan-2 xenografts in nude mice demonstrated that these small peptides are able to penetrate tumor tissue after intravenous injections and bind specifically to irradiated tumor cells. Conclusion: These data suggest that peptides can be identified that target tumors with radiation-induced cell markers and may be clinically useful.

  20. Induction and identification of rabbit peripheral blood derived dendritic cells

    NASA Astrophysics Data System (ADS)

    Zhou, Jing; Yang, FuYuan; Chen, WenLi

    2012-03-01

    Purpose: To study a method of the induction of dendritic cells (DCs) from rabbit peripheral blood. Methods: Peripheral blood cells were removed from rabbit, filtered through nylon mesh. Peripheral blood mononuclear cells (PBMC) were isolated from the blood cells by Ficoll-Hypaque centrifugation (density of 1.077g/cm3).To obtain DCs, PBMC were cultured in RPMI1640 medium containing 10% fetal calf serum, 50U/mL penicillin and streptomycin, referred to subsequently as complete medium, at 37°C in 5% CO2 atmosphere for 4 hours. Nonadherent cells were aspirated, adherent cells were continued incubated in complete medium, supplemented with granulocyte/macrophage colony-stimulating factor (GM-CSF, 50ng/ml),and interleukin 4 (IL-4, 50ng/ml) for 9 days. Fluorescein labeled antibodies(anti-CD14, anti-HLA-DR, anti-CD86) were used to sign cells cultured for 3,6,9 days respectively, Then flow cytometry was performed. Results: Ratio of anti-HLA-DR and anti-CD86 labeled cells increased with induction time extension, in contrast with anti-CD14. Conclusion: Dendritic cells can be effectively induced by the method of this experiment, cell maturation status increased with induction time extension.

  1. Identification of molecular pathways facilitating glioma cell invasion in situ.

    PubMed

    Nevo, Ido; Woolard, Kevin; Cam, Maggie; Li, Aiguo; Webster, Joshua D; Kotliarov, Yuri; Kim, Hong Sug; Ahn, Susie; Walling, Jennifer; Kotliarova, Svetlana; Belova, Galina; Song, Hua; Bailey, Rolanda; Zhang, Wei; Fine, Howard A

    2014-01-01

    Gliomas are mostly incurable secondary to their diffuse infiltrative nature. Thus, specific therapeutic targeting of invasive glioma cells is an attractive concept. As cells exit the tumor mass and infiltrate brain parenchyma, they closely interact with a changing micro-environmental landscape that sustains tumor cell invasion. In this study, we used a unique microarray profiling approach on a human glioma stem cell (GSC) xenograft model to explore gene expression changes in situ in Invading Glioma Cells (IGCs) compared to tumor core, as well as changes in host cells residing within the infiltrated microenvironment relative to the unaffected cortex. IGCs were found to have reduced expression of genes within the extracellular matrix compartment, and genes involved in cell adhesion, cell polarity and epithelial to mesenchymal transition (EMT) processes. The infiltrated microenvironment showed activation of wound repair and tissue remodeling networks. We confirmed by protein analysis the downregulation of EMT and polarity related genes such as CD44 and PARD3 in IGCs, and EFNB3, a tissue-remodeling agent enriched at the infiltrated microenvironment. OLIG2, a proliferation regulator and glioma progenitor cell marker upregulated in IGCs was found to function in enhancing migration and stemness of GSCs. Overall, our results unveiled a more comprehensive picture of the complex and dynamic cell autonomous and tumor-host interactive pathways of glioma invasion than has been previously demonstrated. This suggests targeting of multiple pathways at the junction of invading tumor and microenvironment as a viable option for glioma therapy.

  2. Identification of a putative azadirachtin-binding complex from Drosophila Kc167 cells.

    PubMed

    Robertson, Susan L; Ni, Weiting; Dhadialla, Tarlochan S; Nisbet, Alasdair J; McCusker, Catherine; Ley, Steven V; Mordue, William; Mordue 'Luntz', A Jennifer

    2007-04-01

    Cell-proliferation in Drosophila Kc167 cells was inhibited by 50% when cell cultures contained 1.7 x 10(-7) M azadirachtin for 48 h (a tertranortriterpenoid from the neem tree Azadirachta indica). Drosophila Kc167 cells exhibited direct nuclear damage within 6-h exposure to azadirachtin (5 x 10(-7) M and above) or within 24 h when lower concentrations were used (1 x 10(-9) M). Fractionation of an extract of Drosophila Kc167 cells combined with ligand overlay technique resulted in the identification of a putative azadirachtin binding complex. Identification of the members of this complex by Peptide Mass Fingerprinting (PMF) and N-terminal sequencing identified heat shock protein 60 (hsp60) as one of its components.

  3. An Improved Algorithm of Congruent Matching Cells (CMC) Method for Firearm Evidence Identifications

    PubMed Central

    Tong, Mingsi; Song, John; Chu, Wei

    2015-01-01

    The Congruent Matching Cells (CMC) method was invented at the National Institute of Standards and Technology (NIST) for firearm evidence identifications. The CMC method divides the measured image of a surface area, such as a breech face impression from a fired cartridge case, into small correlation cells and uses four identification parameters to identify correlated cell pairs originating from the same firearm. The CMC method was validated by identification tests using both 3D topography images and optical images captured from breech face impressions of 40 cartridge cases fired from a pistol with 10 consecutively manufactured slides. In this paper, we discuss the processing of the cell correlations and propose an improved algorithm of the CMC method which takes advantage of the cell correlations at a common initial phase angle and combines the forward and backward correlations to improve the identification capability. The improved algorithm is tested by 780 pairwise correlations using the same optical images and 3D topography images as the initial validation. PMID:26958441

  4. Fired Cartridge Case Identification Using Optical Images and the Congruent Matching Cells (CMC) Method

    PubMed Central

    Tong, Mingsi; Song, John; Chu, Wei; Thompson, Robert M

    2014-01-01

    The Congruent Matching Cells (CMC) method for ballistics identification was invented at the National Institute of Standards and Technology (NIST). The CMC method is based on the correlation of pairs of small correlation cells instead of the correlation of entire images. Four identification parameters – TCCF, Tθ, Tx and Ty are proposed for identifying correlated cell pairs originating from the same firearm. The correlation conclusion (matching or non-matching) is determined by whether the number of CMC is ≥ 6. This method has been previously validated using a set of 780 pair-wise 3D topography images. However, most ballistic images stored in current local and national databases are in an optical intensity (grayscale) format. As a result, the reliability of applying the CMC method on optical intensity images is an important issue. In this paper, optical intensity images of breech face impressions captured on the same set of 40 cartridge cases are correlated and analyzed for the validation test of CMC method using optical images. This includes correlations of 63 pairs of matching images and 717 pairs of non-matching images under top ring lighting. Tests of the method do not produce any false identification (false positive) or false exclusion (false negative) results, which support the CMC method and the proposed identification criterion, C = 6, for firearm breech face identifications using optical intensity images. PMID:26601045

  5. Identification of a novel EGF-sensitive cell cycle checkpoint

    SciTech Connect

    Walker, Francesca . E-mail: francesca.walker@ludwig.edu.au; Zhang Huihua; Burgess, Antony W.

    2007-02-01

    The site of action of growth factors on mammalian cell cycle has been assigned to the boundary between the G1 and S phases. We show here that Epidermal Growth Factor (EGF) is also required for mitosis. BaF/3 cells expressing the EGFR (BaF/wtEGFR) synthesize DNA in response to EGF, but arrest in S-phase. We have generated a cell line (BaF/ERX) with defective downregulation of the EGFR and sustained activation of EGFR signalling pathways: these cells undergo mitosis in an EGF-dependent manner. The transit of BaF/ERX cells through G2/M strictly requires activation of EGFR and is abolished by AG1478. This phenotype is mimicked by co-expression of ErbB2 in BaF/wtEGFR cells, and abolished by inhibition of the EGFR kinase, suggesting that sustained signalling of the EGFR, through impaired downregulation of the EGFR or heterodimerization, is required for completion of the cycle. We have confirmed the role of EGFR signalling in the G2/M phase of the cell cycle using a human tumor cell line which overexpresses the EGFR and is dependent on EGFR signalling for growth. These findings unmask an EGF-sensitive checkpoint, helping to understand the link between sustained EGFR signalling, proliferation and the acquisition of a radioresistant phenotype in cancer cells.

  6. Identification of Mouse Mesenteric and Subcutaneous in vitro Adipogenic Cells.

    PubMed

    Miyata, Yugo; Otsuki, Michio; Kita, Shunbun; Shimomura, Iichiro

    2016-02-17

    Fat accumulation and the dysfunction of visceral white adipose tissue (WAT), but not subcutaneous WAT, cause abnormalities in whole body metabolic homeostasis. However, no current drugs specifically target visceral WAT. The primary reason for this is that a practical in vitro culture system for mesenteric adipocytes has not been established. To resolve this issue, we sought to identify in vitro adipogenic cells in mesenteric and subcutaneous WATs. First, we examined the expression pattern of surface antigens in stromal-vascular fraction (SVF) cells from mouse mesenteric and subcutaneous WATs, and found the expression of 30 stem cell-related surface antigens. Then, to evaluate the adipogenic ability of each fraction, we performed in vitro screening, and identified five candidate markers for mesenteric adipogenic cells and one candidate marker for subcutaneous adipogenic cells. To investigate whether in vitro adipogenic ability accurately reflects the conditions in vivo, we performed transplantation experiments, and identified CD9(-) CD201(+) Sca-1(-) cells and CD90(+) cells as mesenteric and subcutaneous in vitro adipogenic cells, respectively. Furthermore, mature adipocytes derived from mesenteric and subcutaneous adipogenic cells maintained each characteristic phenotype in vitro. Thus, our study should contribute to the development of a useful culture system for visceral adipocytes.

  7. Identification of Mouse Mesenteric and Subcutaneous in vitro Adipogenic Cells

    PubMed Central

    Miyata, Yugo; Otsuki, Michio; Kita, Shunbun; Shimomura, Iichiro

    2016-01-01

    Fat accumulation and the dysfunction of visceral white adipose tissue (WAT), but not subcutaneous WAT, cause abnormalities in whole body metabolic homeostasis. However, no current drugs specifically target visceral WAT. The primary reason for this is that a practical in vitro culture system for mesenteric adipocytes has not been established. To resolve this issue, we sought to identify in vitro adipogenic cells in mesenteric and subcutaneous WATs. First, we examined the expression pattern of surface antigens in stromal-vascular fraction (SVF) cells from mouse mesenteric and subcutaneous WATs, and found the expression of 30 stem cell-related surface antigens. Then, to evaluate the adipogenic ability of each fraction, we performed in vitro screening, and identified five candidate markers for mesenteric adipogenic cells and one candidate marker for subcutaneous adipogenic cells. To investigate whether in vitro adipogenic ability accurately reflects the conditions in vivo, we performed transplantation experiments, and identified CD9− CD201+ Sca-1− cells and CD90+ cells as mesenteric and subcutaneous in vitro adipogenic cells, respectively. Furthermore, mature adipocytes derived from mesenteric and subcutaneous adipogenic cells maintained each characteristic phenotype in vitro. Thus, our study should contribute to the development of a useful culture system for visceral adipocytes. PMID:26884347

  8. Prostate cancer stem/progenitor cells: identification, characterization, and implications.

    PubMed

    Tang, Dean G; Patrawala, Lubna; Calhoun, Tammy; Bhatia, Bobby; Choy, Grace; Schneider-Broussard, Robin; Jeter, Collene

    2007-01-01

    Several solid tumors have now been shown to contain stem cell-like cells called cancer stem cells (CSC). These cells, although generally rare, appear to be highly tumorigenic and may be the cells that drive tumor formation, maintain tumor homeostasis, and mediate tumor metastasis. In this Perspective, we first provide our insight on how a CSC should be defined. We then summarize our current knowledge of stem/progenitor cells in the normal human prostate (NHP), an organ highly susceptible to hyperproliferative diseases such as benign prostate hyperplasia (BPH) and prostate cancer (PCa). We further review the evidence that cultured PCa cells, xenograft prostate tumors, and patient tumors may contain stem/progenitor cells. Along with our discussion, we present several methodologies that can be potentially used to identify putative tumor-reinitiating CSC. Finally, we present a hypothetical model for the hierarchical organization of human PCa cells and discuss the implications of this model in helping understand prostate carcinogenesis and design novel diagnostic, prognostic, and therapeutic approaches.

  9. In vivo cell characteristic extraction and identification by photoacoustic flow cytography

    PubMed Central

    He, Guo; Xu, Dong; Qin, Huan; Yang, Sihua; Xing, Da

    2015-01-01

    We present a photoacoustic flow cytography with fast cross-sectional (B-scan) imaging to precisely identify specific cells in vivo. The B-scan imaging speed of the system is up to 200 frame/s with a lateral resolution of 1.5 μm, which allows to dynamically image the flowing cells within the microvascular. The shape, size and photoacoustic intensity of the target cells are extracted from streaming images and integrated into a standard pattern to distinguish cell types. Circulating red blood cells and melanoma cells in blood vessels are simultaneously identified on melanoma-bearing mouse model. The results demonstrate that in vivo photoacoustic flow cytography can provide cells characteristics analysis and cell type’s visual identification, which will be applied for noninvasively monitoring circulating tumor cells (CTCs) and analyzing hematologic diseases. PMID:26504626

  10. Parameter identification for model of T cell proliferation in lymphopenia conditions.

    PubMed

    Ayoub, H; Ainseba, B E; Langlais, M; Hogan, T; Callard, R; Seddon, B; Thiébaut, R

    2014-05-01

    The number of T Lymphocytes (T cells) in the body is under homeostatic control. At equilibrium, the majority of naive T cells are non-dividing and express low levels of the surface protein CD44. In conditions of T cell deficiency (lymphopenia), naive T cells enter into a proliferative phase, undergoing cell division accompanied by a subtle change in their surface expression of CD44. In this study, we use a mathematical modelling approach to analyse the proliferative response of transgenic T cells in lymphopenic conditions. Our nonlinear model is composed of ordinary differential equations and partial differential equations structured by age (maturity of cell) and CD44 expression. To better understand the evolution of CD44 expression on the surface of T cells during cell division, we present a numerical analysis to solve a parameter identification problem. Finally, we show the parameters and the simulations that we obtain from the model and compare them to experimental data.

  11. Identification of inflammatory cells in bovine milk by flow cytometry.

    PubMed

    Redelman, D; Butler, S; Robison, J; Garner, D

    1988-09-01

    Cells recovered from normal or mastitic bovine milk were examined by flow cytometry. All milk samples contained particulate material that was heterogeneous in size and that produced a right-angle light-scatter signal equal to or greater than that produced by human or bovine neutrophils. Although this material labeled with Hoechst 33342, it produced fluorescence intensities below that of intact bovine cells, suggesting that it consisted of cell fragments. Mastitic milk additionally contained other populations of cells that were poorly resolved from the normal particulate material by size (electronic volume sensor) and right-angle light scatter. In order to improve this resolution, the milk cells were incubated with carboxydimethylfluorescein diacetate (CMFDA) to label intact cells. When milk samples labeled with CMFDA were examined by dual-parameter analysis using green fluorescence and right-angle light scatter, five or more populations of cells could be identified in mastitic milk. These populations included intact and degenerate neutrophils, lymphocytes, including both small and activated cells, monocytes, and large activated macrophages containing many vacuoles and phagocytosed particles. Using this procedure, all the animals in the University of Nevada-Reno Holstein dairy herd were tested once a month for 6 months. In addition, individual animals with mastitis were examined one or more times each day during the course of the inflammatory process. In the routine screening, the flow cytometric examination detected mastitis before overt symptoms developed. In cows identified to have mastitis, the flow cytometric examination provided prognostic information regarding the success of treatments.

  12. Identification of functional progenitor cells in the pulmonary vasculature

    PubMed Central

    Firth, Amy L.; Yuan, Jason X. -J.

    2012-01-01

    The pulmonary vasculature comprises a complex network of branching arteries and veins all functioning to reoxygenate the blood for circulation around the body. The cell types of the pulmonary artery are able to respond to changes in oxygen tension in order to match ventilation to perfusion. Stem and progenitor cells in the pulmonary vasculature are also involved, be it in angiogenesis, endothelial dysfunction or formation of vascular lesions. Stem and progenitor cells may be circulating around the body, residing in the pulmonary artery wall or stimulated for release from a central niche like the bone marrow and home to the pulmonary vasculature along a chemotactic gradient. There may currently be some controversy over the pathogenic versus therapeutic roles of stem and progenitor cells and, indeed, it is likely both chains of evidence are correct due to the specific influence of the immediate environmental niche a progenitor cell may be in. Due to their great plasticity and a lack of specific markers for stem and progenitor cells, they can be difficult to precisely identify. This review discusses the methodological approaches used to validate the presence of and subtype of progenitors cells in the pulmonary vasculature while putting it in context of the current knowledge of the therapeutic and pathogenic roles for such progenitor cells. PMID:22558524

  13. Isolation, identification, and characterization of cancer stem cells: A review.

    PubMed

    Abbaszadegan, Mohammad Reza; Bagheri, Vahid; Razavi, Mahya Shariat; Momtazi, Amir Abbas; Sahebkar, Amirhossein; Gholamin, Mehran

    2017-08-01

    Cancer stem cells (CSCs) or tumor-initiating cells (TICs) as a small subset of neoplastic cells are able to produce a tumor (tumorigenesis), maintain the population of tumorigenic cells (self-renewal), and generate the heterogeneous cells constructing the entire tumor (pluripotency). The research on stationary and circulating CSCs due to resistance to conventional therapies and inability in complete eradication of cancer is critical for developing novel therapeutic strategies for a more effective reduction in the risk of tumor metastasis and cancer recurrence. This review compiles information about different methods of detection and dissociation, side population, cellular markers, and establishment culture of CSCs, as well as characteristics of CSCs such as tumorigenicity, and signaling pathways associated with self-renewal and the capability of the same histological tumor regeneration in various cancers. © 2016 Wiley Periodicals, Inc.

  14. Identification of molecular markers of bipolar cells in the murine retina

    PubMed Central

    Kim, Douglas S; Ross, Sarah E; Trimarchi, Jeffrey M; Aach, John; Greenberg, Michael E; Cepko, Constance L

    2008-01-01

    Retinal bipolar neurons serve as relay interneurons that connect rod and cone photoreceptor cells to amacrine and ganglion cells. They exhibit diverse morphologies essential for correct routing of photoreceptor cell signals to specific postsynaptic amacrine and ganglion cells. The development and physiology of these interneurons have not been completely defined molecularly. Despite previous identification of genes expressed in several bipolar cell subtypes, molecules that mark each bipolar cell type still await discovery. In this report, novel genetic markers of murine bipolar cells were found. Candidates were initially generated by using microarray analysis of single bipolar cells and mining of retinal serial analysis of gene expression (SAGE) data. These candidates were subsequently tested for expression in bipolar cells by RNA in situ hybridization. Ten new molecular markers were identified, five of which are highly enriched in their expression in bipolar cells within the adult retina. Double-labeling experiments using probes for previously characterized subsets of bipolar cells were performed to identify the subtypes of bipolar cells that express the novel markers. Additionally, the expression of bipolar cell genes was analyzed in Bhlhb4 knockout retinas, in which rod bipolar cells degenerate postnatally, to delineate further the identity of bipolar cells in which novel markers are found. From the analysis of Bhlhb4 mutant retinas, cone bipolar cell gene expression appears to be relatively unaffected by the degeneration of rod bipolar cells. Identification of molecular markers for the various subtypes of bipolar cells will lead to greater insights into the development and function of these diverse interneurons. PMID:18260140

  15. Identification of autoreactive B cells with labeled nucleosomes.

    PubMed

    Gies, Vincent; Wagner, Alain; Seifert, Cécile; Guffroy, Aurélien; Fauny, Jean-D; Knapp, Anne-M; Pasquali, Jean-L; Martin, Thierry; Dumortier, Hélène; Korganow, Anne-S; Soulas-Sprauel, Pauline

    2017-04-04

    The pathogenesis of autoimmune diseases has not been completely elucidated yet, and only a few specific treatments have been developed so far. In autoimmune diseases mediated by pathogenic autoantibodies, such as systemic lupus erythematosus, the specific detection and analysis of autoreactive B cells is crucial for a better understanding of the physiopathology. Biological characterization of these cells may help to define new therapeutic targets. Very few techniques allowing the precise detection of autoreactive B cells have been described so far. Herein we propose a new flow cytometry technique for specific detection of anti-nucleosome B cells, which secrete autoantibodies in systemic lupus erythematosus, using labeled nucleosomes. We produced different fluorochrome-labeled nucleosomes, characterized them, and finally tested them in flow cytometry. Nucleosomes labeled via the cysteines present in H3 histone specifically bind to autoreactive B cells in the anti-DNA transgenic B6.56R mice model. The present work validates the use of fluorochrome-labeled nucleosomes via cysteines to identify anti-nucleosome B cells and offers new opportunities for the description of autoreactive B cell phenotype.

  16. Identification and characterization of cell-bound membrane vesicles.

    PubMed

    Tang, Qisheng; Zhang, Xiaojun; Zhang, Wendiao; Zhao, Siyuan; Chen, Yong

    2017-05-01

    In contrast to the released/circulating membrane vesicles (extracellular vesicles), cell-bound membrane vesicles are poorly identified and characterized. In this study, cell-bound membrane vesicles on human umbilical vein endothelial cells (HUVECs) and human hepatoma HepG-2 cells were investigated. We identified that cell-bound membrane vesicles are not co-localized with the major markers for extracellular vesicles (e.g. phosphatidylserine, CD63, CD107α, CD31, and DNA fragments for the three well-known types of extracellular vesicles) and for intracellular organelles with similar sizes (e.g. MitoTracker and LAMP1/LAMP3 for mitochondria and multivesicular bodies or lysosomes, respectively). The data imply that cell-bound membrane vesicles are neither the precursors of extracellular vesicles nor a false structure pushed up by an intracellular organelle but probably a novel unknown structure in the plasma membrane. Moreover, we revealed that cell-bound membrane vesicles are resistant to various detergents including but probably not limited to Triton X-100, SDS, and saponin. We further characterized that these unique vesicles are soluble in organic solvents (e.g. chloroform-methanol mixture and ethanol) which can be prevented by a lipid-stabilizing fixative (e.g. OsO4) and that they are co-localized with, but do not monopolize, the major markers (e.g. caveolin-1 and GM1) for lipid rafts (a nano-sized detergent-resistant domains in the plasma membrane). The data imply that cell-bound membrane vesicles contain the lipid component and lipid rafts. Involvement of other specific unknown components might explain the detergent resistance of cell-bound membrane vesicles. Further research will mainly depend on the establishment of an effective approach for isolation/purification of these vesicles from the plasma membrane. Copyright © 2017 Elsevier B.V. All rights reserved.

  17. Laser rastering flow cytometry: fast cell counting and identification

    NASA Astrophysics Data System (ADS)

    Vacca, G.; Junnarkar, M. R.; Goldblatt, N. R.; Yee, M. W.; Van Slyke, B. M.; Briese, T. C.

    2009-02-01

    We describe the concept of laser rastering flow cytometry, where a rapidly scanning laser beam allows counting and classification of cells at much higher rates than currently possible. Modifications to existing flow cytometers to implement the concept include an acousto-optic deflector, fast analog-to-digital conversion, and a two-step digital-signal-processing scheme that handles the high data rates and provides key assay information. Results are shown that prove the concept, demonstrating the ability to resolve closely spaced cells and to measure cells at rates more than an order of magnitude faster than on conventional flow-cytometer-based hematology analyzers.

  18. Identification of Senescent Cells in the Bone Microenvironment

    PubMed Central

    Farr, Joshua N; Fraser, Daniel G; Wang, Haitao; Jaehn, Katharina; Ogrodnik, Mikolaj B; Weivoda, Megan M; Drake, Matthew T; Tchkonia, Tamara; LeBrasseur, Nathan K; Kirkland, James L; Bonewald, Lynda F; Pignolo, Robert J; Monroe, David G; Khosla, Sundeep

    2017-01-01

    Cellular senescence is a fundamental mechanism by which cells remain metabolically active yet cease dividing and undergo distinct phenotypic alterations, including upregulation of p16Ink4a, profound secretome changes, telomere shortening, and decondensation of pericentromeric satellite DNA. Because senescent cells accumulate in multiple tissues with aging, these cells and the dysfunctional factors they secrete, termed the senescence-associated secretory phenotype (SASP), are increasingly recognized as promising therapeutic targets to prevent age-related degenerative pathologies, including osteoporosis. However, the cell type(s) within the bone microenvironment that undergoes senescence with aging in vivo has remained poorly understood, largely because previous studies have focused on senescence in cultured cells. Thus in young (age 6 months) and old (age 24 months) mice, we measured senescence and SASP markers in vivo in highly enriched cell populations, all rapidly isolated from bone/marrow without in vitro culture. In both females and males, p16Ink4a expression by real-time quantitative polymerase chain reaction (rt-qPCR) was significantly higher with aging in B cells, T cells, myeloid cells, osteoblast progenitors, osteoblasts, and osteocytes. Further, in vivo quantification of senescence-associated distension of satellites (SADS), ie, large-scale unraveling of pericentromeric satellite DNA, revealed significantly more senescent osteocytes in old compared with young bone cortices (11% versus 2%, p < 0.001). In addition, primary osteocytes from old mice had sixfold more (p < 0.001) telomere dysfunction-induced foci (TIFs) than osteocytes from young mice. Corresponding with the age-associated accumulation of senescent osteocytes was significantly higher expression of multiple SASP markers in osteocytes from old versus young mice, several of which also showed dramatic age-associated upregulation in myeloid cells. These data show that with aging, a subset of cells

  19. Identification of holocarboxylase synthetase chromatin binding sites in human mammary cell lines using the DNA adenine methyltransferase identification technology.

    PubMed

    Singh, Dipika; Pannier, Angela K; Zempleni, Janos

    2011-06-01

    Holocarboxylase synthetase (HCS) is a chromatin protein that is essential for mediating the covalent binding of biotin to histones. Biotinylation of histones plays crucial roles in the repression of genes and repeats in the human genome. We tested the feasibility of DNA adenine methyltransferase identification (DamID) technology to map HCS binding sites in human mammary cell lines. Full-length HCS was fused to DNA adenine methyltransferase (Dam) for subsequent transfection into breast cancer (MCF-7) and normal breast (MCF-10A) cells. HCS docking sites in chromatin were identified by using the unique adenine methylation sites established by Dam in the fusion construct; docking sites were unambiguously identified using methylation-sensitive digestion, cloning, and sequencing. In total, 15 novel HCS binding sites were identified in the two cell lines, and the following 4 of the 15 overlapped between MCF-7 and MCF-10A cells: inositol polyphosphate-5-phosphatase A, corticotropin hormone precursor, ribosome biogenesis regulatory protein, and leptin precursor. We conclude that DamID is a useful technology to map HCS binding sites in human chromatin and propose that the entire set of HCS binding sites could be mapped by combining DamID with microarray technology.

  20. PCR identification of Salmonella cells in food and stool samples after immunomagnetic separation.

    PubMed

    Spanová, A; Rittich, B; Karpísková, R; Cechová, L; Skapová, D

    2000-01-01

    The purpose of the present study was to investigate the application of various sample preparation methods (cell washing before lysis, purification of DNA using phenol extraction method, immunomagnetic separation-IMS) for the final PCR identification of Salmonella cells. The presence of PCR inhibitors in processed food products (milk powder and dried eggs) can be the cause of false-negative results in PCR without IMS of target cells. It was also demonstrated that IMS-PCR was successfully used for identification and quick confirmation of untypical Salmonella strains isolated from human stool samples and rabbit meat. However, IMS cannot eliminate intracellular PCR inhibitors present in immunoseparated Salmonella cells. These inhibitors must be taken into consideration in evaluation of PCR procedure.

  1. Generation and identification of tumor-evoked regulatory B cells

    PubMed Central

    Biragyn, Arya; Lee-Chang, Catalina; Bodogai, Monica

    2014-01-01

    The involvement of Bregs in cancer remains poorly understood despite their well-documented regulation of responses to the self and protection from harmful autoimmunity. We recently discovered a unique regulatory B cell subset evoked by breast cancer to mediate protection of metastasizing cancer cells. These results together with the wealth of findings of the last 40 years on B cells in tumorigenesis suggest the existence of additional cancer Bregs modulating anticancer responses. To facilitate the search for them, here we provide our detailed protocol for the characterization and generation of tumor-evoked regulatory B cells. Wherever applicable, we also discuss nuances and uniqueness of a Breg study in cancer to warn potential pitfalls. PMID:25015287

  2. [Identification of proliferating cells in Taenia solium cysts].

    PubMed

    Orrego-Solano, Miguel Ángel; Cangalaya, Carla; Nash, Theodore E; Guerra-Giraldez, Cristina

    2014-01-01

    Neoblasts are totipotent cells, solely responsible for the proliferation and maturation of tissues in free-living flatworms. Similar cells have been isolated from parasitic flatworms such as Echinococcus. Taenia solium causes human taeniasis (intestinal) and cysticercosis in humans and pigs. Brain infection with larvae (cysts) of T. solium results in neurocysticercosis which is hyperendemic in Peru, and its treatment is associated with serious neurological symptoms. The proliferative capacity and development stages of T. solium have not been described and the neoblasts of this parasite have not been characterized We looked for cell proliferation in T. solium cysts collected from an infected pig, which were identified when replicating and incorporating bromodeoxyuridine nucleotide detected with a monoclonal antibody. A stable cell line of neoblasts would be useful for systematic in vitro studies on drug efficacy and the biology of T. solium.

  3. Identification, Characterization, and Utilization of Adult Meniscal Progenitor Cells

    DTIC Science & Technology

    2016-09-01

    understanding of the molecular and cellular events that underlie tissue repair is essential for addressing pathologies associated with injury, disease and...aging in the musculoskeletal system. Little information exists about resident stem cell populations in the mouse meniscus, a model increasingly used to...for meniscal pathologies . Analysis of meniscus derived-stem cells in an animal model such as the mouse will allow for detailed studies of their

  4. Identification of the Molecular Determinants of Breast Epithelial Cell Polarity

    DTIC Science & Technology

    2004-10-01

    most apical part of lateral membranes, indicating that apico-basal polarity is established even in malignant T4 cells. In 3D lrBM culture, desmosomes ...and hemidesmosomes are highly visible in lateral and basal domains of S I cells, respectively. On the other hand, desmosomes and hemidesmosomes are...restored distinct localizations of desmosomes and hemidesmosomes and thus established basal polarity. TJs are difficult to identify in Fig. 3B as they are

  5. Identification and characterization of latency-associated peptide-expressing γδ T cells

    PubMed Central

    Rezende, Rafael M.; da Cunha, Andre P.; Kuhn, Chantal; Rubino, Stephen; M'Hamdi, Hanane; Gabriely, Galina; Vandeventer, Tyler; Liu, Shirong; Cialic, Ron; Pinheiro-Rosa, Natalia; Oliveira, Rafael P.; Gaublomme, Jellert T.; Obholzer, Nikolaus; Kozubek, James; Pochet, Nathalie; Faria, Ana M. C.; Weiner, Howard L.

    2015-01-01

    γδ T cells are a subset of lymphocytes specialized in protecting the host against pathogens and tumours. Here we describe a subset of regulatory γδ T cells that express the latency-associated peptide (LAP), a membrane-bound TGF-β1. Thymic CD27+IFN-γ+CCR9+α4β7+TCRγδ+ cells migrate to the periphery, particularly to Peyer's patches and small intestine lamina propria, where they upregulate LAP, downregulate IFN-γ via ATF-3 expression and acquire a regulatory phenotype. TCRγδ+LAP+ cells express antigen presentation molecules and function as antigen presenting cells that induce CD4+Foxp3+ regulatory T cells, although TCRγδ+LAP+ cells do not themselves express Foxp3. Identification of TCRγδ+LAP+ regulatory cells provides an avenue for understanding immune regulation and biologic processes linked to intestinal function and disease. PMID:26644347

  6. Identification and characterization of pig adipose-derived progenitor cells.

    PubMed

    Zhang, Shuang; Bai, Chunyu; Zheng, Dong; Gao, Yuhua; Fan, Yanan; Li, Lu; Guan, Weijun; Ma, Yuehui

    2016-10-01

    Adipose-derived stem cells (ADSCs) are multipotent, and can be differentiated into many cell types in vitro. In this study, tissues from pigs were chosen to identify and characterize ADSCs. Primary ADSCs were sub-cultured to passage 28. The surface markers of ADSCs: CD29, CD71, CD73, CD90, and CD166 were detected by reverse-transcription polymerase chain reaction assays and the markers CD29, CD44, CD105, and vimentin were detected by immunofluorescence. Growth curves and the capacity of clone-forming were performed to test the proliferation of ADSCs. Karyotype analysis showed that ADSCs cultured in vitro were genetically stable. To assess the differentiation capacity of the ADSCs, cells were induced to differentiate into osteoblasts, adipocytes, epithelial cells, neural cells, and hepatocyte-like cells. The results suggest that ADSCs from pigs showed similar biological characteristics with those separated from other species, and their multi-lineage differentiation shows potential as an application for cellular therapy in an animal model.

  7. Systematic identification of cell size regulators in budding yeast

    PubMed Central

    Soifer, Ilya; Barkai, Naama

    2014-01-01

    Cell size is determined by a complex interplay between growth and division, involving multiple cellular pathways. To identify systematically processes affecting size control in G1 in budding yeast, we imaged and analyzed the cell cycle of millions of individual cells representing 591 mutants implicated in size control. Quantitative metric distinguished mutants affecting the mechanism of size control from the majority of mutants that have a perturbed size due to indirect effects modulating cell growth. Overall, we identified 17 negative and dozens positive size control regulators, with the negative regulators forming a small network centered on elements of mitotic exit network. Some elements of the translation machinery affected size control with a notable distinction between the deletions of parts of small and large ribosomal subunit: parts of small ribosomal subunit tended to regulate size control, while parts of the large subunit affected cell growth. Analysis of small cells revealed additional size control mechanism that functions in G2/M, complementing the primary size control in G1. Our study provides new insights about size control mechanisms in budding yeast. PMID:25411401

  8. Identification of Factors Produced and Secreted by Mesenchymal Stromal Cells with the SILAC Method.

    PubMed

    Rocha, Beatriz; Calamia, Valentina; Blanco, Francisco J; Ruiz-Romero, Cristina

    2016-01-01

    Mesenchymal stromal cells (MSCs) secrete a large variety of proteins and factors, which shape the secretome. These proteins participate in multiple cellular functions, including the promotion of regenerative processes in the damaged tissue. Secretomes derived from either undifferentiated MSCs or these cells undergoing osteogenic, chondrogenic, or adipogenic differentiation have been characterized using different liquid chromatography tandem mass spectrometry (LC-MS/MS)-based quantitative proteomic approaches. In this chapter, we describe the use of the Stable Isotope Labeling by Amino Acids in Cell culture (SILAC) strategy for the identification and relative quantification of the mesenchymal stromal cell secretome, specifically during chondrogenesis.

  9. Identification and visualization of multidimensional antigen-specific T-cell populations in polychromatic cytometry data.

    PubMed

    Lin, Lin; Frelinger, Jacob; Jiang, Wenxin; Finak, Greg; Seshadri, Chetan; Bart, Pierre-Alexandre; Pantaleo, Giuseppe; McElrath, Julie; DeRosa, Steve; Gottardo, Raphael

    2015-07-01

    An important aspect of immune monitoring for vaccine development, clinical trials, and research is the detection, measurement, and comparison of antigen-specific T-cells from subject samples under different conditions. Antigen-specific T-cells compose a very small fraction of total T-cells. Developments in cytometry technology over the past five years have enabled the measurement of single-cells in a multivariate and high-throughput manner. This growth in both dimensionality and quantity of data continues to pose a challenge for effective identification and visualization of rare cell subsets, such as antigen-specific T-cells. Dimension reduction and feature extraction play pivotal role in both identifying and visualizing cell populations of interest in large, multi-dimensional cytometry datasets. However, the automated identification and visualization of rare, high-dimensional cell subsets remains challenging. Here we demonstrate how a systematic and integrated approach combining targeted feature extraction with dimension reduction can be used to identify and visualize biological differences in rare, antigen-specific cell populations. By using OpenCyto to perform semi-automated gating and features extraction of flow cytometry data, followed by dimensionality reduction with t-SNE we are able to identify polyfunctional subpopulations of antigen-specific T-cells and visualize treatment-specific differences between them.

  10. Automated imaging, identification, and counting of similar cells from digital hologram reconstructions.

    PubMed

    Mihailescu, Mona; Scarlat, Mihaela; Gheorghiu, Alexandru; Costescu, Julia; Kusko, Mihai; Paun, Irina Alexandra; Scarlat, Eugen

    2011-07-10

    This paper presents our method, which simultaneously combines automatic imaging, identification, and counting with the acquisition of morphological information for at least 1000 blood cells from several three-dimensional images of the same sample. We started with seeking parameters to differentiate between red blood cells that are similar but different with respect to their development stage, i.e., mature or immature. We highlight that these cells have different diffractive patterns with complementary central intensity distribution in a given plane along the propagation axis. We use the Fresnel approximation to simulate propagation through cells modeled as spheroid-shaped phase objects and to find the cell property that has the dominant influence on this behavior. Starting with images obtained in the reconstruction step of the digital holographic microscopy technique, we developed a code for automated simultaneous individual cell image separation, identification, and counting, even when the cells are partially overlapped on a slide, and accurate measuring of their morphological features. To find the centroids of each cell, we propose a method based on analytical functions applied at threshold intervals. Our procedure separates the mature from the immature red blood cells and from the white blood cells through a decision based on gradient and radius values. © 2011 Optical Society of America

  11. Automated imaging, identification, and counting of similar cells from digital hologram reconstructions

    NASA Astrophysics Data System (ADS)

    Mihailescu, Mona; Scarlat, Mihaela; Gheorghiu, Alexandru; Costescu, Julia; Kusko, Mihai; Paun, Irina Alexandra; Scarlat, Eugen

    2011-07-01

    This paper presents our method, which simultaneously combines automatic imaging, identification, and counting with the acquisition of morphological information for at least 1000 blood cells from several three-dimensional images of the same sample. We started with seeking parameters to differentiate between red blood cells that are similar but different with respect to their development stage, i.e., mature or immature. We highlight that these cells have different diffractive patterns with complementary central intensity distribution in a given plane along the propagation axis. We use the Fresnel approximation to simulate propagation through cells modeled as spheroid-shaped phase objects and to find the cell property that has the dominant influence on this behavior. Starting with images obtained in the reconstruction step of the digital holographic microscopy technique, we developed a code for automated simultaneous individual cell image separation, identification, and counting, even when the cells are partially overlapped on a slide, and accurate measuring of their morphological features. To find the centroids of each cell, we propose a method based on analytical functions applied at threshold intervals. Our procedure separates the mature from the immature red blood cells and from the white blood cells through a decision based on gradient and radius values.

  12. Identification of leukemia cell surface proteins in clams

    SciTech Connect

    Reinisch, C.L.; Smolowitz, R.; Miosky, D. Marine Biological Lab., Woods Hole, MA )

    1988-09-01

    Soft-shell clams, Mya arenaria, develop leukemias which, in the advanced stages of disease, kill the host. The authors laboratory has developed an extensive panel of murine monoclonal antibodies to leukemia cells of Mya, and has used these powerful reagents to diagnose the disease with extreme accuracy. They have now ascertained that one membrane-associated protein of approximately 200kD is immunodominant. The function of this protein, regulation of its production and potential site of synthesis are being evaluated. Monoclonal antibodies have also permitted the exploration of the mechanism of leukemogensis. They have evaluated the specific staining pattern of one monoclonal antibody, and have concluded that at least one ontogenic source of leukemic cells may be connective tissue cells lining the sinusoids. Whether or not exposure to severely polluted sites such as New Bedford Harbor stimulates the export of immature hemocytes which then become transformed is at least one possibility amenable to testing using the monoclonal reagents.

  13. Ultrastructural identification of Langerhans cells in normal swine epidermis.

    PubMed Central

    Romano, J; Balaguer, L

    1991-01-01

    Langerhans cells of the epidermis of 6-month-old white crossbred farm pigs were identified by electron microscopy. Ultrastructurally they were similar to those described in other mammals. They were present in basal and suprabasal layers and were characterised by a lobulated nucleus and an electrolucent cytoplasm with occasional dendritic processes, and the absence of tonofilaments and specialised unions with surrounding keratinocytes. They were specifically identified by the presence of characteristic rod or racquet-shaped intracytoplasmic granules. Intraepidermal clear cells without specific granules were present, although no melanocytes were observed. This is the first report of the presence of Birbeck granules in porcine Langerhans cells. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 PMID:1817140

  14. Bacterial cell identification in differential interference contrast microscopy images.

    PubMed

    Obara, Boguslaw; Roberts, Mark A J; Armitage, Judith P; Grau, Vicente

    2013-04-23

    Microscopy image segmentation lays the foundation for shape analysis, motion tracking, and classification of biological objects. Despite its importance, automated segmentation remains challenging for several widely used non-fluorescence, interference-based microscopy imaging modalities. For example in differential interference contrast microscopy which plays an important role in modern bacterial cell biology. Therefore, new revolutions in the field require the development of tools, technologies and work-flows to extract and exploit information from interference-based imaging data so as to achieve new fundamental biological insights and understanding. We have developed and evaluated a high-throughput image analysis and processing approach to detect and characterize bacterial cells and chemotaxis proteins. Its performance was evaluated using differential interference contrast and fluorescence microscopy images of Rhodobacter sphaeroides. Results demonstrate that the proposed approach provides a fast and robust method for detection and analysis of spatial relationship between bacterial cells and their chemotaxis proteins.

  15. Transgenic systems for unequivocal identification of cardiac myocyte nuclei and analysis of cardiomyocyte cell cycle status.

    PubMed

    Raulf, Alexandra; Horder, Hannes; Tarnawski, Laura; Geisen, Caroline; Ottersbach, Annika; Röll, Wilhelm; Jovinge, Stefan; Fleischmann, Bernd K; Hesse, Michael

    2015-05-01

    Even though the mammalian heart has been investigated for many years, there are still uncertainties in the fields of cardiac cell biology and regeneration with regard to exact fractions of cardiomyocytes (CMs) at different developmental stages, their plasticity after cardiac lesion and also their basal turnover rate. A main shortcoming is the accurate identification of CM and the demonstration of CM division. Therefore, an in vivo model taking advantage of a live reporter-based identification of CM nuclei and their cell cycle status is needed. In this technical report, we describe the generation and characterization of embryonic stem cells and transgenic mice expressing a fusion protein of human histone 2B and the red fluorescence protein mCherry under control of the CM specific αMHC promoter. This fluorescence label allows unequivocal identification and quantitation of CM nuclei and nuclearity in isolated cells and native tissue slices. In ventricles of adults, we determined a fraction of <20 % CMs and binucleation of 77-90 %, while in atria a CM fraction of 30 % and a binucleation index of 14 % were found. We combined this transgenic system with the CAG-eGFP-anillin transgene, which identifies cell division and established a novel screening assay for cell cycle-modifying substances in isolated, postnatal CMs. Our transgenic live reporter-based system enables reliable identification of CM nuclei and determination of CM fractions and nuclearity in heart tissue. In combination with CAG-eGFP-anillin-mice, the cell cycle status of CMs can be monitored in detail enabling screening for proliferation-inducing substances in vitro and in vivo.

  16. Potentials of single-cell biology in identification and validation of disease biomarkers.

    PubMed

    Niu, Furong; Wang, Diane C; Lu, Jiapei; Wu, Wei; Wang, Xiangdong

    2016-09-01

    Single-cell biology is considered a new approach to identify and validate disease-specific biomarkers. However, the concern raised by clinicians is how to apply single-cell measurements for clinical practice, translate the message of single-cell systems biology into clinical phenotype or explain alterations of single-cell gene sequencing and function in patient response to therapies. This study is to address the importance and necessity of single-cell gene sequencing in the identification and development of disease-specific biomarkers, the definition and significance of single-cell biology and single-cell systems biology in the understanding of single-cell full picture, the development and establishment of whole-cell models in the validation of targeted biological function and the figure and meaning of single-molecule imaging in single cell to trace intra-single-cell molecule expression, signal, interaction and location. We headline the important role of single-cell biology in the discovery and development of disease-specific biomarkers with a special emphasis on understanding single-cell biological functions, e.g. mechanical phenotypes, single-cell biology, heterogeneity and organization of genome function. We have reason to believe that such multi-dimensional, multi-layer, multi-crossing and stereoscopic single-cell biology definitely benefits the discovery and development of disease-specific biomarkers. © 2016 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.

  17. Identification of Two Saccharomyces cerevisiae Cell Wall Mannan Chemotypes

    PubMed Central

    Cawley, T. N.; Ballou, Clinton E.

    1972-01-01

    We have obtained evidence for two structurally and antigenically different Saccharomyces cerevisiae cell wall mannans. One, which occurs widely and is found in S. cerevisiae strain 238C, is already known to be a neutral mannan which yields mannose, mannobiose, mannotriose, and mannotetraose on acetolysis of the (1 → 6)-linked backbone. The other, which was found in S. cerevisiae brewer's strains, is a phosphomannan with a structure very similar to that of Kloeckera brevis mannan. S. cerevisiae (brewer's yeast strain) was agglutinated by antiserum prepared against Kloeckera brevis cells. The mannan, isolated from a proteolytic digest of the cell wall of the former, did not react with S. cerevisiae 238C antiserum, whereas it cross-reacted strongly with K. brevis antiserum. Controlled acetolysis cleaved the (1 → 6)-linkages in the polysaccharide backbone and released mannose, mannobiose, mannotriose, and mannotriose phosphate. Mild acid treatment of the phosphomannan hydrolyzed the phosphodiester linkage, yielding phosphomonoester mannan and mannose. The resulting phosphomonoester mannan reacted with antiserum prepared against K. brevis possessing monoester phosphate groups on the cell surface. α-d-Mannose-1-phosphate completely inhibited the precipitin reaction between brewer's yeast mannan and the homologous antiserum. Flocculent and nonflocculent strains of this yeast were shown to have similar structural and immunological properties. PMID:4559821

  18. Ligament-Derived Stem Cells: Identification, Characterisation, and Therapeutic Application

    PubMed Central

    Clegg, Peter David; Comerford, Eithne Josephine; Canty-Laird, Elizabeth Gail

    2017-01-01

    Ligament is prone to injury and degeneration and has poor healing potential and, with currently ineffective treatment strategies, stem cell therapies may provide an exciting new treatment option. Ligament-derived stem cell (LDSC) populations have been isolated from a number of different ligament types with the majority of studies focussing on periodontal ligament. To date, only a few studies have investigated LDSC populations in other types of ligament, for example, intra-articular ligaments; however, this now appears to be a developing field. This literature review aims to summarise the current information on nondental LDSCs including in vitro characteristics of LDSCs and their therapeutic potential. The stem cell niche has been shown to be vital for stem cell survival and function in a number of different physiological systems; therefore, the LDSC niche may have an impact on LDSC phenotype. The role of the LDSC niche on LDSC viability and function will be discussed as well as the therapeutic potential of LDSC niche modulation. PMID:28386284

  19. Identification of nuclear. tau. isoforms in human neuroblastoma cells

    SciTech Connect

    Loomis, P.A.; Howard, T.H.; Castleberry, R.P.; Binder, L.I. )

    1990-11-01

    The {tau} proteins have been reported only in association with microtubules and with ribosomes in situ, in the normal central nervous system. In addition, {tau} has been shown to be an integral component of paired helical filaments, the principal constituent of the neurofibrillary tangles found in brains of patients with Alzheimer's disease and of most aged individuals with Down syndrome (trisomy 21). The authors report here the localization of the well-characterized Tau-1 monoclonal antibody to the nucleolar organizer regions of the acrocentric chromosomes and to their interphase counterpart, the fibrillar component of the nucleolus, in human neuroblastoma cells. Similar localization to the nucleolar organizer regions was also observed in other human cell lines and in one monkey kidney cell line but was not seen in non-primate species. Immunochemically, they further demonstrated the existence of the entire {tau} molecule in the isolated nuclei of neuroblastoma cells. Nuclear {tau} proteins, like the {tau} proteins of the paired helical filaments, cannot be extracted in standard SDS-containing electrophoresis sample buffer but require pretreatment with formic acid prior to immunoblot analysis. This work indicates that {tau} may function in processes not directly associated with microtubules and that highly insoluble complexes of {tau} may also play a role in normal cellular physiology.

  20. Identification of Stem Leydig Cells Derived from Pig Testicular Interstitium

    PubMed Central

    Yu, Shuai; Zhang, Pengfei; Dong, Wuzi; Zeng, Wenxian

    2017-01-01

    Stem Leydig cells (SLCs), located in the testicular interstitial compartment in the mammalian testes, are capable of differentiating to testosterone-synthesizing Leydig cells (LCs), thus providing a new strategy for treating testosterone deficiency. However, no previous reports have identified and cultured SLCs derived from the pig. The aim of the current study was to isolate, identify, and culture SLCs from pigs. Haematoxylin and eosin staining and immunochemical analysis showed that SLCs were present and that PDGFRα was mainly expressed in the pig testicular interstitium, indicating that PDGFRα was a marker for SLCs in the neonatal pig. In addition, reverse transcription-PCR results showed that SLC markers were expressed in primary isolated LCs, indicating that they were putative SLCs. The putative SLCs were subsequently cultured with a testicular fluid of piglets (pTF) medium. Clones formed after 7 days and the cells expressed PDGFRα. However, no clones grew in the absence of pTF, but the cells expressed CYP17A1, indicating that pTF could sustain the features of porcine SLCs. To summarize, we isolated porcine SLCs and identified their basic characteristics. Taken together, these results may help lay the foundation for research in the clinical application of porcine SLCs. PMID:28243257

  1. Higher positive identification of malignant CSF cells using the cytocentrifuge than the Suta chamber.

    PubMed

    Almeida, Sérgio Monteiro de; Rotta, Indianara; Conto, Arnaldo José de; Antonelli, Dario; Roda, Carlos Dabdoub; Yamada, Edna Yoshiko; Singer, Gisele M B

    2016-10-01

    To define how to best handle cerebrospinal fluid (CSF) specimens to obtain the highest positivity rate for the diagnosis of malignancy, comparing two different methods of cell concentration, sedimentation and cytocentrifugation. A retrospective analysis of 411 CSF reports. This is a descriptive comparative study. The positive identification of malignant CSF cells was higher using the centrifuge than that using the Suta chamber (27.8% vs. 19.0%, respectively; p = 0.038). Centrifuge positively identified higher numbers of malignant cells in samples with a normal concentration of white blood cells (WBCs) (< 5 cells/mm3) and with more than 200 cells/mm3, although this was not statistically significant. There was no lymphocyte loss using either method. Cytocentrifugation positively identified a greater number of malignant cells in the CSF than cytosedimentation with the Suta chamber. However, there was no difference between the methods when the WBC counts were within the normal range.

  2. Active centromere and chromosome identification in fixed cell lines.

    PubMed

    Beh, Thian T; MacKinnon, Ruth N; Kalitsis, Paul

    2016-01-01

    The centromere plays a crucial role in ensuring the fidelity of chromosome segregation during cell divisions. However, in cancer and constitutional disorders, the presence of more than one active centromere on a chromosome may be a contributing factor to chromosome instability and could also have predictive value in disease progression, making the detection of properly functioning centromeres important. Thus far, antibodies that are widely used for functional centromere detection mainly work on freshly harvested cells whereas most cytogenetic samples are stored long-term in methanol-acetic acid fixative. Hence, we aimed to identify antibodies that would recognise active centromere antigens on methanol-acetic acid fixed cells. A panel of active centromere protein antibodies was tested and we found that a rabbit monoclonal antibody against human CENP-C recognises the active centromeres of cells fixed in methanol-acetic acid. We then tested and compared combinations of established methods namely centromere fluorescence in situ hybridisation (cenFISH), centromere protein immunofluorescence (CENP-IF) and multicolour FISH (mFISH), and showed the usefulness of CENP-IF together with cenFISH followed by mFISH (CENP-IF-cenFISH-mFISH) with the aforementioned anti-CENP-C antibody. We further demonstrated the utility of our method in two cancer cell lines with high proportion of centromere defects namely neocentromere and functional dicentric. We propose the incorporation of the CENP-IF-cenFISH-mFISH method using a commercially available rabbit monoclonal anti-CENP-C into established methods such as dicentric chromosome assay (DCA), prenatal karyotype screening in addition to constitutional and cancer karyotyping. This method will provide a more accurate assessment of centromere abnormality status in chromosome instability disorders.

  3. High-Throughput Single-Cell Derived Sphere Formation for Cancer Stem-Like Cell Identification and Analysis

    PubMed Central

    Chen, Yu-Chih; Ingram, Patrick N.; Fouladdel, Shamileh; McDermott, Sean P.; Azizi, Ebrahim; Wicha, Max S.; Yoon, Euisik

    2016-01-01

    Considerable evidence suggests that many malignancies are driven by a cellular compartment that displays stem cell properties. Cancer stem-like cells (CSCs) can be identified by expression of cell surface markers or enzymatic activity, but these methods are limited by phenotypic heterogeneity and plasticity of CSCs. An alternative phenotypic methodology based on in-vitro sphere formation has been developed, but it is typically labor-intensive and low-throughput. In this work, we present a 1,024-microchamber microfluidic platform for single-cell derived sphere formation. Utilizing a hydrodynamic capturing scheme, more than 70% of the microchambers capture only one cell, allowing for monitoring of sphere formation from heterogeneous cancer cell populations for identification of CSCs. Single-cell derived spheres can be retrieved and dissociated for single-cell analysis using a custom 96-gene panel to probe heterogeneity within the clonal CSC spheres. This microfluidic platform provides reliable and high-throughput sphere formation for CSC identification and downstream clonal analysis. PMID:27292795

  4. High-Throughput Single-Cell Derived Sphere Formation for Cancer Stem-Like Cell Identification and Analysis

    NASA Astrophysics Data System (ADS)

    Chen, Yu-Chih; Ingram, Patrick N.; Fouladdel, Shamileh; McDermott, Sean P.; Azizi, Ebrahim; Wicha, Max S.; Yoon, Euisik

    2016-06-01

    Considerable evidence suggests that many malignancies are driven by a cellular compartment that displays stem cell properties. Cancer stem-like cells (CSCs) can be identified by expression of cell surface markers or enzymatic activity, but these methods are limited by phenotypic heterogeneity and plasticity of CSCs. An alternative phenotypic methodology based on in-vitro sphere formation has been developed, but it is typically labor-intensive and low-throughput. In this work, we present a 1,024-microchamber microfluidic platform for single-cell derived sphere formation. Utilizing a hydrodynamic capturing scheme, more than 70% of the microchambers capture only one cell, allowing for monitoring of sphere formation from heterogeneous cancer cell populations for identification of CSCs. Single-cell derived spheres can be retrieved and dissociated for single-cell analysis using a custom 96-gene panel to probe heterogeneity within the clonal CSC spheres. This microfluidic platform provides reliable and high-throughput sphere formation for CSC identification and downstream clonal analysis.

  5. Daytime identification of summer hailstorm cells from MSG data

    NASA Astrophysics Data System (ADS)

    Merino, A.; López, L.; Sánchez, J. L.; García-Ortega, E.; Cattani, E.; Levizzani, V.

    2014-04-01

    Identifying deep convection is of paramount importance, as it may be associated with extreme weather phenomena that have significant impact on the environment, property and populations. A new method, the hail detection tool (HDT), is described for identifying hail-bearing storms using multispectral Meteosat Second Generation (MSG) data. HDT was conceived as a two-phase method, in which the first step is the convective mask (CM) algorithm devised for detection of deep convection, and the second a hail mask algorithm (HM) for the identification of hail-bearing clouds among cumulonimbus systems detected by CM. Both CM and HM are based on logistic regression models trained with multispectral MSG data sets comprised of summer convective events in the middle Ebro Valley (Spain) between 2006 and 2010, and detected by the RGB (red-green-blue) visualization technique (CM) or C-band weather radar system of the University of León. By means of the logistic regression approach, the probability of identifying a cumulonimbus event with CM or a hail event with HM are computed by exploiting a proper selection of MSG wavelengths or their combination. A number of cloud physical properties (liquid water path, optical thickness and effective cloud drop radius) were used to physically interpret results of statistical models from a meteorological perspective, using a method based on these "ingredients". Finally, the HDT was applied to a new validation sample consisting of events during summer 2011. The overall probability of detection was 76.9 % and the false alarm ratio 16.7 %.

  6. Identification of aromatase activity in rodent pituitary cell strains.

    PubMed

    Callard, G V; Petro, Z; Tashjian, A H

    1983-07-01

    To date, biochemical evidence has been presented for hypophysial aromatization in only one species, a teleost fish, although the pituitary glands of several mammals have been reported to be aromatase negative. To reinvestigate this problem, established clonal strains of rodent pituitary cells (GH3, GH4C1, and AtT20/D16) were incubated at 37 C for 6-48 h in serum-less medium containing [7-3H]androstenedione. Radiolabeled metabolites were isolated by solvent extraction, thin layer chromatography, and phenolic partition. The authenticity of the estrogenic products in both cells and incubation medium was verified by methylation and recrystallization to constant specific activity. Measurement of androgen metabolites was also validated by recrystallization of selected samples. Authentic estrone and 17 beta-estradiol were identified in cultures of the two PRL- and GH-secreting clones, and there were strain differences in the quantity of estrogen produced (GH3 greater than GH4C1). Under the same conditions, aromatization was not detectable in the ACTH-secreting line (AtT20/D16). A time-yield analysis of androgen metabolism in GH4C1 cells showed that aromatization was linear for 12 h after labeling, but that substrate was diverted mainly to 5 alpha-reducing pathways. Large amounts of highly polar metabolites accumulated 24 and 48 h after the addition of [3H]androgen, and subsequent hydrolysis revealed that these were sulfo- and glucuronoconjugates. The metabolic fate of estrogen in GH4C1 cultures was investigated indirectly by adding a radioinert estrone trap together with the radiolabeled androgen substrate and was also tested in separate cultures by adding [3H]estrone and [3H]estradiol directly. Although the two estrogens were interconverted, there was no evidence that formed or added estrogen was extensively metabolized or conjugated. We conclude that the expression of aromatase activity in hypophysial cells is not a property of all transformed lines but may be dictated

  7. Identification of mouse mammary epithelial cells by immunofluorescence with rabbit and guinea pig antikeratin antisera.

    PubMed Central

    Asch, B B; Burstein, N A; Vidrich, A; Sun, T T

    1981-01-01

    Few markers are available to identify the three types of mammary epithelial cells--ductal, alveolar, and myoepithelial--especially in pathological conditions and in cell cultures. We have used antisera to human keratins in immunofluorescence to facilitate the identification of the three mouse mammary epithelial cell types. In frozen tissue sections and primary cell cultures, a rabbit antikeratin antiserum specifically stained cytoplasmic filaments in all three types of epithelial cells. A guinea pig antiserum against the same keratin preparation, however, reacted preferentially with filaments in myoepithelial cells and readily detected this cell type in normal, dysplastic, and malignant mammary tissues and cell cultures. Neither antisera reacted with fibroblasts or any other mesenchymal cells. The combined use of the two antikeratin antisera thereby permits rapid surveys of tissue sections and cultures for the localization of not only all epithelial cells but also the subpopulation of myoepithelial cells. Moreover, when mammary cultures established from late-pregnant or lactating mice were stained simultaneously with guinea pig antikeratin and rabbit anticasein antisera, three populations of epithelial cells were mutually exclusive: those stained by anticasein antiserum, those stained by guinea pig antikeratin antiserum, and those stained by neither, consistent with properties of alveolar, myoepithelial, and ductal cells, respectively. These antisera thus offer a tool for studying different epithelial cell types during mammary development, tumorigenesis, and malignant progression. Images PMID:6170984

  8. Functional classification of cellular proteome profiles support the identification of drug resistance signatures in melanoma cells.

    PubMed

    Paulitschke, Verena; Haudek-Prinz, Verena; Griss, Johannes; Berger, Walter; Mohr, Thomas; Pehamberger, Hubert; Kunstfeld, Rainer; Gerner, Christopher

    2013-07-05

    Drug resistance is a major obstacle in melanoma treatment. Recognition of specific resistance patterns, the understanding of the patho-physiology of drug resistance, and identification of remaining options for individual melanoma treatment would greatly improve therapeutic success. We performed mass spectrometry-based proteome profiling of A375 melanoma cells and HeLa cells characterized as sensitive to cisplatin in comparison to cisplatin resistant M24met and TMFI melanoma cells. Cells were fractionated into cytoplasm, nuclei and secretome and the proteome profiles classified according to Gene Ontology. The cisplatin resistant cells displayed increased expression of lysosomal as well as Ca²⁺ ion binding and cell adherence proteins. These findings were confirmed using Lysotracker Red staining and cell adhesion assays with a panel of extracellular matrix proteins. To discriminate specific survival proteins, we selected constitutively expressed proteins of resistant M24met cells which were found expressed upon challenging the sensitive A375 cells. Using the CPL/MUW proteome database, the selected lysosomal, cell adherence and survival proteins apparently specifying resistant cells were narrowed down to 47 proteins representing a potential resistance signature. These were tested against our proteomics database comprising more than 200 different cell types/cell states for its predictive power. We provide evidence that this signature enables the automated assignment of resistance features as readout from proteome profiles of any human cell type. Proteome profiling and bioinformatic processing may thus support the understanding of drug resistance mechanism, eventually guiding patient tailored therapy.

  9. Identification of oxytocin and vasopressin from neurohypophyseal cell culture.

    PubMed

    Janáky, T; Szabó, P; Kele, Z; Baláspiri, L; Varga, C; Gálfi, M; Vecsernyés, M; Gáspár, L; Juhász, A; László, F A

    1998-01-01

    Our observation that dispersed cultures of neurohypophysis obtained from adult rats are capable of synthesizing and releasing oxytocin and vasopressin is unexpected, because in whole animals these hormones are known only to be stored, not to be produced in the posterior lobe of the pituitary. The hormone content of cell culture medium was elevated from 0 to 129 +/- 14 pg/mg protein for oxytocin and from 0 to 42 +/- 4 pg/mg protein for vasopressin during two weeks as determined by specific radioimmunoassay. By molecular mass and structure determination (tandem mass spectrometry) we have proved that the supernatant of the cell cultures contains not only immunologically but mass spectrometrically identified neurohypophyseal hormones.

  10. Identification, Characterization, and Utilization of Adult Meniscal Progenitor Cells

    DTIC Science & Technology

    2015-09-01

    signature gene Lox, an enzyme responsible for collagen cross-links in the musculoskeletal system. In addition, cells from young and aged mice expressed...unique platform to study synovial joint development and OA pathology due to its short lifespan, low cost of maintenance and availability for genetic...meniscus developmental biology and OA pathology using trans- genic or surgery-induced OA models. Conflict of interest statement The authors of this study

  11. Immunological identification and isolation of phosphotyrosyl proteins in cultured cells

    SciTech Connect

    Martensen, T.M.; Madoff, D.H.; Lane, M.D.

    1987-05-01

    Affinity-purified sheep anti-phosphotyrosine antibodies were utilized to probe for phosphotyrosyl proteins from cultured cells. Western blots of cell extract proteins separated by SDS PAGE were incubated with anti-Tyr-P antibodies followed by peroxidase labeled anti-sheep antibodies or ( SVI)protein G to decorate the immune complexes. Normal, retrovirus transformed, and preadipocyte fibroblasts showed a variety of phosphotyrosyl proteins. Specific binding was indicated by the ability of Tyr-P but not Ser-P or Thr-P to inhibit the immunolocalization. Anti-phosphotyrosine antibodies covalently coupled to Sepharose were used to isolate phosphotyrosyl proteins from cultured cells. The binding capacity of the gel was determined by the quantity of unlabeled phosphotyrosyl protein needed to displace (TSP)labeled phosphotyrosyl protein binding. (TSP)labeled insulin receptor from 3T3-11 adipocytes could be immuno-adsorbed in high yield. Desorption was achieved in good yields under native conditions by incubation with Tyr-P. Treatment of (TSP)insulin receptor with SDS prior to incubation with immobilized antibody did not inhibit immuno-adsorption or -elution. This feature enabled antiphosphotyrosine antibodies to distinguish phosphotyrosyl proteins whose modified residues appear to be inaccessible for antibody binding in the native state.

  12. Identification of novel dendritic cell subset markers in human blood.

    PubMed

    Schütz, Fabian; Hackstein, Holger

    2014-01-10

    Human dendritic cells (DC) are key regulators of innate and adaptive immunity that can be divided in at least three major subpopulations: plasmacytoid DC (pDC), myeloid type 1 DC (mDC1) and myeloid type 2 DC (mDC2) exhibiting different functions. However, research, diagnostic and cell therapeutic studies on human DC subsets are limited because only few DC subset markers have been identified so far. Especially mDC2 representing the rarest blood DC subset are difficult to be separated from mDC1 and pDC due to a paucity of mDC2 markers. We have combined multiparameter flow cytometry analysis of human blood DC subsets with systematic expression analysis of 332 surface antigens in magnetic bead-enriched blood DC samples. The initial analysis revealed eight novel putative DC subset markers CD26, CD85a, CD109, CD172a, CD200, CD200R, CD275 and CD301 that were subsequently tested in bulk peripheral blood mononuclear cell (PBMC) samples from healthy blood donors. Secondary analysis of PBMC samples confirmed three novel DC subset markers CD26 (dipeptidyl peptidase IV), CD85a (Leukocyte immunoglobulin-like receptor B3) and CD275 (inducible costimulator ligand). CD85a is specifically expressed in mDC1 and CD26 and CD275 represent novel mDC2 markers. These markers will facilitate human DC subset discrimination and additionally provide insight into potentially novel DC subset-specific functions.

  13. The minotaur proteome: avoiding cross-species identifications deriving from bovine serum in cell culture models.

    PubMed

    Bunkenborg, Jakob; García, Guadalupe Espadas; Paz, Marcia Ivonne Peña; Andersen, Jens S; Molina, Henrik

    2010-08-01

    Cell culture is a fundamental tool in proteomics where mammalian cells are cultured in vitro using a growth medium often supplemented with 5-15% FBS. Contamination by bovine proteins is difficult to avoid because of adherence to the plastic vessel and the cultured cells. We have generated peptides from bovine serum using four sample preparation methods and analyzed the peptides by high mass accuracy LC-MS/MS. Distinguishing between bovine and human peptides is difficult because of a considerable overlap of identical tryptic peptide sequences. Pitfalls in interpretation, different database search strategies to minimize erroneous identifications and an augmented contaminant database are presented.

  14. Vibrational spectroscopy--a powerful tool for the rapid identification of microbial cells at the single-cell level.

    PubMed

    Harz, M; Rösch, P; Popp, J

    2009-02-01

    Rapid microbial detection and identification with a high grade of sensitivity and selectivity is a great and challenging issue in many fields, primarily in clinical diagnosis, pharmaceutical, or food processing technology. The tedious and time-consuming processes of current microbiological approaches call for faster ideally on-line identification techniques. The vibrational spectroscopic techniques IR absorption and Raman spectroscopy are noninvasive methods yielding molecular fingerprint information; thus, allowing for a fast and reliable analysis of complex biological systems such as bacterial or yeast cells. In this short review, we discuss recent vibrational spectroscopic advances in microbial identification of yeast and bacterial cells for bulk environment and single-cell analysis. IR absorption spectroscopy enables a bulk analysis whereas micro-Raman-spectroscopy with excitation in the near infrared or visible range has the potential for the analysis of single bacterial and yeast cells. The inherently weak Raman signal can be increased up to several orders of magnitude by applying Raman signal enhancement methods such as UV-resonance Raman spectroscopy with excitation in the deep UV region, surface enhanced Raman scattering, or tip-enhanced Raman scattering. Copyright 2008 International Society for Advancement of Cytometry

  15. CD44 Is a Negative Cell Surface Marker for Pluripotent Stem Cell Identification during Human Fibroblast Reprogramming

    PubMed Central

    Vaz, Candida; Tanavde, Vivek; Lakshmipathy, Uma

    2014-01-01

    Induced pluripotent stem cells (iPSCs) are promising tools for disease research and cell therapy. One of the critical steps in establishing iPSC lines is the early identification of fully reprogrammed colonies among unreprogrammed fibroblasts and partially reprogrammed intermediates. Currently, colony morphology and pluripotent stem cell surface markers are used to identify iPSC colonies. Through additional clonal characterization, we show that these tools fail to distinguish partially reprogrammed intermediates from fully reprogrammed iPSCs. Thus, they can lead to the selection of suboptimal clones for expansion. A subsequent global transcriptome analysis revealed that the cell adhesion protein CD44 is a marker that differentiates between partially and fully reprogrammed cells. Immunohistochemistry and flow cytometry confirmed that CD44 is highly expressed in the human parental fibroblasts used for the reprogramming experiments. It is gradually lost throughout the reprogramming process and is absent in fully established iPSCs. When used in conjunction with pluripotent cell markers, CD44 staining results in the clear identification of fully reprogrammed cells. This combination of positive and negative surface markers allows for easier and more accurate iPSC detection and selection, thus reducing the effort spent on suboptimal iPSC clones. PMID:24416407

  16. Identification of autocrine growth factors secreted by CHO cells for applications in single-cell cloning media.

    PubMed

    Lim, U Ming; Yap, Miranda Gek Sim; Lim, Yoon Pin; Goh, Lin-Tang; Ng, Say Kong

    2013-07-05

    Chinese hamster ovary (CHO) cell lines are widely used for the expression of therapeutic recombinant proteins, including monoclonal antibodies and other biologics. For manufacturing, cells derived from a single-cell clone are typically used to ensure product consistency. Presently, fetal bovine serum (FBS) is commonly used to support low cell density cultures to obtain clonal cell populations because cells grow slowly, or even do not survive at low cell densities in protein-free media. However, regulatory authorities have discouraged the use of FBS to reduce the risk of contamination by adventitious agents from animal-derived components. In this study, we demonstrated how a complementary mass spectrometry-based shotgun proteomics strategy enabled the identification of autocrine growth factors in CHO cell-conditioned media, which has led to the development of a fully defined single-cell cloning media that is serum and animal component-free. Out of 290 secreted proteins that were identified, eight secreted growth factors were reported for the first time from CHO cell cultures. By supplementing a combination of these growth factors to protein-free basal media, single cell growth of CHO cells was improved with cloning efficiencies of up to 30%, a 2-fold improvement compared to unsupplemented basal media. Complementary effects of these autocrine growth factors with other paracrine growth factors were also demonstrated when the mixture improved cloning efficiency to 42%, similar to that for the conditioned medium.

  17. Identification and Characterization of Renal Cell Carcinoma Gene Markers

    PubMed Central

    Dalgin, Gul S.; Holloway, Dustin T.; Liou, Louis S.; DeLisi, Charles

    2007-01-01

    Microarray gene expression profiling has been used to distinguish histological subtypes of renal cell carcinoma (RCC), and consequently to identify specific tumor markers. The analytical procedures currently in use find sets of genes whose average differential expression across the two categories differ significantly. In general each of the markers thus identified does not distinguish tumor from normal with 100% accuracy, although the group as a whole might be able to do so. For the purpose of developing a widely used economically viable diagnostic signature, however, large groups of genes are not likely to be useful. Here we use two different methods, one a support vector machine variant, and the other an exhaustive search, to reanalyze data previously generated in our Lab (Lenburg et al. 2003). We identify 158 genes, each having an expression level that is higher (lower) in every tumor sample than in any normal sample, and each having a minimum differential expression across the two categories at a significance of 0.01. The set is highly enriched in cancer related genes (p = 1.6 × 10−12), containing 43 genes previously associated with either RCC or other types of cancer. Many of the biomarkers appear to be associated with the central alterations known to be required for cancer transformation. These include the oncogenes JAZF1, AXL, ABL2; tumor suppressors RASD1, PTPRO, TFAP2A, CDKN1C; and genes involved in proteolysis or cell-adhesion such as WASF2, and PAPPA. PMID:19455236

  18. Identification of novel Mycobacterium tuberculosis CD4 T-cell antigens via high throughput proteome screening

    PubMed Central

    Nayak, Kaustuv; Jing, Lichen; Russell, Ronnie M.; Davies, D. Huw; Hermanson, Gary; Molina, Douglas M.; Liang, Xiaowu; Sherman, David R.; Kwok, William W.; Yang, Junbao; Kenneth, John; Ahamed, Syed F.; Chandele, Anmol; Kaja, Murali-Krishna; Koelle, David M.

    2015-01-01

    Elicitation of CD4 IFN-gamma T cell responses to Mycobacterium tuberculosis (MTB) is a rational vaccine strategy to prevent clinical tuberculosis. Diagnosis of MTB infection is based on T-cell immune memory to MTB antigens. The MTB proteome contains over four thousand open reading frames (ORFs). We conducted a pilot antigen identification study using 164 MTB proteins and MTB-specific T-cells expanded in vitro from 12 persons with latent MTB infection. Enrichment of MTB-reactive T-cells from PBMC used cell sorting or an alternate system compatible with limited resources. MTB proteins were used as single antigens or combinatorial matrices in proliferation and cytokine secretion readouts. Overall, our study found that 44 MTB proteins were antigenic, including 27 not previously characterized as CD4 T-cell antigens. Antigen truncation, peptide, NTM homology, and HLA class II tetramer studies confirmed malate synthase G (encoded by gene Rv1837) as a CD4 T-cell antigen. This simple, scalable system has potential utility for the identification of candidate MTB vaccine and biomarker antigens. PMID:25857935

  19. Low power proton exchange membrane fuel cell system identification and adaptive control

    NASA Astrophysics Data System (ADS)

    Yang, Yee-Pien; Wang, Fu-Cheng; Chang, Hsin-Ping; Ma, Ying-Wei; Weng, Biing-Jyh

    This paper proposes a systematic method of system identification and control of a proton exchange membrane (PEM) fuel cell. This fuel cell can be used for low-power communication devices involving complex electrochemical reactions of nonlinear and time-varying dynamic properties. From a system point of view, the dynamic model of PEM fuel cell is reduced to a configuration of two inputs, hydrogen and air flow rates, and two outputs, cell voltage and current. The corresponding transfer functions describe linearized subsystem dynamics with finite orders and time-varying parameters, which are expressed as discrete-time auto-regression moving-average with auxiliary input models for system identification by the recursive least square algorithm. In the experiments, a pseudo-random binary sequence of hydrogen or air flow rate is fed to a single fuel cell device to excite its dynamics. By measuring the corresponding output signals, each subsystem transfer function of reduced order is identified, while the unmodeled, higher-order dynamics and disturbances are described by the auxiliary input term. This provides a basis of adaptive control strategy to improve the fuel cell performance in terms of efficiency, as well as transient and steady state specifications. Simulation shows that adaptive controller is robust to the variation of fuel cell system dynamics, and it has proved promising from the experimental results.

  20. Colors Identification for Blind People using Cell Phone

    NASA Astrophysics Data System (ADS)

    Dominguez, A. L.; Graffigna, J. P.

    2011-12-01

    Assistive Technology (AT) is an interdisciplinary research area that allows finding solutions to the individual with disability [1] by easing or improving the functions or the skills for accomplishing daily activities. A technology can be considered "assistive" if it is fit for the needs, skills and capabilities of the person, taking into account mainly the intended activity and the limitations of the context and environs where the person performs such activity. The current work intends to solve the problems of vision impaired persons to recognize colors. To this aim, a Java application for cell phones has been made which lets complement the mobiles' technology with that of image processing. The means to obtain the colors from a view are based on analysing the different color models join to a mechanism to reduce the collected data. This paper describes preliminary experiences, methodology and results considering the user perception.

  1. Identification of albumin-binding proteins in capillary endothelial cells

    PubMed Central

    1988-01-01

    Isolated fat tissue microvessels and lung, whose capillary endothelia express in situ specific binding sites for albumin, were homogenized and subjected to SDS-gel electrophoresis and electroblotting. The nitrocellulose strips were incubated with either albumin-gold (Alb-Au) and directly visualized, or with [125I]albumin (monomeric or polymeric) and autoradiographed. The extracts of both microvascular endothelium and the lung express albumin-binding proteins (ABPs) represented by two pairs of polypeptides with major components of molecular mass 31 and 18 kD. The ABP peptides have pIs 8.05 to 8.75. Rabbit aortic endothelium, used as control, does not express detectable amounts of ABPs. The ABPs subjected to electrophoresis bind specifically and with high affinity (Kd = approximately 60 X 10(-9)M) both monomeric and polymeric albumin: the binding is saturable at approximately 80 nM concentration and 50% inhibition is reached at 5.5 micrograms/ml albumin concentration. Sulfhydryl-reducing agents beta-mercaptoethanol and dithiothreitol do not markedly affect the ABPs electrophoretic mobility and binding properties. As indicated by cell surface iodination of isolated capillary endothelium followed by electroblotting, autoradiography, and incubation with Alb-Au, the bands specifically stained by this ligand are also labeled with radioiodine. PMID:2839518

  2. Hematologic Assessment in Pet Rats, Mice, Hamsters, and Gerbils: Blood Sample Collection and Blood Cell Identification.

    PubMed

    Lindstrom, Nicole M; Moore, David M; Zimmerman, Kurt; Smith, Stephen A

    2015-09-01

    Hamsters, gerbils, rats, and mice are presented to veterinary clinics and hospitals for prophylactic care and treatment of clinical signs of disease. Physical examination, history, and husbandry practice information can be supplemented greatly by assessment of hematologic parameters. As a resource for veterinarians and their technicians, this article describes the methods for collection of blood, identification of blood cells, and interpretation of the hemogram in mice, rats, gerbils, and hamsters.

  3. Hematologic assessment in pet rats, mice, hamsters, and gerbils: blood sample collection and blood cell identification.

    PubMed

    Lindstrom, Nicole M; Moore, David M; Zimmerman, Kurt; Smith, Stephen A

    2015-01-01

    Hamsters, gerbils, rats, and mice are presented to veterinary clinics and hospitals for prophylactic care and treatment of clinical signs of disease. Physical examination, history, and husbandry practice information can be supplemented greatly by assessment of hematologic parameters. As a resource for veterinarians and their technicians, this article describes the methods for collection of blood, identification of blood cells, and interpretation of the hemogram in mice, rats, gerbils, and hamsters.

  4. Effects of radiation from a radiofrequency identification (RFID) microchip on human cancer cells.

    PubMed

    Lai, Henry C; Chan, Ho Wing; Singh, Narendra P

    2016-01-01

    Radiofrequency identification (RFID) microchips are used to remotely identify objects, e.g. an animal in which a chip is implanted. A passive RFID microchip absorbs energy from an external source and emits a radiofrequency identification signal which is then decoded by a detector. In the present study, we investigated the effect of the radiofrequency energy emitted by a RFID microchip on human cancer cells. Molt-4 leukemia, BT474 breast cancer, and HepG2 hepatic cancer cells were exposed in vitro to RFID microchip-emitted radiofrequency field for 1 h. Cells were counted before and after exposure. Effects of pretreatment with the spin-trap compound N-tert-butyl-alpha-phenylnitrone or the iron-chelator deferoxamine were also investigated. Results We found that the energy effectively killed/retarded the growth of the three different types of cancer cells, and the effect was blocked by the spin-trap compound or the iron-chelator, whereas an inactive microchip and energy from the external source had no significant effect on the cells. Conclusions Data of the present study suggest that radiofrequency field from the microchip affects cancer cells via the Fenton Reaction. Implantation of RFID microchips in tumors may provide a new method for cancer treatment.

  5. Identification of rolling circulating tumor cells using photoacoustic time-of-flight method

    NASA Astrophysics Data System (ADS)

    Sarimollaoglu, Mustafa; Nedosekin, Dmitry A.; Galanzha, Ekaterina I.; Zharov, Vladimir P.

    2013-03-01

    Existing optical techniques for in vivo measurement of blood flow velocity are not quite applicable for determination of velocity of individual cells or nanoparticles. A time-of-flight photoacoustic (PA) technique can solve this problem by measuring the transient PA signal width, which is related to the cell velocity passing the laser beam. This technique was demonstrated in vivo using an animal (mouse) model by estimating the velocity of nanoparticles, and red and white blood cells labeled with conjugated gold nanorods (GNRs) in the bloodstream. Here we describe the features and the parameters of novel modifications to the PA time-of-flight method and its new application for real-time monitoring of circulating tumor cells (CTCs), such as B16F10 melanoma. This method provided, for the first time, identification of rolling CTCs in analogy to rolling white blood cells and CTC aggregates. Specifically, monitoring of PA signal widths from CTCs in mouse ear microvessels revealed double maxima in peak-width histograms associated with the fast moving portion of CTCs in central flow and slowly rolling CTCs in analogy to white blood cells. We also developed a two-parameter plot representing PA peak amplitude and peak widths. This method allowed identification of fast-moving individual CTCs, CTC aggregates, and rolling CTCs. The discovery of rolling CTCs in relatively large blood vessels indicates a higher probability of CTC extravasations, further increasing the possibility of metastasis through rolling mechanism in addition to mechanical capturing of CTCs in small vessels.

  6. Identification of individual biofilm-forming bacterial cells using Raman tweezers.

    PubMed

    Samek, Ota; Bernatová, Silvie; Ježek, Jan; Šiler, Martin; Šerý, Mojmir; Krzyžánek, Vladislav; Hrubanová, Kamila; Zemánek, Pavel; Holá, Veronika; Růžička, Filip

    2015-05-01

    A method for in vitro identification of individual bacterial cells is presented. The method is based on a combination of optical tweezers for spatial trapping of individual bacterial cells and Raman microspectroscopy for acquisition of spectral “Raman fingerprints” obtained from the trapped cell. Here, Raman spectra were taken from the biofilm-forming cells without the influence of an extracellular matrix and were compared with biofilm-negative cells. Results of principal component analyses of Raman spectra enabled us to distinguish between the two strains of Staphylococcus epidermidis. Thus, we propose that Raman tweezers can become the technique of choice for a clearer understanding of the processes involved in bacterial biofilms which constitute a highly privileged way of life for bacteria, protected from the external environment.

  7. Identification of individual biofilm-forming bacterial cells using Raman tweezers

    NASA Astrophysics Data System (ADS)

    Samek, Ota; Bernatová, Silvie; Ježek, Jan; Šiler, Martin; Šerý, Mojmir; Krzyžánek, Vladislav; Hrubanová, Kamila; Zemánek, Pavel; Holá, Veronika; Růžička, Filip

    2015-05-01

    A method for in vitro identification of individual bacterial cells is presented. The method is based on a combination of optical tweezers for spatial trapping of individual bacterial cells and Raman microspectroscopy for acquisition of spectral "Raman fingerprints" obtained from the trapped cell. Here, Raman spectra were taken from the biofilm-forming cells without the influence of an extracellular matrix and were compared with biofilm-negative cells. Results of principal component analyses of Raman spectra enabled us to distinguish between the two strains of Staphylococcus epidermidis. Thus, we propose that Raman tweezers can become the technique of choice for a clearer understanding of the processes involved in bacterial biofilms which constitute a highly privileged way of life for bacteria, protected from the external environment.

  8. Identification of Cell Type-Specific Differences in Erythropoietin Receptor Signaling in Primary Erythroid and Lung Cancer Cells

    PubMed Central

    Salopiata, Florian; Depner, Sofia; Wäsch, Marvin; Böhm, Martin E.; Mücke, Oliver; Plass, Christoph; Lehmann, Wolf D.; Kreutz, Clemens; Timmer, Jens; Klingmüller, Ursula

    2016-01-01

    Lung cancer, with its most prevalent form non-small-cell lung carcinoma (NSCLC), is one of the leading causes of cancer-related deaths worldwide, and is commonly treated with chemotherapeutic drugs such as cisplatin. Lung cancer patients frequently suffer from chemotherapy-induced anemia, which can be treated with erythropoietin (EPO). However, studies have indicated that EPO not only promotes erythropoiesis in hematopoietic cells, but may also enhance survival of NSCLC cells. Here, we verified that the NSCLC cell line H838 expresses functional erythropoietin receptors (EPOR) and that treatment with EPO reduces cisplatin-induced apoptosis. To pinpoint differences in EPO-induced survival signaling in erythroid progenitor cells (CFU-E, colony forming unit-erythroid) and H838 cells, we combined mathematical modeling with a method for feature selection, the L1 regularization. Utilizing an example model and simulated data, we demonstrated that this approach enables the accurate identification and quantification of cell type-specific parameters. We applied our strategy to quantitative time-resolved data of EPO-induced JAK/STAT signaling generated by quantitative immunoblotting, mass spectrometry and quantitative real-time PCR (qRT-PCR) in CFU-E and H838 cells as well as H838 cells overexpressing human EPOR (H838-HA-hEPOR). The established parsimonious mathematical model was able to simultaneously describe the data sets of CFU-E, H838 and H838-HA-hEPOR cells. Seven cell type-specific parameters were identified that included for example parameters for nuclear translocation of STAT5 and target gene induction. Cell type-specific differences in target gene induction were experimentally validated by qRT-PCR experiments. The systematic identification of pathway differences and sensitivities of EPOR signaling in CFU-E and H838 cells revealed potential targets for intervention to selectively inhibit EPO-induced signaling in the tumor cells but leave the responses in erythroid

  9. Identification and developmental analysis of endothelin receptor type-A expressing cells in the mouse kidney.

    PubMed

    Kitazawa, Taro; Sato, Takahiro; Nishiyama, Koichi; Asai, Rieko; Arima, Yuichiro; Uchijima, Yasunobu; Kurihara, Yukiko; Kurihara, Hiroki

    2011-10-01

    The endothelin (Edn) system plays pleiotropic roles in renal function and various disease processes through two distinct G protein-coupled receptors, Edn receptors type-A (Ednra) and type-B (Ednrb). However, difficulties in the accurate identification of receptor-expressing cells in situ have made it difficult to dissect their diverse action in renal (patho)physiology. We have recently established mouse lines in which lacZ and EGFP are 'knocked-in' to the Ednra locus to faithfully mark Ednra-expressing cells. Here we analyzed these mice for their expression in the kidney to characterize Ednra-expressing cells. Ednra expression was first observed in undifferentiated mesenchymal cells around the ureteric bud at E12.5. Thereafter, Ednra expression was widely observed in vascular smooth muscle cells, JG cells and mesenchymal cells in the interstitium. After growth, the expression became confined to vascular smooth muscle cells, pericytes and renin-producing JG cells. By contrast, most cells in the nephron and vascular endothelial cells did not express Ednra. These results indicate that Ednra expression may be linked with non-epithelial fate determination and differentiation of metanephric mesenchyme. Ednra-lacZ/EGFP knock-in mice may serve as a useful tool in studies on renal function and pathophysiology of various renal diseases.

  10. Raman-based identification of circulating tumor cells for cancer diagnosis

    NASA Astrophysics Data System (ADS)

    Krafft, Christoph; Beleites, Claudia; Schie, Iwan W.; Clement, Joachim H.; Popp, Jürgen

    2016-03-01

    Circulating tumor cells (CTCs) that can be extracted from body fluids offer new prospects in cancer diagnostics. An overview about our recent achievements is presented to use Raman-based methodologies to distinguish cancer cells from normal blood cells. In a first approach, a microfluidic chip was developed to collect Raman spectra from optically trapped cells. Whereas sensitivities and specificities were promising, the throughput was not compatible with the expected low number of CTCs per million white blood cells. A second strategy immobilized up to 200,000 cells onto a microhole array made of silicon nitride. Rapid microscopic screening can be applied to pre-select a subset of cells from which Raman spectra are collected for specific CTC identification. As this approach is compatible with living cells and Raman spectroscopy with 785 nm excitation is non-destructive, a robotic arm can select positively identified CTCs for in-depth biochemical assessment. Finally, an in vivo approach directly collects CTCs from the blood stream. This way reduces the cell number to a manageable size that is subjected to Raman spectroscopy for cell typing and enumeration. An integrated acquisition mode was introduced to further increase the throughput and robustness of single cell classification.

  11. Performance of mid infrared spectroscopy in skin cancer cell type identification

    NASA Astrophysics Data System (ADS)

    Kastl, Lena; Kemper, Björn; Lloyd, Gavin R.; Nallala, Jayakrupakar; Stone, Nick; Naranjo, Valery; Penaranda, Francisco; Schnekenburger, Jürgen

    2017-02-01

    Marker free optical spectroscopy is a powerful tool for the rapid inspection of pathologically suspicious skin lesions and the non-invasive detection of early skin tumors. This goal can be reached by the combination of signal localization and the spectroscopical detection of chemical cell signatures. We here present the development and application of mid infrared spectroscopy (midIR) for the analysis of skin tumor cell types and three dimensional tissue phantoms towards the application of midIR spectroscopy for fast and reliable skin diagnostics. We developed standardized in vitro skin systems with increasing complexity, from single skin cell types as fibroblasts, keratinocytes and melanoma cells, to mixtures of these and finally three dimensional skin cancer phantoms. The cell systems were characterized with different systems in the midIR range up to 12 μm. The analysis of the spectra by novel data processing algorithms demonstrated the clear separation of all cell types, especially melanoma cells. Special attention and algorithm training was required for closely related mesenchymal cell types as dedifferentiated melanoma cells and fibroblasts. Proof of concept experiments with mixtures of in vivo fluorescence labelled skin cell types allowed the test of the new algorithms performance for the identification of specific cell types. The intense training of the software systems with various samples resulted in a increased sensitivity and specificity of the combined midIR and software system. These data highlight the potential of midIR spectroscopy as sensitive and specific future optical biopsy technology.

  12. A method for structure analysis of nanomaterials by electron diffraction: phase identification and unit cell determination.

    PubMed

    Honglong, Shi; Guling, Zhang; Bin, Zou; Minting, Luo; Wenzhong, Wang

    2013-06-01

    We report a quick and easy method for a random selected area electron diffraction (SAED) pattern without rotating and tilting the specimen to perform phase identification and unit cell determination by combined with the XRD softwares. If your TEM is well aligned and camera length is carefully corrected, two-dimensional (2D)-SAED pattern can be directly transformed to 1D-profile after the center determination of pattern, this profile is then imported to XRD analysis packages. Finally, phase identification and unit cell determination can be performed after peak search or precise peak position determined by profile fitting. Two examples, flaky-like TiO2 nanomaterial and TiO2 nanotubes precipitated by the silver nanoparticles, were tested and verified for the validation of phase identification and unit cell determination using this method; the successful crystallographic analysis of one single gold nanocrystal indicates it is still validate for the nanocrystals with the smaller diffraction volume, but need two or more random tilt SAED patterns. This method could be further used in the quantitative phase analysis, structure determination and Rietveld refinement for the nanomaterials if the reliable integrated intensity can be extracted. Copyright © 2013 Wiley Periodicals, Inc.

  13. Pitfalls and fallacies interfering with correct identification of embryonic stem cells implanted into the brain after experimental traumatic injury.

    PubMed

    Molcanyi, Marek; Bosche, Bert; Kraitsy, Klaus; Patz, Silke; Zivcak, Jozef; Riess, Peter; El Majdoub, Faycal; Hescheler, Jürgen; Goldbrunner, Roland; Schäfer, Ute

    2013-04-30

    Cell-therapy was proposed to be a promising tool in case of death or impairment of specific cell types. Correct identification of implanted cells became crucial when evaluating the success of transplantation therapy. Various methods of cell labeling have been employed in previously published studies. The use of intrinsic signaling of green fluorescent protein (GFP) has led to a well known controversy in the field of cardiovascular research. We encountered similar methodological pitfalls after transplantation of GFP-transfected embryonic stem cells into rat brains following traumatic brain injury (TBI). As the identification of implanted graft by intrinsic autofluorescence failed, anti-GFP labeling coupled to fluorescent and conventional antibodies was needed to visualize the implanted cells. Furthermore, different cell types with strong intrinsic autofluorescence were found at the sites of injury and transplantation, thus mimicking the implanted stem cells. GFP-positive stem cells were correctly localized, using advanced histological techniques. The activation of microglia/macrophages, accompanying the transplantation post TBI, was shown to be a significant source of artefacts, interfering with correct identification of implanted stem cells. Dependent on the strategy of stem cell tracking, the phagocytosis of implanted cells as observed in this study, might also impede the interpretation of results. Critical appraisal of previously published data as well as a review of different histological techniques provide tools for a more accurate identification of transplanted stem cells.

  14. Lgr5-positive cells are cancer stem cells in skin squamous cell carcinoma.

    PubMed

    Liu, Shunli; Gong, Zhenyu; Chen, Mingrui; Liu, Benli; Bian, Donghui; Wu, Kai

    2014-11-01

    Cancer stem cells (CSCs) in most human tumors are commonly identified and enriched using similar strategies for identifying normal stem cells, including flow cytometry assays for side population, high aldehyde dehydrogenase (ALDH) activity, and CD133 positivity. Thus, development of a method for isolating a specific cancer using cancer-specific characteristic appears to be potentially important. Here, we reported extremely high Lgr5 levels in the specimen from skin squamous cell carcinoma (SCC) in patients. Using SCC cell line A431, we detected high Lgr5 and CD133 levels in ALDH-high or side population from these cancer cells. To figure out whether Lgr5 is a marker of CSCs in SCC, we transfected A431 cells with a Lgr5-creERT-2A-DTR/Cag-Loxp-GFP-STOP-Loxp-RFP plasmid and purified transfected cells (tA431) based on GFP by flow cytometry. 4-Hydroxytamoxifen (4-OHT) was given to label Lgr5-positive cells with RFP, for comparison to GFP-positive Lgr5-negative cells. Lgr5-positive cells grew significantly faster than Lgr5-negative cells, and the fold increase in growth of Lgr5-positive vs Lgr5-negative cells is significantly higher than SP vs non-SP, or ALDH-high vs ALDH-low, or CD133-positive vs CD133-negative cells. Moreover, in Lgr5-negative population, Lgr5-positive re-appeared in culture with time, suggesting that Lgr5-positive cells can be regenerated from Lgr5-negative cells. Furthermore, the growth of tA431 cells significantly decreased upon a single dose of diphtheria toxin (DT)/4-OHT to eliminate Lgr5-positive cell lineage, while multiple doses of DT/4-OHT nearly completely inhibited tA431 cell growth. Taken together, our data provide compelling data to demonstrate that Lgr5-positive cells are CSCs in skin SCC.

  15. Systematic evaluation of markers used for the identification of human induced pluripotent stem cells

    PubMed Central

    Bharathan, Sumitha Prameela; Manian, Kannan Vrindavan; Aalam, Syed Mohammed Musheer; Palani, Dhavapriya; Deshpande, Prashant Ajit; Pratheesh, Mankuzhy Damodaran; Srivastava, Alok

    2017-01-01

    ABSTRACT Low efficiency of somatic cell reprogramming and heterogeneity among human induced pluripotent stem cells (hiPSCs) demand extensive characterization of isolated clones before their use in downstream applications. By monitoring human fibroblasts undergoing reprogramming for their morphological changes and expression of fibroblast (CD13), pluripotency markers (SSEA-4 and TRA-1-60) and a retrovirally expressed red fluorescent protein (RV-RFP), we compared the efficiency of these features to identify bona fide hiPSC colonies. The co-expression kinetics of fibroblast and pluripotency markers in the cells being reprogrammed and the emerging colonies revealed the heterogeneity within SSEA-4+ and TRA-1-60+ cells, and the inadequacy of these commonly used pluripotency markers for the identification of bona fide hiPSC colonies. The characteristic morphological changes in the emerging hiPSC colonies derived from fibroblasts expressing RV-RFP showed a good correlation between hiPSC morphology acquisition and silencing of RV-RFP and facilitated the easy identification of hiPSCs. The kinetics of retroviral silencing and pluripotency marker expression in emerging colonies suggested that combining both these markers could demarcate the stages of reprogramming with better precision than with pluripotency markers alone. Our results clearly demonstrate that the pluripotency markers that are routinely analyzed for the characterization of established iPSC colonies are not suitable for the isolation of pluripotent cells in the early stages of reprogramming, and silencing of retrovirally expressed reporter genes helps in the identification of colonies that have attained a pluripotent state and the morphology of human embryonic stem cells (hESCs). PMID:28089995

  16. Quantitative flow cytometric identification of aberrant T cell clusters in erythrodermic cutaneous T cell lymphoma. Implications for staging and prognosis.

    PubMed

    Horna, Pedro; Deaver, Darcie M; Qin, Dahui; Moscinski, Lynn C; Sotomayor, Eduardo M; Glass, L Frank; Sokol, Lubomir

    2014-05-01

    Assessment of peripheral blood tumour burden for staging of cutaneous T cells lymphoma is most often accomplished by flow cytometry (FC) using various non-standarised strategies. We report the results of calculating absolute Sezary cell counts (SCCs) by FC, based on the identification of aberrant T cell clusters on a virtual 6-dimensional space and independently of the expected immunophenotype (6D-FC SCC). 6D-FC SCCs were calculated on 65 peripheral blood specimens from 28 patients with erythrodermic cutaneous T cells lymphoma (stage III or IV). Comparisons were made with recommended FC strategies and correlations with overall mortality were studied. At first visit, 17 of 28 patients (61%) had 6D-FC SCCs meeting current criteria for Stage IV disease (≥1000 SC/μL); while only 2 patients (7%) met Stage IV criteria on other tissues. As defined by comprehensive staging using clinicomorphological criteria and 6D-FC SCCs, Stage IV disease identified a subgroup of patients with worse overall survival (p=0.0227). Residual non-aberrant CD4 T cells were markedly decreased in Stage IV disease (p=0.018). Among 65 specimens, discrepancies were observed between 6D-FC SCCs and usual FC thresholds for Stage IV disease, namely a CD4:CD8 ratio ≥10:1 (9 discrepancies, 14%), and ≥40% aberrant CD4 T cells (4 discrepancies, 6%). Surprisingly, 8 cases (12%) from 6 patients exhibited two distinctively separate clusters of aberrant CD4 T cells with different CD7 and/or CD26 expression. Visual 6-dimensional identification of aberrant T cell clusters by FC allows for the calculation of clinically significant SCCs. Simplified gating strategies and relative quantitative values might underestimate the immunophenotypical complexity of Sezary cells.

  17. Rapid Identification of Cell-Specific, Internalizing RNA Aptamers with Bioinformatics Analyses of a Cell-Based Aptamer Selection

    PubMed Central

    Thiel, William H.; Bair, Thomas; Peek, Andrew S.; Liu, Xiuying; Dassie, Justin; Stockdale, Katie R.; Behlke, Mark A.; Miller, Francis J.; Giangrande, Paloma H.

    2012-01-01

    Background The broad applicability of RNA aptamers as cell-specific delivery tools for therapeutic reagents depends on the ability to identify aptamer sequences that selectively access the cytoplasm of distinct cell types. Towards this end, we have developed a novel approach that combines a cell-based selection method (cell-internalization SELEX) with high-throughput sequencing (HTS) and bioinformatics analyses to rapidly identify cell-specific, internalization-competent RNA aptamers. Methodology/Principal Findings We demonstrate the utility of this approach by enriching for RNA aptamers capable of selective internalization into vascular smooth muscle cells (VSMCs). Several rounds of positive (VSMCs) and negative (endothelial cells; ECs) selection were performed to enrich for aptamer sequences that preferentially internalize into VSMCs. To identify candidate RNA aptamer sequences, HTS data from each round of selection were analyzed using bioinformatics methods: (1) metrics of selection enrichment; and (2) pairwise comparisons of sequence and structural similarity, termed edit and tree distance, respectively. Correlation analyses of experimentally validated aptamers or rounds revealed that the best cell-specific, internalizing aptamers are enriched as a result of the negative selection step performed against ECs. Conclusions and Significance We describe a novel approach that combines cell-internalization SELEX with HTS and bioinformatics analysis to identify cell-specific, cell-internalizing RNA aptamers. Our data highlight the importance of performing a pre-clear step against a non-target cell in order to select for cell-specific aptamers. We expect the extended use of this approach to enable the identification of aptamers to a multitude of different cell types, thereby facilitating the broad development of targeted cell therapies. PMID:22962591

  18. Cell Lineage Identification and Stem Cell Culture in a Porcine Model for the Study of Intestinal Epithelial Regeneration

    PubMed Central

    Gonzalez, Liara M.; Williamson, Ian; Piedrahita, Jorge A.; Blikslager, Anthony T.; Magness, Scott T.

    2013-01-01

    Significant advances in intestinal stem cell biology have been made in murine models; however, anatomical and physiological differences between mice and humans limit mice as a translational model for stem cell based research. The pig has been an effective translational model, and represents a candidate species to study intestinal epithelial stem cell (IESC) driven regeneration. The lack of validated reagents and epithelial culture methods is an obstacle to investigating IESC driven regeneration in a pig model. In this study, antibodies against Epithelial Adhesion Molecule 1 (EpCAM) and Villin marked cells of epithelial origin. Antibodies against Proliferative Cell Nuclear Antigen (PCNA), Minichromosome Maintenance Complex 2 (MCM2), Bromodeoxyuridine (BrdU) and phosphorylated Histone H3 (pH3) distinguished proliferating cells at various stages of the cell cycle. SOX9, localized to the stem/progenitor cells zone, while HOPX was restricted to the +4/‘reserve’ stem cell zone. Immunostaining also identified major differentiated lineages. Goblet cells were identified by Mucin 2 (MUC2); enteroendocrine cells by Chromogranin A (CGA), Gastrin and Somatostatin; and absorptive enterocytes by carbonic anhydrase II (CAII) and sucrase isomaltase (SIM). Transmission electron microscopy demonstrated morphologic and sub-cellular characteristics of stem cell and differentiated intestinal epithelial cell types. Quantitative PCR gene expression analysis enabled identification of stem/progenitor cells, post mitotic cell lineages, and important growth and differentiation pathways. Additionally, a method for long-term culture of porcine crypts was developed. Biomarker characterization and development of IESC culture in the porcine model represents a foundation for translational studies of IESC-driven regeneration of the intestinal epithelium in physiology and disease. PMID:23840480

  19. Identification of a breached fuel pin in the Interim Examination and Maintenance Cell

    SciTech Connect

    McGuiness, P.W.; Kalk, J.J.; Hicks, D.F.

    1987-09-01

    At the Interim Examination and Maintenance (IEM) Cell, experiments are routinely disassembled and examined following irradiation in the Fast Flux Test Facility (FFTF). Recently and for the first time, a fueled experiment which had breached its cladding during irradiation was disassembled in the cell. The processing objective was to locate and identify the one pin (out of 217 pins) with breached cladding, and recover selected test pins for further examination. Identification of the breached pin proved to be challenging. After all pins were weighed the data were inconclusive, and alternate procedures had to be developed and implemented. Ultimately, four independent methods were used to pinpoint the breached pin.

  20. Identification of Meflin as a Potential Marker for Mesenchymal Stromal Cells

    PubMed Central

    Maeda, Keiko; Enomoto, Atsushi; Hara, Akitoshi; Asai, Naoya; Kobayashi, Takeshi; Horinouchi, Asuka; Maruyama, Shoichi; Ishikawa, Yuichi; Nishiyama, Takahiro; Kiyoi, Hitoshi; Kato, Takuya; Ando, Kenju; Weng, Liang; Mii, Shinji; Asai, Masato; Mizutani, Yasuyuki; Watanabe, Osamu; Hirooka, Yoshiki; Goto, Hidemi; Takahashi, Masahide

    2016-01-01

    Bone marrow-derived mesenchymal stromal cells (BM-MSCs) in culture are derived from BM stromal cells or skeletal stem cells. Whereas MSCs have been exploited in clinical medicine, the identification of MSC-specific markers has been limited. Here, we report that a cell surface and secreted protein, Meflin, is expressed in cultured MSCs, fibroblasts and pericytes, but not other types of cells including epithelial, endothelial and smooth muscle cells. In vivo, Meflin is expressed by immature osteoblasts and chondroblasts. In addition, Meflin is found on stromal cells distributed throughout the BM, and on pericytes and perivascular cells in multiple organs. Meflin maintains the undifferentiated state of cultured MSCs and is downregulated upon their differentiation, consistent with the observation that Meflin-deficient mice exhibit increased number of osteoblasts and accelerated bone development. In the bone and BM, Meflin is more highly expressed in primitive stromal cells that express platelet-derived growth factor receptor α and Sca-1 than the Sca-1-negative adipo-osteogenic progenitors, which create a niche for hematopoiesis. Those results are consistent with a decrease in the number of clonogenic colony-forming unit-fibroblasts within the BM of Meflin-deficient mice. These preliminary data suggest that Meflin is a potential marker for cultured MSCs and their source cells in vivo. PMID:26924503

  1. Strategies to regenerate hair cells: identification of progenitors and critical genes.

    PubMed

    Breuskin, Ingrid; Bodson, Morgan; Thelen, Nicolas; Thiry, Marc; Nguyen, Laurent; Belachew, Shibeshih; Lefebvre, Philippe P; Malgrange, Brigitte

    2008-02-01

    Deafness commonly results from a lesion of the sensory cells and/or of the neurons of the auditory part of the inner ear. There are currently no treatments designed to halt or reverse the progression of hearing loss. A key goal in developing therapy for sensorineural deafness is the identification of strategies to replace lost hair cells. In amphibians and birds, a spontaneous post-injury regeneration of all inner ear sensory hair cells occurs. In contrast, in the mammalian cochlea, hair cells are only produced during embryogenesis. Many studies have been carried out in order to demonstrate the persistence of endogenous progenitors. The present review is first focused on the occurrence of spontaneous supernumerary hair cells and on nestin positive precursors found in the organ of Corti. A second approach to regenerating hair cells would be to find genes essential for their differentiation. This review will also focus on critical genes for embryonic hair cell formation such as the cell cycle related proteins, the Atoh1 gene and the Notch signaling pathway. Understanding mechanisms that underlie hair cell production is an essential prerequisite to defining therapeutic strategies to regenerate hair cells in the mature inner ear.

  2. Cutting edge: identification and characterization of human intrahepatic CD49a+ NK cells.

    PubMed

    Marquardt, Nicole; Béziat, Vivien; Nyström, Sanna; Hengst, Julia; Ivarsson, Martin A; Kekäläinen, Eliisa; Johansson, Helene; Mjösberg, Jenny; Westgren, Magnus; Lankisch, Tim O; Wedemeyer, Heiner; Ellis, Ewa C; Ljunggren, Hans-Gustaf; Michaëlsson, Jakob; Björkström, Niklas K

    2015-03-15

    Although NK cells are considered innate, recent studies in mice revealed the existence of a unique lineage of hepatic CD49a(+)DX5(-) NK cells with adaptive-like features. Development of this NK cell lineage is, in contrast to conventional NK cells, dependent on T-bet but not Eomes. In this study, we describe the identification of a T-bet(+)Eomes(-)CD49a(+) NK cell subset readily detectable in the human liver, but not in afferent or efferent hepatic venous or peripheral blood. Human intrahepatic CD49a(+) NK cells express killer cell Ig-like receptor and NKG2C, indicative of having undergone clonal-like expansion, are CD56(bright), and express low levels of CD16, CD57, and perforin. After stimulation, CD49a(+) NK cells express high levels of inflammatory cytokines but degranulate poorly. CD49a(+) NK cells retain their phenotype after expansion in long-term in vitro cultures. These results demonstrate the presence of a likely human counterpart of mouse intrahepatic NK cells with adaptive-like features. Copyright © 2015 by The American Association of Immunologists, Inc.

  3. Non-destructive Identification of Individual Leukemia Cells by Optical Trapping Raman Spectroscopy

    SciTech Connect

    Chan, J W; Taylor, D S; Lane, S; Zwerdling, T; Tuscano, J; Huser, T

    2007-03-05

    Currently, a combination of technologies is typically required to assess the malignancy of cancer cells. These methods often lack the specificity and sensitivity necessary for early, accurate diagnosis. Here we demonstrate using clinical samples the application of laser trapping Raman spectroscopy as a novel approach that provides intrinsic biochemical markers for the noninvasive detection of individual cancer cells. The Raman spectra of live, hematopoietic cells provide reliable molecular fingerprints that reflect their biochemical composition and biology. Populations of normal T and B lymphocytes from four healthy individuals, and cells from three leukemia patients were analyzed, and multiple intrinsic Raman markers associated with DNA and protein vibrational modes have been identified that exhibit excellent discriminating power for cancer cell identification. A combination of two multivariate statistical methods, principal component analysis (PCA) and linear discriminant analysis (LDA), was used to confirm the significance of these markers for identifying cancer cells and classifying the data. The results indicate that, on average, 95% of the normal cells and 90% of the patient cells were accurately classified into their respective cell types. We also provide evidence that these markers are unique to cancer cells and not purely a function of differences in their cellular activation.

  4. Got black swimming dots in your cell culture? Identification of Achromobacter as a novel cell culture contaminant.

    PubMed

    Gray, Jennifer Sue; Birmingham, Janette Marie; Fenton, Jenifer Imig

    2010-03-01

    Cell culture model systems are utilized for their ease of use, relative inexpensiveness, and potentially limitless sample size. Reliable results cannot be obtained, however, when cultures contain contamination. This report discusses the observation and identification of mobile black specks observed in multiple cell lines. Cultures of the contamination were grown, and DNA was purified from isolated colonies. The 16S rDNA gene was PCR amplified using primers that will amplify the gene from many genera, and then sequenced. Sequencing results matched the members of the genus Achromobacter, bacteria common in the environment. Achromobacter species have been shown to be resistant to multiple antibiotics. Attempts to decontaminate the eukaryotic cell culture used multiple antibiotics at different concentrations. The contaminating Achromobacter was eventually eliminated, without permanently harming the eukaryotic cells, using a combination of the antibiotics ciprofloxacin and piperacillin. (c) 2009 The International Association for Biologicals. Published by Elsevier Ltd. All rights reserved.

  5. Got black swimming dots in your cell culture? Identification of Achromobacter as a novel cell culture contaminant

    PubMed Central

    Gray, Jennifer Sue; Birmingham, Janette Marie; Fenton, Jenifer Imig

    2009-01-01

    ARTICLE SUMMARY Cell culture model systems are utilized for their ease of use, relative inexpensiveness, and potentially limitless sample size. Reliable results cannot be obtained, however, when cultures contain contamination. This report discusses the observation and identification of mobile black specks observed in multiple cell lines. Cultures of the contamination were grown, and DNA was purified from isolated colonies. The 16S rDNA gene was PCR amplified using primers that will amplify the gene from many genera, and then sequenced. Sequencing results matched the members of the genus Achromobacter, bacteria common in the environment. Achromobacter species have been shown to be resistant to multiple antibiotics. Attempts to decontaminate the eukaryotic cell culture used multiple antibiotics at different concentrations. The contaminating Achromobacter was eventually eliminated, without permanently harming the eukaryotic cells, using a combination of the antibiotics ciprofloxacin and piperacillin. PMID:19926304

  6. Cell line name recognition in support of the identification of synthetic lethality in cancer from text

    PubMed Central

    Kaewphan, Suwisa; Van Landeghem, Sofie; Ohta, Tomoko; Van de Peer, Yves; Ginter, Filip; Pyysalo, Sampo

    2016-01-01

    Motivation: The recognition and normalization of cell line names in text is an important task in biomedical text mining research, facilitating for instance the identification of synthetically lethal genes from the literature. While several tools have previously been developed to address cell line recognition, it is unclear whether available systems can perform sufficiently well in realistic and broad-coverage applications such as extracting synthetically lethal genes from the cancer literature. In this study, we revisit the cell line name recognition task, evaluating both available systems and newly introduced methods on various resources to obtain a reliable tagger not tied to any specific subdomain. In support of this task, we introduce two text collections manually annotated for cell line names: the broad-coverage corpus Gellus and CLL, a focused target domain corpus. Results: We find that the best performance is achieved using NERsuite, a machine learning system based on Conditional Random Fields, trained on the Gellus corpus and supported with a dictionary of cell line names. The system achieves an F-score of 88.46% on the test set of Gellus and 85.98% on the independently annotated CLL corpus. It was further applied at large scale to 24 302 102 unannotated articles, resulting in the identification of 5 181 342 cell line mentions, normalized to 11 755 unique cell line database identifiers. Availability and implementation: The manually annotated datasets, the cell line dictionary, derived corpora, NERsuite models and the results of the large-scale run on unannotated texts are available under open licenses at http://turkunlp.github.io/Cell-line-recognition/. Contact: sukaew@utu.fi PMID:26428294

  7. Cell line name recognition in support of the identification of synthetic lethality in cancer from text.

    PubMed

    Kaewphan, Suwisa; Van Landeghem, Sofie; Ohta, Tomoko; Van de Peer, Yves; Ginter, Filip; Pyysalo, Sampo

    2016-01-15

    The recognition and normalization of cell line names in text is an important task in biomedical text mining research, facilitating for instance the identification of synthetically lethal genes from the literature. While several tools have previously been developed to address cell line recognition, it is unclear whether available systems can perform sufficiently well in realistic and broad-coverage applications such as extracting synthetically lethal genes from the cancer literature. In this study, we revisit the cell line name recognition task, evaluating both available systems and newly introduced methods on various resources to obtain a reliable tagger not tied to any specific subdomain. In support of this task, we introduce two text collections manually annotated for cell line names: the broad-coverage corpus Gellus and CLL, a focused target domain corpus. We find that the best performance is achieved using NERsuite, a machine learning system based on Conditional Random Fields, trained on the Gellus corpus and supported with a dictionary of cell line names. The system achieves an F-score of 88.46% on the test set of Gellus and 85.98% on the independently annotated CLL corpus. It was further applied at large scale to 24 302 102 unannotated articles, resulting in the identification of 5 181 342 cell line mentions, normalized to 11 755 unique cell line database identifiers. The manually annotated datasets, the cell line dictionary, derived corpora, NERsuite models and the results of the large-scale run on unannotated texts are available under open licenses at http://turkunlp.github.io/Cell-line-recognition/. sukaew@utu.fi. © The Author 2015. Published by Oxford University Press.

  8. Phage-AgNPs complex as SERS probe for U937 cell identification.

    PubMed

    Lentini, Germana; Fazio, Enza; Calabrese, Federica; De Plano, Laura M; Puliafico, Maria; Franco, Domenico; Nicolò, Marco S; Carnazza, Santina; Trusso, Sebastiano; Allegra, Alessandro; Neri, Fortunato; Musolino, Caterina; Guglielmino, Salvatore P P

    2015-12-15

    The early diagnosis of malignancy is the most critical factor for patient survival and the treatment of cancer. In particular, leukemic cells are highly heterogeneous, and there is a need to develop new rapid and accurate detection systems for early diagnosis and monitoring of minimal residual disease. This study reports the utilization of molecular networks consisting of entire bacteriophage structure, displaying specific peptides, directly assembled with silver nanoparticles as a new Surface Enhanced Raman Spectroscopy (SERS) probe for U937 cells identification in vitro. A 9-mer pVIII M13 phage display library is screened against U937 to identify peptides that selectively recognize these cells. Then, phage clone is assembled with silver nanoparticles and the resulting network is used to obtain a SERS signal on cell-type specific molecular targets. The proposed strategy could be a very sensitive tool for the design of biosensors for highly specific and selective identification of hematological cancer cells and for detection of minimal residual disease in a significant proportion of human blood malignancy. Copyright © 2015 Elsevier B.V. All rights reserved.

  9. Automated Cell Identification and Tracking Using Nanoparticle Moving-Light-Displays

    PubMed Central

    Tonkin, James A.; Rees, Paul; Brown, Martyn R.; Errington, Rachel J.; Smith, Paul J.; Chappell, Sally C.; Summers, Huw D.

    2012-01-01

    An automated technique for the identification, tracking and analysis of biological cells is presented. It is based on the use of nanoparticles, enclosed within intra-cellular vesicles, to produce clusters of discrete, point-like fluorescent, light sources within the cells. Computational analysis of these light ensembles in successive time frames of a movie sequence, using k-means clustering and particle tracking algorithms, provides robust and automated discrimination of live cells and their motion and a quantitative measure of their proliferation. This approach is a cytometric version of the moving light display technique which is widely used for analyzing the biological motion of humans and animals. We use the endocytosis of CdTe/ZnS, core-shell quantum dots to produce the light displays within an A549, epithelial, lung cancer cell line, using time-lapse imaging with frame acquisition every 5 minutes over a 40 hour time period. The nanoparticle moving light displays provide simultaneous collection of cell motility data, resolution of mitotic traversal dynamics and identification of familial relationships allowing construction of multi-parameter lineage trees. PMID:22829889

  10. Identification and quantitation of morphological cell types in electrophoretically separated human embryonic kidney cell cultures

    NASA Technical Reports Server (NTRS)

    Williams, K. B.; Kunze, M. E.; Todd, P. W.

    1985-01-01

    Four major cell types were identified by phase microscopy in early passage human embryonic kidney cell cultures. They are small and large epithelioid, domed, and fenestrated cells. Fibroblasts are also present in some explants. The percent of each cell type changes with passage number as any given culture grows. As a general rule, the fraction of small epithelioid cells increases, while the fraction of fenestrated cells, always small, decreases further. When fibroblasts are present, they always increase in percentage of the total cell population. Electrophoretic separation of early passage cells showed that the domed cells have the highest electrophoretic mobility, fibroblasts have an intermediate high mobility, small epithelioid cells have a low mobility, broadly distributed, and fenestrated cells have the lowest mobility. All cell types were broadly distributed among electrophoretic subfractions, which were never pure but only enriched with respect to a given cell type.

  11. Identification and quantitation of morphological cell types in electrophoretically separated human embryonic kidney cell cultures

    NASA Technical Reports Server (NTRS)

    Williams, K. B.; Kunze, M. E.; Todd, P. W.

    1985-01-01

    Four major cell types were identified by phase microscopy in early passage human embryonic kidney cell cultures. They are small and large epithelioid, domed, and fenestrated cells. Fibroblasts are also present in some explants. The percent of each cell type changes with passage number as any given culture grows. As a general rule, the fraction of small epithelioid cells increases, while the fraction of fenestrated cells, always small, decreases further. When fibroblasts are present, they always increase in percentage of the total cell population. Electrophoretic separation of early passage cells showed that the domed cells have the highest electrophoretic mobility, fibroblasts have an intermediate high mobility, small epithelioid cells have a low mobility, broadly distributed, and fenestrated cells have the lowest mobility. All cell types were broadly distributed among electrophoretic subfractions, which were never pure but only enriched with respect to a given cell type.

  12. Identification of CCDC6-RET fusion in the human lung adenocarcinoma cell line, LC-2/ad.

    PubMed

    Matsubara, Daisuke; Kanai, Yoshihiko; Ishikawa, Shumpei; Ohara, Shiori; Yoshimoto, Taichiro; Sakatani, Takashi; Oguni, Sachiko; Tamura, Tomoko; Kataoka, Hiroaki; Endo, Shunsuke; Murakami, Yoshinori; Aburatani, Hiroyuki; Fukayama, Masashi; Niki, Toshiro

    2012-12-01

    Rearranged during transfection (RET) fusions have been newly identified in approximately 1% of patients with primary lung tumors. However, patient-derived lung cancer cell lines harboring RET fusions have not yet been established or identified, and therefore, the effectiveness of an RET inhibitor on lung tumors with endogenous RET fusion has not yet been studied. In this study, we report identification of CCDC6-RET fusion in the human lung adenocarcinoma cell line LC-2/ad. LC-2/ad showed distinctive sensitivity to the RET inhibitor, vandetanib, among 39 non-small lung cancer cell lines. The xenograft tumor of LC-2/ad showed cribriform acinar structures, a morphologic feature of primary RET fusion-positive lung adenocarcinomas. LC-2/ad cells could provide useful resources to analyze molecular functions of RET-fusion protein and its response to RET inhibitors.

  13. Identification of cell cycle-regulated genes by convolutional neural network.

    PubMed

    Liu, Chenglin; Cui, Peng; Huang, Tao

    2017-04-17

    The cell cycle-regulated genes express periodically with the cell cycle stages, and the identification and study of these genes can provide a deep understanding of the cell cycle process. Large false positives and low overlaps are big problems in cell cycle-regulated gene detection. Here, a computational framework called DLGene was proposed for cell cycle-regulated gene detection. It is based on the convolutional neural network, a deep learning algorithm representing raw form of data pattern without assumption of their distribution. First, the expression data was transformed to categorical state data to denote the changing state of gene expression, and four different expression patterns were revealed for the reported cell cycle-regulated genes. Then, DLGene was applied to discriminate the non-cell cycle gene and the four subtypes of cell cycle genes. Its performances were compared with six traditional machine learning methods. At last, the biological functions of representative cell cycle genes for each subtype were analyzed. Our method showed better and more balanced performance of sensitivity and specificity comparing to other machine learning algorithms. The cell cycle genes had very different expression pattern with non-cell cycle genes and among the cell-cycle genes, there were four subtypes. Our method not only detects the cell cycle genes, but also describes its expression pattern, such as when its highest expression level is reached and how it changes with time. For each type, we analyzed the biological functions of the representative genes and such results provided novel insight of the cell cycle mechanisms. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

  14. Identification of transcription factors linked to cell cycle regulation in Arabidopsis.

    PubMed

    Dehghan Nayeri, Fatemeh

    2014-01-01

    Cell cycle is an essential process in growth and development of living organisms consists of the replication and mitotic phases separated by 2 gap phases; G1 and G2. It is tightly controlled at the molecular level and especially at the level of transcription. Precise regulation of the cell cycle is of central significance for plant growth and development and transcription factors are global regulators of gene expression playing essential roles in cell cycle regulation. This study has uncovered TFs that are involved in the control of cell cycle progression. With the aid of multi-parallel quantitative RT-PCR, the expression changes of 1880 TFs represented in the Arabidopsis TF platform was monitored in Arabidopsis synchronous MM2d cells during a 19 h period representing different time points corresponding to the 4 cell cycle phases after treatment of MM2d cells with Aphidicolin. Comparative TF expression analyses performed on synchronous cells resulted in the identification of 239 TFs differentially expressed during the cell cycle, while about one third of TFs were constitutively expressed through all time points. Phase-specific TFs were also identified.

  15. Identification of transcription factors linked to cell cycle regulation in Arabidopsis

    PubMed Central

    Dehghan Nayeri, Fatemeh

    2014-01-01

    Cell cycle is an essential process in growth and development of living organisms consists of the replication and mitotic phases separated by 2 gap phases; G1 and G2. It is tightly controlled at the molecular level and especially at the level of transcription. Precise regulation of the cell cycle is of central significance for plant growth and development and transcription factors are global regulators of gene expression playing essential roles in cell cycle regulation. This study has uncovered TFs that are involved in the control of cell cycle progression. With the aid of multi-parallel quantitative RT-PCR, the expression changes of 1880 TFs represented in the Arabidopsis TF platform was monitored in Arabidopsis synchronous MM2d cells during a 19 h period representing different time points corresponding to the 4 cell cycle phases after treatment of MM2d cells with Aphidicolin. Comparative TF expression analyses performed on synchronous cells resulted in the identification of 239 TFs differentially expressed during the cell cycle, while about one third of TFs were constitutively expressed through all time points. Phase-specific TFs were also identified. PMID:25482767

  16. Efficient DNA fingerprinting method for the identification of cross-culture contamination of cell lines.

    PubMed

    Matsuo, Y; Nishizaki, C; Drexler, H G

    1999-09-01

    In order to identify cross-culture contamination of cell lines, we applied DNA fingerprinting using variable number of tandem repeat (VNTR) loci and short tandem repeat (STR) loci amplified by polymerase chain reaction (PCR) instead of a radioisotope labeled multilocus probe. Eleven cell lines were used for the Apo B and D1S80 loci detection, and twelve cell lines were examined in the Y-chromosome analysis. The data obtained from the sister cell lines NALM-6 and B85, two MOLM-1 cultures from two cryopreserved tubes, and four subclones of BALM-9 and its sister cell line BALM-10, displayed clear and distinct bands of each PCR product for both Apo B and D1S80. Detection of a Y-chromosome DNA sequence is another very informative marker for the identification of cell lines, if the Y-chromosome is present. We examined eight cell lines for the expression of four STR loci; the data thus generated were compared with the results previously reported from other laboratories. The resulting electrophoretic banding patterns showed that our "home-made" STR detection system is a useful and efficient tool for the authentication of cell lines. PCR detection of VNTR and STR loci represents a simple, rapid and powerful DNA fingerprinting technique to authenticate human cell lines and to detect cross-culture contamination. This PCR technique may be used in lieu of the more time-consuming, labor-intensive and radioactive Southern blot multilocus method.

  17. Lymphocyte imprinting with melanoma antigens acquired by trogocytosis facilitates identification of tumor-reactive T cells

    PubMed Central

    Eisenberg, Galit; Uzana, Ronny; Pato, Aviad; Frankenburg, Shoshana; Merims, Sharon; Yefenof, Eitan; Ferrone, Soldano; Peretz, Tamar; Machlenkin, Arthur; Lotem, Michal

    2013-01-01

    Trogocytosis is a contact-dependent inter-cellular transfer of membrane fragments and associated molecules from antigen presenting cells to effector lymphocytes. We previously demonstrated that trogocytosis also occurs between tumor target and cognate melanoma antigen-specific cytotoxic T cells (CTL). Here we show that, following trogocytosis, immune effector cells acquire molecular components of the tumor, including surface antigens, which are detectable by specific monoclonal antibodies. We demonstrate that CD8+ and CD4+ T cells from melanoma patients’ PBMC and tumor infiltrating lymphocytes (TIL) capture melanoma antigens, enabling identification of trogocytosing lymphocytes by staining with antigen-specific antibodies. This finding circumvents the necessity of tumor pre-labeling, which in the past was mandatory to detect membrane-capturing T cells. Through the detection of melanoma antigens on TIL, we sorted trogocytosing T cells and verified their preferential reactivity and cytotoxicity. Furthermore, tumor-antigen imprinted T cells were detected at low frequency in fresh TIL cultures shortly after extraction from the tumor. Thus, T cell imprinting by tumor antigens may allow the enrichment of melanoma antigen-specific T cells for research and potentially even for the adoptive immunotherapy of patients with cancer. PMID:23626012

  18. Identification of an IL-4-Inducible Gene Expressed in Differentiating Lymphocytes and Male Germ Cells

    PubMed Central

    Nabavi, Nasrin; Grusby, Michael J.; Finn, Patricia W.; Wolgemuth, Debra J.; Glimcher, Laurie H.

    1990-01-01

    Interleukin 4 (IL-4) is a cytokine that is involved in the differentiation of B and T lymphocytes. In this report, we describe the identification of a novel gene, N.52, which was cloned from the murine pre-B cell line R8205 grown in the presence of IL-4 for 48 hr. Although N.52 expression is detectable at low levels in unstimulated R8205 cells, the level of N.52 dramatically increases after only .4 hr exposure to IL-4 and remains at a high .level up to 48 hr. Although N.52 expression is low or absent in normal spleen B and T cells, its expression can be induced by the differentiation signals delivered by LPS in B cells and by Con A in T-cell hybrids. While N.52 mRNA is absent in all highly differentiated organs, it is detectable in stem cell harboring lymphoid tissues such as bone marrow, fetal liver, and thymus. Furthermore, N.52 mRNA is expressed at strikingly high levels in the testis, specifically in differentiating male germ cells. It is induced by differentiation signals triggered by the combination of cyclic AMP and retinoic acid in teratocarcinoma F9 cells. Taken together, these data suggest that N.52 is a developmentally regulated gene whose expression in cells of the immune and reproductive systems may be controlled by stimuli that induce differentiation. PMID:2136202

  19. MHC Multimer-Guided and Cell Culture-Independent Isolation of Functional T Cell Receptors from Single Cells Facilitates TCR Identification for Immunotherapy

    PubMed Central

    Dössinger, Georg; Bunse, Mario; Bet, Jeannette; Albrecht, Julia; Paszkiewicz, Paulina J.; Weißbrich, Bianca; Schiedewitz, Isabell; Henkel, Lynette; Schiemann, Matthias; Neuenhahn, Michael; Uckert, Wolfgang; Busch, Dirk H.

    2013-01-01

    Adoptive therapy using T cells redirected to target tumor- or infection-associated antigens is a promising strategy that has curative potential and broad applicability. In order to accelerate the screening process for suitable antigen-specific T cell receptors (TCRs), we developed a new approach circumventing conventional in vitro expansion-based strategies. Direct isolation of paired full-length TCR sequences from non-expanded antigen-specific T cells was achieved by the establishment of a highly sensitive PCR-based T cell receptor single cell analysis method (TCR-SCAN). Using MHC multimer-labeled and single cell-sorted HCMV-specific T cells we demonstrate a high efficacy (approximately 25%) and target specificity of TCR-SCAN receptor identification. In combination with MHC-multimer based pre-enrichment steps, we were able to isolate TCRs specific for the oncogenes Her2/neu and WT1 even from very small populations (original precursor frequencies of down to 0.00005% of CD3+ T cells) without any cell culture step involved. Genetic re-expression of isolated receptors demonstrates their functionality and target specificity. We believe that this new strategy of TCR identification may provide broad access to specific TCRs for therapeutically relevant T cell epitopes. PMID:23637823

  20. Immunocapture and Identification of Cell Membrane Protein Antigenic Targets of Serum Autoantibodies*

    PubMed Central

    Littleton, Edward; Dreger, Mathias; Palace, Jackie; Vincent, Angela

    2009-01-01

    There is increasing interest in the role of antibodies targeting specific membrane proteins in neurological and other diseases. The target(s) of these pathogenic antibodies is known in a few diseases, usually when candidate cell surface proteins have been tested. Approaches for identifying new antigens have mainly resulted in the identification of antibodies to intracellular proteins, which are often very useful as diagnostic markers for disease but unlikely to be directly involved in disease pathogenesis because they are not accessible to circulating antibodies. To identify cell surface antigens, we developed a “conformational membrane antigen isolation and identification” strategy. First, a cell line is identified that reacts with patient sera but not with control sera. Second, intact cells are exposed to sera to allow the binding of presumptive autoantibodies to their cell surface targets. After washing off non-bound serum components, the cells are lysed, and immune complexes are precipitated. Third, the bound surface antigen is identified by mass spectrometry. As a model system we used a muscle cell line, TE671, that endogenously expresses muscle-specific tyrosine receptor kinase (MuSK) and sera or plasmas from patients with a subtype of the autoimmune disease myasthenia gravis in which patients have autoantibodies against MuSK. MuSK was robustly detected as the only membrane protein in immunoprecipitates from all three patient samples tested and not from the three MuSK antibody-negative control samples processed in parallel. Of note, however, there were many intracellular proteins found in the immunoprecipitates from both patients and controls, suggesting that these were nonspecifically immunoprecipitated from cell extracts. The conformational membrane antigen isolation and identification technique should be of value for the detection of highly relevant antigenic targets in the growing number of suspected antibody-mediated autoimmune disorders. The

  1. Sorting, identification and enrichment of side population cells in THP-1 acute monocytic leukemia cells.

    PubMed

    Wang, Yingchao; Yin, Chuyun; Feng, Lei; Ma, Lina; Wei, Yongwei; Sheng, Guangyao

    2013-05-01

    The objective of the present study was to examine and determine whether the human acute monocytic leukemia cell line THP-1 contains side population (SP) cells, and, if so, to increase the proportion of SP cells using arabinosylcytosine (Ara-C). Fluorescent microscopy and flow cytometry were employed to detect the percentage of SP cells in THP-1 cells. Then, SP and non-SP (NSP) cell subpopulations were collected and identified. THP-1 cells were incubated with different concentrations of Ara-C for 24 h and the proportion of SP cells was detected. Our results demonstrated that the percentage of SP cells was 1.81 ± 0.99% in THP-1 cells. A majority of the SP cells remained in the G₀/G₁ phase, and the expression of CD34⁺ and CD34⁺CD38⁻ and the proliferation ability of the SP cells were higher compared to NSP cells (P<0.05). The mRNA expression of multidrug resistance genes (ABCG2 and ABCB1), apoptosis regulation genes (Bcl-2) and the Bcl-2/Bax value of SP cells were higher than those of NSP cells. SP cells have been shown to be more tumorigenic than NSP cells. Following co-culture with Ara-C, the proportion of SP cells increased significantly and subsequently the Ara-C concentration increased. These findings suggest that the THP-1 cell line contains SP cells and that SP cells possess certain intrinsic stem cell properties and may contain a larger proportion of leukemia stem cells (LSCs). The concentrations of SP cells can be increased with Ara-C by co-culture, and this technique is a useful and important application for the study of LSCs.

  2. Developments in techniques for the isolation, enrichment, main culture conditions and identification of spermatogonial stem cells.

    PubMed

    He, Yanan; Chen, Xiaoli; Zhu, Huabin; Wang, Dong

    2015-12-01

    The in vitro culture system of spermatogonial stem cells (SSCs) provides a basis for studies on spermatogenesis, and also contributes to the development of new methods for the preservation of livestock and animal genetic modification. In vitro culture systems have mainly been established for mouse SSCs, but are lacking for farm animals. We reviewed and analyzed the current progress in SSC techniques such as isolation, purification, cultivation and identification. Based on the published studies, we concluded that two-step enzyme digestion and magnetic-activated cell sorting are fast becoming the main methods for isolation and enrichment of SSCs. With regard to the culture systems, serum and feeders were earlier thought to play an important role in the self-renewal and proliferation of SSCs, but serum- and feeder-free culture systems as a means of overcoming the limitations of SSC differentiation in long-term SSC culture are being explored. However, there is still a need to establish more efficient and ideal culture systems that can also be used for SSC culture in larger mammals. Although the lack of SSC-specific surface markers has seriously affected the efficiency of purification and identification, the transgenic study is helpful for our identification of SSCs. Therefore, future studies on SSC techniques should focus on improving serum- and feeder-free culture techniques, and discovering and identifying specific surface markers of SSCs, which will provide new ideas for the optimization of SSC culture systems for mice and promote related studies in farm animals.

  3. Whole cell screen based identification of spiropiperidines with potent antitubercular properties.

    PubMed

    Tantry, Subramanyam J; Degiacomi, Giulia; Sharma, Sreevalli; Jena, Lalit Kumar; Narayan, Ashwini; Guptha, Supreeth; Shanbhag, Gajanan; Menasinakai, Sreenivasaiah; Mallya, Meenakshi; Awasthy, Disha; Balakrishnan, Gayathri; Kaur, Parvinder; Bhattacharjee, Deepa; Narayan, Chandan; Reddy, Jitendar; Naveen Kumar, C N; Shandil, Radha; Boldrin, Francesca; Ventura, Marcello; Manganelli, Riccardo; Hartkoorn, Ruben C; Cole, Stewart T; Panda, Manoranjan; Markad, Shankar D; Ramachandran, Vasanthi; Ghorpade, Sandeep R; Dinesh, Neela

    2015-08-15

    Whole cell based screens to identify hits against Mycobacterium tuberculosis (Mtb), carried out under replicating and non-replicating (NRP) conditions, resulted in the identification of multiple, novel but structurally related spiropiperidines with potent antitubercular properties. These compounds could be further classified into three classes namely 3-(3-aryl-1,2,4-oxadiazol-5-yl)-1'-alkylspiro[indene-1,4'-piperidine] (abbr. spiroindenes), 4-(3-aryl-1,2,4-oxadiazol-5-yl)-1'-alkylspiro[chromene-2,4'-piperidine] (abbr. spirochromenes) and 1'-benzylspiro[indole-1,4'-piperidin]-2(1H)-one (abbr. spiroindolones). Spiroindenes showed ⩾ 4 log10 kill (at 2-12 μM) on replicating Mtb, but were moderately active under non replicating conditions. Whole genome sequencing efforts of spiroindene resistant mutants resulted in the identification of I292L mutation in MmpL3 (Mycobacterial membrane protein Large), required for the assembly of mycolic acid into the cell wall core of Mtb. MIC modulation studies demonstrated that the mutants were cross-resistant to spirochromenes but not to spiroindolones. This Letter describes lead identification efforts to improve potency while reducing the lipophilicity and hERG liabilities of spiroindenes. Additionally, as deduced from the SAR studies, we provide insights regarding the new chemical opportunities that the spiroindolones can offer to the TB drug discovery initiatives.

  4. Identification of a unique B-cell-stimulating factor produced by a cloned dendritic cell.

    PubMed Central

    Clayberger, C; DeKruyff, R H; Fay, R; Cantor, H

    1985-01-01

    We describe a cloned dendritic cell, clone Den-1, which is a potent accessory cell for some B-cell responses. Clone Den-1 produces a unique lymphokine that induces polyclonal B-cell proliferation in the absence of other costimulators. This clone or factors produced by it also stimulate purified B cells to develop plaque-forming cell responses to type 2 antigens. The effect of this factor(s) on various B-cell populations and its relationship to previously described B-cell-stimulating factors is discussed. Images PMID:3871522

  5. Identification of glucagon-producing cells (A cells) in dog gastric mucosa

    PubMed Central

    1976-01-01

    An immunocytochemical technique using specific antiglucagon serum reveals the presence of glucagon-containing cells situated exclusively in the oxyntic glandular mucosa of the dog stomach. Electron microscope examination of the mucosa demonstrated endocrine cells containing secretory granules with a round dense core surrounded by a clear halo, indistinguishable from secretory granules of pancreatic A cells. Like the alpha granules of pancreatic A cells, the granules of these gastric endocrine cells exhibited a peripheral distribution of silver grains after Grimelius silver staining. Moreover, the granules of these cells were found to be specifically labeled with reaction product, using the peroxidase immunocytochemical technique at the ultrastructural level. Accordingly, these cells were named gastric A cells. These data suggest that the gastric oxyntic mucosa contains cells indistinguishable cytologically, cytochemically, and immunocytochemically from pancreatic A cells. It is believed that gastric A cells are responsible for the secretion of the gastric glucagon. PMID:770482

  6. Direct identification of ligand-receptor interactions on living cells and tissues.

    PubMed

    Frei, Andreas P; Jeon, Ock-Youm; Kilcher, Samuel; Moest, Hansjoerg; Henning, Lisa M; Jost, Christian; Plückthun, Andreas; Mercer, Jason; Aebersold, Ruedi; Carreira, Erick M; Wollscheid, Bernd

    2012-10-01

    Many cellular responses are triggered by proteins, drugs or pathogens binding to cell-surface receptors, but it can be challenging to identify which receptors are bound by a given ligand. Here we describe TRICEPS, a chemoproteomic reagent with three moieties--one that binds ligands containing an amino group, a second that binds glycosylated receptors on living cells and a biotin tag for purifying the receptor peptides for identification by quantitative mass spectrometry. We validated this ligand-based, receptor-capture (LRC) technology using insulin, transferrin, apelin, epidermal growth factor, the therapeutic antibody trastuzumab and two DARPins targeting ErbB2. In some cases, we could also determine the approximate ligand-binding sites on the receptors. Using TRICEPS to label intact mature vaccinia viruses, we identified the cell surface proteins AXL, M6PR, DAG1, CSPG4 and CDH13 as binding factors on human cells. This technology enables the identification of receptors for many types of ligands under near-physiological conditions and without the need for genetic manipulations.

  7. A selective hybrid stochastic strategy for fuel-cell multi-parameter identification

    NASA Astrophysics Data System (ADS)

    Guarnieri, Massimo; Negro, Enrico; Di Noto, Vito; Alotto, Piergiorgio

    2016-11-01

    The in situ identification of fuel-cell material parameters is crucial both for guiding the research for advanced functionalized materials and for fitting multiphysics models, which can be used in fuel cell performance evaluation and optimization. However, this identification still remains challenging when dealing with direct measurements. This paper presents a method for achieving this aim by stochastic optimization. Such techniques have been applied to the analysis of fuel cells for ten years, but typically to specific problems and by means of semi-empirical models, with an increased number of articles published in the last years. We present an original formulation that makes use of an accurate zero-dimensional multi-physical model of a polymer electrolyte membrane fuel cell and of two cooperating stochastic algorithms, particle swarm optimization and differential evolution, to extract multiple material parameters (exchange current density, mass transfer coefficient, diffusivity, conductivity, activation barriers …) from the experimental data of polarization curves (i.e. in situ measurements) under some controlled temperature, gas back pressure and humidification. The method is suitable for application in other fields where fitting of multiphysics nonlinear models is involved.

  8. Purification and identification of cell surface antigens using lamprey monoclonal antibodies

    PubMed Central

    Yu, Cuiling; Ali, Shabab; St. Germain, Jonathan; Liu, Yanling; Yu, Xuecong; Jaye, David L.; Moran, Michael F.; Cooper, Max D.; Ehrhardt, Götz R.A.

    2013-01-01

    Variable lymphocyte receptor (VLR) B antibodies of the evolutionary distant sea lamprey are structurally distinct from conventional mammalian antibodies. The different protein architecture and large evolutionary distance of jawless vertebrates suggest that VLR antibodies may represent promising tools for biomarker discovery. Here we report the generation of panels of monoclonal VLR antibodies from lamprey larvae immunized with human T cells and the use of a recombinant monoclonal VLR antibody for antigen purification and mass spectrometric identification. We demonstrate that despite predicted low affinity of individual VLR antigen binding units to the antigen, the high avidity resulting from decameric assembly of secreted VLR antibodies allows for efficient antigen capture and subsequent identification by mass spectometry. We show that VLR antibodies detect their antigens with high specificity and can be used in various standard laboratory application techniques. The lamprey antibodies are novel reagents that can complement conventional monoclonal antibodies in multiple scientific research disciplines. PMID:22964555

  9. Identification of cancer stem cell markers in human malignant mesothelioma cells

    SciTech Connect

    Ghani, Farhana Ishrat; Yamazaki, Hiroto; Iwata, Satoshi; Okamoto, Toshihiro; Aoe, Keisuke; Okabe, Kazunori; Mimura, Yusuke; Fujimoto, Nobukazu; Kishimoto, Takumi; Yamada, Taketo; Xu, C. Wilson; Morimoto, Chikao

    2011-01-14

    Research highlights: {yields} We performed serial transplantation of surgical samples and established new cell lines of malignant mesothelioma. {yields} SP cell and expressions of CD9/CD24/CD26 were often observed in mesothelioma cell lines. {yields} SP and CD24{sup +} cells proliferated by asymmetric cell division-like manner. CD9{sup +} and CD24{sup +} cells have higher potential to generate spheroid colony. {yields} The marker-positive cells have clear tendency to generate larger tumors in mice. -- Abstract: Malignant mesothelioma (MM) is an aggressive and therapy-resistant neoplasm arising from the pleural mesothelial cells and usually associated with long-term asbestos exposure. Recent studies suggest that tumors contain cancer stem cells (CSCs) and their stem cell characteristics are thought to confer therapy-resistance. However, whether MM cell has any stem cell characteristics is not known. To understand the molecular basis of MM, we first performed serial transplantation of surgical samples into NOD/SCID mice and established new cell lines. Next, we performed marker analysis of the MM cell lines and found that many of them contain SP cells and expressed several putative CSC markers such as CD9, CD24, and CD26. Interestingly, expression of CD26 closely correlated with that of CD24 in some cases. Sorting and culture assay revealed that SP and CD24{sup +} cells proliferated by asymmetric cell division-like manner. In addition, CD9{sup +} and CD24{sup +} cells have higher potential to generate spheroid colony than negative cells in the stem cell medium. Moreover, these marker-positive cells have clear tendency to generate larger tumors in mouse transplantation assay. Taken together, our data suggest that SP, CD9, CD24, and CD26 are CSC markers of MM and could be used as novel therapeutic targets.

  10. Identification of Tympanic Border Cells as Slow-Cycling Cells in the Cochlea

    PubMed Central

    Taniguchi, Mirei; Yamamoto, Norio; Nakagawa, Takayuki; Ogino, Eriko; Ito, Juichi

    2012-01-01

    Mammalian cochlear sensory epithelial cells are believed to possess minimal regenerative potential because they halt proliferation during late stage of embryogenesis and never regenerate after birth. This means that sensorineural hearing loss caused by the death of cochlear sensory epithelial cells is a permanent condition. However, stem cells were recently identified in neonatal mice following dissociation of their inner ear organs. This suggests that regenerative therapy for sensorineural hearing loss may be possible. Unfortunately, dissociation distorts the microanatomy of the inner ear, making it difficult to determine the precise location of stem cells in unaltered specimens. To develop new therapeutic approaches based on sensory epithelial cell regeneration, the location of these stem cells must be elucidated. Stem cells normally proliferate at a slow rate in adult organs. In fact, so-called label-retaining cells, or slow-cycling cells, of the brain and skin are recognized as stem cells. In this study, using the exogenous proliferation marker, 5′-bromo-2′-deoxyuridine (BrdU) in combination with the endogenous proliferation marker Ki-67, we identified tympanic border cells. These cells, which are located beneath the basilar membrane in vivo, represent slow-cycling cells of the murine cochlea. Immunohistochemically, these cells stained positive for the immature cell marker Nestin. But it will be difficult to achieve regeneration of the cochlear function because these slow-cycling cells disappear in the mature murine cochlea. PMID:23119055

  11. Identification of a Mouse Cytomegalovirus Gene Selectively Targeting CD86 Expression on Antigen-Presenting Cells

    PubMed Central

    Loewendorf, Andrea; Krüger, Corinna; Borst, Eva Maria; Wagner, Markus; Just, Ursula; Messerle, Martin

    2004-01-01

    We and others have shown that infection of dendritic cells with murine cytomegalovirus (MCMV) leads to severe functional impairment of these antigen-presenting cells (D. M. Andrews, C. E. Andoniou, F. Granucci, P. Ricciardi-Castagnoli, and M. A. Degli-Esposti, Nat. Immunol. 2:1077-1084, 2001; S. Mathys, T. Schroeder, J. Ellwart, U. H. Koszinowski, M. Messerle, and U. Just, J. Infect. Dis. 187:988-999, 2003). Phenotypically, reduced surface expression of costimulatory molecules and major histocompatibility complex molecules was detected. In order to identify the molecular basis for the observed effects, we generated MCMV mutants with large deletions of nonessential genes. The study was facilitated by the finding that a monocyte-macrophage cell line displayed similar phenotypic alterations after MCMV infection. By analyzing the expression of cell surface molecules on infected cells, we identified a mutant virus which is no longer able to downmodulate the expression of the costimulatory molecule CD86. Additional mutants with smaller deletions allowed us to pin down the responsible gene to a certain genomic region. RNA analysis led to the identification of the spliced gene m147.5, encoding a protein with 145 amino acids. Experiments with an m147.5 mutant revealed that the protein affects CD86 expression only, suggesting that additional MCMV genes are responsible for downmodulation of the other surface molecules. Identification of viral gene products interfering with functionally important proteins of antigen-presenting cells will provide the basis to dissect the complex interaction of CMV with these important cells and to evaluate the biological importance of these viral genes in vivo. PMID:15542658

  12. High-resolution Identification and Separation of Living Cell Types by Multiple microRNA-responsive Synthetic mRNAs

    PubMed Central

    Endo, Kei; Hayashi, Karin; Saito, Hirohide

    2016-01-01

    The precise identification and separation of living cell types is critical to both study cell function and prepare cells for medical applications. However, intracellular information to distinguish live cells remains largely inaccessible. Here, we develop a method for high-resolution identification and separation of cell types by quantifying multiple microRNA (miRNA) activities in live cell populations. We found that a set of miRNA-responsive, in vitro synthesized mRNAs identify a specific cell population as a sharp peak and clearly separate different cell types based on less than two-fold differences in miRNA activities. Increasing the number of miRNA-responsive mRNAs enhanced the capability for cell identification and separation, as we precisely and simultaneously distinguished different cell types with similar miRNA profiles. In addition, the set of synthetic mRNAs separated HeLa cells into subgroups, uncovering heterogeneity of the cells and the level of resolution achievable. Our method could identify target live cells and improve the efficiency of cell purification from heterogeneous populations. PMID:26902536

  13. On-Bead Two-Color (OBTC) Cell Screen for Direct Identification of Highly Selective Cell Surface Receptor Ligands

    PubMed Central

    Udugamasooriya, D. Gomika; Kodadek, Thomas

    2012-01-01

    Combinatorial library screens can identify a suitable ligand for a biological target of interest out of thousands or even millions of compounds, and can play a key role in the modern drug development process. While conventional high-throughput cell screens based on functional assays require expensive robotics, simple on-bead combinatorial assays for ligand binding to the target protein can be done far more cheaply. This article describes one such assay, developed using combinatorial peptoid libraries for targeting integral membrane receptors or other cell surface-exposed molecules. In addition to the reduced cost, a unique advantage of this assay is the direct identification of the most selective ligands for a cell surface receptor that is expressed in its natural environment. PMID:22582145

  14. Identification of G1-Regulated Genes in Normally Cycling Human Cells

    PubMed Central

    Beyrouthy, Maroun J.; Alexander, Karen E.; Baldwin, Amy; Whitfield, Michael L.; Bass, Hank W.; McGee, Dan; Hurt, Myra M.

    2008-01-01

    Background Obtaining synchronous cell populations is essential for cell-cycle studies. Methods such as serum withdrawal or use of drugs which block cells at specific points in the cell cycle alter cellular events upon re-entry into the cell cycle. Regulatory events occurring in early G1 phase of a new cell cycle could have been overlooked. Methodology and Findings We used a robotic mitotic shake-off apparatus to select cells in late mitosis for genome-wide gene expression studies. Two separate microarray experiments were conducted, one which involved isolation of RNA hourly for several hours from synchronous cell populations, and one experiment which examined gene activity every 15 minutes from late telophase of mitosis into G1 phase. To verify synchrony of the cell populations under study, we utilized methods including BrdU uptake, FACS, and microarray analyses of histone gene activity. We also examined stress response gene activity. Our analysis enabled identification of 200 early G1-regulated genes, many of which currently have unknown functions. We also confirmed the expression of a set of genes candidates (fos, atf3 and tceb) by qPCR to further validate the newly identified genes. Conclusion and Significance Genome-scale expression analyses of the first two hours of G1 in naturally cycling cells enabled the discovery of a unique set of G1-regulated genes, many of which currently have unknown functions, in cells progressing normally through the cell division cycle. This group of genes may contain future targets for drug development and treatment of human disease. PMID:19079774

  15. Identification of a Functional Plasmodesmal Localization Signal in a Plant Viral Cell-To-Cell-Movement Protein

    PubMed Central

    Yuan, Cheng; Lazarowitz, Sondra G.

    2016-01-01

    ABSTRACT Our fundamental knowledge of the protein-sorting pathways required for plant cell-to-cell trafficking and communication via the intercellular connections termed plasmodesmata has been severely limited by the paucity of plasmodesmal targeting sequences that have been identified to date. To address this limitation, we have identified the plasmodesmal localization signal (PLS) in the Tobacco mosaic virus (TMV) cell-to-cell-movement protein (MP), which has emerged as the paradigm for dissecting the molecular details of cell-to-cell transport through plasmodesmata. We report here the identification of a bona fide functional TMV MP PLS, which encompasses amino acid residues between positions 1 and 50, with residues Val-4 and Phe-14 potentially representing critical sites for PLS function that most likely affect protein conformation or protein interactions. We then demonstrated that this PLS is both necessary and sufficient for protein targeting to plasmodesmata. Importantly, as TMV MP traffics to plasmodesmata by a mechanism that is distinct from those of the three plant cell proteins in which PLSs have been reported, our findings provide important new insights to expand our understanding of protein-sorting pathways to plasmodesmata. PMID:26787834

  16. Identification of stem cells that maintain and regenerate lingual keratinized epithelial cells.

    PubMed

    Tanaka, Toshihiro; Komai, Yoshihiro; Tokuyama, Yoko; Yanai, Hirotsugu; Ohe, Shuichi; Okazaki, Kazuichi; Ueno, Hiroo

    2013-05-01

    Lingual keratinized epithelial cells, which constitute the filiform papillae of the tongue, have one of the most rapid tissue turnover rates in the mammalian body and are thought to be the source of squamous cell carcinoma of the tongue. However, the mechanism of tissue maintenance and regeneration is largely unknown for these cells. Here, we show that stem cells positive for Bmi1, keratin 14 and keratin 5 are present in the base but not at the very bottom of the interpapillary pit (observed most frequently in the second or third layer (position +2 or +3) from the basal cells). Using a multicolour lineage tracing method, we demonstrated that one stem cell per interpapillary pit survives long-term. The cells were shown to be unipotent stem cells for keratinized epithelial cells but not for taste bud cells, and were found to usually be in a slow-growing or resting state; however, on irradiation-induced injury, the cells rapidly entered the cell cycle and regenerated tongue epithelium. The elimination of Bmi1-positive stem cells significantly suppressed the regeneration. Taken together, these results suggest that the stem cells identified in this study are important for tissue maintenance and regeneration of the lingual epithelium.

  17. Identification of different subsets of lung cells using Raman microspectroscopy and whole cell nucleus isolation.

    PubMed

    Pijanka, Jacek K; Stone, Nicholas; Rutter, Abigail V; Forsyth, Nicholas; Sockalingum, Ganesh D; Yang, Ying; Sulé-Suso, Josep

    2013-09-07

    Raman spectroscopy has been widely used to study its possible clinical application in cancer diagnosis. However, in order to make it into clinical practice, it is important that this technique is able not only to identify cancer cells from their normal counterparts, but also from the array of cells present in human tissues. To this purpose, we used Raman spectroscopy to assess whether this technique was able to differentiate not only between lung cancer cells and lung epithelial cells but also from lung fibroblasts. Furthermore, we studied whether the differences were due to cell lineage (epithelial versus fibroblast) or to different proliferative characteristics of cells, and where in the cell compartment these differences might reside. To answer these questions we studied cell cytoplasm, cell nucleus and isolated whole cell nuclei. Our data suggests that Raman spectroscopy can differentiate between lung cancer, lung epithelial cells and lung fibroblasts. More important, it can also differentiate between 2 cells from the same lineage (fibroblast) but with one of them rendered immortal and with an increased proliferative activity. Finally, it seems that the main spectral differences reside in the cell nucleus and that the study of isolated nuclei strengthens the differences between cells.

  18. Identification Of Cells With A Compact Microscope Imaging System With Intelligent Controls

    NASA Technical Reports Server (NTRS)

    McDowell, Mark (Inventor)

    2006-01-01

    A Microscope Imaging System (CMIS) with intelligent controls is disclosed that provides techniques for scanning, identifying, detecting and tracking mic?oscopic changes in selected characteristics or features of various surfaces including, but not limited to, cells, spheres, and manufactured products subject to difficult-to-see imperfections. The practice of the present invention provides applications that include colloidal hard spheres experiments, biological cell detection for patch clamping, cell movement and tracking, as well as defect identification in products, such as semiconductor devices, where surface damage can be significant, but difficult to detect. The CMIS system is a machine vision system, which combines intelligent image processing with remote control capabilities and provides the ability to autofocus on a microscope sample, automatically scan an image, and perform machine vision analysis on multiple samples simultaneously.

  19. Identification and Characterization of CD133(pos) Subpopulation Cells From a Human Laryngeal Cancer Cell Line.

    PubMed

    Qiu, Hai-ou; Wang, Huifang; Che, Na; Li, Dong; Mao, Yong; Zeng, Qiao; Ge, Rongming

    2016-04-06

    Recent research indicates that CD133 are expressed in several kinds of stem cells, among which, its high expression in laryngeal carcinoma has caused wide concern. To further explore efficaciously targeting drugs to laryngeal carcinoma stem cells (CSCs), we transplanted a solid tumor from CSCs into abdominal subcutaneous tissue of nude mice, and then compared the biological characteristics of laryngeal solid tumors with or without cisplatin intervention. In this study, the expression of CD133 was detected in the Hep-2 cell line by flow cytometry. By applying magnetic cell sorting (MACS) technology, we reported the results of purifying CD133-positive cells from a Hep-2 cell line. Cell proliferation, colony formation, and tumor-forming ability were examined in vitro and in vivo to identify the marker of CSCs in Hep-2 cell line. Upon flow cytometry analysis, CD133 was expressed constantly on 40.12±1.32% in Hep-2 cell line. Cell proliferation and colony formation ability were higher in CD133-positive cells compared to CD133-negative cells, and the in vivo tumorigenesis experiment showed the same results as in vitro assay. The 2 subpopulations cells were both sensitive to DDP, among which, the effect of DPP on proliferation ability and tumor-forming ability of CD133-positive cells was obviously greater than that of CD133-negative cells. Above all, our study revealed that CD133-positive cells have properties of higher proliferation, colony formation, and tumorigenesis in Hep-2 cell line, indicating that CD133 could be a marker to characterize laryngeal cancer stem cells.

  20. Identification of specific relaxin-binding cells in the human female.

    PubMed

    Kohsaka, T; Min, G; Lukas, G; Trupin, S; Campbell, E T; Sherwood, O D

    1998-10-01

    Relaxin is secreted during pregnancy, but it has no verified effects in humans. The objective of the present study was to identify the cells containing specific relaxin-binding sites in the uterine cervix, vagina, uterus, mammary glands, mammary nipples, and term placenta in the human. The uterine cervix, vagina, and uterus were obtained from hysterectomy specimens. Mammary glands and nipples were obtained after modified radical mastectomy. Placenta was obtained after normal delivery. Tissue samples were cut into slices (0.5-3 cm3), frozen in liquid nitrogen, and cryosectioned (8 microm). Cells that bind relaxin were identified by sequential application of biotinylated porcine relaxin probe, antibiotin immunoglobulin G conjugated to 1 nm colloidal gold, and silver enhancement for signal amplification. Relaxin bound with specificity to epithelial cells, smooth muscle cells, and blood vessels in the cervix, vagina, uterus, and mammary nipples; to epithelial cells and blood vessels in the mammary glands; and to skin of the mammary nipples. In addition, relaxin bound to individual cell types within the term placenta (amnion epithelium, syncytiotrophoblasts, blood vessels), and to sebaceous glands within the nipples. We conclude that the specific relaxin-binding cells probably contain relaxin receptors. Identification of putative relaxin receptors may provide insight into physiological and/or therapeutic roles of relaxin in the human.

  1. Machine Learning Approach to Automated Quality Identification of Human Induced Pluripotent Stem Cell Colony Images

    PubMed Central

    Haponen, Markus; Rasku, Jyrki

    2016-01-01

    The focus of this research is on automated identification of the quality of human induced pluripotent stem cell (iPSC) colony images. iPS cell technology is a contemporary method by which the patient's cells are reprogrammed back to stem cells and are differentiated to any cell type wanted. iPS cell technology will be used in future to patient specific drug screening, disease modeling, and tissue repairing, for instance. However, there are technical challenges before iPS cell technology can be used in practice and one of them is quality control of growing iPSC colonies which is currently done manually but is unfeasible solution in large-scale cultures. The monitoring problem returns to image analysis and classification problem. In this paper, we tackle this problem using machine learning methods such as multiclass Support Vector Machines and several baseline methods together with Scaled Invariant Feature Transformation based features. We perform over 80 test arrangements and do a thorough parameter value search. The best accuracy (62.4%) for classification was obtained by using a k-NN classifier showing improved accuracy compared to earlier studies. PMID:27493680

  2. Cell-impedance-based label-free technology for the identification of new drugs.

    PubMed

    Lundstrom, Kenneth

    2017-04-01

    Drug discovery has progressed from relatively simple binding or activity screening assays to high-throughput screening of sophisticated compound libraries with emphasis on miniaturization and automation. The development of functional assays has enhanced the success rate in discovering novel drug molecules. Many technologies, originally based on radioactive labeling, have sequentially been replaced by methods based on fluorescence labeling. Recently, the focus has switched to label-free technologies in cell-based screening assays. Areas covered: Label-free, cell-impedance-based methods comprise of different technologies including surface plasmon resonance, mass spectrometry and biosensors applied for screening of anticancer drugs, G protein-coupled receptors, receptor tyrosine kinase and virus inhibitors, drug and nanoparticle cytotoxicity. Many of the developed methods have been used for high-throughput screening in cell lines. Cell viability and morphological damage prediction have been monitored in three-dimensional spheroid human HT-29 carcinoma cells and whole Schistosomula larvae. Expert opinion: Progress in label-free, cell-impedance-based technologies has facilitated drug screening and may enhance the discovery of potential novel drug molecules through, and improve target molecule identification in, alternative signal pathways. The variety of technologies to measure cellular responses through label-free cell-impedance based approaches all support future drug development and should provide excellent assets for finding better medicines.

  3. Identification and characterization of Arabidopsis thaliana genes involved in xylem secondary cell walls.

    PubMed

    Yokoyama, Ryusuke; Nishitani, Kazuhiko

    2006-05-01

    The xylem of higher plants offers support to aerial portions of the plant body and serves as conduit for the translocation of water and nutrients. Terminal differentiation of xylem cells typically involves deposition of thick secondary cell walls. This is a dynamic cellular process accompanied by enhanced rates of cellulose deposition and the induction of synthesis of specific secondary-wall matrix polysaccharides and lignin. The secondary cell wall is essential for the function of conductive and supportive xylem tissues. Recently, significant progress has been made in identifying the genes responsible for xylem secondary cell wall formation. However, our present knowledge is still insufficient to account for the molecular processes by which this complex system operates. To acquire further information about xylem secondary cell walls, we initially focused our research effort on a set of genes specifically implicated in secondary cell wall formation, as well as on loss-of-function mutants. Results from two microarray screens identified several key candidate genes responsible for secondary cell wall formation. Reverse genetic analyses led to the identification of a glycine-rich protein involved in maintaining the stable structure of protoxylem, which is essential for the transport of water and nutrients. A combination of expression analyses and reverse genetics allows us to systematically identify new genes required for the development of physical properties of the xylem secondary wall.

  4. Identification of Distinct Tumor Subpopulations in Lung Adenocarcinoma via Single-Cell RNA-seq.

    PubMed

    Min, Jae-Woong; Kim, Woo Jin; Han, Jeong A; Jung, Yu-Jin; Kim, Kyu-Tae; Park, Woong-Yang; Lee, Hae-Ock; Choi, Sun Shim

    2015-01-01

    Single-cell sequencing, which is used to detect clinically important tumor subpopulations, is necessary for understanding tumor heterogeneity. Here, we analyzed transcriptomic data obtained from 34 single cells from human lung adenocarcinoma (LADC) patient-derived xenografts (PDXs). To focus on the intrinsic transcriptomic signatures of these tumors, we filtered out genes that displayed extensive expression changes following xenografting and cell culture. Then, we performed clustering analysis using co-regulated gene modules rather than individual genes to minimize read drop-out errors associated with single-cell sequencing. This combined approach revealed two distinct intra-tumoral subgroups that were primarily distinguished by the gene module G64. The G64 module was predominantly composed of cell-cycle genes. E2F1 was found to be the transcription factor that most likely mediates the expression of the G64 module in single LADC cells. Interestingly, the G64 module also indicated inter-tumoral heterogeneity based on its association with patient survival and other clinical variables such as smoking status and tumor stage. Taken together, these results demonstrate the feasibility of single-cell RNA sequencing and the strength of our analytical pipeline for the identification of tumor subpopulations.

  5. Identification of internalizing human single-chain antibodies targeting brain tumor sphere cells.

    PubMed

    Zhu, Xiaodong; Bidlingmaier, Scott; Hashizume, Rintaro; James, C David; Berger, Mitchel S; Liu, Bin

    2010-07-01

    Glioblastoma multiforme (GBM) is the most common and aggressive form of primary brain tumor for which there is no curative treatment to date. Resistance to conventional therapies and tumor recurrence pose major challenges to treatment and management of this disease, and therefore new therapeutic strategies need to be developed. Previous studies by other investigators have shown that a subpopulation of GBM cells can grow as neurosphere-like cells when cultured in restrictive medium and exhibits enhanced tumor-initiating ability and resistance to therapy. We report here the identification of internalizing human single-chain antibodies (scFv) targeting GBM tumor sphere cells. We selected a large naive phage antibody display library on the glycosylation-dependent CD133 epitope-positive subpopulation of GBM cells grown as tumor spheres and identified internalizing scFvs that target tumor sphere cells broadly, as well as scFvs that target the CD133-positive subpopulation. These scFvs were found to be efficiently internalized by GBM tumor sphere cells. One scFv GC4 inhibited self-renewal of GBM tumor sphere cells in vitro. We have further developed a full-length human IgG1 based on this scFv, and found that it potently inhibits proliferation of GBM tumor sphere cells and GBM cells grown in regular nonselective medium. Taken together, these results show that internalizing human scFvs targeting brain tumor sphere cells can be readily identified from a phage antibody display library, which could be useful for further development of novel therapies that target subpopulations of GBM cells to combat recurrence and resistance to treatment. (c)2010 AACR.

  6. Identification of genes associated with tumor development in CaSki cells in the cosmic space.

    PubMed

    Guo, Fengjie; Li, Yalin; Liu, Yan; Huang, Jian; Zhang, Zhijie; Wang, Jiajia; Li, Yuehui; Hu, Jinyue; Li, Guancheng

    2012-06-01

    It is important to understand the mechanisms of tumor development for curing cervical cancer. However, the molecular basis determining the different characteristics of tumor remains unclear. Space environment as a special study model can expand the study field of tumor development. To approach this, after human cervical carcinoma CaSki cells were flown on “Shen Zhou IV” space shuttle mission, the cell morphology and proliferation was investigated after flying to ground. We found that the growth of 48A9 CaSki cell (flight group) became slow compared with ground groups. Observation of cells by light microscopy revealed differences in cell morphology between ground controls and flight groups, and the flight group exhibited morphologic differences, characterized by rounder, smoother, decreased, smaller and low-adhension cells. Transmission electron microscope images showed the structure of the ultrastructural characteristics of 48A9 CaSki cells were clearly distinct from those of the ground CaSki cells in aspects of mitochondrion, cytoplasm, nucleus and ribosomes. MTT and soft agar assay showed that 48A9 CaSki cells grew slowly compared to ground control. Furthermore, suppression subtractive hybridization combining with reverse Northern blot was used to identify differently expression genes between flight and ground groups. These differentially expressed genes included cytoskeleton, cell differentiation, cell apoptosis, signal transduction, DNA repair, protein synthesis, substance metabolism, and antigen presentation. The identification of differently expressed genes which is likely to increase our understanding of the molecular processes underlying tumor development will provide new insight into tumor development mechanisms, and may facilitate the development of new anticancer strategies.

  7. Identification of internalizing human single chain antibodies targeting brain tumor sphere cells

    PubMed Central

    Zhu, Xiaodong; Bidlingmaier, Scott; Hashizume, Rintaro; James, C. David; Berger, Mitchel S.; Liu, Bin

    2010-01-01

    Glioblastoma multiforme (GBM) is the most common and aggressive form of primary brain tumor and there is no curative treatment to date. Resistance to conventional therapies and tumor recurrence pose major challenges to treatment and management of this disease, and therefore new therapeutic strategies need to be developed. Previous studies by other investigators have shown that a subpopulation of GBM cells can grow as neurosphere-like cells when cultured in restrictive media, and exhibit enhanced tumor initiating ability and resistance to therapy. We report here the identification of internalizing human single chain antibodies (scFvs) targeting GBM tumor sphere cells. We selected a large naive phage antibody display library on the glycosylation-dependent CD133 epitope-positive subpopulation of GBM cells grown as tumor spheres and identified internalizing scFvs that target tumor sphere cells broadly, as well as scFvs that target the CD133 positive subpopulation. These scFvs were found to be efficiently internalized by GBM tumor sphere cells. One scFv GC4 inhibited self-renewal of GBM tumor sphere cells in vitro. We have further developed a full-length human IgG1 based on this scFv and found that it potently inhibits proliferation of GBM tumor sphere cells and GBM cells grown in regular non-selective media. Taken together, these results show that internalizing human scFvs targeting brain tumor sphere cells can be readily identified from a phage antibody display library, which could be useful for further development of novel therapies that target subpopulations of GBM cells to combat recurrence and resistance to treatment. PMID:20587664

  8. Identification of cell-type-specific mutations in nodal T-cell lymphomas

    PubMed Central

    Nguyen, T B; Sakata-Yanagimoto, M; Asabe, Y; Matsubara, D; Kano, J; Yoshida, K; Shiraishi, Y; Chiba, K; Tanaka, H; Miyano, S; Izutsu, K; Nakamura, N; Takeuchi, K; Miyoshi, H; Ohshima, K; Minowa, T; Ogawa, S; Noguchi, M; Chiba, S

    2017-01-01

    Recent genetic analysis has identified frequent mutations in ten-eleven translocation 2 (TET2), DNA methyltransferase 3A (DNMT3A), isocitrate dehydrogenase 2 (IDH2) and ras homolog family member A (RHOA) in nodal T-cell lymphomas, including angioimmunoblastic T-cell lymphoma and peripheral T-cell lymphoma, not otherwise specified. We examined the distribution of mutations in these subtypes of mature T-/natural killer cell neoplasms to determine their clonal architecture. Targeted sequencing was performed for 71 genes in tumor-derived DNA of 87 cases. The mutations were then analyzed in a programmed death-1 (PD1)-positive population enriched with tumor cells and CD20-positive B cells purified by laser microdissection from 19 cases. TET2 and DNMT3A mutations were identified in both the PD1+ cells and the CD20+ cells in 15/16 and 4/7 cases, respectively. All the RHOA and IDH2 mutations were confined to the PD1+ cells, indicating that some, including RHOA and IDH2 mutations, being specific events in tumor cells. Notably, we found that all NOTCH1 mutations were detected only in the CD20+ cells. In conclusion, we identified both B- as well as T-cell-specific mutations, and mutations common to both T and B cells. These findings indicate the expansion of a clone after multistep and multilineal acquisition of gene mutations. PMID:28157189

  9. Bioinformatics-Based Identification of Candidate Genes from QTLs Associated with Cell Wall Traits in Populus

    SciTech Connect

    Ranjan, Priya; Yin, Tongming; Zhang, Xinye; Kalluri, Udaya C; Yang, Xiaohan; Jawdy, Sara; Tuskan, Gerald A

    2009-11-01

    Quantitative trait locus (QTL) studies are an integral part of plant research and are used to characterize the genetic basis of phenotypic variation observed in structured populations and inform marker-assisted breeding efforts. These QTL intervals can span large physical regions on a chromosome comprising hundreds of genes, thereby hampering candidate gene identification. Genome history, evolution, and expression evidence can be used to narrow the genes in the interval to a smaller list that is manageable for detailed downstream functional genomics characterization. Our primary motivation for the present study was to address the need for a research methodology that identifies candidate genes within a broad QTL interval. Here we present a bioinformatics-based approach for subdividing candidate genes within QTL intervals into alternate groups of high probability candidates. Application of this approach in the context of studying cell wall traits, specifically lignin content and S/G ratios of stem and root in Populus plants, resulted in manageable sets of genes of both known and putative cell wall biosynthetic function. These results provide a roadmap for future experimental work leading to identification of new genes controlling cell wall recalcitrance and, ultimately, in the utility of plant biomass as an energy feedstock.

  10. Integrative framework for identification of key cell identity genes uncovers determinants of ES cell identity and homeostasis.

    PubMed

    Cinghu, Senthilkumar; Yellaboina, Sailu; Freudenberg, Johannes M; Ghosh, Swati; Zheng, Xiaofeng; Oldfield, Andrew J; Lackford, Brad L; Zaykin, Dmitri V; Hu, Guang; Jothi, Raja

    2014-04-22

    Identification of genes associated with specific biological phenotypes is a fundamental step toward understanding the molecular basis underlying development and pathogenesis. Although RNAi-based high-throughput screens are routinely used for this task, false discovery and sensitivity remain a challenge. Here we describe a computational framework for systematic integration of published gene expression data to identify genes defining a phenotype of interest. We applied our approach to rank-order all genes based on their likelihood of determining ES cell (ESC) identity. RNAi-mediated loss-of-function experiments on top-ranked genes unearthed many novel determinants of ESC identity, thus validating the derived gene ranks to serve as a rich and valuable resource for those working to uncover novel ESC regulators. Underscoring the value of our gene ranks, functional studies of our top-hit Nucleolin (Ncl), abundant in stem and cancer cells, revealed Ncl's essential role in the maintenance of ESC homeostasis by shielding against differentiation-inducing redox imbalance-induced oxidative stress. Notably, we report a conceptually novel mechanism involving a Nucleolin-dependent Nanog-p53 bistable switch regulating the homeostatic balance between self-renewal and differentiation in ESCs. Our findings connect the dots on a previously unknown regulatory circuitry involving genes associated with traits in both ESCs and cancer and might have profound implications for understanding cell fate decisions in cancer stem cells. The proposed computational framework, by helping to prioritize and preselect candidate genes for tests using complex and expensive genetic screens, provides a powerful yet inexpensive means for identification of key cell identity genes.

  11. Identification of ostruthin from Peucedanum ostruthium rhizomes as an inhibitor of vascular smooth muscle cell proliferation.

    PubMed

    Joa, Helge; Vogl, Sylvia; Atanasov, Atanas G; Zehl, Martin; Nakel, Thomas; Fakhrudin, Nanang; Heiss, Elke H; Picker, Paolo; Urban, Ernst; Wawrosch, Christoph; Saukel, Johannes; Reznicek, Gottfried; Kopp, Brigitte; Dirsch, Verena M

    2011-06-24

    Inhibition of vascular smooth muscle cell (VSMC) proliferation is of substantial interest in combating cardiovascular disease. A dichloromethane extract from the rhizomes of Peucedanum ostruthium, a traditionally used Austrian medicinal plant with anti-inflammatory properties, was examined for a putative antiproliferative activity in rat aortic VSMC. This extract inhibited serum (10%)-induced VSMC proliferation concentration dependently. Further identification and biological testing of its major constituents revealed that the coumarin ostruthin (7) is the major antiproliferative substance. In summary, a new bioactivity of P. ostruthium rhizomes is described, and 7 has been identified as the responsible compound.

  12. Identification of a bacteriolysis-associated virulence factor against lung epithelial cells in Pseudomonas aeruginosa PAO-1 cell lysate.

    PubMed

    Shinagawa, Masaaki; Kobayashi, Daisuke; Goto, Maki; Tanaka, Maki; Kuribayashi, Kageaki; Yanagihara, Nozomi; Watanabe, Naoki

    2014-10-01

    The precise identities of the virulence factors of Pseudomonas aeruginosa after bacteriolysis are still unknown. In the present study, we identified PA0423 protein, which was isolated from the Pseudomonas PAO-1 strain, as the factor responsible for cytotoxicity in lung epithelial cells. Whole bacterial cell lysate of P. aeruginosa PAO-1 caused cytotoxicity in A549 lung epithelial cells. This cytotoxic factor could be partially purified via gel-filtration and anion-exchange column chromatography, and its activity was attenuated by proteinase K treatment. The cytotoxic fraction increased caspase-3/7 activity in A549 cells, suggesting the induction of apoptosis. This fraction was then subjected to amino-acid sequence analysis by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, resulting in the identification of 7 matches, 4 of which were with known proteins (PA0122, PA2687, PA3406, and PA0423). Deletion mutant analysis of these 7 candidates revealed that only the PA0423 mutation led to reduced cytotoxicity, indicating that this protein is the virulence factor. Furthermore, PA0423 recombinant protein was constructed, purified, and refolded. Transduction of recombinant PA0423, but not PA0122, into A549 cells engendered a dose-dependent cytotoxic effect. These results show the first evidence that specific bacteriolysis-induced virulence factor PA0423 from Pseudomonas is toxic to lung epithelial cells. Copyright © 2014 Elsevier Ltd. All rights reserved.

  13. Effects of green tea extract on lung cancer A549 cells: proteomic identification of proteins associated with cell migration.

    PubMed

    Lu, Qing-Yi; Yang, Yanan; Jin, Yu Sheng; Zhang, Zuo-Feng; Heber, David; Li, Frederick P; Dubinett, Steven M; Sondej, Melissa A; Loo, Joseph A; Rao, Jian Yu

    2009-02-01

    Green tea polyphenols exhibit multiple antitumor activities, and the mechanisms of action are not completely understood. Previously, we reported that green tea extract (GTE)-induced actin remolding is associated with increased cell adhesion and decreased motility in A549 lung cancer cells. To identify the cellular targets responsible for green tea-induced actin remodeling, we performed 2-DE LC-MS/MS of A549 cells before and after GTE exposure. We have identified 14 protein spots that changed in expression (> or =2-fold) after GTE treatment. These proteins are involved in calcium-binding, cytoskeleton and motility, metabolism, detoxification, or gene regulation. In particular we found upregulation of several genes that modulate actin remodeling and cell migration, including lamin A/C. Our data indicated that GTE-induced lamin A/C upregulation appears to be at the transcriptional level and the increased expression results in the decrease in cell motility, as confirmed by siRNA. The result of the study demonstrates that GTE alters the levels of many proteins involved in growth, motility and apoptosis of A549 cells and their identification may explain the multiple antitumor activities of GTE.

  14. Accounting for cell lineage and sex effects in the identification of cell-specific DNA methylation using a Bayesian model selection algorithm.

    PubMed

    White, Nicole; Benton, Miles; Kennedy, Daniel; Fox, Andrew; Griffiths, Lyn; Lea, Rodney; Mengersen, Kerrie

    2017-01-01

    Cell- and sex-specific differences in DNA methylation are major sources of epigenetic variation in whole blood. Heterogeneity attributable to cell type has motivated the identification of cell-specific methylation at the CpG level, however statistical methods for this purpose have been limited to pairwise comparisons between cell types or between the cell type of interest and whole blood. We developed a Bayesian model selection algorithm for the identification of cell-specific methylation profiles that incorporates knowledge of shared cell lineage and allows for the identification of differential methylation profiles in one or more cell types simultaneously. Under the proposed methodology, sex-specific differences in methylation by cell type are also assessed. Using publicly available, cell-sorted methylation data, we show that 51.3% of female CpG markers and 61.4% of male CpG markers identified were associated with differential methylation in more than one cell type. The impact of cell lineage on differential methylation was also highlighted. An evaluation of sex-specific differences revealed differences in CD56+NK methylation, within both single and multi- cell dependent methylation patterns. Our findings demonstrate the need to account for cell lineage in studies of differential methylation and associated sex effects.

  15. [Identification of cell division of CD8+ NKT cells in vitro].

    PubMed

    Guo, Ye-Lei; Chen, Yu; Zhong, Jiang; Zhang, Shi-Lun

    2011-02-01

    To measure the in vitro cell division ability of CD8(+);NKT cells by CFSE staining and flow cytometry(FCM). Fresh spleen lymphocytes of C57BL/J mice were separated and stained with CFSE, and then stimulated by ConA and LPS for 3 d, and by SEB for 5 d and 10 d respectively. The stimulated cells were harvested and analyzed for CD69 expression on the cell surface and the ability of cell division using FCM. The SEB-activated effector cells for 10 d further stimulated with IL-2 for the consecutive 10 days, and were analyzed for their cell division ability, CD69 expression and NKT cell subsets by FCM. ConA, LPS and SEB stimulated the proliferation of spleen cells. ConA and LPS made the cells divide 3 times within 3 d, and increased CD69 expression up to 74.19% and 41.56% respectively. SEB made the cells divide 5 times within 5 d and 7 times within 10 d respectively, with increased CD69 expression of 32.09% and 48.66% respectively. Ten-day IL-2 stimulation of SEB-activated cells caused population expansion for 7 times with the CD8(+);NKT cell subsets significantly increased from 0.36% to 38.58% and CD69 expression significantly increased from 0.11% to 83.74%. The SEB-activated CD8(+);NKT cells proliferated in vitro and their cell division capability could be determined by CFSE staining and FCM.

  16. Detection and identification of putative bacterial endosymbionts and endogenous viruses in tick cell lines.

    PubMed

    Alberdi, M Pilar; Dalby, Matthew J; Rodriguez-Andres, Julio; Fazakerley, John K; Kohl, Alain; Bell-Sakyi, Lesley

    2012-06-01

    As well as being vectors of many viral, bacterial, and protozoan pathogens of medical and veterinary importance, ticks harbour a variety of microorganisms which are not known to be pathogenic for vertebrate hosts. Continuous cell lines established from ixodid and argasid ticks could be infected with such endosymbiotic bacteria and endogenous viruses, but to date very few cell lines have been examined for their presence. DNA and RNA extracted from over 50 tick cell lines deposited in the Roslin Wellcome Trust Tick Cell Biobank (http://tickcells.roslin.ac.uk) were screened for presence of bacteria and RNA viruses, respectively. Sequencing of PCR products amplified using pan-16S rRNA primers revealed the presence of DNA sequences from bacterial endosymbionts in several cell lines derived from Amblyomma and Dermacentor spp. ticks. Identification to species level was attempted using Rickettsia- and Francisella-specific primers. Pan-Nairovirus primers amplified PCR products of uncertain specificity in cell lines derived from Rhipicephalus, Hyalomma, Ixodes, Carios, and Ornithodoros spp. ticks. Further characterisation attempted with primers specific for Crimean-Congo haemorrhagic fever virus segments confirmed the absence of this arbovirus in the cells. A set of pan-Flavivirus primers did not detect endogenous viruses in any of the cell lines. Transmission electron microscopy revealed the presence of endogenous reovirus-like viruses in many of the cell lines; only 4 of these lines gave positive results with primers specific for the tick Orbivirus St Croix River virus, indicating that there may be additional, as yet undescribed 'tick-only' viruses inhabiting tick cell lines.

  17. Identification and genetic analysis of cancer cells with PCR-activated cell sorting

    PubMed Central

    Eastburn, Dennis J.; Sciambi, Adam; Abate, Adam R.

    2014-01-01

    Cell sorting is a central tool in life science research for analyzing cellular heterogeneity or enriching rare cells out of large populations. Although methods like FACS and FISH-FC can characterize and isolate cells from heterogeneous populations, they are limited by their reliance on antibodies, or the requirement to chemically fix cells. We introduce a new cell sorting technology that robustly sorts based on sequence-specific analysis of cellular nucleic acids. Our approach, PCR-activated cell sorting (PACS), uses TaqMan PCR to detect nucleic acids within single cells and trigger their sorting. With this method, we identified and sorted prostate cancer cells from a heterogeneous population by performing >132 000 simultaneous single-cell TaqMan RT-PCR reactions targeting vimentin mRNA. Following vimentin-positive droplet sorting and downstream analysis of recovered nucleic acids, we found that cancer-specific genomes and transcripts were significantly enriched. Additionally, we demonstrate that PACS can be used to sort and enrich cells via TaqMan PCR reactions targeting single-copy genomic DNA. PACS provides a general new technical capability that expands the application space of cell sorting by enabling sorting based on cellular information not amenable to existing approaches. PMID:25030902

  18. Identification and Targeting of Candidate Pre-Existing Lurker Cells that Give Rise to Castration-Resistant Prostate Cancer

    DTIC Science & Technology

    2014-10-01

    propagation. Lgr5+ intestinal stem cells can initiate and maintain murine intestinal adenomas (6, 7). In mouse models of skin cancer, hair follicle bulge...AWARD NUMBER: W81XWH-13-1-0470 TITLE: Identification and Targeting of Candidate Pre... Targeting of Candidate Pre-Existing Lurker Cells that Give Rise to 5a. CONTRACT NUMBER Castration-Resistant Prostate Cancer 5b

  19. Efficient identification of mutated cancer antigens recognized by T cells associated with durable tumor regressions.

    PubMed

    Lu, Yong-Chen; Yao, Xin; Crystal, Jessica S; Li, Yong F; El-Gamil, Mona; Gross, Colin; Davis, Lindy; Dudley, Mark E; Yang, James C; Samuels, Yardena; Rosenberg, Steven A; Robbins, Paul F

    2014-07-01

    Cancer immunotherapy with adoptive transfer of tumor-infiltrating lymphocytes (TIL) represents an effective treatment for patients with metastatic melanoma, with the objective regressions in up to 72% of patients in three clinical trials. However, the antigen targets recognized by these effective TILs remain largely unclear. Melanoma patients 2359 and 2591 both experienced durable complete regressions of metastases ongoing beyond five years following adoptive TIL transfer. Two conventional screening approaches were carried out to identify the antigens recognized by these clinically effective TILs. In addition, a novel approach was developed in this study to identify mutated T-cell antigens by screening a tandem minigene library, which comprised nonsynonymous mutation sequences identified by whole-exome sequencing of autologous tumors. Screening of an autologous melanoma cDNA library using a conventional approach led to the identification of previously undescribed nonmutated targets recognized by TIL 2359 or TIL 2591. In contrast, screening of tandem minigene libraries encoding tumor-specific mutations resulted in the identification of mutated kinesin family member 2C (KIF2C) antigen as a target of TIL 2359, and mutated DNA polymerase alpha subunit B (POLA2) antigen as a target of TIL 2591. Both KIF2C and POLA2 have been found to play important roles in cell proliferation. These findings suggest that the minigene screening approach can facilitate the antigen repertoire analysis of tumor reactive T cells, and lead to the development of new adoptive cell therapies with purified T cells that recognize candidate-mutated antigens derived from genes essential for the carcinogenesis. ©2014 American Association for Cancer Research.

  20. Efficient identification of mutated cancer antigens recognized by T cells associated with durable tumor regressions

    PubMed Central

    Lu, Yong-Chen; Yao, Xin; Crystal, Jessica S.; Li, Yong F.; El-Gamil, Mona; Gross, Colin; Davis, Lindy; Dudley, Mark E.; Yang, James C.; Samuels, Yardena; Rosenberg, Steven A.; Robbins, Paul F.

    2014-01-01

    Purpose Cancer immunotherapy with adoptive transfer of tumor infiltrating lymphocytes (TILs) represents an effective treatment for patients with metastatic melanoma, with the objective regressions in up to 72% of patients in three clinical trials. However, the antigen targets recognized by these effective TILs remain largely unclear. Experimental Design Melanoma patients 2359 and 2591 both experienced durable complete regressions of metastases ongoing beyond five years following adoptive TIL transfer. Two conventional screening approaches were carried out to identify the antigens recognized by these clinically effective TILs. In addition, a novel approach was developed in this study to identify mutated T-cell antigens by screening a tandem minigene library, which comprised non-synonymous mutation sequences identified by whole-exome sequencing of autologous tumors. Results The autologous melanoma cDNA library screening led to the identification of previously undescribed non-mutated targets recognized by TIL 2359 or TIL 2591. On the other hand, the screening of tandem minigene libraries resulted in the identification of mutated kinesin family member 2C (KIF2C) antigen as a target of TIL 2359, and mutated DNA polymerase alpha subunit B (POLA2) antigen as a target of TIL 2591. Both KIF2C and POLA2 have been found to play important roles in cell proliferation. Conclusions These findings suggest that the minigene screening approach can facilitate the antigen repertoire analysis of tumor reactive T cells, and lead to the development of new adoptive cell therapies with purified T cells that recognize candidate mutated antigens derived from genes essential for the carcinogenesis. PMID:24987109

  1. Identification of tumor cells infiltrating into connective tissue in esophageal cancer by multiphoton microscopy

    NASA Astrophysics Data System (ADS)

    Xu, Jian; Jiang, Liwei; Kang, Deyong; Wu, Xuejing; Xu, Meifang; Zhuo, Shuangmu; Zhu, Xiaoqin; Lin, Jiangbo; Chen, Jianxin

    2016-10-01

    Esophageal cancer is one of the most common malignancies of the gastrointestinal cancers and carries poorer prognosis than other gastrointestinal cancers. In general practice, the depth of tumor infiltration in esophageal wall is crucial to establishing appropriate treatment plan which is established by detecting the tumor infiltration depth. Connective tissue is one of the main structures that form the esophageal wall. So, identification of tumor cells infiltrating into connective tissue is helping for detecting the tumor infiltration depth. Our aim is to evaluate whether multiphoton microscopy (MPM) can be used to detect tumor cells infiltrating into connective tissue in the esophageal cancer. MPM is well-suited for real-time detecting morphologic and cellular changes in fresh tissues since many endogenous fluorophores of fresh tissues are excited through two-photon excited fluorescence (TPEF) and second harmonic generation (SHG). In this work, microstructure of tumor cells and connective tissue are first studied. Then, morphological changes of collagen fibers after the infiltration of tumor cells are shown. These results show that MPM has the ability to detect tumor cells infiltrating into connective tissue in the esophageal cancer. In the future, MPM may be a promising imaging technique for detecting tumor cells in esophageal cancer.

  2. Identification of Cross-Reactive Norovirus CD4+ T Cell Epitopes ▿

    PubMed Central

    LoBue, Anna D.; Lindesmith, Lisa C.; Baric, Ralph S.

    2010-01-01

    Immune responses and the components of protective immunity following norovirus infection in humans are poorly understood. Although antibody responses following norovirus infection have been partially characterized, T cell responses in humans remain largely undefined. In contrast, T cells have been shown to be essential for viral clearance of mouse norovirus (MNV) infection. In this paper, we demonstrate that CD4+ T cells secrete gamma interferon (IFN-γ) in response to stimulation with MNV virus-like particles (VLPs) after MNV infection, supporting earlier reports for norovirus-infected mice and humans. Utilizing this model, we immunized mice with alphavirus vectors (Venezuelan equine encephalitis [VEE] virus replicon particles [VRPs]) expressing Norwalk virus (NV) or Farmington Hills virus (FH) virus-like particles to evaluate T cell epitopes shared between human norovirus strains. Stimulation of splenocytes from norovirus VRP-immunized mice with overlapping peptides from complete libraries of the NV or FH capsid proteins revealed specific amino acid sequences containing T cell epitopes that were conserved within genoclusters and genogroups. Immunization with heterologous norovirus VRPs resulted in specific cross-reactive IFN-γ secretion profiles following stimulation with NV and FH peptides in the mouse. Identification of unique strain-specific and cross-reactive epitopes may provide insight into homologous and heterologous T cell-mediated norovirus immunity and provide a platform for the study of norovirus-induced cellular immunity in humans. PMID:20573810

  3. Identification of a nucleoside analog active against adenosine kinase–expressing plasma cell malignancies

    PubMed Central

    Sadek, Jouliana; Hernandez-Hopkins, Denise; Akar, Gunkut; Barelli, Peter J.; Sahai, Michelle A.; Zhou, Hufeng; Totonchy, Jennifer; Jayabalan, David; Niesvizky, Ruben; Guasparri, Ilaria; Liu, Yifang; Sei, Shizuko; Shoemaker, Robert H.; Elemento, Olivier; Kaye, Kenneth M.

    2017-01-01

    Primary effusion lymphoma (PEL) is a largely incurable malignancy of B cell origin with plasmacytic differentiation. Here, we report the identification of a highly effective inhibitor of PEL. This compound, 6-ethylthioinosine (6-ETI), is a nucleoside analog with toxicity to PEL in vitro and in vivo, but not to other lymphoma cell lines tested. We developed and performed resistome analysis, an unbiased approach based on RNA sequencing of resistant subclones, to discover the molecular mechanisms of sensitivity. We found different adenosine kinase–inactivating (ADK-inactivating) alterations in all resistant clones and determined that ADK is required to phosphorylate and activate 6-ETI. Further, we observed that 6-ETI induces ATP depletion and cell death accompanied by S phase arrest and DNA damage only in ADK-expressing cells. Immunohistochemistry for ADK served as a biomarker approach to identify 6-ETI–sensitive tumors, which we documented for other lymphoid malignancies with plasmacytic features. Notably, multiple myeloma (MM) expresses high levels of ADK, and 6-ETI was toxic to MM cell lines and primary specimens and had a robust antitumor effect in a disseminated MM mouse model. Several nucleoside analogs are effective in treating leukemias and T cell lymphomas, and 6-ETI may fill this niche for the treatment of PEL, plasmablastic lymphoma, MM, and other ADK-expressing cancers. PMID:28504647

  4. Identification of embryonic stem cell-derived midbrain dopaminergic neurons for engraftment.

    PubMed

    Ganat, Yosif M; Calder, Elizabeth L; Kriks, Sonja; Nelander, Jenny; Tu, Edmund Y; Jia, Fan; Battista, Daniela; Harrison, Neil; Parmar, Malin; Tomishima, Mark J; Rutishauser, Urs; Studer, Lorenz

    2012-08-01

    Embryonic stem cells (ESCs) represent a promising source of midbrain dopaminergic (DA) neurons for applications in Parkinson disease. However, ESC-based transplantation paradigms carry a risk of introducing inappropriate or tumorigenic cells. Cell purification before transplantation may alleviate these concerns and enable identification of the specific DA neuron stage most suitable for cell therapy. Here, we used 3 transgenic mouse ESC reporter lines to mark DA neurons at 3 stages of differentiation (early, middle, and late) following induction of differentiation using Hes5::GFP, Nurr1::GFP, and Pitx3::YFP transgenes, respectively. Transplantation of FACS-purified cells from each line resulted in DA neuron engraftment, with the mid-stage and late-stage neuron grafts being composed almost exclusively of midbrain DA neurons. Mid-stage neuron cell grafts had the greatest amount of DA neuron survival and robustly induced recovery of motor deficits in hemiparkinsonian mice. Our data suggest that the Nurr1+ stage (middle stage) of neuronal differentiation is particularly suitable for grafting ESC-derived DA neurons. Moreover, global transcriptome analysis of progeny from each of the ESC reporter lines revealed expression of known midbrain DA neuron genes and also uncovered previously uncharacterized midbrain genes. These data demonstrate remarkable fate specificity of ESC-derived DA neurons and outline a sequential stage-specific ESC reporter line paradigm for in vivo gene discovery.

  5. Lattice Symmetry and Identification-The Fundamental Role of Reduced Cells in Materials Characterization.

    PubMed

    Mighell, A D

    2001-01-01

    In theory, physical crystals can be represented by idealized mathematical lattices. Under appropriate conditions, these representations can be used for a variety of purposes such as identifying, classifying, and understanding the physical properties of materials. Critical to these applications is the ability to construct a unique representation of the lattice. The vital link that enabled this theory to be realized in practice was provided by the 1970 paper on the determination of reduced cells. This seminal paper led to a mathematical approach to lattice analysis initially based on systematic reduction procedures and the use of standard cells. Subsequently, the process evolved to a matrix approach based on group theory and linear algebra that offered a more abstract and powerful way to look at lattices and their properties. Application of the reduced cell to both database work and laboratory research at NIST was immediately successful. Currently, this cell and/or procedures based on reduction are widely and routinely used by the general scientific community: (i) for calculating standard cells for the reporting of crystalline materials, (ii) for classifying materials, (iii) in crystallographic database work (iv) in routine x-ray and neutron diffractometry, and (v) in general crystallographic research. Especially important is its use in symmetry determination and in identification. The focus herein is on the role of the reduced cell in lattice symmetry determination.

  6. Identification of innate lymphoid cells in single-cell RNA-Seq data.

    PubMed

    Suffiotti, Madeleine; Carmona, Santiago J; Jandus, Camilla; Gfeller, David

    2017-07-01

    Innate lymphoid cells (ILCs) consist of natural killer (NK) cells and non-cytotoxic ILCs that are broadly classified into ILC1, ILC2, and ILC3 subtypes. These cells recently emerged as important early effectors of innate immunity for their roles in tissue homeostasis and inflammation. Over the last few years, ILCs have been extensively studied in mouse and human at the functional and molecular level, including gene expression profiling. However, sorting ILCs with flow cytometry for gene expression analysis is a delicate and time-consuming process. Here we propose and validate a novel framework for studying ILCs at the transcriptomic level using single-cell RNA-Seq data. Our approach combines unsupervised clustering and a new cell type classifier trained on mouse ILC gene expression data. We show that this approach can accurately identify different ILCs, especially ILC2 cells, in human lymphocyte single-cell RNA-Seq data. Our new model relies only on genes conserved across vertebrates, thereby making it in principle applicable in any vertebrate species. Considering the rapid increase in throughput of single-cell RNA-Seq technology, our work provides a computational framework for studying ILC2 cells in single-cell transcriptomic data and may help exploring their conservation in distant vertebrate species.

  7. [BIOLOGICAL FEATURES AND IDENTIFICATION OF ENDOTHELIAL PROGENITOR CELLS FROM PERIPHERAL BLOOD].

    PubMed

    Qiao, Wei; Zhou, Min; Liu, Changjian; Qiao, Tong

    2015-07-01

    To compare the biological features of early and late endothelial progenitor cells (EPCs) by isolating and culturing early and late EPCs from the human peripheral blood so as to find some unique properties of EPCs and to propose a suitable strategy for EPCs identification. Mononuclear cells were isolated from the human peripheral blood using density gradient centrifugation. Then, the cells were inoculated in human fibronectin-coated culture flasks and cultured in endothelial cell basal medium 2. After 4-7 days and 2-3 weeks culture, early and late EPCs were obtained respectively. The morphology, proliferation potential, surface markers, cytokine secretion, angiogenic ability, and nitric oxide (NO) release were compared between 2 types of EPCs. Meanwhile, the human aortic endothelial cells (HAECs) were used as positive control. The morphology of early and late EPCs was different: early EPCs formed a cell cluster with a spindle shape after 4-7 days of culture, and late EPCs showed a cobblestone appearance. Late EPCs were characterized by high proliferation potential and were able to form capillary tubes on Matrigel, but early EPCs did not have this feature. Both types EPCs could ingest acetylated low density lipoprotein and combine with ulex europaeus I. Flow cytometry analysis showed that early EPCs did not express CD34 and CD133, but expressed the CD14 and CD45 of the hematopoietic stem cell markers; however, late EPCs expressed CD31 and CD34 of the endothelial cell markers, but did not express CD14, CD45, and CD133. By RT-PCR analysis, the expressions of vascular endothelial growth receptor 2 and vascular endothelial cadherin in early EPCs were significantly lower than those in the late EPCs and HAECs (P < 0.05), but no significant difference was found in the expression of von Willebrand factor and endothelial nitric oxide synthase (eNOS) between 2 type EPCs (P > 0.05). The concentrations of vascular endothelial growth factor, granulocyte colony-stimulating factor

  8. Prostate stem cell antigen: Identification of immunogenic peptides and assessment of reactive CD8+ T cells in prostate cancer patients.

    PubMed

    Kiessling, Andrea; Schmitz, Marc; Stevanovic, Stefan; Weigle, Bernd; Hölig, Kristina; Füssel, Monika; Füssel, Susanne; Meye, Axel; Wirth, Manfred P; Rieber, Ernst Peter

    2002-12-01

    Identification of TAAs recognized by CD8(+) CTLs paved the way for new concepts in cancer therapy. In view of the heterogeneity of tumors and their diverse escape mechanisms, CTL-based cancer therapy largely depends on an appropriate number of TAAs. In prostate cancer, the number of antigens defined as suitable targets of CTLs remains rather limited. PSCA is widely distributed in prostate cancer. In this report, we define immunogenic peptides of PSCA which are recognized by circulating CD8(+) T cells from prostate cancer patients and able to activate CTLs in vitro. Screening the amino acid sequence of PSCA for peptides containing a binding motif for HLA-A*0201 resulted in 8 candidate peptides. Specificity and affinity of peptide binding were verified in a competition assay. Frequencies of CD8(+) T lymphocytes reactive against selected epitopes were determined in the blood of prostate cancer patients using the ELISPOT assay. Increased frequencies were revealed for CD8(+) T cells recognizing the peptides ALQPGTALL and AILALLPAL. CTLs from prostate cancer patients were raised against these 2 peptides in vitro when presented by autologous DCs. They specifically recognized peptide-pulsed T2 target cells and prostate cancer cells that were HLA-A*0201- and PSCA-positive, indicating that these peptides were naturally generated by tumor cells. These data suggest that PSCA is a promising target for the immunotherapy of prostate cancer.

  9. Identification of Salmonella functions critical for bacterial cell division within eukaryotic cells.

    PubMed

    Henry, T; García-Del Portillo, F; Gorvel, J P

    2005-04-01

    Salmonella typhimurium multiplication inside eukaryotic host cells is critical for virulence. Salmonella typhimurium strain SL1344 appears as filaments upon growth in macrophages and MelJuSo cells, a human melanoma cell line, indicating a specific blockage in the bacterial cell division process. Several studies have investigated the host cell response impairing bacterial division. However, none looked at the bacterial factors involved in inhibition of Salmonella division inside eukaryotic cells. We show here that blockage in the bacterial division process is sulA-independent and takes place after FtsZ-ring assembly. Salmonella typhimurium genes in which mutations lead to filamentous growth within host cells were identified by a large scale mutagenesis approach on strain 12023, revealing bacterial functions crucial for cell division within eukaryotic cells. We finally demonstrate that SL1344 filamentation is a result of hisG mutation, requires the activity of an enzyme of the histidine biosynthetic pathway HisFH and is specific for the vacuolar environment.

  10. Prospective identification of functionally distinct stem cells and neurosphere-initiating cells in adult mouse forebrain

    PubMed Central

    Mich, John K; Signer, Robert AJ; Nakada, Daisuke; Pineda, André; Burgess, Rebecca J; Vue, Tou Yia; Johnson, Jane E; Morrison, Sean J

    2014-01-01

    Neurosphere formation is commonly used as a surrogate for neural stem cell (NSC) function but the relationship between neurosphere-initiating cells (NICs) and NSCs remains unclear. We prospectively identified, and isolated by flow cytometry, adult mouse lateral ventricle subventricular zone (SVZ) NICs as GlastmidEGFRhighPlexinB2highCD24−/lowO4/PSA-NCAM−/lowTer119/CD45− (GEPCOT) cells. They were highly mitotic and short-lived in vivo based on fate-mapping with Ascl1CreERT2 and Dlx1CreERT2. In contrast, pre-GEPCOT cells were quiescent, expressed higher Glast, and lower EGFR and PlexinB2. Pre-GEPCOT cells could not form neurospheres but expressed the stem cell markers Slc1a3-CreERT, GFAP-CreERT2, Sox2CreERT2, and Gli1CreERT2 and were long-lived in vivo. While GEPCOT NICs were ablated by temozolomide, pre-GEPCOT cells survived and repopulated the SVZ. Conditional deletion of the Bmi-1 polycomb protein depleted pre-GEPCOT and GEPCOT cells, though pre-GEPCOT cells were more dependent upon Bmi-1 for Cdkn2a (p16Ink4a) repression. Our data distinguish quiescent NSCs from NICs and make it possible to study their properties in vivo. DOI: http://dx.doi.org/10.7554/eLife.02669.001 PMID:24843006

  11. Identification of a cell-surface antigen selectively expressed on the natural killer cell

    PubMed Central

    1977-01-01

    We have studied the cell-surface phenotype of natural killer (NK) cells of NZB and B6 mice which react to an MuLV+ lymphoid tumor. (a) NK cells do not express Thy1, Ly2, or Ig surface markers. (b) NK cells express an antigen recognized by C3H anti-CE antiserum ('anti-Ly1.2 antiserum'). Inasmuch as NK activity of spleen cells from B6 and B6/Ly1.1 congenic strains were both equally sensitive to C3H anti-CE antiserum, the NK antigen is distinct from Ly1.2. This point was confirmed by the observation that alphaNK activity was removed by absorption of C3H anti-CE antiserum with spleen cells from either B6 or B6/Ly1.1 congenic strains. Absorption of C3H alphaCE serum with BALB/c thymocytes and spleen cells (which are Ly1.2+NK-) removed anti-Ly1.2 activity and left anti-NK activity intact. This absorption step could be circumvented by inserting the BALB/c genotype into the recipient immunized to CE cells (i.e., (C3H X BALB/c)F1 alphaCE spleen cells). This antiserum, provisionally termed 'anti-NK', defines a new subclass of lymphocytes which may play a central role in the immunosurveillance against tumors. PMID:187714

  12. Identification of a receptor necessary for Nogo-B stimulated chemotaxis and morphogenesis of endothelial cells

    PubMed Central

    Miao, Robert Qing; Gao, Yuan; Harrison, Kenneth D.; Prendergast, Jay; Acevedo, Lisette M.; Yu, Jun; Hu, Fenghua; Strittmatter, Stephen M.; Sessa, William C.

    2006-01-01

    Nogo isoforms (Nogo-A and -B) have been implicated in regulating neural and cardiovascular functions, such as cell spreading and chemotaxis. Unlike the loop domain (Nogo-66) found in all Nogo isoforms that can interact with a neural-specific Nogo-66 receptor, the receptor for the amino terminus of Nogo-B that mediates vascular function is unknown. Here, we identify a previously uncharacterized Nogo-B receptor specific for the amino terminus of Nogo-B and show that Nogo-B receptor localizes with the ligand Nogo-B during VEGF and wound healing angiogenesis in vivo, mediates chemotaxis in a heterologous expression system and chemotaxis, and 3D tube formation in native endothelial cells. Thus, identification of this receptor may lead to the discovery of agonists or antagonists of this pathway to regulate vascular remodeling and angiogenesis. PMID:16835300

  13. Identification of ADAM 31: a protein expressed in Leydig cells and specialized epithelia.

    PubMed

    Liu, L; Smith, J W

    2000-06-01

    A family of proteins containing a disintegrin and metalloproteinase domain (ADAMs) has been identified recently. Here, we report the identification of a novel member of the ADAM protein family from mouse. This protein is designated ADAM 31. The complementary DNA sequence of ADAM 31 predicts a transmembrane protein with metalloproteinase, disintegrin, cysteine-rich, and cytoplasmic domains. Messenger RNA encoding ADAM 31 was most abundant in testes, but was also detected in many other tissues. More significantly, the antibodies raised against ADAM 31 reveal that the protein has a unique and restricted expression pattern. ADAM 31 is expressed in Leydig cells of the testes, but unlike many other ADAMs, it is not found on developing sperm. Furthermore, ADAM 31 is highly expressed on four types of specialized epithelia: the cauda epididymidis, the vas deferens, the convoluted tubules of the kidney, and the parietal cells of the stomach.

  14. Identification of candidate microbial sequences from inflammatory lesion of giant cell arteritis.

    PubMed

    Gordon, Lynn K; Goldman, Melissa; Sandusky, Hallie; Ziv, Nurit; Hoffman, Gary S; Goodglick, Todd; Goodglick, Lee

    2004-06-01

    Giant cell arteritis (GCA) is a granulomatous inflammatory disease of medium and large arteries which is prevalent in the elderly population. The etiology of GCA is unknown, although the immunologic features suggest the possible presence of a microorganism. Our group has examined whether microbial DNA fragments were present at GCA lesions and whether such microbial fragments could be associated with disease pathogenesis. Initial identification of microbial sequences was performed using genomic representational difference analysis (RDA). Laser dissecting microscopy was used to isolate cells from GCA lesions and adjacent uninvolved temporal artery. Using genomic RDA, we isolated 10 gene fragments; three of these sequences had high homology with prokaryotic genes and were considered high-priority candidates for further study. An examination of serum from GCA(+) individuals (in contrast to healthy age-matched controls) showed the presence of IgG which recognized in vitro translated proteins from these clones.

  15. Detection and identification of Vibrio species using whole-cell protein pattern analysis.

    PubMed

    Lee, Chae-Yoon; Hong, Yeun; Ryu, Jio; Kim, Young-Rok; Oh, Sang-Suk; Lee, Soon-Ho; Hwang, In-Gyun; Kim, Hae-Yeong

    2012-08-01

    Outbreaks of foodborne diseases associated with Vibrio species such as V. parahaemolyticus, V. vulnificus, and V. cholerae frequently occur in countries having a dietary habit of raw seafood consumption. For rapid identification of different Vibrio species involved in foodborne diseases, whole-cell protein pattern analysis for 13 type strains of 12 Vibrio species was performed using SDS-PAGE analysis. Pathogenic Vibrio species such as V. parahaemolyticus, V. vulnificus, V. cholerae, V. alginolyticus, V. fluvialis, and V. mimicus were included in the 12 Vibrio species used in this study. Each of the 12 Vibrio species showed clearly specific band patterns of its own. Two different strains of V. parahaemolyticus showed two different SDS-PAGE wholecell protein patterns, giving the possibility of categorizing isolated strains in the same V. parahaemolyticus species into two subgroups. The 36 Vibrio isolates collected from sushi restaurants in Busan were all identified as V. parahaemolyticus by comparing their protein patterns with those of Vibrio type strains. The identified isolates were categorized into two different subgroups of V. parahaemolyticus. The whole-cell protein pattern analysis by SDS-PAGE can be used as a specific, rapid, and simple identification method for Vibrio spp. involved in foodborne diseases at the subspecies level.

  16. Comprehensive Identification of Meningococcal Genes and Small Noncoding RNAs Required for Host Cell Colonization

    PubMed Central

    Capel, Elena; Zomer, Aldert L.; Nussbaumer, Thomas; Bole, Christine; Izac, Brigitte; Frapy, Eric; Meyer, Julie; Bouzinba-Ségard, Haniaa; Bille, Emmanuelle; Jamet, Anne; Cavau, Anne; Letourneur, Franck; Bourdoulous, Sandrine; Rattei, Thomas; Coureuil, Mathieu

    2016-01-01

    ABSTRACT Neisseria meningitidis is a leading cause of bacterial meningitis and septicemia, affecting infants and adults worldwide. N. meningitidis is also a common inhabitant of the human nasopharynx and, as such, is highly adapted to its niche. During bacteremia, N. meningitidis gains access to the blood compartment, where it adheres to endothelial cells of blood vessels and causes dramatic vascular damage. Colonization of the nasopharyngeal niche and communication with the different human cell types is a major issue of the N. meningitidis life cycle that is poorly understood. Here, highly saturated random transposon insertion libraries of N. meningitidis were engineered, and the fitness of mutations during routine growth and that of colonization of endothelial and epithelial cells in a flow device were assessed in a transposon insertion site sequencing (Tn-seq) analysis. This allowed the identification of genes essential for bacterial growth and genes specifically required for host cell colonization. In addition, after having identified the small noncoding RNAs (sRNAs) located in intergenic regions, the phenotypes associated with mutations in those sRNAs were defined. A total of 383 genes and 8 intergenic regions containing sRNA candidates were identified to be essential for growth, while 288 genes and 33 intergenic regions containing sRNA candidates were found to be specifically required for host cell colonization. PMID:27486197

  17. Label-free identification of white blood cell using optical diffraction tomography (Conference Presentation)

    NASA Astrophysics Data System (ADS)

    Yoon, Jonghee; Kim, Kyoohyun; Kim, Min-hyeok; Kang, Suk-Jo; Park, YongKeun

    2016-03-01

    White blood cells (WBC) have crucial roles in immune systems which defend the host against from disease conditions and harmful invaders. Various WBC subsets have been characterized and reported to be involved in many pathophysiologic conditions. It is crucial to isolate a specific WBC subset to study its pathophysiological roles in diseases. Identification methods for a specific WBC population are rely on invasive approaches, including Wright-Gimesa staining for observing cellular morphologies and fluorescence staining for specific protein markers. While these methods enable precise classification of WBC populations, they could disturb cellular viability or functions. In order to classify WBC populations in a non-invasive manner, we exploited optical diffraction tomography (ODT). ODT is a three-dimensional (3-D) quantitative phase imaging technique that measures 3-D refractive index (RI) distributions of individual WBCs. To test feasibility of label-free classification of WBC populations using ODT, we measured four subtypes of WBCs, including B cell, CD4 T cell, CD8 T cell, and natural killer (NK) cell. From measured 3-D RI tomograms of WBCs, we obtain quantitative structural and biochemical information and classify each WBC population using a machine learning algorithm.

  18. Flow Cytometric Single-Cell Identification of Populations in Synthetic Bacterial Communities.

    PubMed

    Rubbens, Peter; Props, Ruben; Boon, Nico; Waegeman, Willem

    2017-01-01

    Bacterial cells can be characterized in terms of their cell properties using flow cytometry. Flow cytometry is able to deliver multiparametric measurements of up to 50,000 cells per second. However, there has not yet been a thorough survey concerning the identification of the population to which bacterial single cells belong based on flow cytometry data. This paper not only aims to assess the quality of flow cytometry data when measuring bacterial populations, but also suggests an alternative approach for analyzing synthetic microbial communities. We created so-called in silico communities, which allow us to explore the possibilities of bacterial flow cytometry data using supervised machine learning techniques. We can identify single cells with an accuracy >90% for more than half of the communities consisting out of two bacterial populations. In order to assess to what extent an in silico community is representative for its synthetic counterpart, we created so-called abundance gradients, a combination of synthetic (i.e., in vitro) communities containing two bacterial populations in varying abundances. By showing that we are able to retrieve an abundance gradient using a combination of in silico communities and supervised machine learning techniques, we argue that in silico communities form a viable representation for synthetic bacterial communities, opening up new opportunities for the analysis of synthetic communities and bacterial flow cytometry data in general.

  19. Identification of human T cell antigens for the development of vaccines against Mycobacterium tuberculosis.

    PubMed

    Bertholet, Sylvie; Ireton, Gregory C; Kahn, Maria; Guderian, Jeffrey; Mohamath, Raodoh; Stride, Nicole; Laughlin, Elsa M; Baldwin, Susan L; Vedvick, Thomas S; Coler, Rhea N; Reed, Steven G

    2008-12-01

    Development of a subunit vaccine for Mycobacterium tuberculosis (Mtb) depends on the identification of Ags that induce appropriate T cell responses. Using bioinformatics, we selected a panel of 94 Mtb genes based on criteria that included growth in macrophages, up- or down-regulation under hypoxic conditions, secretion, membrane association, or because they were members of the PE/PPE or EsX families. Recombinant proteins encoded by these genes were evaluated for IFN-gamma recall responses using PBMCs from healthy subjects previously exposed to Mtb. From this screen, dominant human T cell Ags were identified and 49 of these proteins, formulated in CpG, were evaluated as vaccine candidates in a mouse model of tuberculosis. Eighteen of the individual Ags conferred partial protection against challenge with virulent Mtb. A combination of three of these Ags further increased protection against Mtb to levels comparable to those achieved with bacillus Calmette-Guérin vaccination. Vaccine candidates that led to reduction in lung bacterial burden following challenge-induced pluripotent CD4 and CD8 T cells, including Th1 cell responses characterized by elevated levels of Ag-specific IgG2c, IFN-gamma, and TNF. Priority vaccine Ags elicited pluripotent CD4 and CD8 T responses in purified protein derivative-positive donor PBMCs. This study identified numerous novel human T cell Ags suitable to be included in subunit vaccines against tuberculosis.

  20. Identification of Human T Cell Antigens for the Development of Vaccines Against Mycobacterium Tuberculosis

    PubMed Central

    Bertholet, Sylvie; Ireton, Gregory C; Kahn, Maria; Guderian, Jeffrey; Mohamath, Raodoh; Stride, Nicole; Laughlin, Elsa M.; Baldwin, Susan L.; Vedvick, Thomas S.; Coler, Rhea N.; Reed, Steven G.

    2008-01-01

    Development of a subunit vaccine for Mycobacterium tuberculosis (Mtb) depends on the identification of antigens that induce appropriate T cell responses. Using bioinformatics, we selected a panel of 94 Mtb genes based on criteria which included growth in macrophages, up- or down-regulation under hypoxic conditions, secretion, membrane association, or because they were members of the PE/PPE or EsX families. Recombinant proteins encoded by these genes were evaluated for IFN-γ recall responses using PBMC from healthy subjects previously exposed to Mtb. From this screen, dominant human T-cell antigens were identified and 49 of these proteins, formulated in CpG, were evaluated as vaccine candidates in a mouse model of tuberculosis (TB). Eighteen of the individual antigens conferred partial protection against challenge with virulent Mtb. A combination of three of these antigens further increased protection against Mtb to levels comparable to those achieved with BCG vaccination. Vaccine candidates that led to reduction in lung bacterial burden following challenge induced pluripotent CD4 and CD8 T cells, including TH1 cell responses characterized by elevated levels of antigen-specific IgG2c, IFN-γ and TNF. Priority vaccine antigens elicited pluripotent CD4 and CD8 T responses in PPD+ donor PBMC. This study identified numerous novel human T cell antigens suitable to be included in subunit vaccines against TB. PMID:19017986

  1. Identification and characterization of [6]-shogaol from ginger as inhibitor of vascular smooth muscle cell proliferation

    PubMed Central

    Liu, Rongxia; Heiss, Elke H; Sider, Nadine; Schinkovitz, Andreas; Gröblacher, Barbara; Guo, Dean; Bucar, Franz; Bauer, Rudolf; Dirsch, Verena M; Atanasov, Atanas G

    2015-01-01

    Scope Vascular smooth muscle cell (VSMC) proliferation is involved in the pathogenesis of cardiovascular disease, making the identification of new counteracting agents and their mechanisms of action relevant. Ginger and its constituents have been reported to improve cardiovascular health, but no studies exist addressing a potential interference with VSMC proliferation. Methods and results The dichloromethane extract of ginger inhibited VSMC proliferation when monitored by resazurin metabolic conversion (IC50 = 2.5 μg/mL). The examination of major constituents from ginger yielded [6]-shogaol as the most active compound (IC50 = 2.7 μM). In the tested concentration range [6]-shogaol did not exhibit cytotoxicity toward VSMC and did not interfere with endothelial cell proliferation. [6]-shogaol inhibited DNA synthesis and induced accumulation of the VSMC in the G0/G1 cell-cycle phase accompanied with activation of the nuclear factor-erythroid 2-related factor 2 (Nrf2)/HO-1 pathway. Since [6]-shogaol lost its antiproliferative activity in the presence of the heme oxygenase-1 (HO-1) inhibitor tin protoporphyrin IX, HO-1 induction appears to contribute to the antiproliferative effect. Conclusion This study demonstrates for the first time inhibitory potential of ginger constituents on VSMC proliferation. The presented data suggest that [6]-shogaol exerts its antiproliferative effect through accumulation of cells in the G0/G1 cell-cycle phase associated with activation of the Nrf2/HO-1 pathway. PMID:25631547

  2. Flow Cytometric Single-Cell Identification of Populations in Synthetic Bacterial Communities

    PubMed Central

    Boon, Nico; Waegeman, Willem

    2017-01-01

    Bacterial cells can be characterized in terms of their cell properties using flow cytometry. Flow cytometry is able to deliver multiparametric measurements of up to 50,000 cells per second. However, there has not yet been a thorough survey concerning the identification of the population to which bacterial single cells belong based on flow cytometry data. This paper not only aims to assess the quality of flow cytometry data when measuring bacterial populations, but also suggests an alternative approach for analyzing synthetic microbial communities. We created so-called in silico communities, which allow us to explore the possibilities of bacterial flow cytometry data using supervised machine learning techniques. We can identify single cells with an accuracy >90% for more than half of the communities consisting out of two bacterial populations. In order to assess to what extent an in silico community is representative for its synthetic counterpart, we created so-called abundance gradients, a combination of synthetic (i.e., in vitro) communities containing two bacterial populations in varying abundances. By showing that we are able to retrieve an abundance gradient using a combination of in silico communities and supervised machine learning techniques, we argue that in silico communities form a viable representation for synthetic bacterial communities, opening up new opportunities for the analysis of synthetic communities and bacterial flow cytometry data in general. PMID:28122063

  3. Identification of stromal cell products that interact with pre-B cells

    PubMed Central

    1996-01-01

    Our understanding of lympho-hematopoietic microenvironments is incomplete, and a new cloning strategy was developed to identify molecules that bind to B lineage lymphocyte precursors. A cell sorting procedure was used for initial enrichment of cDNAs from stromal cell mRNA that contained signal sequences and were therefore likely to encode transmembrane or secreted proteins. A second step involved expression of the library as soluble Ig fusion proteins. Finally, pools representing these proteins were screened for the ability to recognize pre-B cells. This approach resulted in the cloning of biglycan, syndecan 4, collagen type I, clusterin, matrix glycoprotein sc1, osteonectin, and one unknown molecule (designated SIM). The full-length cDNA of SIM revealed that it is a type I transmembrane protein, and its intracellular domain has weak homology with myosin heavy chain and related proteins. Staining of established cell lines and freshly isolated hematopoietic cells with the Ig fusion proteins revealed distinct patterns of reactivity and differential dependence on divalent cations. Biglycan-, sc1-, and SIM-Ig fusion proteins selectively increased interleukin 7-dependent proliferation of pre-B cells. Overexpression of the entire SIM protein affected the morphology of 293T cells, while expression of just the extracellular portion was without effect. Thus, a series of stromal cell surface molecules has been identified that interact with blood cell precursors. Three of them promoted the survival and/or proliferation of pre-B cells in culture, and all merit further study in relation to lympho-hematopoiesis. PMID:8707854

  4. Identification of New Hematopoietic Cell Subsets with a Polyclonal Antibody Library Specific for Neglected Proteins

    PubMed Central

    Creo, Pasquale; Gallina, Pierangela; Curti, Serena; Sugliano, Elisa; Scavelli, Rossana; Cattaneo, Davide; Canidio, Elena; Marconi, Maurizio; Rebulla, Paolo; Sarmientos, Paolo; Viale, Giuseppe; Pagani, Massimiliano; Abrignani, Sergio

    2012-01-01

    The identification of new markers, the expression of which defines new phenotipically and functionally distinct cell subsets, is a main objective in cell biology. We have addressed the issue of identifying new cell specific markers with a reverse proteomic approach whereby approximately 1700 human open reading frames encoding proteins predicted to be transmembrane or secreted have been selected in silico for being poorly known, cloned and expressed in bacteria. These proteins have been purified and used to immunize mice with the aim of obtaining polyclonal antisera mostly specific for linear epitopes. Such a library, made of about 1600 different polyclonal antisera, has been obtained and screened by flow cytometry on cord blood derived CD34+CD45dim cells and on peripheral blood derived mature lymphocytes (PBLs). We identified three new proteins expressed by fractions of CD34+CD45dim cells and eight new proteins expressed by fractions of PBLs. Remarkably, we identified proteins the presence of which had not been demonstrated previously by transcriptomic analysis. From the functional point of view, looking at new proteins expressed on CD34+CD45dim cells, we identified one cell surface protein (MOSC-1) the expression of which on a minority of CD34+ progenitors marks those CD34+CD45dim cells that will go toward monocyte/granulocyte differentiation. In conclusion, we show a new way of looking at the membranome by assessing expression of generally neglected proteins with a library of polyclonal antisera, and in so doing we have identified new potential subsets of hematopoietic progenitors and of mature PBLs. PMID:22496798

  5. Identification of chimeric antigen receptors that mediate constitutive or inducible proliferation of T cells.

    PubMed

    Frigault, Matthew J; Lee, Jihyun; Basil, Maria Ciocca; Carpenito, Carmine; Motohashi, Shinichiro; Scholler, John; Kawalekar, Omkar U; Guedan, Sonia; McGettigan, Shannon E; Posey, Avery D; Ang, Sonny; Cooper, Laurence J N; Platt, Jesse M; Johnson, F Brad; Paulos, Chrystal M; Zhao, Yangbing; Kalos, Michael; Milone, Michael C; June, Carl H

    2015-04-01

    This study compared second-generation chimeric antigen receptors (CAR) encoding signaling domains composed of CD28, ICOS, and 4-1BB (TNFRSF9). Here, we report that certain CARs endow T cells with the ability to undergo long-term autonomous proliferation. Transduction of primary human T cells with lentiviral vectors encoding some of the CARs resulted in sustained proliferation for up to 3 months following a single stimulation through the T-cell receptor (TCR). Sustained numeric expansion was independent of cognate antigen and did not require the addition of exogenous cytokines or feeder cells after a single stimulation of the TCR and CD28. Results from gene array and functional assays linked sustained cytokine secretion and expression of T-bet (TBX21), EOMES, and GATA-3 to the effect. Sustained expression of the endogenous IL2 locus has not been reported in primary T cells. Sustained proliferation was dependent on CAR structure and high expression, the latter of which was necessary but not sufficient. The mechanism involves constitutive signaling through NF-κB, AKT, ERK, and NFAT. The propagated CAR T cells retained a diverse TCR repertoire, and cellular transformation was not observed. The CARs with a constitutive growth phenotype displayed inferior antitumor effects and engraftment in vivo. Therefore, the design of CARs that have a nonconstitutive growth phenotype may be a strategy to improve efficacy and engraftment of CAR T cells. The identification of CARs that confer constitutive or nonconstitutive growth patterns may explain observations that CAR T cells have differential survival patterns in clinical trials. ©2015 American Association for Cancer Research.

  6. Spermatogonial stem cells specific marker identification in channel catfish, Ictalurus punctatus and blue catfish, I. furcatus.

    PubMed

    Shang, Mei; Su, Baofeng; Lipke, Elizabeth A; Perera, Dayan A; Li, Chao; Qin, Zhenkui; Li, Yun; Dunn, David A; Cek, Sehriban; Peatman, Eric; Dunham, Rex A

    2015-12-01

    Testicular germ cells of channel catfish, Ictalurus punctatus, and blue catfish, I. furcatus were separated into four layers with Percoll density gradient centrifugation, containing different cell types (40% in the first layer were spermatogonial stem cells, SSCs). Expression of seventeen genes was analyzed for cells from different layers by real-time quantitative PCR. Pfkfb4, Urod, Plzf, Integrin6, IntegrinV, Thy1 and Cdh1 genes showed the same expression change pattern in both channel and blue catfish as these genes were down-regulated in the spermatocytes and even more so in spermatids. Plzf and Integrin6 had especially high expression in SSCs and can be used as SSCs specific markers. Sox2 gene was up-regulated in spermatocytes and even more highly up-regulated in spermatids, which indicated it could be a spermatid marker. In contrast to channel catfish, Id4, Smad5 and Prdm14 gene expressions were strongly down-regulated in spermatocyte cells, but up-regulated in spermatid cells in blue catfish. Smad5 gene was down-regulated in spermatocytes, but up-regulated in both spermatogonia and spermatids, allowing identification as a marker for spermatocytes in blue catfish. Oct4, Id4, Gfrα2, Pum2 and Prdm14 genes showed different expression patterns in the testicular germ cells of channel and blue catfish. This may be a partial explanation to the differing responses of channel catfish and blue catfish to induced spawning technologies. The SSCs specific markers can be used for further SSCs labeling, which can increase the SSCs sorting efficiency and be applied in various studies involving SSCs and other germ cells.

  7. Phylogenetic identification and in situ detection of individual microbial cells without cultivation.

    PubMed Central

    Amann, R I; Ludwig, W; Schleifer, K H

    1995-01-01

    The frequent discrepancy between direct microscopic counts and numbers of culturable bacteria from environmental samples is just one of several indications that we currently know only a minor part of the diversity of microorganisms in nature. A combination of direct retrieval of rRNA sequences and whole-cell oligonucleotide probing can be used to detect specific rRNA sequences of uncultured bacteria in natural samples and to microscopically identify individual cells. Studies have been performed with microbial assemblages of various complexities ranging from simple two-component bacterial endosymbiotic associations to multispecies enrichments containing magnetotactic bacteria to highly complex marine and soil communities. Phylogenetic analysis of the retrieved rRNA sequence of an uncultured microorganism reveals its closest culturable relatives and may, together with information on the physicochemical conditions of its natural habitat, facilitate more directed cultivation attempts. For the analysis of complex communities such as multispecies biofilms and activated-sludge flocs, a different approach has proven advantageous. Sets of probes specific to different taxonomic levels are applied consecutively beginning with the more general and ending with the more specific (a hierarchical top-to-bottom approach), thereby generating increasingly precise information on the structure of the community. Not only do rRNA-targeted whole-cell hybridizations yield data on cell morphology, specific cell counts, and in situ distributions of defined phylogenetic groups, but also the strength of the hybridization signal reflects the cellular rRNA content of individual cells. From the signal strength conferred by a specific probe, in situ growth rates and activities of individual cells might be estimated for known species. In many ecosystems, low cellular rRNA content and/or limited cell permeability, combined with background fluorescence, hinders in situ identification of autochthonous

  8. Identification of miRNAs differentially expressed in human epilepsy with or without granule cell pathology.

    PubMed

    Zucchini, Silvia; Marucci, Gianluca; Paradiso, Beatrice; Lanza, Giovanni; Roncon, Paolo; Cifelli, Pierangelo; Ferracin, Manuela; Giulioni, Marco; Michelucci, Roberto; Rubboli, Guido; Simonato, Michele

    2014-01-01

    The microRNAs (miRNAs) are small size non-coding RNAs that regulate expression of target mRNAs at post-transcriptional level. miRNAs differentially expressed under pathological conditions may help identifying mechanisms underlying the disease and may represent biomarkers with prognostic value. However, this kind of studies are difficult in the brain because of the cellular heterogeneity of the tissue and of the limited access to fresh tissue. Here, we focused on a pathology affecting specific cells in a subpopulation of epileptic brains (hippocampal granule cells), an approach that bypasses the above problems. All patients underwent surgery for intractable temporal lobe epilepsy and had hippocampal sclerosis associated with no granule cell pathology in half of the cases and with type-2 granule cell pathology (granule cell layer dispersion or bilamination) in the other half. The expression of more than 1000 miRNAs was examined in the laser-microdissected dentate granule cell layer. Twelve miRNAs were differentially expressed in the two groups. One of these, miR487a, was confirmed to be expressed at highly differential levels in an extended cohort of patients, using RT-qPCR. Bioinformatics searches and RT-qPCR verification identified ANTXR1 as a possible target of miR487a. ANTXR1 may be directly implicated in granule cell dispersion because it is an adhesion molecule that favors cell spreading. Thus, miR487a could be the first identified element of a miRNA signature that may be useful for prognostic evaluation of post-surgical epilepsy and may drive mechanistic studies leading to the identification of therapeutic targets.

  9. Identification of miRNAs Differentially Expressed in Human Epilepsy with or without Granule Cell Pathology

    PubMed Central

    Paradiso, Beatrice; Lanza, Giovanni; Roncon, Paolo; Cifelli, Pierangelo; Ferracin, Manuela; Giulioni, Marco; Michelucci, Roberto; Simonato, Michele

    2014-01-01

    The microRNAs (miRNAs) are small size non-coding RNAs that regulate expression of target mRNAs at post-transcriptional level. miRNAs differentially expressed under pathological conditions may help identifying mechanisms underlying the disease and may represent biomarkers with prognostic value. However, this kind of studies are difficult in the brain because of the cellular heterogeneity of the tissue and of the limited access to fresh tissue. Here, we focused on a pathology affecting specific cells in a subpopulation of epileptic brains (hippocampal granule cells), an approach that bypasses the above problems. All patients underwent surgery for intractable temporal lobe epilepsy and had hippocampal sclerosis associated with no granule cell pathology in half of the cases and with type-2 granule cell pathology (granule cell layer dispersion or bilamination) in the other half. The expression of more than 1000 miRNAs was examined in the laser-microdissected dentate granule cell layer. Twelve miRNAs were differentially expressed in the two groups. One of these, miR487a, was confirmed to be expressed at highly differential levels in an extended cohort of patients, using RT-qPCR. Bioinformatics searches and RT-qPCR verification identified ANTXR1 as a possible target of miR487a. ANTXR1 may be directly implicated in granule cell dispersion because it is an adhesion molecule that favors cell spreading. Thus, miR487a could be the first identified element of a miRNA signature that may be useful for prognostic evaluation of post-surgical epilepsy and may drive mechanistic studies leading to the identification of therapeutic targets. PMID:25148080

  10. Identification and characterization of CD133+CD44+ cancer stem cells from human laryngeal squamous cell carcinoma cell lines

    PubMed Central

    Wang, Jue; Wu, Yongyan; Gao, Wei; Li, Fei; Bo, Yunfeng; Zhu, Meixia; Fu, Rong; Liu, Qingqing; Wen, Shuxin; Wang, Binquan

    2017-01-01

    Background: Laryngeal squamous cell carcinoma ranks second among head and neck squamous-cell carcinomas. Cancer stem cells can support cancer growth and malignant behavior. Therefore, cancer stem cells isolated from laryngeal squamous cell carcinoma tissue could be used to investigate the initiation, progression, and treatment strategies of this cancer. Methods: We isolated CD133-CD44-, CD133-CD44+, CD133+CD44- and CD133+CD44+ cell populations from laryngeal squamous-cell carcinoma cell lines Hep2 and TU-177 by magnetic-activated cell sorting. Sphere formation, cell proliferation, migration, invasion, colony formation, resistance to radio- and chemotherapy, and in vivo tumorigenicity of these populations were evaluated. Moreover, we investigated the expression of the stem-cell markers (sex determining region Y)-box 2 (SOX2) and octamer-binding transcription factor 4 (OCT4) in CD133-CD44-, CD133-CD44+, CD133+CD44-, CD133+CD44+ cell populations and parental Hep2 and TU-177 cells. Results: As compared with CD133-CD44-, CD133-CD44+, CD133+CD44- populations and parental cells, CD133+CD44+ cells showed higher cell viability, migration and invasive capability and colony formation ability as well as stronger resistance to cisplatin and irradiation. Moreover, levels of SOX2 and OCT4 and tumorigenicity in nude mice were greater in CD133+CD44+ Hep2 and TU-177 cells than other cell populations and parental cells. Conclusion: The CD133+CD44+ population of laryngeal squamous-cell carcinoma Hep2 and TU-177 cells have stem cell properties and showed more malignant features than CD133+CD44- and CD133-CD44+ cell populations. CD133+CD44+ cancer stem cells may be a promising target for developing anticancer drugs and treatment strategies for laryngeal squamous cell carcinoma. PMID:28261352

  11. Multicolor Immunofluorescent Imaging of Complex Cellular Mixtures on Micropallet Arrays Enables the Identification of Single Cells of Defined Phenotype.

    PubMed

    Westerhof, Trisha M; Li, Guann-Pyng; Bachman, Mark; Nelson, Edward L

    2016-04-06

    A Micropallet-Array-based strategy allowing the identification of cells of defined phenotype in complex mixtures, such as would be obtained from a tissue biopsy, is presented. Following the distribution of single adherent cells from the mixture on individual pedestals, termed "micropallets", immunofluorescent confocal imaging is applied to interrogate the expression of five cell surface molecules. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  12. Bioluminescent Orthotopic Mouse Models of Human Localized Non-Small Cell Lung Cancer: Feasibility and Identification of Circulating Tumour Cells

    PubMed Central

    Lahon, Benoit; Castier, Yves; Lesèche, Guy; Soria, Jean-Charles; Vozenin, Marie-Catherine; Decraene, Charles; Deutsch, Eric

    2011-01-01

    Background Preclinical models of non-small cell lung cancer (NSCLC) require better clinical relevance to study disease mechanisms and innovative therapeutics. We sought to compare and refine bioluminescent orthotopic mouse models of human localized NSCLC. Methods Athymic nude mice underwent subcutaneous injection (group 1-SC, n = 15, control), percutaneous orthotopic injection (group 2-POI, n = 30), surgical orthotopic implantation of subcutaneously grown tumours (group 3-SOI, n = 25), or transpleural orthotopic injection (group 4-TOI, n = 30) of A549-luciferase cells. Bioluminescent in vivo imaging was then performed weekly. Circulating tumour cells (CTCs) were searched using Cellsearch® system in SC and TOI models. Results Group 2-POI was associated with unexpected direct pleural spreading of the cellular solution in 53% of the cases, forbidding further evaluation of any localized lung tumour. Group 3-SOI was characterized by high perioperative mortality, initially localized lung tumours, and local evolution. Group 4-TOI was associated with low perioperative mortality, initially localized lung tumours, loco regional extension, and distant metastasis. CTCs were detected in 83% of nude mice bearing subcutaneous or orthotopic NSCLC tumours. Conclusions Transpleural orthotopic injection of A549-luc cells in nude mouse lung induces localized tumour, followed by lymphatic extension and specific mortality, and allowed the first time identification of CTCs in a NSCLC mice model. PMID:22022511

  13. Identification of T cell-signaling pathways that stimulate latent HIV in primary cells

    PubMed Central

    Brooks, David G.; Arlen, Philip A.; Gao, Lianying; Kitchen, Christina M. R.; Zack, Jerome A.

    2003-01-01

    Eradication of HIV infection depends on the elimination of a small, but stable population of latently infected T cells. After the discontinuation of therapy, activation of latent virus can rekindle infection. To purge this reservoir, it is necessary to define cellular signaling pathways that lead to activation of latent HIV. We used the SCID-hu (Thy/Liv) mouse model of HIV latency to analyze a broad array of T cell-signaling pathways and show in primary, quiescent cells that viral induction depends on the activation of two primary intracellular signaling pathways, protein kinase C or nuclear factor of activated T cells (NF-AT). In contrast, inhibition or activation of other important T cell stimulatory pathways (such as mitogen-activated protein kinase, calcium flux, or histone deacetylation) do not significantly induce virus expression. We found that the activation of NF-κB is critical to viral reactivation; however, all pathways that stimulate NF-κBdonot reactivate latent virus. Our studies further show that inhibition of NF-κB does not prevent activation of HIV by NF-AT, indicating that these pathways can function independently to activate the HIV LTR. Thus, we define several molecular pathways that trigger HIV reactivation from latency and provide evidence that latent HIV infection is maintained by the functional lack of particular transcription factors in quiescent cells. PMID:14569007

  14. Identification of a novel gene expressed in activated natural killer cells and T cells

    SciTech Connect

    Dahl, C.A.; Schall, R.P.; He, H.; Cairns, J.S. )

    1992-01-15

    The authors have isolated a cDNA clone from a human activated NK cell-derived cDNA library that identifies a transcript [NK4] that is selectively expressed in lymphocytes. The expression of this transcript is increased after activation of T cells by mitogens or activation of NK cells by IL-2 (lymphokine-activated killer cells). The transcript levels demonstrated by Northern blot analysis increase by 12 h after activation, remain high for at least 48 h, and require protein synthesis for expression. Southern blot analysis of B lymphoblastoid lines derived from 18 unrelated individuals reveal variable banding patterns suggestive of polymorphism within the NK4 gene. No homology was found between the sequence of the coding region of this transcript and any sequences in the GenBank data base. Sequence homology to the U1 small nuclear RNA was found within the 3[prime] untranslated region immediately upstream of the site of polyadenylation, suggesting a possible role for U1 in the polyadenylation process. Sequence analysis indicates the transcript would encode a protein having a mass of 27 kDa. The presence of a signal sequence and lack of a transmembrane region suggests that the protein is secreted. In addition, the protein contains an RGD sequence that may be involved in cellular adhesion. This transcript appears to encode a novel product common to the activation pathways of both NK cells and T cells. 50 refs., 8 figs.

  15. Identification of chimeric antigen receptors that mediate constitutive or inducible proliferation of T cells

    PubMed Central

    Frigault, Matthew J; Lee, Jihyun; Basil, Maria Ciocca; Carpenito, Carmine; Motohashi, Shinichiro; Scholler, John; Kawalekar, Omkar U.; Guedan, Sonia; McGettigan, Shannon E.; Posey, Avery D.; Ang, Sonny; Cooper, Laurence J. N.; Platt, Jesse M.; Johnson, F. Brad; Paulos, Chrystal M; Zhao, Yangbing; Kalos, Michael; Milone, Michael C.; June, Carl H.

    2015-01-01

    This study compared second generation chimeric antigen receptors encoding signaling domains composed of CD28, ICOS and 4-1BB. Here we report that certain CARs endow T cells with the ability to undergo long-term autonomous proliferation. Transduction of primary human T-cell with lentiviral vectors encoding some of the CARs resulted in sustained proliferation for up to three months following a single stimulation through the TCR. Sustained numeric expansion was independent of cognate antigen and did not require the addition of exogenous cytokines or feeder cells after a single stimulation of the TCR and CD28. Results from gene array and functional assays linked sustained cytokine secretion and expression of T-bet, EOMES and GATA-3 to the effect. Sustained expression of the endogenous IL2 locus has not been reported in primary T cells. Sustained proliferation was dependent on CAR structure and high expression, the latter of which was necessary but not sufficient. The mechanism involves constitutive signaling through NF-kB, Akt, Erk and NFAT. The propagated CAR T cells retained a diverse TCR repertoire and cellular transformation was not observed. The CARs with a constitutive growth phenotype displayed inferior antitumor effects and engraftment in vivo. Therefore the design of CARs that have a non-constitutive growth phenotype may be a strategy to improve efficacy and engraftment of CAR T cells. The identification of CARs that confer constitutive or non-constitutive growth patterns may explain observations that CAR T cells have differential survival patterns in clinical trials. PMID:25600436

  16. Identification of Long Non-Coding RNAs Deregulated in Multiple Myeloma Cells Resistant to Proteasome Inhibitors

    PubMed Central

    Malek, Ehsan; Kim, Byung-Gyu; Driscoll, James J.

    2016-01-01

    While the clinical benefit of proteasome inhibitors (PIs) for multiple myeloma (MM) treatment remains unchallenged, dose-limiting toxicities and the inevitable emergence of drug resistance limit their long-term utility. Disease eradication is compromised by drug resistance that is either present de novo or therapy-induced, which accounts for the majority of tumor relapses and MM-related deaths. Non-coding RNAs (ncRNAs) are a broad class of RNA molecules, including long non-coding RNAs (lncRNAs), that do not encode proteins but play a major role in regulating the fundamental cellular processes that control cancer initiation, metastasis, and therapeutic resistance. While lncRNAs have recently attracted significant attention as therapeutic targets to potentially improve cancer treatment, identification of lncRNAs that are deregulated in cells resistant to PIs has not been previously addressed. We have modeled drug resistance by generating three MM cell lines with acquired resistance to either bortezomib, carfilzomib, or ixazomib. Genome-wide profiling identified lncRNAs that were significantly deregulated in all three PI-resistant cell lines relative to the drug-sensitive parental cell line. Strikingly, certain lncRNAs deregulated in the three PI-resistant cell lines were also deregulated in MM plasma cells isolated from newly diagnosed patients compared to healthy plasma cells. Taken together, these preliminary studies strongly suggest that lncRNAs represent potential therapeutic targets to prevent or overcome drug resistance. More investigations are ongoing to expand these initial studies in a greater number of MM patients to better define lncRNAs signatures that contribute to PI resistance in MM. PMID:27782060

  17. Identification of fetal mesenchymal stem cells in maternal blood: implications for non-invasive prenatal diagnosis.

    PubMed

    O'Donoghue, K; Choolani, M; Chan, J; de la Fuente, J; Kumar, S; Campagnoli, C; Bennett, P R; Roberts, I A G; Fisk, N M

    2003-08-01

    Strategies for genetic prenatal diagnosis on fetal cells in the maternal circulation have been limited by lack of a cell type present only in fetal blood. However, the recent identification of mesenchymal stem cells (MSC) in first trimester fetal blood offers the prospect of targeting MSC for non-invasive prenatal diagnosis. We developed protocols for fetal MSC enrichment from maternal blood and determined sensitivity and specificity in mixing experiments of male fetal MSC added to female blood, in dilutions from 1 in 10(5) to 10(8). We then used the optimal protocol to isolate fetal MSC from maternal blood in the first trimester, using blood taken after surgical termination of pregnancy as a model of increased feto-maternal haemorrhage. In model mixtures, we could amplify one male fetal MSC in 2.5 x 10(7) adult female nucleated cells, yielding a 100% pure population of fetal cells, but not one fetal MSC in 10(8) nucleated cells. Fetal MSC were identified in one of 20 post-termination maternal blood samples and confirmed as fetal MSC by XY fluorescence in-situ hybridization (FISH), immunophenotyping and osteogenic and adipogenic differentiation. We report the isolation of fetal MSC from maternal blood; however, their rarity in post-termination blood suggests they are unlikely to have a role in non-invasive prenatal diagnosis. Failure to locate these cells routinely may be attributed to their low frequency in maternal blood, to sensitivity limitations of enrichment technology, and/or to their engraftment in maternal tissues soon after transplacental passage. We speculate that gender microchimerism in post-reproductive maternal tissues might result from feto-maternal trafficking of MSC in early pregnancy.

  18. Identification of Lactobacillus plantarum genes modulating the cytokine response of human peripheral blood mononuclear cells

    PubMed Central

    2010-01-01

    Background Modulation of the immune system is one of the most plausible mechanisms underlying the beneficial effects of probiotic bacteria on human health. Presently, the specific probiotic cell products responsible for immunomodulation are largely unknown. In this study, the genetic and phenotypic diversity of strains of the Lactobacillus plantarum species were investigated to identify genes of L. plantarum with the potential to influence the amounts of cytokines interleukin 10 (IL-10) and IL-12 and the ratio of IL-10/IL-12 produced by peripheral blood mononuclear cells (PBMCs). Results A total of 42 Lactobacillus plantarum strains isolated from diverse environmental and human sources were evaluated for their capacity to stimulate cytokine production in PBMCs. The L. plantarum strains induced the secretion of the anti-inflammatory cytokine IL-10 over an average 14-fold range and secretion of the pro-inflammatory cytokine IL-12 over an average 16-fold range. Comparisons of the strain-specific cytokine responses of PBMCs to comparative genome hybridization profiles obtained with L. plantarum WCFS1 DNA microarrays (also termed gene-trait matching) resulted in the identification of 6 candidate genetic loci with immunomodulatory capacities. These loci included genes encoding an N-acetyl-glucosamine/galactosamine phosphotransferase system, the LamBDCA quorum sensing system, and components of the plantaricin (bacteriocin) biosynthesis and transport pathway. Deletion of these genes in L. plantarum WCFS1 resulted in growth phase-dependent changes in the PBMC IL-10 and IL-12 cytokine profiles compared with wild-type cells. Conclusions The altered PBMC cytokine profiles obtained with the L. plantarum WCFS1 mutants were in good agreement with the predictions made by gene-trait matching for the 42 L. plantarum strains. This study therefore resulted in the identification of genes present in certain strains of L. plantarum which might be responsible for the stimulation of anti

  19. Identification of a Transcription Factor That Regulates Host Cell Exit and Virulence of Mycobacterium tuberculosis

    PubMed Central

    Srinivasan, Lalitha; Gurses, Serdar A.; Hurley, Benjamin E.; Miller, Jessica L.; Karakousis, Petros C.; Briken, Volker

    2016-01-01

    The interaction of Mycobacterium tuberculosis (Mtb) with host cell death signaling pathways is characterized by an initial anti-apoptotic phase followed by a pro-necrotic phase to allow for host cell exit of the bacteria. The bacterial modulators regulating necrosis induction are poorly understood. Here we describe the identification of a transcriptional repressor, Rv3167c responsible for regulating the escape of Mtb from the phagosome. Increased cytosolic localization of MtbΔRv3167c was accompanied by elevated levels of mitochondrial reactive oxygen species and reduced activation of the protein kinase Akt, and these events were critical for the induction of host cell necrosis and macroautophagy. The increase in necrosis led to an increase in bacterial virulence as reflected in higher bacterial burden and reduced survival of mice infected with MtbΔRv3167c. The regulon of Rv3167c thus contains the bacterial mediators involved in escape from the phagosome and host cell necrosis induction, both of which are crucial steps in the intracellular lifecycle and virulence of Mtb. PMID:27191591

  20. Identification of Polo-like kinase 1 interaction inhibitors using a novel cell-based assay

    PubMed Central

    Normandin, Karine; Lavallée, Jean-François; Futter, Marie; Beautrait, Alexandre; Duchaine, Jean; Guiral, Sébastien; Marinier, Anne; Archambault, Vincent

    2016-01-01

    Polo-like kinase 1 (Plk1) plays several roles in cell division and it is a recognized cancer drug target. Plk1 levels are elevated in cancer and several types of cancer cells are hypersensitive to Plk1 inhibition. Small molecule inhibitors of the kinase domain (KD) of Plk1 have been developed. Their selectivity is limited, which likely contributes to their toxicity. Polo-like kinases are characterized by a Polo-Box Domain (PBD), which mediates interactions with phosphorylation substrates or regulators. Inhibition of the PBD could allow better selectivity or result in different effects than inhibition of the KD. In vitro screens have been used to identify PBD inhibitors with mixed results. We developed the first cell-based assay to screen for PBD inhibitors, using Bioluminescence Resonance Energy Transfer (BRET). We screened through 112 983 compounds and characterized hits in secondary biochemical and biological assays. Subsequent Structure-Activity Relationship (SAR) analysis on our most promising hit revealed that it requires an alkylating function for its activity. In addition, we show that the previously reported PBD inhibitors thymoquinone and Poloxin are also alkylating agents. Our cell-based assay is a promising tool for the identification of new PBD inhibitors with more drug-like profiles using larger and more diverse chemical libraries. PMID:27874094

  1. Identification and discrimination of extracellularly active cathepsins B and L in high-invasive melanoma cells.

    PubMed

    Klose, Anke; Zigrino, Paola; Dennhöfer, Ralf; Mauch, Cornelia; Hunzelmann, Nicolas

    2006-06-01

    We established a novel protocol for lithium dodecyl sulfate (LDS) gelatin zymography, which operates under reducing conditions and at a slightly acidic pH value (6.5). This zymographic assay is based on polyacrylamide gel electrophoresis and facilitates the electrophoretic separation of human cathepsins in an active state. By this technique, activity of purified human liver cathepsin B was detected at a concentration as low as 50 ng and was blocked only in the presence of the cysteine protease inhibitor E-64 and the specific cathepsin B inhibitor CA-074 but not by aspartate, serine, or matrix metalloprotease inhibitors. The method was applied to analyze cathepsin activities in cell culture supernatants of the high-invasive melanoma cell line MV3. Interestingly, LDS zymography of MV3 cell supernatants in combination with specific inhibitors of cathepsins B and L identified three forms of extracellularly active cathepsin B and two forms of proteolytically active cathepsin L. We herein describe the generation and biochemical significance of acidic LDS zymography. This novel method permits not only the enzymatic analysis of purified cysteine proteases but also the identification and discrimination of different cathepsin activities in biological fluids, cell lysates, or supernatants, especially of cathepsins B and L, which are closely linked to major inflammatory and malignant processes.

  2. Identification of hematic cells by spectroscopic analysis of the intrinsic fluorescence

    NASA Astrophysics Data System (ADS)

    Monici, Monica; Agati, Giovanni; Fusi, Franco; Bernabei, Pietro A.; Caporale, Roberto; Ferrini, Pierluigi R.; Croce, Anna C.; Bottiroli, Giovanni F.; Cioncolini, Stefano; Innocenti, Alberto; Pratesi, Riccardo

    1994-12-01

    The determination of blood cell composition has been a valuable tool in diagnoses. In particular, both total and differential counts are considered the basic parameters that characterize the leukocyte population. Since 100 years ago, manual techniques were introduced that allow a morphological examination of blood smears. At present, the automated analysis has been proved to be particularly difficult to standardize. In fact, the identification and count of the five leukocyte populations are not completely solved problems in routine methods for hematological analysis. Optoelectronics could have a decisive role in the development of new techniques that can ensure characteristics of automation, reliability, accuracy and rapidity of execution. Fluorescence spectroscopy techniques could represent a valid approach. Recently, the evaluation of tissue and cell autofluorescence has been applied to the diagnosis of solid tissue neoplasies. In this work, we have considered the possibility to develop a reliable method of leukocyte analysis based on their intrinsic fluorescence emission properties. The study has been performed by applying both spectrofluorometric techniques to enriched suspensions of cells and microspectrofluorometric techniques to single leukocytes. The results obtained have shown the possibility to recognize some cell populations on the grounds of the intrinsic fluorescence characteristics.

  3. Identification of Key Proteins in Human Epithelial Cells Responding to Bystander Signals From Irradiated Trout Skin

    PubMed Central

    Smith, Richard; Wang, Jiaxi; Seymour, Colin; Mothersill, Carmel; Howe, Orla

    2015-01-01

    Radiation-induced bystander signaling has been found to occur in live rainbow trout fish (Oncorhynchus mykiss). This article reports identification of key proteomic changes in a bystander reporter cell line (HaCaT) grown in low-dose irradiated tissue-conditioned media (ITCM) from rainbow trout fish. In vitro explant cultures were generated from the skin of fish previously exposed to low doses (0.1 and 0.5 Gy) of X-ray radiation in vivo. The ITCM was harvested from all donor explant cultures and placed on recipient HaCaT cells to observe any change in protein expression caused by the bystander signals. Proteomic methods using 2-dimensional (2D) gel electrophoresis and mass spectroscopy were employed to screen for novel proteins expressed. The proteomic changes measured in HaCaT cells receiving the ITCM revealed that exposure to 0.5 Gy induced an upregulation of annexin A2 and cingulin and a downregulation of Rho-GDI2, F-actin-capping protein subunit beta, microtubule-associated protein RP/EB family member, and 14-3-3 proteins. The 0.1 Gy dose also induced a downregulation of Rho-GDI2, hMMS19, F-actin-capping protein subunit beta, and microtubule-associated protein RP/EB family member proteins. The proteins reported may influence apoptotic signaling, as the results were suggestive of an induction of cell communication, repair mechanisms, and dysregulation of growth signals. PMID:26673684

  4. Identification of EDIL3 on extracellular vesicles involved in breast cancer cell invasion.

    PubMed

    Lee, Jeong-Eun; Moon, Pyong-Gon; Cho, Young-Eun; Kim, Young-Bum; Kim, In-San; Park, Hoyong; Baek, Moon-Chang

    2016-01-10

    Cancer cell-derived extracellular vesicles have been linked to the pathogenesis of various cancers; however, the role of extracellular vesicles in tumorigenesis remains unclear. To identify extracellular vesicle proteins involved in cancer metastasis, quantitative proteomic analyses were performed on extracellular vesicles derived from two representative breast cancer cell lines: the less invasive MCF-7 and the invasive MDA-MB-231. Proteomic analysis allowed for the identification of 270 proteins in the extracellular vesicles. Here we report a new function of EDIL3 on extracellular vesicles, which are sufficient for enhancement of cell invasion and for acceleration of lung metastasis in vivo. This invasion is most likely mediated via the integrin-FAK signaling cascade in breast cancer cells. However, these effects are suppressed when EDIL3 is inactivated, providing evidence for a critical role of EDIL3 in development of cancer. Consistently, in human patients with metastatic breast cancer, the levels of EDIL3 on circulating extracellular vesicles are significantly elevated. This information is a remarkable breakthrough in understanding of the molecular mechanism underlying metastasis of breast cancer as well as in the research for cancer biomarkers using circulating extracellular vesicles. Furthermore, targeting EDIL3 on extracellular vesicles may lead to a new therapeutic option for treatment of breast cancer. Copyright © 2015 Elsevier B.V. All rights reserved.

  5. Genetic identification and cloning of a gene required for developmental cell interactions in Myxococcus xanthus.

    PubMed Central

    Gill, R E; Cull, M G; Fly, S

    1988-01-01

    Developmental mutants of Myxococcus xanthus have been previously described which appear to be defective in required cell-cell interactions. These mutants fall into four phenotypic classes, Asg, Bsg, Csg, and Dsg, each of which is unable to differentiate into spores but can be rescued by extracellular complementation by wild-type cells or by mutants of a different class. We report the identification of one of the loci in which mutations result in a Bsg phenotype. The cloned locus was contained on a 12-kilobase EcoRI fragment and then localized by subcloning and a combination of in vitro and transposon mutagenesis. All mutations in this locus behave as a single complementation group, which we designate bsgA (formerly ssbA). Each of the bsgA mutations results in a nonsporulating phenotype, which can be rescued by extracellular complementation. Furthermore, we report that the bsgA mutants have a distinctive interaction with wild-type cells when vegetatively growing, swarming colonies converge. Images PMID:2846514

  6. Identification of proteins enriched in rice egg or sperm cells by single-cell proteomics.

    PubMed

    Abiko, Mafumi; Furuta, Kensyo; Yamauchi, Yoshio; Fujita, Chiharu; Taoka, Masato; Isobe, Toshiaki; Okamoto, Takashi

    2013-01-01

    In angiosperms, female gamete differentiation, fertilization, and subsequent zygotic development occur in embryo sacs deeply embedded in the ovaries. Despite their importance in plant reproduction and development, how the egg cell is specialized, fuses with the sperm cell, and converts into an active zygote for early embryogenesis remains unclear. This lack of knowledge is partly attributable to the difficulty of direct analyses of gametes in angiosperms. In the present study, proteins from egg and sperm cells obtained from rice flowers were separated by one-dimensional polyacrylamide gel electrophoresis and globally identified by highly sensitive liquid chromatography coupled with tandem mass spectroscopy. Proteome analyses were also conducted for seedlings, callus, and pollen grains to compare their protein expression profiles to those of gametes. The proteomics data have been deposited to the ProteomeXchange with identifier PXD000265. A total of 2,138 and 2,179 expressed proteins were detected in egg and sperm cells, respectively, and 102 and 77 proteins were identified as preferentially expressed in egg and sperm cells, respectively. Moreover, several rice or Arabidopsis lines with mutations in genes encoding the putative gamete-enriched proteins showed clear phenotypic defects in seed set or seed development. These results suggested that the proteomic data presented in this study are foundational information toward understanding the mechanisms of reproduction and early development in angiosperms.

  7. Identification of Proteins Enriched in Rice Egg or Sperm Cells by Single-Cell Proteomics

    PubMed Central

    Abiko, Mafumi; Furuta, Kensyo; Yamauchi, Yoshio; Fujita, Chiharu; Taoka, Masato; Isobe, Toshiaki; Okamoto, Takashi

    2013-01-01

    In angiosperms, female gamete differentiation, fertilization, and subsequent zygotic development occur in embryo sacs deeply embedded in the ovaries. Despite their importance in plant reproduction and development, how the egg cell is specialized, fuses with the sperm cell, and converts into an active zygote for early embryogenesis remains unclear. This lack of knowledge is partly attributable to the difficulty of direct analyses of gametes in angiosperms. In the present study, proteins from egg and sperm cells obtained from rice flowers were separated by one-dimensional polyacrylamide gel electrophoresis and globally identified by highly sensitive liquid chromatography coupled with tandem mass spectroscopy. Proteome analyses were also conducted for seedlings, callus, and pollen grains to compare their protein expression profiles to those of gametes. The proteomics data have been deposited to the ProteomeXchange with identifier PXD000265. A total of 2,138 and 2,179 expressed proteins were detected in egg and sperm cells, respectively, and 102 and 77 proteins were identified as preferentially expressed in egg and sperm cells, respectively. Moreover, several rice or Arabidopsis lines with mutations in genes encoding the putative gamete-enriched proteins showed clear phenotypic defects in seed set or seed development. These results suggested that the proteomic data presented in this study are foundational information toward understanding the mechanisms of reproduction and early development in angiosperms. PMID:23936051

  8. Identification of cytotoxic drugs that selectively target tumor cells with MYC overexpression.

    PubMed

    Frenzel, Anna; Zirath, Hanna; Vita, Marina; Albihn, Ami; Henriksson, Marie Arsenian

    2011-01-01

    Expression of MYC is deregulated in a wide range of human cancers, and is often associated with aggressive disease and poorly differentiated tumor cells. Identification of compounds with selectivity for cells overexpressing MYC would hence be beneficial for the treatment of these tumors. For this purpose we used cell lines with conditional MYCN or c-MYC expression, to screen a library of 80 conventional cytotoxic compounds for their ability to reduce tumor cell viability and/or growth in a MYC dependent way. We found that 25% of the studied compounds induced apoptosis and/or inhibited proliferation in a MYC-specific manner. The activities of the majority of these were enhanced both by c-MYC or MYCN over-expression. Interestingly, these compounds were acting on distinct cellular targets, including microtubules (paclitaxel, podophyllotoxin, vinblastine) and topoisomerases (10-hydroxycamptothecin, camptothecin, daunorubicin, doxorubicin, etoposide) as well as DNA, RNA and protein synthesis and turnover (anisomycin, aphidicholin, gliotoxin, MG132, methotrexate, mitomycin C). Our data indicate that MYC overexpression sensitizes cells to disruption of specific pathways and that in most cases c-MYC and MYCN overexpression have similar effects on the responses to cytotoxic compounds. Treatment of the cells with topoisomerase I inhibitors led to down-regulation of MYC protein levels, while doxorubicin and the small molecule MYRA-A was found to disrupt MYC-Max interaction. We conclude that the MYC pathway is only targeted by a subset of conventional cytotoxic drugs currently used in the clinic. Elucidating the mechanisms underlying their specificity towards MYC may be of importance for optimizing treatment of tumors with MYC deregulation. Our data also underscores that MYC is an attractive target for novel therapies and that cellular screenings of chemical libraries can be a powerful tool for identifying compounds with a desired biological activity.

  9. Dynamic fuel cell stack model for real-time simulation based on system identification

    NASA Astrophysics Data System (ADS)

    Meiler, M.; Schmid, O.; Schudy, M.; Hofer, E. P.

    The authors have been developing an empirical mathematical model to predict the dynamic behaviour of a polymer electrolyte membrane fuel cell (PEMFC) stack. Today there is a great number of models, describing steady-state behaviour of fuel cells by estimating the equilibrium voltage for a certain set of operating parameters, but models capable of predicting the transient process between two steady-state points are rare. However, in automotive applications round about 80% of operating situations are dynamic. To improve the reliability of fuel cell systems by model-based control for real-time simulation dynamic fuel cell stack model is needed. Physical motivated models, described by differential equations, usually are complex and need a lot of computing time. To meet the real-time capability the focus is set on empirical models. Fuel cells are highly nonlinear systems, so often used auto-regressive (AR), output-error (OE) or Box-Jenkins (BJ) models do not accomplish satisfying accuracy. Best results are achieved by splitting the behaviour into a nonlinear static and a linear dynamic subsystem, a so-called Uryson-Model. For system identification and model validation load steps with different amplitudes are applied to the fuel cell stack at various operation points and the voltage response is recorded. The presented model is implemented in MATLAB environment and has a computing time of less than 1 ms per step on a standard desktop computer with a 2.8 MHz CPU and 504 MB RAM. Lab tests are carried out at DaimlerChrysler R&D Centre with DaimlerChrysler PEMFC hardware and a good agreement is found between model simulations and lab tests.

  10. Identification of cell-specific targets of sumoylation during mouse spermatogenesis

    PubMed Central

    Xiao, Yuxuan; Pollack, Daniel; Andrusier, Miriam; Levy, Avi; Callaway, Myrasol; Nieves, Edward; Reddi, Prabhakara; Vigodner, Margarita

    2015-01-01

    Recent findings suggest diverse and potentially multiple roles of SUMO in testicular function and spermatogenesis. However, SUMO targets remain uncharacterized in the testis due to the complex multicellular nature of testicular tissue, the inability to maintain and manipulate spermatogenesis in vitro, and the technical challenges involved in identifying low-abundance endogenous SUMO targets. In this study, we performed cell-specific identification of sumoylated proteins using concentrated cell lysates prepared with de-sumoylation inhibitors from freshly purified spermatocytes and spermatids. One-hundred and twenty proteins were uniquely identified in the spermatocyte and/or spermatid fractions. The identified proteins are involved in the regulation of transcription, stress response, microRNA biogenesis, regulation of major enzymatic pathways, nuclear-cytoplasmic transport, cell cycle control, acrosome biogenesis, and other processes. Several proteins with important roles during spermatogenesis were chosen for further characterization by co-immunoprecipitation, co-localization and in-vitro sumoylation studies. GPS-SUMO software was used to identify consensus and non-consensus sumoylation sites within the amino acid sequences of the proteins. The analyses confirmed the cell-specific sumoylation and/or SUMO interaction of several novel, previously uncharacterized SUMO targets such as CDK1, RNAP II, CDC5, MILI, DDX4, TDP-43 and STK31. Furthermore, several proteins that were previously identified as SUMO targets in somatic cells (e.g., KAP1, MDC1) were identified as SUMO targets in germ cells. Many of these proteins have a unique role in spermatogenesis and during meiotic progression. This research opens a novel avenue for further studies of SUMO at the level of individual targets. PMID:26701181

  11. Identification of cell-specific targets of sumoylation during mouse spermatogenesis.

    PubMed

    Xiao, Yuxuan; Pollack, Daniel; Andrusier, Miriam; Levy, Avi; Callaway, Myrasol; Nieves, Edward; Reddi, Prabhakara; Vigodner, Margarita

    2016-02-01

    Recent findings suggest diverse and potentially multiple roles of small ubiquitin-like modifier (SUMO) in testicular function and spermatogenesis. However, SUMO targets remain uncharacterized in the testis due to the complex multicellular nature of testicular tissue, the inability to maintain and manipulate spermatogenesis in vitro, and the technical challenges involved in identifying low-abundance endogenous SUMO targets. In this study, we performed cell-specific identification of sumoylated proteins using concentrated cell lysates prepared with de-sumoylation inhibitors from freshly purified spermatocytes and spermatids. One-hundred and twenty proteins were uniquely identified in the spermatocyte and/or spermatid fractions. The identified proteins are involved in the regulation of transcription, stress response, microRNA biogenesis, regulation of major enzymatic pathways, nuclear-cytoplasmic transport, cell-cycle control, acrosome biogenesis, and other processes. Several proteins with important roles during spermatogenesis were chosen for further characterization by co-immunoprecipitation, co-localization, and in vitro sumoylation studies. GPS-SUMO Software was used to identify consensus and non-consensus sumoylation sites within the amino acid sequences of the proteins. The analyses confirmed the cell-specific sumoylation and/or SUMO interaction of several novel, previously uncharacterized SUMO targets such as CDK1, RNAP II, CDC5, MILI, DDX4, TDP-43, and STK31. Furthermore, several proteins that were previously identified as SUMO targets in somatic cells (KAP1 and MDC1) were identified as SUMO targets in germ cells. Many of these proteins have a unique role in spermatogenesis and during meiotic progression. This research opens a novel avenue for further studies of SUMO at the level of individual targets.

  12. Development and feasibility of an electronic white blood cell identification trainer.

    PubMed

    Haun, Daniel E; Foley, Angela B; Jarreau, Patsy C

    2013-01-01

    A prototype computer-based training tool to improve WBC identification skills was developed. Students were assigned to complete five simulated WBC differentials but were allowed ample free time to use the tool at will to complete additional cases and to use the software in two alternative learning modes. The assignment was made at the end of the traditional WBC differential training activities in the first semester of hematology in the clinical laboratory science curriculum. The tool recorded usage data during the one month that students had access. Student performance was compared to the consensus results from an expert panel of hematology instructors. Usage tracking data was extracted and reviewed. The performance data indicated that students varied in WBC identification skill on the assignment. The usage tracking data showed that students used the tool only slightly more than the assigned cases and did not use alternative learning modes. Data from the expert panel indicated that the experts varied greatly in the number of discrepancies from the consensus opinion. Item analysis indicated the cell types that were most problematic. The prototype experience prompted the creation of a revised subsequent version of the trainer that is now being evaluated in our CLS program. The new trainer is web-based offering personal computer and mobile device access.

  13. Identification of Arsenic Direct-Binding Proteins in Acute Promyelocytic Leukaemia Cells

    PubMed Central

    Zhang, Tao; Lu, Haojie; Li, Weijun; Hu, Ronggui; Chen, Zi

    2015-01-01

    The identification of arsenic direct-binding proteins is essential for determining the mechanism by which arsenic trioxide achieves its chemotherapeutic effects. At least two cysteines close together in the amino acid sequence are crucial to the binding of arsenic and essential to the identification of arsenic-binding proteins. In the present study, arsenic binding proteins were pulled down with streptavidin and identified using a liquid chromatograph-mass spectrometer (LC-MS/MS). More than 40 arsenic-binding proteins were separated, and redox-related proteins, glutathione S-transferase P1 (GSTP1), heat shock 70 kDa protein 9 (HSPA9) and pyruvate kinase M2 (PKM2), were further studied using binding assays in vitro. Notably, PKM2 has a high affinity for arsenic. In contrast to PKM2, GSTP1and HSPA9 did not combine with arsenic directly in vitro. These observations suggest that arsenic-mediated acute promyelocytic leukaemia (APL) suppressive effects involve PKM2. In summary, we identified several arsenic binding proteins in APL cells and investigated the therapeutic mechanisms of arsenic trioxide for APL. Further investigation into specific signal pathways by which PKM2 mediates APL developments may lead to a better understanding of arsenic effects on APL. PMID:26569224

  14. Isolation of epithelial cells from tooth brush and gender identification by amplification of SRY gene

    PubMed Central

    Reddy, Vikram Simha A; Sriram, G; Saraswathi, TR; Sivapathasundharam, B

    2011-01-01

    Introduction: This study determines the importance of tooth brush from which DNA can be isolated and used for sex determination in forensic analysis. Materials and Methods: A total of 30 samples were collected and stored at room temperature for different periods of time interval. The epithelial cells adhered to the bristles of tooth brush were collected and genomic DNA was extracted and quantified using Nanodrop 1000 spectrophotometer. Results: Gender identification was done by amplification of sex determining region on Y chromosome (SRY) gene using real-time polymerase chain reaction and minimal amount of DNA (in pico grams) with 100% sensitivity and 73.3% specificity, i.e., all male samples showed positive results and out of 15 female samples 4 showed false positive results, i.e wrongly identified as males. Conclusion: With this study, we conclude that PCR is a valuable and sensitive procedure where minute contamination may cause alteration in the result, i.e, 4 females showed false positive result. Minute amount of DNA in picograms, which was collected at different intervals is enough for amplification of SRY gene and tooth brush can be used as one of the very valuable sources of gender identification. PMID:22022136

  15. Identification of novel bacterial histidine biosynthesis inhibitors using docking, ensemble rescoring and whole cell assays

    PubMed Central

    Henriksen, S. T.; Liu, J.; Estiu, G.; Oltvai, Z. N.; Wiest, O.

    2010-01-01

    The rapid spread on multi-drug resistant strains of Staphylococcus aureus requires not just novel treatment options, but the development of faster methods for the identification of new hits for drug development. The exponentially increasing speed of computational methods makes a more extensive use in the early stages of drug discovery attractive if sufficient accuracy can be achieved. Computational target identification using systems-level methods suggested the histidine biosynthesis pathway as an attractive target against S. aureus. Potential inhibitors for the pathway were identified through docking, followed by ensemble rescoring that is sufficiently accurate to justify immediate testing of the identified compounds by whole cell assays, avoiding the need for time-consuming and often difficult intermediary enzyme assays. This novel strategy is demonstrated for three key enzymes of the S. aureus histidine biosynthesis pathway, which is predicted to be essential for bacterial biomass productions. Virtual screening of a library of ~106 compounds identified 49 potential inhibitors of three enzymes of this pathway. 18 representative compounds were directly tested on three S. aureus-and two E. coli strains in standard disc inhibition assays. 13 compounds are inhibitors of some or all of the S. aureus strains, while 14 compounds weakly inhibit growth in one or both E. coli strains. The high hit rate obtained from a fast virtual screen demonstrates the applicability of this novel strategy to the histidine biosynthesis pathway. PMID:20573514

  16. Identification of novel bacterial histidine biosynthesis inhibitors using docking, ensemble rescoring, and whole-cell assays.

    PubMed

    Henriksen, S T; Liu, J; Estiu, G; Oltvai, Z N; Wiest, O

    2010-07-15

    The rapid spread on multidrug-resistant strains of Staphylococcus aureus requires not just novel treatment options, but the development of faster methods for the identification of new hits for drug development. The exponentially increasing speed of computational methods makes a more extensive use in the early stages of drug discovery attractive if sufficient accuracy can be achieved. Computational target identification using systems-level methods suggested the histidine biosynthesis pathway as an attractive target against S. aureus. Potential inhibitors for the pathway were identified through docking, followed by ensemble rescoring, that is sufficiently accurate to justify immediate testing of the identified compounds by whole-cell assays, avoiding the need for time-consuming and often difficult intermediary enzyme assays. This novel strategy is demonstrated for three key enzymes of the S. aureus histidine biosynthesis pathway, which is predicted to be essential for bacterial biomass productions. Virtual screening of a library of approximately 10(6) compounds identified 49 potential inhibitors of three enzymes of this pathway. Eighteen representative compounds were directly tested on three S. aureus- and two Escherichia coli strains in standard disk inhibition assays. Thirteen compounds are inhibitors of some or all of the S. aureus strains, while 14 compounds weakly inhibit growth in one or both E. coli strains. The high hit rate obtained from a fast virtual screen demonstrates the applicability of this novel strategy to the histidine biosynthesis pathway.

  17. Identification of inhibitors using a cell based assay for monitoring golgi-resident protease activity

    PubMed Central

    Coppola, Julia M.; Hamilton, Christin A.; Bhojani, Mahaveer S.; Larsen, Martha J.; Ross, Brian D.; Rehemtulla, Alnawaz

    2007-01-01

    Non-invasive real time quantification of cellular protease activity allows monitoring of enzymatic activity and identification of activity modulators within the protease’s natural milieu. We developed a protease-activity assay based on differential localization of a recombinant reporter consisting of a Golgi retention signal and a protease cleavage sequence fused to alkaline phosphatase (AP). When expressed in mammalian cells, this protein localizes to Golgi bodies and, upon protease mediated cleavage, AP translocates to the extracellular medium where its activity is measured. We used this system to monitor the Golgi-associated protease furin, a pluripotent enzyme with a key role in tumorigenesis, viral propagation of avian influenza, ebola, and HIV, and in activation of anthrax, pseudomonas, and diphtheria toxins. This technology was adapted for high throughput screening of 30,000 compound small molecule libraries, leading to identification of furin inhibitors. Further, this strategy was utilized to identify inhibitors of another Golgi protease, the β-site APP-cleaving enzyme (BACE). BACE cleavage of the amyloid precursor protein leads to formation of the Aβ peptide, a key event that leads to Alzheimer’s disease. In conclusion, we describe a customizable, non-invasive technology for real time assessment of Golgi protease activity used to identify inhibitors of furin and BACE. PMID:17316541

  18. Identification of Host Cell Factors Associated with Astrovirus Replication in Caco-2 Cells

    PubMed Central

    Murillo, Andrea; Vera-Estrella, Rosario; Barkla, Bronwyn J.; Méndez, Ernesto

    2015-01-01

    ABSTRACT Astroviruses are small, nonenveloped viruses with a single-stranded positive-sense RNA genome causing acute gastroenteritis in children and immunocompromised patients. Since positive-sense RNA viruses have frequently been found to replicate in association with membranous structures, in this work we characterized the replication of the human astrovirus serotype 8 strain Yuc8 in Caco-2 cells, using density gradient centrifugation and free-flow zonal electrophoresis (FFZE) to fractionate cellular membranes. Structural and nonstructural viral proteins, positive- and negative-sense viral RNA, and infectious virus particles were found to be associated with a distinct population of membranes separated by FFZE. The cellular proteins associated with this membrane population in infected and mock-infected cells were identified by tandem mass spectrometry. The results indicated that membranes derived from multiple cell organelles were present in the population. Gene ontology and protein-protein interaction network analysis showed that groups of proteins with roles in fatty acid synthesis and ATP biosynthesis were highly enriched in the fractions of this population in infected cells. Based on this information, we investigated by RNA interference the role that some of the identified proteins might have in the replication cycle of the virus. Silencing of the expression of genes involved in cholesterol (DHCR7, CYP51A1) and fatty acid (FASN) synthesis, phosphatidylinositol (PI4KIIIβ) and inositol phosphate (ITPR3) metabolism, and RNA helicase activity (DDX23) significantly decreased the amounts of Yuc8 genomic and antigenomic RNA, synthesis of the structural protein VP90, and virus yield. These results strongly suggest that astrovirus RNA replication and particle assembly take place in association with modified membranes potentially derived from multiple cell organelles. IMPORTANCE Astroviruses are common etiological agents of acute gastroenteritis in children and

  19. Identification of a long non-coding RNA gene, growth hormone secretagogue receptor opposite strand, which stimulates cell migration in non-small cell lung cancer cell lines.

    PubMed

    Whiteside, Eliza J; Seim, Inge; Pauli, Jana P; O'Keeffe, Angela J; Thomas, Patrick B; Carter, Shea L; Walpole, Carina M; Fung, Jenny N T; Josh, Peter; Herington, Adrian C; Chopin, Lisa K

    2013-08-01

    The molecular mechanisms involved in non‑small cell lung cancer tumourigenesis are largely unknown; however, recent studies have suggested that long non-coding RNAs (lncRNAs) are likely to play a role. In this study, we used public databases to identify an mRNA-like, candidate long non-coding RNA, GHSROS (GHSR opposite strand), transcribed from the antisense strand of the ghrelin receptor gene, growth hormone secretagogue receptor (GHSR). Quantitative real-time RT-PCR revealed higher expression of GHSROS in lung cancer tissue compared to adjacent, non-tumour lung tissue. In common with many long non-coding RNAs, GHSROS is 5' capped and 3' polyadenylated (mRNA-like), lacks an extensive open reading frame and harbours a transposable element. Engineered overexpression of GHSROS stimulated cell migration in the A549 and NCI-H1299 non-small cell lung cancer cell lines, but suppressed cell migration in the Beas-2B normal lung-derived bronchoepithelial cell line. This suggests that GHSROS function may be dependent on the oncogenic context. The identification of GHSROS, which is expressed in lung cancer and stimulates cell migration in lung cancer cell lines, contributes to the growing number of non-coding RNAs that play a role in the regulation of tumourigenesis and metastatic cancer progression.

  20. Identification of metabolites of the cell-differentiating agent hexamethylene bisacetamide in humans.

    PubMed

    Callery, P S; Egorin, M J; Geelhaar, L A; Nayar, M S

    1986-10-01

    Hexamethylene bisacetamide, a compound which in vitro induces differentiation in a wide variety of human and animal cancer cell lines, is being investigated in phase I clinical trials. After i.v. administration of hexamethylene bisacetamide to humans, urine contained the parent compound and at least five metabolites formed by deacetylation and oxidation pathways. Identification of urinary metabolites was accomplished by gas chromatography-mass spectrometric analysis after isolation by ion exchange chromatography or extraction with ethyl acetate. Metabolites with amino or alcohol groups were trifluoroacetylated and acidic functional groups were esterified with 2,2,2-trifluoroethanol or methanol. The structure of each metabolite was confirmed by comparison with authentic standards. Metabolites identified included the major metabolite, 6-acetamidohexanoic acid; the monodeacetylated product, N-acetyl-1,6-diaminohexane; the bis-deacetylated diamine, 1,6-diaminohexane; and the amino acid, 6-aminohexanoic acid and its lactam, caprolactam.

  1. Identification of a breached fuel pin in the IEM (Interim Examination and Maintenance) cell

    SciTech Connect

    McGuinness, P.W.; Kalk, J.J.; Hicks, D.F.

    1987-01-01

    Novel methods were successfully employed to identify one breached fuel pin in a 217-pin fuel assembly. The assembly was an experiment that had been irradiated at the Fast Flux Test Facility (FFTF), an experimental liquid-metal reactor operated by Westinghouse Hanford Company for the US Department of Energy. A fuel assembly known to contain breached fuel pins was removed from the sodium-cooled FFTF reactor in November 1984. Later, this assembly was brought into the FFTF's Interim Examination and Maintenance (IEM) cell to be disassembled and, for the first time ever at FFTF, to identify a breached fuel pin. The synergistic evaluation of the four different verification techniques - visual examination, cladding swipe activity, wash water radiochemistry, and pin weight - provided rapid and positive identification. The capability to perform future detective work of this kind has been conclusively demonstrated.

  2. Identification of cardiomyocyte nuclei and assessment of ploidy for the analysis of cell turnover

    SciTech Connect

    Bergmann, Olaf; Zdunek, Sofia; Alkass, Kanar; Druid, Henrik; Bernard, Samuel; Frisen, Jonas

    2011-01-15

    Assays to quantify myocardial renewal rely on the accurate identification of cardiomyocyte nuclei. We previously {sup 14}C birth dated human cardiomyocytes based on the nuclear localization of cTroponins T and I. A recent report by Kajstura et al. suggested that cTroponin I is only localized to the nucleus in a senescent subpopulation of cardiomyocytes, implying that {sup 14}C birth dating of cTroponin T and I positive cell populations underestimates cardiomyocyte renewal in humans. We show here that the isolation of cell nuclei from the heart by flow cytometry with antibodies against cardiac Troponins T and I, as well as pericentriolar material 1 (PCM-1), allows for isolation of close to all cardiomyocyte nuclei, based on ploidy and marker expression. We also present a reassessment of cardiomyocyte ploidy, which has important implications for the analysis of cell turnover, and iododeoxyuridine (IdU) incorporation data. These data provide the foundation for reliable analysis of cardiomyocyte turnover in humans.

  3. A portable Raman acoustic levitation spectroscopic system for the identification and environmental monitoring of algal cells.

    PubMed

    Wood, Bayden R; Heraud, Philip; Stojkovic, Slobodanka; Morrison, Danielle; Beardall, John; McNaughton, Don

    2005-08-01

    We report the coupling of a portable Raman spectrometer to an acoustic levitation device to enable environmental monitoring and the potential taxonomic identification of microalgae. Spectra of living cells were recorded at 785 nm using a fiber-optic probe coupled to a portable Raman spectrometer. The spectra exhibit an excellent signal-to-noise ratio and clearly show bands from chlorophyll a and beta-carotene. Spectra of levitated photobleached microalgae clearly show a reduction in chlorophyll a concentration relative to beta-carotene after 10 min of exposure to a quartz halogen lamp. Spectra recorded from levitated nitrogen-limited cells also show a significant reduction in bands associated with chlorophyll a, as compared to nitrogen-replete cells. To investigate the diagnostic capability of the technique, four species of microalgae were analyzed. Good quality spectra of all four species were obtained showing varying ratios of beta-carotene to chlorophyll. The combination of an acoustic levitation device and a portable Raman spectrometer shows potential as a taxonomic and environmental monitoring tool with direct application to field studies in remote environments.

  4. Identification and characterization of a resident vascular stem/progenitor cell population in preexisting blood vessels.

    PubMed

    Naito, Hisamichi; Kidoya, Hiroyasu; Sakimoto, Susumu; Wakabayashi, Taku; Takakura, Nobuyuki

    2012-02-15

    Vasculogenesis, the in-situ assembly of angioblast or endothelial progenitor cells (EPCs), may persist into adult life, contributing to new blood vessel formation. However, EPCs are scattered throughout newly developed blood vessels and cannot be solely responsible for vascularization. Here, we identify an endothelial progenitor/stem-like population located at the inner surface of preexisting blood vessels using the Hoechst method in which stem cell populations are identified as side populations. This population is dormant in the steady state but possesses colony-forming ability, produces large numbers of endothelial cells (ECs) and when transplanted into ischaemic lesions, restores blood flow completely and reconstitutes de-novo long-term surviving blood vessels. Moreover, although surface markers of this population are very similar to conventional ECs, and they reside in the capillary endothelium sub-population, the gene expression profile is completely different. Our results suggest that this heterogeneity of stem-like ECs will lead to the identification of new targets for vascular regeneration therapy.

  5. Identification of retinal cells in in-vivo high resolution images

    NASA Astrophysics Data System (ADS)

    Rangel-Fonseca, Piero; Gómez-Vieyra, Armando; Malacara-Hernández, Daniel; Estrada-Rico, Julio C.; Hernández-Gómez, Geovanni

    2013-11-01

    Recent advances in the acquisition of in-vivo high resolution retinal images through the use of Adaptive Optics (AO) have allowed the identification of cellular structures such as cones and rods, in and out of the fovea, in such a way that their histological characteristics can be studied in-vivo and later compared to data obtained post-mortem. In this work, an algorithm is proposed for the detection of photoreceptors; it consists of two stages: Early Cell Detection (ECD), to detect all candidate cells, and Refinement of Cell Detection (RCD), to reduce over-detection of photoreceptors. The algorithm has been tested using synthetic and real images, the latter acquired with an Adaptive Optics Scanning Light Ophthalmoscope (AOSLO). The proposed algorithm was compared against the one developed by Li and Roorda, and both algorithms were tested on synthetic and real images, yielding similar algorithm performance on both kinds of images when they had only cones; however, the algorithm developed by Li and Roorda, when applied to real images having cones and rods, identifies photoreceptors in vascular tissue, in addition to showing low rod detection.

  6. Identification of Pancreatic Cancer Specific Cell-Surface Markers for Development of Targeting Ligands

    PubMed Central

    Morse, David L.; Hostetter, Galen; Balagurunathan, Yoganand; Gillies, Robert J.; Han, Haiyong

    2014-01-01

    Pancreatic cancer is generally detected at later stages with a poor prognosis and a high-mortality rate. Development of theranostic imaging agents that non-invasively target pancreatic cancer by gene expression and deliver therapies directly to malignant cells could greatly improve therapeutic outcomes. Small-peptide ligands that bind cell-surface proteins and are conjugated to imaging moieties have demonstrated efficacy in cancer imaging. Identification of cancer specific targets is a major bottleneck in the development of such agents. Herein, a method is presented that uses DNA microarray expression profiling of large sets of normal and cancer tissues to identify targets expressed in cancer but not expressed in relevant normal tissues. Identified targets are subsequently validated for protein expression using tissue microarray. Further validations are performed by quantifying expression in pancreatic cancer cells by quantitative real-time reverse-transcription polymerase chain reaction (qRT-PCR), by immunocytochemistry and immunohistochemistry and by reviewing data and literature in public databases. Validated targets are selected for ligand development based on the existence of a known ligand or by known structure activity relationships useful for development of novel ligands. PMID:20217597

  7. Identification of Genetic and Chemical Modulators of Zebrafish Mechanosensory Hair Cell Death

    PubMed Central

    Roberts, Brock; Linbo, Tor; Coffin, Allison B.; Knisely, Anna J.; Simon, Julian A.; Rubel, Edwin W.; Raible, David W.

    2008-01-01

    Inner ear sensory hair cell death is observed in the majority of hearing and balance disorders, affecting the health of more than 600 million people worldwide. While normal aging is the single greatest contributor, exposure to environmental toxins and therapeutic drugs such as aminoglycoside antibiotics and antineoplastic agents are significant contributors. Genetic variation contributes markedly to differences in normal disease progression during aging and in susceptibility to ototoxic agents. Using the lateral line system of larval zebrafish, we developed an in vivo drug toxicity interaction screen to uncover genetic modulators of antibiotic-induced hair cell death and to identify compounds that confer protection. We have identified 5 mutations that modulate aminoglycoside susceptibility. Further characterization and identification of one protective mutant, sentinel (snl), revealed a novel conserved vertebrate gene. A similar screen identified a new class of drug-like small molecules, benzothiophene carboxamides, that prevent aminoglycoside-induced hair cell death in zebrafish and in mammals. Testing for interaction with the sentinel mutation suggests that the gene and compounds may operate in different pathways. The combination of chemical screening with traditional genetic approaches is a new strategy for identifying drugs and drug targets to attenuate hearing and balance disorders. PMID:18454195

  8. Rapid single-cell detection and identification of pathogens by using surface-enhanced Raman spectroscopy.

    PubMed

    Dina, N E; Zhou, H; Colniţă, A; Leopold, N; Szoke-Nagy, T; Coman, C; Haisch, C

    2017-05-21

    For the successful treatment of infections, real-time analysis and enhanced multiplex capacity, sensitivity and cost-effectiveness of the developed detection method are critical. In this work, surface-enhanced Raman scattering (SERS) was employed with the final aim of identification and discrimination of pathogenic bacteria, based on their detected SERS fingerprint at the single-cell level. Several genera of bacteria that are found in most of the isolated infections in bacteraemia were successfully identified in less than 5 minutes without the use of antibodies or other specific receptors. The key element of the SERS direct detection platform is the SERS substrate, which combines easy production at low costs with a high enhancement enabling single-cell detection. The innovative approach of detection required the in situ synthesis of silver nanoparticles (NPs), ensuring an intimate contact with the bacterial membrane. This protocol provided a good reproducibility of the single-cell SERS spectra and was successfully applied both on Gram-negative and Gram-positive microorganisms (E. coli, M. morganii, E. lactis, L. casei). Thus, a label-free SERS-based biosensor for pathogen detection was developed with low costs, minimal sample preparation, high-accuracy and a very short analysis time of less than 5 min, which is crucial for infection diagnosis.

  9. Identification of Two New Mechanisms That Regulate Fruit Growth by Cell Expansion in Tomato

    PubMed Central

    Musseau, Constance; Just, Daniel; Jorly, Joana; Gévaudant, Frédéric; Moing, Annick; Chevalier, Christian; Lemaire-Chamley, Martine; Rothan, Christophe; Fernandez, Lucie

    2017-01-01

    Key mechanisms controlling fruit weight and shape at the levels of meristem, ovary or very young fruit have already been identified using natural tomato diversity. We reasoned that new developmental modules prominent at later stages of fruit growth could be discovered by using new genetic and phenotypic diversity generated by saturated mutagenesis. Twelve fruit weight and tissue morphology mutants likely affected in late fruit growth were selected among thousands of fruit size and shape EMS mutants available in our tomato EMS mutant collection. Their thorough characterization at organ, tissue and cellular levels revealed two major clusters controlling fruit growth and tissue morphogenesis either through (i) the growth of all fruit tissues through isotropic cell expansion or (ii) only the growth of the pericarp through anisotropic cell expansion. These likely correspond to new cell expansion modules controlling fruit growth and tissue morphogenesis in tomato. Our study therefore opens the way for the identification of new gene regulatory networks controlling tomato fruit growth and morphology. PMID:28659942

  10. In Situ Identification of CD44+/CD24− Cancer Cells in Primary Human Breast Carcinomas

    PubMed Central

    Perrone, Giuseppe; Gaeta, Laura Maria; Zagami, Mariagiovanna; Nasorri, Francesca; Coppola, Roberto; Borzomati, Domenico; Bartolozzi, Francesco; Altomare, Vittorio; Trodella, Lucio; Tonini, Giuseppe; Santini, Daniele; Cavani, Andrea; Muda, Andrea Onetti

    2012-01-01

    Breast cancer cells with the CD44+/CD24− phenotype have been reported to be tumourigenic due to their enhanced capacity for cancer development and their self-renewal potential. The identification of human tumourigenic breast cancer cells in surgical samples has recently received increased attention due to the implications for prognosis and treatment, although limitations exist in the interpretation of these studies. To better identify the CD44+/CD24− cells in routine surgical specimens, 56 primary breast carcinoma cases were analysed by immunofluorescence and confocal microscopy, and the results were compared using flow cytometry analysis to correlate the amount and distribution of the CD44+/CD24− population with clinicopathological features. Using these methods, we showed that the breast carcinoma cells displayed four distinct sub-populations based on the expression pattern of CD44 and CD24. The CD44+/CD24− cells were found in 91% of breast tumours and constituted an average of 6.12% (range, 0.11%–21.23%) of the tumour. A strong correlation was found between the percentage of CD44+/CD24− cells in primary tumours and distant metastasis development (p = 0.0001); in addition, there was an inverse significant association with ER and PGR status (p = 0.002 and p = 0.001, respectively). No relationship was evident with tumour size (T) and regional lymph node (N) status, differentiation grade, proliferative index or HER2 status. In a multivariate analysis, the percentage of CD44+/CD24− cancer cells was an independent factor related to metastasis development (p = 0.004). Our results indicate that confocal analysis of fluorescence-labelled breast cancer samples obtained at surgery is a reliable method to identify the CD44+/CD24− tumourigenic cell population, allowing for the stratification of breast cancer patients into two groups with substantially different relapse rates on the basis of CD44+/CD24− cell percentage. PMID:23028444

  11. Identification of microprocessor-dependent cancer cells allows screening for growth-sustaining micro-RNAs.

    PubMed

    Peric, D; Chvalova, K; Rousselet, G

    2012-04-19

    Micro-RNAs are deregulated in cancer cells, and some are either tumor suppressive or oncogenic. In addition, a link has been established between decreased expression of micro-RNAs and transformation, and several proteins of the RNA interference pathway have been shown to be haploinsufficient tumor suppressors. Oncogenic micro-RNAs (oncomiRs) could represent new therapeutic targets, and their identification is therefore crucial. However, structural and functional redundancy between micro-RNAs hampers approaches relying on individual micro-RNA inhibition. We reasoned that in cancer cells that depend on oncomiRs, impairing the micro-RNA pathway could lead to growth perturbation rather than increased tumorigenesis. Identifying such cells could allow functional analyses of individual micro-RNAs by complementation of the phenotypes observed upon global micro-RNA inhibition. Therefore, we developed episomal vectors coding for small hairpin RNAs targeting either Drosha or DGCR8, the two components of the microprocessor, the nuclear micro-RNA maturation complex. We identified cancer cell lines in which both vectors induced colony growth arrest. We then screened for individual micro-RNAs complementing this growth arrest, and identified miR-19a, miR-19b, miR-20a and miR-27b as major growth-sustaining micro-RNAs. However, the effect of miR-19a and miR-19b was only transient. In addition, embryonic stem cell-derived micro-RNAs with miR-20a seeds were much less efficient than miR-20a in sustaining cancer cell growth, a finding that contrasted with results obtained in stem cells. Finally, we showed that the tumor suppressor phosphatase and tensin homologue deleted on chromosome 10, a shared target of miR-19 and miR-20, was functionally involved in the growth arrest induced by microprocessor inhibition. We conclude that our approach allowed to identify microprocessor-dependent cancer cells, which could be used to screen for growth-sustaining micro-RNAs. This complementation screen

  12. Identification of Targetable HER2 Aberrations in Head and Neck Squamous Cell Carcinoma

    PubMed Central

    Birkeland, Andrew C.; Yanik, Megan; Tillman, Brittny N.; Scott, Megan V.; Foltin, Susan K.; Mann, Jacqueline E.; Michmerhuizen, Nicole L.; Ludwig, Megan L.; Sandelski, Morgan M.; Komarck, Christine M.; Carey, Thomas E.; Prince, Mark E.P.; Bradford, Carol R.; McHugh, Jonathan B.; Spector, Matthew E.; Brenner, J. Chad

    2016-01-01

    Importance HER2 is an important drug target in breast cancer, where anti-HER2 therapy has been shown to lead to improvements in disease recurrence and overall survival. HER2 status in head and neck squamous cell carcinoma (HNSCC) has not been well studied. Identification of HER2 positive tumors and characterization of response to HER2 therapy could lead to targeted treatment options in HNSCC. Objective To identify HER2 aberrations in HNSCCs and investigate potential for HER2 targeted therapy in HNSCCs. Design, Setting, and Participants Retrospective case series of patients with laryngeal and oral cavity SCC enrolled in the University of MichiganSPORE. Publically available sequencing data(TCGA) was reviewed to identify additional mutations and overexpression in HER2 in HNSCC. Established HNSCC cell lines were used for follow-up in vitro analysis. Interventions Using targeted, amplicon-based sequencing with the Oncomine Cancer Panel, we assessed the copy number and mutation status of commonly altered genes in HNSCCs. Immunohistochemical staining was performed on tissue microarrays of HNSCCs to assess expression of HER2. Western blotting for HNSCC cell line HER2 expression, and cell survival assays after treatment with HER2 inhibitors were performed. Main Outcomes and Measures Prevalence of HER2 genetic aberrations and HER2 overexpression in laryngeal and oral cavity squamous cell carcinomas (SCCs). Prevalence of HER2 aberrations in HNSCC in TCGA. HER2 protein expression in HNSCC cell lines. Response of HNSCC cell lines to targeted HER2 inhibitors. Results Forty-two laryngeal SCC samples were screened by targeted sequencing, of which 4 were positive for HER2 amplification. Two samples identified with sequencing showed HER2 overexpression on immunohistochemistry. Two of 94 oral cavity SCC samples were positive for HER2 on immunohistochemistry. Analysis of 288 patients from publicly available HNSCC sequencing data revealed 9 amplifications in HER2. Protein expression

  13. Flow cytometric analysis of mast cells from normal and pathological human bone marrow samples: identification and enumeration.

    PubMed Central

    Orfao, A.; Escribano, L.; Villarrubia, J.; Velasco, J. L.; Cerveró, C.; Ciudad, J.; Navarro, J. L.; San Miguel, J. F.

    1996-01-01

    In the present paper we have used a three-color immunofluorescence procedure combined with flow cytometry cell analysis and sorting for the identification and enumeration of human mast cells in both normal and pathological bone marrow samples. Our results show that bone marrow mast cells are clearly identifiable on the basis of their light-scatter properties and strong CD117 expression. These cells were negative for the CD34, CD38, and BB4 antigens. In addition, they were CD33+ and displayed a high reactivity for the anti-IgE monoclonal antibody. The identity of the CD117-strong+ cells (mast cells) was confirmed by both microscopic examination and flow cytometry analysis. The overall frequency of mast cells in the bone marrow samples analyzed in the present study was constantly lower than 1%. The lowest frequencies corresponded to normal human bone marrow samples (0.0080 +/- 0.0082%) and the highest to those patients suffering from indolent systemic mast cell disease (0.40 +/- 0.13%). In summary, our results show that the identification and enumeration of bone marrow mast cells can be achieved using multiparametric flow cytometry. Moreover, once identified, mast cells are suitable for being characterized from the phenotypic and the functional point of view, facilitating the comparison between normal and abnormal mast cells. Images Figure 3 PMID:8909239

  14. Identification of a Soluble Factor that Induces Cell Death and Growth Inhibition in Prostate Carcinoma Cells

    DTIC Science & Technology

    2010-02-01

    person shall be subject to any penalty for failing to comply with a collection of information if it does not display a currently valid OMB control number...investigation based upon their abundance, secretary potential and reported roles in cell growth control (these proteins are listed in Table 1 in this report...i.e., calcyclin, calvasculin, ubiquitin carboxyl-terminal hydrolase isozyme L1, transgelin-3, odorant -binding protein 2a precursor, tubulin-specific

  15. Identification and characterization of [6]-shogaol from ginger as inhibitor of vascular smooth muscle cell proliferation.

    PubMed

    Liu, Rongxia; Heiss, Elke H; Sider, Nadine; Schinkovitz, Andreas; Gröblacher, Barbara; Guo, Dean; Bucar, Franz; Bauer, Rudolf; Dirsch, Verena M; Atanasov, Atanas G

    2015-05-01

    Vascular smooth muscle cell (VSMC) proliferation is involved in the pathogenesis of cardiovascular disease, making the identification of new counteracting agents and their mechanisms of action relevant. Ginger and its constituents have been reported to improve cardiovascular health, but no studies exist addressing a potential interference with VSMC proliferation. The dichloromethane extract of ginger inhibited VSMC proliferation when monitored by resazurin metabolic conversion (IC50 = 2.5 μg/mL). The examination of major constituents from ginger yielded [6]-shogaol as the most active compound (IC50 = 2.7 μM). In the tested concentration range [6]-shogaol did not exhibit cytotoxicity toward VSMC and did not interfere with endothelial cell proliferation. [6]-shogaol inhibited DNA synthesis and induced accumulation of the VSMC in the G0 /G1 cell-cycle phase accompanied with activation of the nuclear factor-erythroid 2-related factor 2 (Nrf2)/HO-1 pathway. Since [6]-shogaol lost its antiproliferative activity in the presence of the heme oxygenase-1 (HO-1) inhibitor tin protoporphyrin IX, HO-1 induction appears to contribute to the antiproliferative effect. This study demonstrates for the first time inhibitory potential of ginger constituents on VSMC proliferation. The presented data suggest that [6]-shogaol exerts its antiproliferative effect through accumulation of cells in the G0 /G1 cell-cycle phase associated with activation of the Nrf2/HO-1 pathway. © 2015 The Authors. Molecular Nutrition & Food Research published by Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  16. 3D high-content screening for the identification of compounds that target cells in dormant tumor spheroid regions

    SciTech Connect

    Wenzel, Carsten; Riefke, Björn; Gründemann, Stephan; Krebs, Alice; Christian, Sven; Prinz, Florian; Osterland, Marc; Golfier, Sven; Räse, Sebastian; Ansari, Nariman; Esner, Milan; Bickle, Marc; Pampaloni, Francesco; Mattheyer, Christian; Stelzer, Ernst H.; Parczyk, Karsten; Prechtl, Stefan; Steigemann, Patrick

    2014-04-15

    Cancer cells in poorly vascularized tumor regions need to adapt to an unfavorable metabolic microenvironment. As distance from supplying blood vessels increases, oxygen and nutrient concentrations decrease and cancer cells react by stopping cell cycle progression and becoming dormant. As cytostatic drugs mainly target proliferating cells, cancer cell dormancy is considered as a major resistance mechanism to this class of anti-cancer drugs. Therefore, substances that target cancer cells in poorly vascularized tumor regions have the potential to enhance cytostatic-based chemotherapy of solid tumors. With three-dimensional growth conditions, multicellular tumor spheroids (MCTS) reproduce several parameters of the tumor microenvironment, including oxygen and nutrient gradients as well as the development of dormant tumor regions. We here report the setup of a 3D cell culture compatible high-content screening system and the identification of nine substances from two commercially available drug libraries that specifically target cells in inner MCTS core regions, while cells in outer MCTS regions or in 2D cell culture remain unaffected. We elucidated the mode of action of the identified compounds as inhibitors of the respiratory chain and show that induction of cell death in inner MCTS core regions critically depends on extracellular glucose concentrations. Finally, combinational treatment with cytostatics showed increased induction of cell death in MCTS. The data presented here shows for the first time a high-content based screening setup on 3D tumor spheroids for the identification of substances that specifically induce cell death in inner tumor spheroid core regions. This validates the approach to use 3D cell culture screening systems to identify substances that would not be detectable by 2D based screening in otherwise similar culture conditions. - Highlights: • Establishment of a novel method for 3D cell culture based high-content screening. • First reported high

  17. Molecular approaches for forensic cell type identification: On mRNA, miRNA, DNA methylation and microbial markers.

    PubMed

    Sijen, Titia

    2015-09-01

    Human biological traces have the potential to present strong evidence for placing a suspect at a crime scene. In cases, the activity that led to deposition of an individual's cellular material is increasingly disputed, for which the identification of cell types could be crucial. This review aims to give an overview of the possibilities of the employment of mRNA, miRNA, DNA methylation and microbial markers for tissue identification in a forensic context. The biological background that renders these markers tissue-specificity is considered, as this can affect data interpretation. Furthermore, the forensic relevance of inferring certain cell types is discussed, as are the various methodologies that can be applied. Forensic stains can carry minute amounts of cell material that may be degraded or polluted and most likely cell material of multiple sources will be present. The interpretational challenges that are imposed by this compromised state will be discussed as well.

  18. Identification of co-expressed gene signatures in mouse B1, marginal zone and B2 B-cell populations

    PubMed Central

    Mabbott, Neil A; Gray, David

    2014-01-01

    In mice, three major B-cell subsets have been identified with distinct functionalities: B1 B cells, marginal zone B cells and follicular B2 B cells. Here, we used the growing body of publicly available transcriptomics data to create an expression atlas of 84 gene expression microarray data sets of distinct mouse B-cell subsets. These data were subjected to network-based cluster analysis using BioLayout Express3D. Using this analysis tool, genes with related functions clustered together in discrete regions of the network graph and enabled the identification of transcriptional networks that underpinned the functional activity of distinct cell populations. Some gene clusters were expressed highly by most of the cell populations included in this analysis (such as those with activity related to house-keeping functions). Others contained genes with expression patterns specific to distinct B-cell subsets. While these clusters contained many genes typically associated with the activity of the cells they were specifically expressed in, many novel B-cell-subset-specific candidate genes were identified. A large number of uncharacterized genes were also represented in these B-cell lineage-specific clusters. Further analysis of the activities of these uncharacterized candidate genes will lead to the identification of novel B-cell lineage-specific transcription factors and regulators of B-cell function. We also analysed 36 microarray data sets from distinct human B-cell populations. These data showed that mouse and human germinal centre B cells shared similar transcriptional features, whereas mouse B1 B cells were distinct from proposed human B1 B cells. PMID:24032749

  19. Identification of Vulnerable Cell Types in Major Brain Disorders Using Single Cell Transcriptomes and Expression Weighted Cell Type Enrichment.

    PubMed

    Skene, Nathan G; Grant, Seth G N

    2016-01-01

    The cell types that trigger the primary pathology in many brain diseases remain largely unknown. One route to understanding the primary pathological cell type for a particular disease is to identify the cells expressing susceptibility genes. Although this is straightforward for monogenic conditions where the causative mutation may alter expression of a cell type specific marker, methods are required for the common polygenic disorders. We developed the Expression Weighted Cell Type Enrichment (EWCE) method that uses single cell transcriptomes to generate the probability distribution associated with a gene list having an average level of expression within a cell type. Following validation, we applied EWCE to human genetic data from cases of epilepsy, Schizophrenia, Autism, Intellectual Disability, Alzheimer's disease, Multiple Sclerosis and anxiety disorders. Genetic susceptibility primarily affected microglia in Alzheimer's and Multiple Sclerosis; was shared between interneurons and pyramidal neurons in Autism and Schizophrenia; while intellectual disabilities and epilepsy were attributable to a range of cell-types, with the strongest enrichment in interneurons. We hypothesized that the primary cell type pathology could trigger secondary changes in other cell types and these could be detected by applying EWCE to transcriptome data from diseased tissue. In Autism, Schizophrenia and Alzheimer's disease we find evidence of pathological changes in all of the major brain cell types. These findings give novel insight into the cellular origins and progression in common brain disorders. The methods can be applied to any tissue and disorder and have applications in validating mouse models.

  20. Markers for the identification of tendon-derived stem cells in vitro and tendon stem cells in situ - update and future development.

    PubMed

    Lui, Pauline Po Yee

    2015-06-02

    The efficacy of tendon-derived stem cells (TDSCs) for the promotion of tendon and tendon-bone junction repair has been reported in animal studies. Modulation of the tendon stem cell niche in vivo has also been reported to influence tendon structure. There is a need to have specific and reliable markers that can define TDSCs in vitro and tendon stem cells in situ for several reasons: to understand the basic biology of TDSCs and their subpopulations in vitro; to understand the identity, niches and functions of tendon/progenitor stem cells in vivo; to meet the governmental regulatory requirements for quality of TDSCs when translating the exciting preclinical findings into clinical trial/practice; and to develop new treatment strategies for mobilizing endogenous stem/progenitor cells in tendon. TDSCs were reported to express the common mesenchymal stem cell (MSC) markers and some embryonic stem cell (ESC) markers, and there were attempts to use these markers to label tendon stem cells in situ. Are these stem cell markers useful for the identification of TDSCs in vitro and tracking of tendon stem cells in situ? This review aims to discuss the values of the panel of MSC, ESC and tendon-related markers for the identification of TDSCs in vitro. Important factors influencing marker expression by TDSCs are discussed. The usefulness and limitations of the panel of MSC, ESC and tendon-related markers for tracking stem cells in tendon, especially tendon stem cells, in situ are then reviewed. Future research directions are proposed.

  1. Computational Identification of Key Regulators in Two Different Colorectal Cancer Cell Lines

    PubMed Central

    Wlochowitz, Darius; Haubrock, Martin; Arackal, Jetcy; Bleckmann, Annalen; Wolff, Alexander; Beißbarth, Tim; Wingender, Edgar; Gültas, Mehmet

    2016-01-01

    Transcription factors (TFs) are gene regulatory proteins that are essential for an effective regulation of the transcriptional machinery. Today, it is known that their expression plays an important role in several types of cancer. Computational identification of key players in specific cancer cell lines is still an open challenge in cancer research. In this study, we present a systematic approach which combines colorectal cancer (CRC) cell lines, namely 1638N-T1 and CMT-93, and well-established computational methods in order to compare these cell lines on the level of transcriptional regulation as well as on a pathway level, i.e., the cancer cell-intrinsic pathway repertoire. For this purpose, we firstly applied the Trinity platform to detect signature genes, and then applied analyses of the geneXplain platform to these for detection of upstream transcriptional regulators and their regulatory networks. We created a CRC-specific position weight matrix (PWM) library based on the TRANSFAC database (release 2014.1) to minimize the rate of false predictions in the promoter analyses. Using our proposed workflow, we specifically focused on revealing the similarities and differences in transcriptional regulation between the two CRC cell lines, and report a number of well-known, cancer-associated TFs with significantly enriched binding sites in the promoter regions of the signature genes. We show that, although the signature genes of both cell lines show no overlap, they may still be regulated by common TFs in CRC. Based on our findings, we suggest that canonical Wnt signaling is activated in 1638N-T1, but inhibited in CMT-93 through cross-talks of Wnt signaling with the VDR signaling pathway and/or LXR-related pathways. Furthermore, our findings provide indication of several master regulators being present such as MLK3 and Mapk1 (ERK2) which might be important in cell proliferation, migration, and invasion of 1638N-T1 and CMT-93, respectively. Taken together, we provide

  2. A simple metal staining procedure for identification and visualization of single cells by LA-ICP-MS.

    PubMed

    Herrmann, A J; Techritz, S; Jakubowski, N; Haase, A; Luch, A; Panne, U; Mueller, L

    2017-05-21

    High lateral resolution of metal detection in single cells by use of laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) demands powerful staining methods. In this work different staining procedures for the single cell analysis with LA-ICP-MS were optimized. An iridium intercalator was utilized to stain the cell nuclei whereas the whole cell was stained by the use of maleimido-mono-amide-DOTA (mDOTA) complexing lanthanide(iii) ions. The content of the artificially introduced metals per cell was quantified using a matrix matched calibration approach based on cellulose membranes onto which standards were spotted by a microarray spotter. Absolute metal stain amounts in the range of 2.34 to 9.81 femtomole per cell were determined. The metal staining procedures allow direct identification and visualization of single cells and their cell compartments by element microscopy without the use of bright field images of the sample.

  3. Utility of CD54, CD229, and CD319 for the identification of plasma cells in patients with clonal plasma cell diseases.

    PubMed

    Pojero, Fanny; Flores-Montero, Juan; Sanoja, Luzalba; Pérez, José Juan; Puig, Noemí; Paiva, Bruno; Bottcher, Sebastian; van Dongen, Jacques J M; Orfao, Alberto

    2016-01-01

    Multiparameter flow cytometry (MFC) identification and characterization of plasma cells (PCs) is a useful tool to support diagnosis, prognostication, and monitoring of PC diseases (PCD). Currently, the number of MFC markers suited for the identification of PC remains limited. Moreover, antibody therapies against PC-associated markers further compromise the utility of the most widely used reagents (e.g., CD38). Despite markers other than CD38 and CD138 are recognized as potentially useful PC-identification markers, no study has comparatively evaluated their performance in combination with CD38 and CD138. Here we compared the utility of CD229, CD54, and CD319 for the identification of normal and aberrant PCs. Bone marrow (BM) samples from 5 healthy controls, two noninfiltrated nonHodgkin lymphoma cases and 46 PCD patients plus 3 extraosseous plasmocytomas, and normal peripheral blood (PB) specimens, were studied. Our results showed adequate performance of all three markers once combined with CD38. In contrast, when combined with CD138 for the identification of PC, only CD229 provided a good discrimination between PCs and all other cells for all BM and PB samples analyzed; in contrast, CD54 and CD319 showed limited utility for the identification of PCs, mainly because of significant overlap of the staining for these two markers on PCs and other myeloid cells in the sample. From the three markers evaluated, CD229 may be considered as the most reliable marker to replace CD38 or CD138 for the identification of PCs in patients undergoing anti-CD38 or anti-CD138 therapy, until a better alternative is available. © 2015 International Clinical Cytometry Society.

  4. Advances towards reliable identification and concentration determination of rare cells in peripheral blood

    NASA Astrophysics Data System (ADS)

    Alemany Server, R.; Martens, D.; Jans, K.; Bienstman, P.; Hill, D.

    2016-03-01

    Through further development, integration and validation of micro-nano-bio and biophotonics systems FP7 CanDo is developing an instrument that will permit highly reproducible and reliable identification and concentration determination of rare cells in peripheral blood for two key societal challenges, early and low cost anti-cancer drug efficacy determination and cancer diagnosis/monitoring. A cellular link between the primary malignant tumour and the peripheral metastases, responsible for 90% of cancerrelated deaths, has been established in the form of circulating tumour cells (CTCs) in peripheral blood. Furthermore, the relatively short survival time of CTCs in peripheral blood means that their detection is indicative of tumour progression thereby providing in addition to a prognostic value an evaluation of therapeutic efficacy and early recognition of tumour progression in theranostics. In cancer patients however blood concentrations are very low (=1 CTC/1E9 cells) and current detection strategies are too insensitive, limiting use to prognosis of only those with advanced metastatic cancer. Similarly, problems occur in therapeutics with anti-cancer drug development leading to lengthy and costly trials often preventing access to market. The novel cell separation/Raman analysis technologies plus nucleic acid based molecular characterization of the CanDo platform will provide an accurate CTC count with high throughput and high yield meeting both key societal challenges. Being beyond the state of art it will lead to substantial share gains not just in the high end markets of drug discovery and cancer diagnostics but due to modular technologies also in others. Here we present preliminary DNA hybridization sensing results.

  5. Identification and Validation of Housekeeping Genes for Gene Expression Analysis of Cancer Stem Cells.

    PubMed

    Lemma, Silvia; Avnet, Sofia; Salerno, Manuela; Chano, Tokuhiro; Baldini, Nicola

    2016-01-01

    The characterization of cancer stem cell (CSC) subpopulation, through the comparison of the gene expression signature in respect to the native cancer cells, is particularly important for the identification of novel and more effective anticancer strategies. However, CSC have peculiar characteristics in terms of adhesion, growth, and metabolism that possibly implies a different modulation of the expression of the most commonly used housekeeping genes (HKG), like b-actin (ACTB). Although it is crucial to identify which are the most stable HKG genes to normalize the data derived from quantitative Real-Time PCR analysis to obtain robust and consistent results, an exhaustive validation of reference genes in CSC is still missing. Here, we isolated CSC spheres from different musculoskeletal sarcomas and carcinomas as a model to investigate on the stability of the mRNA expression of 15 commonly used HKG, in respect to the native cells. The selected genes were analysed for the variation coefficient and compared using the popular algorithms NormFinder and geNorm to evaluate stability ranking. As a result, we found that: 1) Tata Binding Protein (TBP), Tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein zeta polypeptide (YWHAZ), Peptidylprolyl isomerase A (PPIA), and Hydroxymethylbilane synthase (HMBS) are the most stable HKG for the comparison between CSC and native cells; 2) at least four reference genes should be considered for robust results; 3) the use of ACTB should not be recommended, 4) specific HKG should be considered for studies that are focused only on a specific tumor type, like sarcoma or carcinoma. Our results should be taken in consideration for all the studies of gene expression analysis of CSC, and will substantially contribute for future investigations aimed to identify novel anticancer therapy based on CSC targeting.

  6. Identification, expansion and characterization of cancer cells with stem cell properties from head and neck squamous cell carcinomas.

    PubMed

    Kaseb, Hatem O; Fohrer-Ting, Helene; Lewis, Dale W; Lagasse, Eric; Gollin, Susanne M

    2016-10-15

    Head and neck squamous cell carcinoma (HNSCC) is a major public health concern. Recent data indicate the presence of cancer stem cells (CSC) in many solid tumors, including HNSCC. Here, we assessed the stem cell (SC) characteristics, including cell surface markers, radioresistance, chromosomal instability, and in vivo tumorigenic capacity of CSC isolated from HNSCC patient specimens. We show that spheroid enrichment of CSC from early and short-term HNSCC cell cultures was associated with increased expression of CD44, CD133, SOX2 and BMI1 compared with normal oral epithelial cells. On immunophenotyping, five of 12 SC/CSC markers were homogenously expressed in all tumor cultures, while one of 12 was negative, four of 12 showed variable expression, and two of the 12 were expressed heterogeneously. We showed that irradiated CSCs survived and retained their self-renewal capacity across different ionizing radiation (IR) regimens. Fluorescence in situ hybridization (FISH) analyses of parental and clonally-derived tumor cells revealed different chromosome copy numbers from cell to cell, suggesting the presence of chromosomal instability in HNSCC CSC. Further, our in vitro and in vivo mouse engraftment studies suggest that CD44+/CD66- is a promising, consistent biomarker combination for HNSCC CSC. Overall, our findings add further evidence to the proposed role of HNSCC CSCs in therapeutic resistance. Copyright © 2016 Elsevier Inc. All rights reserved.

  7. [Species-identification and antimicrobial susceptibility tests by the fully automated RAISUS using an early-harvested cell suspension].

    PubMed

    Nakasone, Isamu; Kisanuki, Kyoko; Higa, Miyako; Kinjo, Tohru; Yamane, Nobuhisa

    2006-01-01

    We evaluated the usefulness of an early-harvested bacterial cell suspension to the fully automated RAISUS (Nissui Pharmaceuticals Co., Ltd., Tokyo) to provide the results of species-identification and antimicrobial susceptibility testings within a day after overnight-incubation of the primary cultures. A single, well-separated colony appeared on the primary culture plate was transferred onto a blood agar or chocolate agar plates, then incubated for 3 to 6 hours. The cell suspension to the RAISUS was properly prepared to the McFarland 0.5 turbidity from the early-harvested bacterial cells. When the five ATCC reference strains, consisting of Staphylococcus aureus ATCC 29213, Enterococcus faecalis ATCC 29212, Streptococcus pneumoniae ATCC 49619, Escherichia coli ATCC 25922 and Pseudomonas aeruginosa ATCC 27853, were repeatedly tested for the species-identification, all the identification results were acceptable. Antimicrobial susceptibility tests were evaluated with the above five strains and Haemophilus influenzae ATCC 49247. The results obtained indicated that the most susceptibility test results were comparable to those MICs obtained by the standard test procedure, but some strains, in particular, H. influenzae and P. aeruginosa gave significantly discrepant MICs for certain antimicrobial agents. The significant discrepancy in MIC determinations regarded the difference of viable cell concentrations in the cell suspension prepared respectively. Through the analysis of laboratory workflow, it became to apparent that 18S to 20S of the tests were completed by 5:00 p.m., and it required to wait until 3:00 a.m. to complete 90S of the tests. With these results, the early-harvested bacterial cell suspension is applicable to species-identification by RAISUS, but it is necessary to adjust viable cell concentrations to antimicrobial susceptibility test. Also, it is urgent to reconstitute a daily workflow to improve the rapidity of RAISUS test function.

  8. Identification of CBX3 and ABCA5 as putative biomarkers for tumor stem cells in osteosarcoma.

    PubMed

    Saini, Vaibhav; Hose, Curtis D; Monks, Anne; Nagashima, Kunio; Han, Bingnan; Newton, Dianne L; Millione, Angelena; Shah, Jalpa; Hollingshead, Melinda G; Hite, Karen M; Burkett, Mark W; Delosh, Rene M; Silvers, Thomas E; Scudiero, Dominic A; Shoemaker, Robert H

    2012-01-01

    Recently, there has been renewed interest in the role of tumor stem cells (TSCs) in tumorigenesis, chemoresistance, and relapse of malignant tumors including osteosarcoma. The potential exists to improve osteosarcoma treatment through characterization of TSCs and identification of therapeutic targets. Using transcriptome, proteome, immunophenotyping for cell-surface markers, and bioinformatic analyses, heterogeneous expression of previously reported TSC or osteosarcoma markers, such as CD133, nestin, POU5F1 (OCT3/4), NANOG, SOX2, and aldehyde dehydrogenase, among others, was observed in vitro. However, consistently significantly lower CD326, CD24, CD44, and higher ABCG2 expression in TSC-enriched as compared with un-enriched osteosarcoma cultures was observed. In addition, consistently higher CBX3 expression in TSC-enriched osteosarcoma cultures was identified. ABCA5 was identified as a putative biomarker of TSCs and/or osteosarcoma. Lastly, in a high-throughput screen we identified epigenetic (5-azacytidine), anti-microtubule (vincristine), and anti-telomerase (3,11-difluoro-6,8,13-trimethyl- 8H-quino [4,3,2-kl] acridinium methosulfate; RHPS4)-targeted therapeutic agents as candidates for TSC ablation in osteosarcoma.

  9. Identification of CBX3 and ABCA5 as Putative Biomarkers for Tumor Stem Cells in Osteosarcoma

    PubMed Central

    Saini, Vaibhav; Hose, Curtis D.; Monks, Anne; Nagashima, Kunio; Han, Bingnan; Newton, Dianne L.; Millione, Angelena; Shah, Jalpa; Hollingshead, Melinda G.; Hite, Karen M.; Burkett, Mark W.; Delosh, Rene M.; Silvers, Thomas E.; Scudiero, Dominic A.; Shoemaker, Robert H.

    2012-01-01

    Recently, there has been renewed interest in the role of tumor stem cells (TSCs) in tumorigenesis, chemoresistance, and relapse of malignant tumors including osteosarcoma. The potential exists to improve osteosarcoma treatment through characterization of TSCs and identification of therapeutic targets. Using transcriptome, proteome, immunophenotyping for cell-surface markers, and bioinformatic analyses, heterogeneous expression of previously reported TSC or osteosarcoma markers, such as CD133, nestin, POU5F1 (OCT3/4), NANOG, SOX2, and aldehyde dehydrogenase, among others, was observed in vitro. However, consistently significantly lower CD326, CD24, CD44, and higher ABCG2 expression in TSC-enriched as compared with un-enriched osteosarcoma cultures was observed. In addition, consistently higher CBX3 expression in TSC-enriched osteosarcoma cultures was identified. ABCA5 was identified as a putative biomarker of TSCs and/or osteosarcoma. Lastly, in a high-throughput screen we identified epigenetic (5-azacytidine), anti-microtubule (vincristine), and anti-telomerase (3,11-difluoro-6,8,13-trimethyl- 8H-quino [4,3,2-kl] acridinium methosulfate; RHPS4)-targeted therapeutic agents as candidates for TSC ablation in osteosarcoma. PMID:22870217

  10. Identification of galectin-7 as a potential biomarker for esophageal squamous cell carcinoma by proteomic analysis

    PubMed Central

    2010-01-01

    Background Esophageal squamous cell carcinoma (ESCC) is one of the most common malignancies. Early diagnosis is critical for guiding the therapeutic management of ESCC. It is imperative to find more effective biomarkers of ESCC. Methods To identify novel biomarkers for esophageal squamous cell carcinoma (ESCC), specimens from 10 patients with ESCC were subjected to a comparative proteomic analysis. The proteomic patterns of ESCC samples and normal esophageal epithelial tissues (NEETs) were compared using two-dimensional gel electrophoresis. And differentially expressed proteins were identified using MALDI-TOF-MS/MS. For further identification of protein in selected spot, western blotting and immunohistochemistry were employed. Results Twelve proteins were up-regulated and fifteen proteins were down-regulated in the ESCC samples compared with the NEET samples. Up-regulation of galectin-7 was further confirmed by western blotting and immunohistochemistry. Furthermore, immunohistochemical staining of galectin-7 was performed on a tissue microarray containing ESCC samples (n = 50) and NEET samples (n = 10). The expression levels of galectin-7 were markedly higher in the ESCC samples than in the NEET samples (P = 0.012). In addition, tissue microarray analysis also showed that the expression level of galectin-7 was related to the differentiation of ESCC. Conclusions The present proteomics analysis revealed that galectin-7 was highly expressed in ESCC tissues. The alteration in the expression of galectin-7 was confirmed using a tissue microarray. These findings suggest that galectin-7 could be used as a potential biomarker for ESCC. PMID:20546628

  11. Cell Lysate-Based AlphaLISA Deubiquitinase Assay Platform for Identification of Small Molecule Inhibitors.

    PubMed

    Ott, Christine A; Baljinnyam, Bolormaa; Zakharov, Alexey V; Jadhav, Ajit; Simeonov, Anton; Zhuang, Zhihao

    2017-09-15

    The deubiquitinases, or DUBs, are associated with various human diseases, including neurological disorders, cancer, and viral infection, making them excellent candidates for pharmacological intervention. Drug discovery campaigns against DUBs require enzymatic deubiquitination assays amenable for high-throughput screening (HTS). Although several DUB substrates and assays have been developed in recent years, they are largely limited to recombinantly purified DUBs. Many DUBs are large multidomain proteins that are difficult to obtain recombinantly in sufficient quantities for HTS. Therefore, an assay that obviates the need of recombinant protein generation and also recapitulates a physiologically relevant environment is highly desirable. Such an assay will open doors for drug discovery against many therapeutically relevant, but currently inaccessible, DUBs. Here, we report a cell lysate DUB assay based on AlphaLISA technology for high throughput screening. This assay platform uses a biotin-tagged ubiquitin probe and a HA-tagged DUB expressed in human cells. The assay was validated and adapted to a 1536-well format, which enabled a screening against UCHL1 as proof of principle using a library of 15 000 compounds. We expect that the new platform can be readily adapted to other DUBs to allow the identification of more potent and selective small molecule inhibitors and chemical probes.

  12. Identification of a unique gene cluster of Brucella spp. that mediates adhesion to host cells.

    PubMed

    Czibener, Cecilia; Ugalde, Juan Esteban

    2012-01-01

    Brucella, the causative agent of brucellosis, a major zoonotic disease affecting a broad range of mammals, is a gram-negative bacterium whose virulence is dependent on the capacity to attach and invade different cells of the host. The bacterium is able to infect through a diverse repertoire of epitheliums: skin, airways or gastric. Although much has been studied on the mechanisms Brucella uses to establish an intracellular replication niche, almost none is known on how the bacterium adheres and invades host cells. We report here the identification of a pathogenicity island that harbors a gene homologous to proteins with bacterial immunoglobulin-like domains present in other pathogens that play a role in attachment and invasion. Deletion of the entire island results in a mutant with a reduced attachment capacity measured by intracellular replication and adhesion assays. Intraperitoneal and oral experimental infection of mice strongly suggests that this island plays a role during the oral infection probably mediating attachment and trespassing of the gastric epithelium to establish a systemic infection. Copyright © 2011 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.

  13. Identification of a unique gene cluster of Brucella spp. that mediates adhesion to host cells

    PubMed Central

    Czibener, Cecilia; Ugalde, Juan Esteban

    2011-01-01

    Brucella, the causative agent of brucellosis, a major zoonotic disease affecting a broad range of mammals, is a gram negative bacterium whose virulence is dependent on the capacity to attach and invade different cells of the host. The bacterium is able to infect through a diverse repertoire of epitheliums: skin, airways or gastric. Although much has been studied on the mechanisms Brucella uses to establish an intracellular replication niche, almost none is known on how the bacterium adheres and invades host cells. We report here the identification of a pathogenicity island that harbors a gene homologous to proteins with bacterial immunoglobulin-like domains present in other pathogens that play a role in attachment and invasion. Deletion of the entire island results in a mutant with a reduced attachment capacity measured by intracellular replication and adhesion assays. Intraperitoneal and oral experimental infection of mice strongly suggests that this island plays a role during the oral infection probably mediating attachment and trespassing of the gastric epithelium to establish a systemic infection. PMID:21911075

  14. Identification of novel transcriptional regulators involved in macrophage differentiation and activation in U937 cells.

    PubMed

    Baek, Young-Sook; Haas, Stefan; Hackstein, Holger; Bein, Gregor; Hernandez-Santana, Maria; Lehrach, Hans; Sauer, Sascha; Seitz, Harald

    2009-04-02

    Monocytes and macrophages play essential role in innate immunity. Understanding the underlying mechanism of macrophage differentiation and the identification of regulatory mechanisms will help to find new strategies to prevent their harmful effects in chronic inflammatory diseases and sepsis. Maturation of blood monocytes into tissue macrophages and subsequent inflammatory response was mimicked in U937 cells of human histocytic lymphoma origin. Whole genome array analysis was employed to evaluate gene expression profile to identify underlying transcriptional networks implicated during the processes of differentiation and inflammation. In addition to already known transcription factors (i.e. MAFB, EGR, IRF, BCL6, NFkB, AP1, Nur77), gene expression analysis further revealed novel genes (i.e. MEF2, BRI, HLX, HDAC5, H2AV, TCF7L2, NFIL3) previously uncharacterized to be involved in the differentiation process. A total of 58 selected genes representing cytokines, chemokines, surface antigens, signaling molecules and transcription factors were validated by real time PCR and compared to primary monocyte-derived macrophages. Beside the verification of several new genes, the comparison reveals individual heterogeneity of blood donors. Up regulation of MEF2 family, HDACs, and H2AV during cell differentiation and inflammation sheds new lights onto regulation events on transcriptional and epigenetic level controlling these processes. Data generated will serve as a source for further investigation of macrophages differentiation pathways and related biological responses.

  15. Identification of Serum Peptidome Signatures of Non-Small Cell Lung Cancer.

    PubMed

    Klupczynska, Agnieszka; Swiatly, Agata; Hajduk, Joanna; Matysiak, Jan; Dyszkiewicz, Wojciech; Pawlak, Krystian; Kokot, Zenon J

    2016-03-31

    Due to high mortality rates of lung cancer, there is a need for identification of new, clinically useful markers, which improve detection of this tumor in early stage of disease. In the current study, serum peptide profiling was evaluated as a diagnostic tool for non-small cell lung cancer patients. The combination of the ZipTip technology with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) for the analysis of peptide pattern of cancer patients (n = 153) and control subjects (n = 63) was presented for the first time. Based on the observed significant differences between cancer patients and control subjects, the classification model was created, which allowed for accurate group discrimination. The model turned out to be robust enough to discriminate a new validation set of samples with satisfactory sensitivity and specificity. Two peptides from the diagnostic pattern for non-small cell lung cancer (NSCLC) were identified as fragments of C3 and fibrinogen α chain. Since ELISA test did not confirm significant differences in the expression of complement component C3, further study will involve a quantitative approach to prove clinical utility of the other proteins from the proposed multi-peptide cancer signature.

  16. RNA-Seq for the identification of novel Mediator transcripts in endothelial progenitor cells.

    PubMed

    Rienzo, Monica; Costa, Valerio; Scarpato, Margherita; Schiano, Concetta; Casamassimi, Amelia; Grimaldi, Vincenzo; Ciccodicola, Alfredo; Napoli, Claudio

    2014-08-15

    Mediator (MED) complex is a multiprotein playing a key role in the eukaryotic transcription. Alteration of MED function may have enormous pathophysiological consequences and several MED genes have been implicated in human diseases. Here, we have combined computational and experimental approaches to identify and characterize, new transcripts generated by alternative splicing (AS) for all MED genes, through the analysis of our recently published RNA-Sequencing datasets of endothelial progenitor cells (EPCs). This combined strategy allowed us to identify novel transcripts for MED4, MED9, MED11, MED14, MED27 and CDK8 most of them generated by AS. All the newly identified transcripts, except MED11, are predicted to encode novel protein isoforms. The identification of novel MED variants could lead to the finding of other MED complexes with different functions depending on their subunit composition. Finally, the expression profile of all MED genes, together with an extensive gene expression analysis, may be useful to better classify the diverse subsets of cell populations that contribute to neovascularization. Copyright © 2014. Published by Elsevier B.V.

  17. Dynamic changes in energy metabolism upon embryonic stem cell differentiation support developmental toxicant identification.

    PubMed

    van Dartel, Dorien A M; Schulpen, Sjors H; Theunissen, Peter T; Bunschoten, Annelies; Piersma, Aldert H; Keijer, Jaap

    2014-10-03

    Embryonic stem cells (ESC) are widely used to study embryonic development and to identify developmental toxicants. Particularly, the embryonic stem cell test (EST) is well known as in vitro model to identify developmental toxicants. Although it is clear that energy metabolism plays a crucial role in embryonic development, the modulation of energy metabolism in in vitro models, such as the EST, is not yet described. The present study is among the first studies that analyses whole genome expression data to specifically characterize metabolic changes upon ESC early differentiation. Our transcriptomic analyses showed activation of glycolysis, truncated activation of the tricarboxylic acid (TCA) cycle, activation of lipid synthesis, as well as activation of glutaminolysis during the early phase of ESC differentiation. Taken together, this energy metabolism profile points towards energy metabolism reprogramming in the provision of metabolites for biosynthesis of cellular constituents. Next, we defined a gene set that describes this energy metabolism profile. We showed that this gene set could be successfully applied in the EST to identify developmental toxicants known to modulate cellular biosynthesis (5-fluorouracil and methoxyacetic acid), while other developmental toxicants or the negative control did not modulate the expression of this gene set. Our description of dynamic changes in energy metabolism during early ESC differentiation, as well as specific identification of developmental toxicants modulating energy metabolism, is an important step forward in the definition of the applicability domain of the EST. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

  18. Rapid identification by surface-enhanced Raman spectroscopy of cancer cells at low concentrations flowing in a microfluidic channel.

    PubMed

    Pallaoro, Alessia; Hoonejani, Mehran R; Braun, Gary B; Meinhart, Carl D; Moskovits, Martin

    2015-01-01

    Reliable identification and collection of cells from bodily fluids is of growing interest for monitoring patient response to therapy and for early detection of disease or its recurrence. We describe a detection platform that combines microfluidics with surface-enhanced Raman spectroscopy (SERS) for the identification of individual mammalian cells continuously flowing in a microfluidics channel. A mixture of cancerous and noncancerous prostate cells was incubated with SERS biotags (SBTs) developed and synthesized by us, then injected into a flow-focused microfluidic channel, which forces the cells into a single file. The spectrally rich SBTs are based on a silver nanoparticle dimer core labeled with a Raman-active small reporter molecule paired with an affinity biomolecule, providing a unique barcode whose presence in a composite SERS spectrum can be deconvoluted. Individual cancer cells passing through the focused laser beam were correctly identified among a proportionally larger number of other cells by their Raman signatures. We examine two deconvolution strategies: principal component analysis and classical least-squares. The deconvolution strategies are used to unmix the overall spectrum to determine the relative contributions between two SBT barcodes, where one SBT barcode indicates neuropilin-1 overexpression, while a second SBT barcode is more universal and indicates unspecific binding to a cell's membrane. Highly reliable results were obtained for all of the cell mixture ratios tested, the lowest being 1 in 100 cells.

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

    PubMed Central

    Kiniwa, Yukiko; Li, Jiang; Wang, Mingjun; Sun, Chuang; Lee, Jeffrey E.; Wang, Rong-Fu; Wang, Helen Y.

    2015-01-01

    Immunotherapy has emerged as a promising strategy for the treatment of metastatic melanoma. Clinical studies have demonstrated the feasibility of cancer immunotherapy using tumor antigens recognized by CD8+ T cells. However, the overall immune responses induced by these antigens are too weak and transient to induce tumor regression in the majority of patients who received immunization. A growing body of evidence suggests that CD4+ T helper (Th) cells play an important role in antitumor immunity. Therefore, the identification of MHC class II-restricted tumor antigens capable of stimulating CD4+ T cells may provide opportunities for developing effective cancer vaccines. To this end, we describe the identification of developmentally regulated GTP-binding protein 1 (DRG-1) as a melanoma-associated antigen recognized by HLA-DR11-restricted CD4+ Th1 cells. Epitope mapping analysis showed that the DRG1248-268 epitope of DRG-1 was required for T cell recognition. Reverse transcription-polymerase chain reaction revealed that DRG-1 was highly expressed in melanoma cell lines but not in normal tissues. DRG-1 knockdown by lentiviral-based shRNA suppressed melanoma cell proliferation and soft agar colony formation. Taken together, these data suggest that DRG-1 plays an important role in melanoma cell growth and transformation, indicating that DRG1 may represent a novel target for CD4+ T cell-mediated immunotherapy in melanoma. PMID:25993655

  20. Techniques for determining pressure in the hydrothermal diamond- anvil cell: behavior and identification of ice polymorphs (I, III, V, VI)

    USGS Publications Warehouse

    Haselton, H.T.; I-Ming, Chou; Shen, A.H.; Bassett, William A.

    1995-01-01

    For H2O densities > 1.0 g/cm3, a determination of the ice melting temperature provides the density information required to calculate the P-T path that the sample in a hydrothermal diamond-anvil cell follows when the sample is heated isochorically. The principal difficulty is the identification of the polymorph because of metastable behavior of ices in the H2O system. Usually, an accurate identification of the liquidus ice phase can be made without analytical instrumentation and requires only careful observations. -Authors

  1. Identification of Protein-Protein Interactions and Topologies in Living Cells with Chemical Cross-linking and Mass Spectrometry

    SciTech Connect

    Zhang, Haizhen; Tang, Xiaoting; Munske, Gerhard R.; Tolic, Nikola; Anderson, Gordon A.; Bruce, James E.

    2009-03-01

    We present results from a novel strategy that enables concurrent identification of protein-protein interactions and topologies in living cells without specific antibodies or genetic manipulations for immuno/affinity purifications. The strategy consists of: (i) chemical cross-linking reaction: intact cell labeling with a novel class of chemical cross-linkers, protein interaction reporters (PIRs); (ii) two-stage mass spectrometric analysis: stage 1 identification of PIR-labeled proteins and construction of a restricted database by 2D-LC/MS/MS; and stage 2 analysis of PIR-labeled peptides by multiplexed LC/FTICR-MS; (iii) data analysis: identification of cross-linked peptides and proteins of origin using accurate mass and other constraints. The primary advantage of the PIR approach and distinction from current technology is that protein interactions together with topologies are detected in native biological systems by stabilizing protein complexes with new covalent bonds while the proteins are present in the original cellular environment. Thus, weak or transient interactions or interactions that require properly folded, localized, or membrane-bound proteins can be labeled and identified through the PIR approach. This strategy was applied to S. oneidensis bacterial cells and initial studies resulted in identification of a set of protein-protein interactions and their contact/binding regions. Furthermore, most identified interactions involved membrane proteins, suggesting the PIR approach is particularly suited for studies of membrane protein-protein interactions, an area under-represented with current widely-used approaches.

  2. Identification of embryonic precursor cells that differentiate into thymic epithelial cells expressing autoimmune regulator

    PubMed Central

    Takizawa, Nobukazu; Miyauchi, Maki; Yanai, Hiromi; Tateishi, Ryosuke; Shinzawa, Miho; Yoshinaga, Riko; Kurihara, Masaaki; Yasuda, Hisataka; Sakamoto, Reiko; Yoshida, Nobuaki

    2016-01-01

    Medullary thymic epithelial cells (mTECs) expressing autoimmune regulator (Aire) are critical for preventing the onset of autoimmunity. However, the differentiation program of Aire-expressing mTECs (Aire+ mTECs) is unclear. Here, we describe novel embryonic precursors of Aire+ mTECs. We found the candidate precursors of Aire+ mTECs (pMECs) by monitoring the expression of receptor activator of nuclear factor-κB (RANK), which is required for Aire+ mTEC differentiation. pMECs unexpectedly expressed cortical TEC molecules in addition to the mTEC markers UEA-1 ligand and RANK and differentiated into mTECs in reaggregation thymic organ culture. Introduction of pMECs in the embryonic thymus permitted long-term maintenance of Aire+ mTECs and efficiently suppressed the onset of autoimmunity induced by Aire+ mTEC deficiency. Mechanistically, pMECs differentiated into Aire+ mTECs by tumor necrosis factor receptor-associated factor 6-dependent RANK signaling. Moreover, nonclassical nuclear factor-κB activation triggered by RANK and lymphotoxin-β receptor signaling promoted pMEC induction from progenitors exhibiting lower RANK expression and higher CD24 expression. Thus, our findings identified two novel stages in the differentiation program of Aire+ mTECs. PMID:27401343

  3. Identification of embryonic precursor cells that differentiate into thymic epithelial cells expressing autoimmune regulator.

    PubMed

    Akiyama, Nobuko; Takizawa, Nobukazu; Miyauchi, Maki; Yanai, Hiromi; Tateishi, Ryosuke; Shinzawa, Miho; Yoshinaga, Riko; Kurihara, Masaaki; Demizu, Yosuke; Yasuda, Hisataka; Yagi, Shintaro; Wu, Guoying; Matsumoto, Mitsuru; Sakamoto, Reiko; Yoshida, Nobuaki; Penninger, Josef M; Kobayashi, Yasuhiro; Inoue, Jun-Ichiro; Akiyama, Taishin

    2016-07-25

    Medullary thymic epithelial cells (mTECs) expressing autoimmune regulator (Aire) are critical for preventing the onset of autoimmunity. However, the differentiation program of Aire-expressing mTECs (Aire(+) mTECs) is unclear. Here, we describe novel embryonic precursors of Aire(+) mTECs. We found the candidate precursors of Aire(+) mTECs (pMECs) by monitoring the expression of receptor activator of nuclear factor-κB (RANK), which is required for Aire(+) mTEC differentiation. pMECs unexpectedly expressed cortical TEC molecules in addition to the mTEC markers UEA-1 ligand and RANK and differentiated into mTECs in reaggregation thymic organ culture. Introduction of pMECs in the embryonic thymus permitted long-term maintenance of Aire(+) mTECs and efficiently suppressed the onset of autoimmunity induced by Aire(+) mTEC deficiency. Mechanistically, pMECs differentiated into Aire(+) mTECs by tumor necrosis factor receptor-associated factor 6-dependent RANK signaling. Moreover, nonclassical nuclear factor-κB activation triggered by RANK and lymphotoxin-β receptor signaling promoted pMEC induction from progenitors exhibiting lower RANK expression and higher CD24 expression. Thus, our findings identified two novel stages in the differentiation program of Aire(+) mTECs.

  4. β -[18F]Fluoro Azomycin Arabinoside (β -[18F]FAZA): Synthesis, Radiofluorination and Preliminary PET Imaging of Murine A431 Tumors.

    PubMed

    Kumar, Piyush; Roselt, Peter; Reischl, Gerald; Cullinane, Carlene; Beiki, Davood; Ehrlichmann, Walter; Binns, David; Naimi, Ebrahim; Yang, Jennifer; Hicks, Rodney; Machulla, Hans-Juergen; Wiebe, Leonard I

    2017-01-01

    1-α-D-(5-Deoxy-5-[18F]fluoroarabinofuranosyl)-2-nitroimidazole([18F] FAZA) is a PET radiotracer that demonstrates excellent potential in imaging regional hypoxia, and is clinically used in diagnosing a wide range of solid tumors in cancer patients. [18F]FAZA, however, is radiofluorinated in only moderate recovered radiochemical yield (rRCY, ~12%). It is postulated that the relative stability of the C1' β-anomeric bond at C5' will make 1-β-D-(5-fluoro-5-deoxyarabinofuranosyl)-2-nitroimidazole (β-FAZA), the β-conformer of FAZA, an attractive candidate for clinical hypoxia imaging. The principle goals were to synthesize β-FAZA and β-Ac2TsAZA, the radiofluorination precursor, to establish the radiofluorination chemistry leading to β-[18F]FAZA, and to investigate the biodistribution of β-[18F]FAZA in an animal tumor-bearing model using PET imaging. The appropriately-protected furanose sugar was coupled with 2-nitroimidazole to afford 1-β-D-(2,3-di-O-acetylarabinofuranosyl)-2-nitroimidazole (β-Ac2AZA). Fluorination of β-Ac2AZA with DAST, followed by alkaline hydrolysis, afforded β-FAZA (21%). The radiolabeling synthon, 1-β-D-(5-O-toluenesulfonyl-2,3-di-O-acetylarabinofuranosyl)-2-nitroimidazole (β-Ac2TsAZA), on radiofluorination using the 18F/K222 complex under various reaction conditions, followed by base-catalyzed deacetylation, afforded β-[18F]FAZA. β-[18F]FAZA was radiochemically stable for at least 8 h when stored in aqueous ethanol (8%) at 22 °C. A preliminary PET imaging-based biodistribution study of β-[18F]FAZA was performed in A431 tumor-bearing nude mice. β-FAZA and β-Ac2TsAZA were synthesized in satisfactory yield. Radiochemistry of [18F]FAZA was established. PET images showed strong uptake in hypoxic regions of the tumor. The synthesis of β-FAZA and β-[18F]FAZA are reported. Radiofluorination of β-Ac2TsAZA and the deprotection of β-Ac2[18F]FAZA were facile, but led to a more complex mixture of radiofluorinated by-products than

  5. Single and multi-subject clustering of flow cytometry data for cell-type identification and anomaly detection.

    PubMed

    Pouyan, Maziyar Baran; Jindal, Vasu; Birjandtalab, Javad; Nourani, Mehrdad

    2016-08-10

    Measurement of various markers of single cells using flow cytometry has several biological applications. These applications include improving our understanding of behavior of cellular systems, identifying rare cell populations and personalized medication. A common critical issue in the existing methods is identification of the number of cellular populations which heavily affects the accuracy of results. Furthermore, anomaly detection is crucial in flow cytometry experiments. In this work, we propose a two-stage clustering technique for cell type identification in single subject flow cytometry data and extend it for anomaly detection among multiple subjects. Our experimentation on 42 flow cytometry datasets indicates high performance and accurate clustering (F-measure > 91 %) in identifying main cellular populations. Furthermore, our anomaly detection technique evaluated on Acute Myeloid Leukemia dataset results in only <2 % false positives.

  6. Context based mixture model for cell phase identification in automated fluorescence microscopy

    PubMed Central

    Wang, Meng; Zhou, Xiaobo; King, Randy W; Wong, Stephen TC

    2007-01-01

    Background Automated identification of cell cycle phases of individual live cells in a large population captured via automated fluorescence microscopy technique is important for cancer drug discovery and cell cycle studies. Time-lapse fluorescence microscopy images provide an important method to study the cell cycle process under different conditions of perturbation. Existing methods are limited in dealing with such time-lapse data sets while manual analysis is not feasible. This paper presents statistical data analysis and statistical pattern recognition to perform this task. Results The data is generated from Hela H2B GFP cells imaged during a 2-day period with images acquired 15 minutes apart using an automated time-lapse fluorescence microscopy. The patterns are described with four kinds of features, including twelve general features, Haralick texture features, Zernike moment features, and wavelet features. To generate a new set of features with more discriminate power, the commonly used feature reduction techniques are used, which include Principle Component Analysis (PCA), Linear Discriminant Analysis (LDA), Maximum Margin Criterion (MMC), Stepwise Discriminate Analysis based Feature Selection (SDAFS), and Genetic Algorithm based Feature Selection (GAFS). Then, we propose a Context Based Mixture Model (CBMM) for dealing with the time-series cell sequence information and compare it to other traditional classifiers: Support Vector Machine (SVM), Neural Network (NN), and K-Nearest Neighbor (KNN). Being a standard practice in machine learning, we systematically compare the performance of a number of common feature reduction techniques and classifiers to select an optimal combination of a feature reduction technique and a classifier. A cellular database containing 100 manually labelled subsequence is built for evaluating the performance of the classifiers. The generalization error is estimated using the cross validation technique. The experimental results show

  7. Registered report: Systematic identification of genomic markers of drug sensitivity in cancer cells

    PubMed Central

    Vanden Heuvel, John P; Bullenkamp, Jessica

    2016-01-01

    The Reproducibility Project: Cancer Biology seeks to address growing concerns about the reproducibility in scientific research by conducting replications of selected experiments from a number of high-profile papers in the field of cancer biology. The papers, which were published between 2010 and 2012, were selected on the basis of citations and Altmetric scores (Errington et al., 2014). This Registered Report describes the proposed replication plan of key experiments from “Systematic identification of genomic markers of drug sensitivity in cancer cells” by Garnett and colleagues, published in Nature in 2012 (Garnett et al., 2012). The experiments to be replicated are those reported in Figures 4C, 4E, 4F, and Supplemental Figures 16 and 20. Garnett and colleagues performed a high throughput screen assessing the effect of 130 drugs on 639 cancer-derived cell lines in order to identify novel interactions for possible therapeutic approaches. They then tested this approach by exploring in more detail a novel interaction they identified in which Ewing’s sarcoma cell lines showed an increased sensitivity to PARP inhibitors (Figure 4C). Mesenchymal progenitor cells (MPCs) transformed with the signature EWS-FLI1 translocation, the hallmark of Ewing’s sarcoma family tumors, exhibited increased sensitivity to the PARP inhibitor olaparib as compared to MPCs transformed with a different translocation (Figure 4E). Knockdown mediated by siRNA of EWS-FLI1 abrogated this sensitivity to olaparib (Figure 4F). The Reproducibility Project: Cancer Biology is a collaboration between the Center for Open Science and Science Exchange, and the results of the replications will be published by eLife. DOI: http://dx.doi.org/10.7554/eLife.13620.001 PMID:27336789

  8. Identification of novel markers that outperform EpCAM in quantifying circulating tumor cells.

    PubMed

    Kim, Min-Ji; Choi, Na Young; Lee, Eun Kyung; Kang, Myung-Soo

    2014-08-01

    Circulating tumor cells (CTCs) can be used to predict the spread of cancer to distant sites, to monitor the clinical response to therapy and to predict patient survival. The currently used EpCAM antibody-mediated identification of CTCs may lead to false negative results due to the low level or absence of EpCAM expression in several types of cancer, thus provoking a need to identify novel CTC markers. The Cancer Cell Line Encyclopedia (CCLE) microarray dataset, storing 18,915 gene expression profiles across 967 cancer cell lines derived from 25 primary sites, was systematically analyzed. The results obtained were cross-validated using an independent microarray dataset generated from 1,911 clinical cancer specimens derived from 15 different cancers. Through bioinformatics analyses we identified, categorized and prioritized three classes of novel markers: pan-CTC markers (n = 45), EpCAM((-/low)) CTC markers (n = 16) and single cancer type-specific markers (n = 74). The pan-CTC markers were significantly, uniformly and constitutively over-expressed in most cancer types, except in cancers of hematopoietic and lymphoid origin. The EpCAM((-/low)) CTC markers were over-expressed in cancers with low or undetectable EpCAM expression levels. Among these, 22 markers were validated in an independent microarray dataset. In addition, 74 markers that were over-expressed in only single cancer types were categorized. The combined use of these novel markers in conjunction with cancer type-specific markers should be able to quantify CTCs that are not captured by EpCAM antibodies, and to enhance the sensitivity and specificity of CTC detection among admixtures containing leucocytes or other types of contaminants.

  9. Identification of target genes of synovial sarcoma-associated fusion oncoprotein using human pluripotent stem cells

    SciTech Connect

    Hayakawa, Kazuo; Ikeya, Makoto; Fukuta, Makoto; Woltjen, Knut; Tamaki, Sakura; Takahara, Naoko; Kato, Tomohisa; Sato, Shingo; Otsuka, Takanobu; Toguchida, Junya

    2013-03-22

    Highlights: ► We tried to identify targets of synovial sarcoma (SS)-associated SYT–SSX fusion gene. ► We established pluripotent stem cell (PSC) lines with inducible SYT–SSX gene. ► SYT–SSX responsive genes were identified by the induction of SYT–SSX in PSC. ► SS-related genes were selected from database by in silico analyses. ► 51 genes were finally identified among SS-related genes as targets of SYT–SSX in PSC. -- Abstract: Synovial sarcoma (SS) is a malignant soft tissue tumor harboring chromosomal translocation t(X; 18)(p11.2; q11.2), which produces SS-specific fusion gene, SYT–SSX. Although precise function of SYT–SSX remains to be investigated, accumulating evidences suggest its role in gene regulation via epigenetic mechanisms, and the product of SYT–SSX target genes may serve as biomarkers of SS. Lack of knowledge about the cell-of-origin of SS, however, has placed obstacle in the way of target identification. Here we report a novel approach to identify SYT–SSX2 target genes using human pluripotent stem cells (hPSCs) containing a doxycycline-inducible SYT–SSX2 gene. SYT–SSX2 was efficiently induced both at mRNA and protein levels within three hours after doxycycline administration, while no morphological change of hPSCs was observed until 24 h. Serial microarray analyses identified genes of which the expression level changed more than twofold within 24 h. Surprisingly, the majority (297/312, 95.2%) were up-regulated genes and a result inconsistent with the current concept of SYT–SSX as a transcriptional repressor. Comparing these genes with SS-related genes which were selected by a series of in silico analyses, 49 and 2 genes were finally identified as candidates of up- and down-regulated target of SYT–SSX, respectively. Association of these genes with SYT–SSX in SS cells was confirmed by knockdown experiments. Expression profiles of SS-related genes in hPSCs and human mesenchymal stem cells (hMSCs) were strikingly

  10. Single cell time-lapse analysis reveals that podoplanin enhances cell survival and colony formation capacity of squamous cell carcinoma cells

    PubMed Central

    Miyashita, Tomoyuki; Higuchi, Youichi; Kojima, Motohiro; Ochiai, Atsushi; Ishii, Genichiro

    2017-01-01

    Tumor initiating cells (TICs) are characterized by high clonal expansion capacity. We previously reported that podoplanin is a TIC-specific marker for the human squamous cell carcinoma cell line A431. The aim of this study is to explore the molecular mechanism underlying the high clonal expansion potential of podoplanin-positive A431cells using Fucci imaging. Single podoplanin-positive cells created large colonies at a significantly higher frequency than single podoplanin-negative cells, whereas no difference was observed between the two types of cells with respect to cell cycle status. Conversely, the cell death ratio of progenies derived from podoplanin-positive single cell was significantly lower than that of cells derived from podoplanin-negative cells. Single A431 cells, whose podoplanin expression was suppressed by RNA interference, exhibited increased cell death ratios and decreased frequency of large colony forming. Moreover, the frequency of large colony forming decreased significantly when podoplanin-positive single cells was treated with a ROCK (Rho-associated coiled-coil kinase) inhibitor, whereas no difference was observed in single podoplanin-negative cells. Our current study cleared that high clonal expansion capacity of podoplanin-positive TICs populations was the result of reduced cell death by podoplanin-mediated signaling. Therefore, podoplanin activity may be a therapeutic target in the treatment of squamous cell carcinomas. PMID:28059107

  11. Corticotropin-Releasing Factor Increases Ca(2+)sub i via Receptor- Mediated Ca(2+) Channels in Human Epidermoid A-431 Cells

    DTIC Science & Technology

    1994-01-01

    8217 • r.006 O’c O"I4 1-s ".4 41 ww(". IRT OATE_- ’ 3 . REPOEr TYPE ANO DAY[S COVERED 4. TITLE AND SUIY171. S. FUNDING NUMBERS Corticotropin-releasing...ORGANIZATION NAM.E(S) AND ADOISES 3 EFRMN RANZTO Walter Reeed Army Institute of Research RIPORT NUMBER WashingtontoDC 20307-5100 9. SPONSORING/ MONITORING AGCNCv...of thetd NsiCd a11i.hamesO)alto• did am Moak the macs Is mC•n Tlacd by CR?. eRFI ab cumid [Cal He coels rate wi1*TMB-6oir Wsyanoim, kibbitaso

  12. Identification on Membrane and Characterization of Phosphoproteins Using an Alkoxide-Bridged Dinuclear Metal Complex as a Phosphate-Binding Tag Molecule

    PubMed Central

    Nakanishi, Tsuyoshi; Ando, Eiji; Furuta, Masaru; Kinoshita, Eiji; Kinoshita-Kikuta, Emiko; Koike, Tohru; Tsunasawa, Susumu; Nishimura, Osamu

    2007-01-01

    We have developed a method for on-membrane direct identification of phosphoproteins, which are detected by a phosphate-binding tag (Phos-tag) that has an affinity to phosphate groups with a chelated Zn2+ ion. This rapid profiling approach for phosphoproteins combines chemical inkjet technology for microdispensing of reagents onto a tiny region of target proteins with mass spectrometry for on-membrane digested peptides. Using this method, we analyzed human epidermoid carcinoma cell lysates of A-431 cells stimulated with epidermal growth factor, and identified six proteins with intense signals upon affinity staining with the phosphate-binding tag. It was already known that these proteins are phosphorylated, and our new approach proved to be effective at rapid profiling of phosphoproteins. Furthermore, we tried to determine their phosphorylation sites by MS/MS analysis after in-gel digestion of the corresponding spots on the 2DE gel to the rapid on-membrane identifications. As one example of use of information gained from the rapid-profiling approach, we successfully characterized a phosphorylation site at Ser-113 on prostaglandin E synthase 3. PMID:18166671

  13. Location and identification of colloidal gold particles on the cell surface with a scanning electron microscope and energy dispersive analyzer

    SciTech Connect

    Eskelinen, S.; Peura, R.

    1988-09-01

    The use of colloidal gold particles for locating cell surface components by scanning electron microscopy (SEM) has been restricted due to difficulties in the identification of these gold particles under SEM. It is shown here how the gold particles bound to cell surfaces can be located and identified under SEM using the secondary electron imaging (SEI) mode with an energy dispersive X-ray microanalyzer (EDS). This enables reliable identification of gold particles and good quality micrographs of the cells to be achieved at the same time. The distribution of receptors for two lectins, concanavalin A (ConA) and wheat germ agglutinin (WGA), on the surface of cultured Raji cells and human erythrocytes is presented as an example. Raji cells and erythrocytes were fixed with glutaraldehyde, post-fixed with a glutaraldehyde-tannic acid mixture and then incubated with ConA- or WGA-coated gold particles. After dehydration and critical point drying, the specimen filters were mounted on copper stubs and coated with carbon. The cells were examined on a JEOL TEMSCAN 100CX II electron microscope. The gold particles could be identified with the EDS analyzer, which was able to detect the Au spectrum when the electron beam was focused on single gold particles using a magnification of 100,000 or more. High-resolution photographs of the same cells were obtained up to the same magnification of 100,000.

  14. Staining histological lung sections with Sudan Black B or Sudan III for automated identification of alveolar epithelial type II cells.

    PubMed

    Schneider, Jan Philipp; Pedersen, Lars; Mühlfeld, Christian; Ochs, Matthias

    2015-10-01

    Alveolar epithelial type II (AE2) cells produce, store and secrete pulmonary surfactant and serve as progenitor cells for the alveolar epithelium. They are thus an interesting target in wide fields of pulmonary research. Stereological methods allow their quantification based on measurements on histological sections. A proper AE2 cell quantification, however, requires a method of tissue processing that results in little tissue shrinkage during processing. It was recently shown that a primary fixation with a mixture of glutaraldehyde and formaldehyde, postfixation with osmium tetroxide and uranyl acetate and embedding in glycol methacrylate fulfills this requirement. However, a proper quantification, furthermore, requires a secure identification of the cells under the microscope. Classical approaches using routine stainings, high magnifications and systematic uniform random sampling can result in a tedious counting procedure. In this article we show that Sudan Black B and Sudan III staining in combination with the previously described "low shrinkage method" of tissue processing result in good staining of lamellar bodies of AE2 cells (their storing organelles of surfactant) and thus provide a good signal of AE2 cells, which allows their easy and secure identification even at rather low magnifications. We further show that this signal enables automated detection of AE2 cells by image analysis, which should make this method a suitable staining method for the recently developed and more efficient proportionator sampling. Copyright © 2015 Elsevier GmbH. All rights reserved.

  15. Identification of aminopyrimidine-sulfonamides as potent modulators of Wag31-mediated cell elongation in mycobacteria.

    PubMed

    Singh, Vinayak; Dhar, Neeraj; Pató, János; Kolly, Gaëlle S; Korduláková, Jana; Forbak, Martin; Evans, Joanna C; Székely, Rita; Rybniker, Jan; Palčeková, Zuzana; Zemanová, Júlia; Santi, Isabella; Signorino-Gelo, François; Rodrigues, Liliana; Vocat, Anthony; Covarrubias, Adrian S; Rengifo, Monica G; Johnsson, Kai; Mowbray, Sherry; Buechler, Joseph; Delorme, Vincent; Brodin, Priscille; Knott, Graham W; Aínsa, José A; Warner, Digby F; Kéri, György; Mikušová, Katarína; McKinney, John D; Cole, Stewart T; Mizrahi, Valerie; Hartkoorn, Ruben C

    2017-01-01

    There is an urgent need to discover new anti-tubercular agents with novel mechanisms of action in order to tackle the scourge of drug-resistant tuberculosis. Here, we report the identification of such a molecule - an AminoPYrimidine-Sulfonamide (APYS1) that has potent, bactericidal activity against M. tuberculosis. Mutations in APYS1-resistant M. tuberculosis mapped exclusively to wag31, a gene that encodes a scaffolding protein thought to orchestrate cell elongation. Recombineering confirmed that a Gln201Arg mutation in Wag31 was sufficient to cause resistance to APYS1, however, neither overexpression nor conditional depletion of wag31 impacted M. tuberculosis susceptibility to this compound. In contrast, expression of the wildtype allele of wag31 in APYS1-resistant M. tuberculosis was dominant and restored susceptibility to APYS1 to wildtype levels. Time-lapse imaging and scanning electron microscopy revealed that APYS1 caused gross malformation of the old pole of M. tuberculosis, with eventual lysis. These effects resembled the morphological changes observed following transcriptional silencing of wag31 in M. tuberculosis. These data show that Wag31 is likely not the direct target of APYS1, but the striking phenotypic similarity between APYS1 exposure and genetic depletion of Wag31 in M. tuberculosis suggests that APYS1 might indirectly affect Wag31 through an as yet unknown mechanism. © 2016 John Wiley & Sons Ltd.

  16. Improved recovery and identification of membrane proteins from rat hepatic cells using a centrifugal proteomic reactor.

    PubMed

    Zhou, Hu; Wang, Fangjun; Wang, Yuwei; Ning, Zhibin; Hou, Weimin; Wright, Theodore G; Sundaram, Meenakshi; Zhong, Shumei; Yao, Zemin; Figeys, Daniel

    2011-10-01

    Despite their importance in many biological processes, membrane proteins are underrepresented in proteomic analysis because of their poor solubility (hydrophobicity) and often low abundance. We describe a novel approach for the identification of plasma membrane proteins and intracellular microsomal proteins that combines membrane fractionation, a centrifugal proteomic reactor for streamlined protein extraction, protein digestion and fractionation by centrifugation, and high performance liquid chromatography-electrospray ionization-tandem MS. The performance of this approach was illustrated for the study of the proteome of ER and Golgi microsomal membranes in rat hepatic cells. The centrifugal proteomic reactor identified 945 plasma membrane proteins and 955 microsomal membrane proteins, of which 63 and 47% were predicted as bona fide membrane proteins, respectively. Among these proteins, >800 proteins were undetectable by the conventional in-gel digestion approach. The majority of the membrane proteins only identified by the centrifugal proteomic reactor were proteins with ≥ 2 transmembrane segments or proteins with high molecular mass (e.g. >150 kDa) and hydrophobicity. The improved proteomic reactor allowed the detection of a group of endocytic and/or signaling receptor proteins on the plasma membrane, as well as apolipoproteins and glycerolipid synthesis enzymes that play a role in the assembly and secretion of apolipoprotein B100-containing very low density lipoproteins. Thus, the centrifugal proteomic reactor offers a new analytical tool for structure and function studies of membrane proteins involved in lipid and lipoprotein metabolism.

  17. In situ detection and identification of hesperidin crystals in satsuma mandarin (Citrus unshiu) peel cells.

    PubMed

    Inoue, Tsuyoshi; Yoshinaga, Arata; Takabe, Keiji; Yoshioka, Terutaka; Ogawa, Kazunori; Sakamoto, Masahiro; Azuma, Jun-ichi; Honda, Yoichi

    2015-01-01

    Hesperidin, a flavonoid known to have important pharmacological effects, accumulates particularly in the peels of satsuma mandarin (Citrus unshiu). Although histochemical studies have suggested that hesperidin forms crystals in some tissues of the Rutaceae and Umbelliferae, there has been no rigorous in situ detection or identification of hesperidin crystals in C. unshiu. To characterise the chemical component of the crystals found in C. unshiu peels using Raman microscopy. Sections of C. unshiu peels were made. The distribution and morphology of crystals in the sections were analysed microscopically. Raman microscopy was used to detect hesperidin in the sections directly. The crystals were more abundant in immature peel and were observed particularly in areas surrounding vascular bundles, around the border between the flavedo and albedo layers and just below the epidermal cells. In the morphological analysis by scanning electron microscopy, needle-shaped crystals aggregated and formed clusters of spherical crystals. Spectra obtained by Raman microscopy of the crystals in the peel sections were consistent with those of the hesperidin standard. This study showed the detailed distribution of crystals in C. unshiu peels and their main component was identified using Raman microscopy to be hesperidin for the first time. Copyright © 2014 John Wiley & Sons, Ltd.

  18. Identification of genes associated with tumorigenesis of meibomian cell carcinoma by microarray analysis.

    PubMed

    Kumar, Arun; Kumar Dorairaj, Syril; Prabhakaran, Venkatesh C; Prakash, D Ravi; Chakraborty, Sanjukta

    2007-11-01

    Meibomian cell carcinoma (MCC) is a malignant tumor of the meibomian glands located in the eyelids. No information exists on the cytogenetic and genetic aspects of MCC. There is no report on the gene expression profile of MCC. Thus there is a need, for both scientific and clinical reasons, to identify genes and pathways that are involved in the development and progression of MCC. We analyzed the gene expression profile of MCC by the microarray technique. Forty-four genes were upregulated and 149 genes were downregulated in MCC. Differential expression data were confirmed for 5 genes by semiquantitative RT-PCR in MCC tumors: GTF2H4, RBM12, UBE2D3, DDX17, and LZTS1. We found dysregulation of two major pathways in MCC: MAPK and JAK/STAT. Clusters of genes on chromosomes 1, 12, and 19 were dysregulated in MCC. The data presented here will facilitate the identification of specific markers and therapeutic targets for the treatment of MCC patients.

  19. Improved Recovery and Identification of Membrane Proteins from Rat Hepatic Cells using a Centrifugal Proteomic Reactor*

    PubMed Central

    Zhou, Hu; Wang, Fangjun; Wang, Yuwei; Ning, Zhibin; Hou, Weimin; Wright, Theodore G.; Sundaram, Meenakshi; Zhong, Shumei; Yao, Zemin; Figeys, Daniel

    2011-01-01

    Despite their importance in many biological processes, membrane proteins are underrepresented in proteomic analysis because of their poor solubility (hydrophobicity) and often low abundance. We describe a novel approach for the identification of plasma membrane proteins and intracellular microsomal proteins that combines membrane fractionation, a centrifugal proteomic reactor for streamlined protein extraction, protein digestion and fractionation by centrifugation, and high performance liquid chromatography-electrospray ionization-tandem MS. The performance of this approach was illustrated for the study of the proteome of ER and Golgi microsomal membranes in rat hepatic cells. The centrifugal proteomic reactor identified 945 plasma membrane proteins and 955 microsomal membrane proteins, of which 63 and 47% were predicted as bona fide membrane proteins, respectively. Among these proteins, >800 proteins were undetectable by the conventional in-gel digestion approach. The majority of the membrane proteins only identified by the centrifugal proteomic reactor were proteins with ≥2 transmembrane segments or proteins with high molecular mass (e.g. >150 kDa) and hydrophobicity. The improved proteomic reactor allowed the detection of a group of endocytic and/or signaling receptor proteins on the plasma membrane, as well as apolipoproteins and glycerolipid synthesis enzymes that play a role in the assembly and secretion of apolipoprotein B100-containing very low density lipoproteins. Thus, the centrifugal proteomic reactor offers a new analytical tool for structure and function studies of membrane proteins involved in lipid and lipoprotein metabolism. PMID:21749988

  20. Proteomics-based identification of plasma biomarkers in oral squamous cell carcinoma.

    PubMed

    Tung, Chun-Liang; Lin, Szu-Ting; Chou, Hsiu-Chuan; Chen, Yi-Wen; Lin, Hwan-Chung; Tung, Chung-Liang; Huang, Kao-Jean; Chen, Yi-Ju; Lee, Ying-Ray; Chan, Hong-Lin

    2013-03-05

    Oral squamous cell carcinoma (OSCC) is an aggressive cancer and its occurrence is closely related to betel nut chewing in Taiwan. However, there are few prognostic and diagnostic biomarkers for this disease especially for its association with betel nut chewing. Recent progresses in quantitative proteomics have offered opportunities to discover plasma proteins as biomarkers for tracking the progression and for understanding the molecular mechanisms of OSCC. In present study, plasma samples from OSCC patients with at least 5-year history of betel nut chewing and healthy donors were analyzed by fluorescence 2D-DIGE-based proteomic analysis. Totally, 38 proteins have been firmly identified representing 13 unique gene products. These proteins mainly function in inflammatory responses (such as fibrinogen gamma chain) and transport (Apolipoprotein A-I). Additionally, the current quantitative proteomic approach has identified numerous OSCC biomarkers including fibrinogen (alpha/beta/gamma) chain, haptoglobin, leucine-rich alpha-2-glycoprotein and ribosomal protein S6 kinase alpha-3 (RSK2) which have not been reported and may be associated with the progression and development of the disease. In summary, this study reports a comprehensive patient-based proteomic approach for the identification of potential plasma biomarkers in OSCC. The potential of utilizing these markers for screening and treating OSCC warrants further investigations.

  1. The use of P63 immunohistochemistry for the identification of squamous cell carcinoma of the lung.

    PubMed

    Conde, Esther; Angulo, Bárbara; Redondo, Pilar; Toldos, Oscar; García-García, Elena; Suárez-Gauthier, Ana; Rubio-Viqueira, Belén; Marrón, Carmen; García-Luján, Ricardo; Sánchez-Céspedes, Montse; López-Encuentra, Angel; Paz-Ares, Luis; López-Ríos, Fernando

    2010-08-17

    While some targeted agents should not be used in squamous cell carcinomas (SCCs), other agents might preferably target SCCs. In a previous microarray study, one of the top differentially expressed genes between adenocarcinomas (ACs) and SCCs is P63. It is a well-known marker of squamous differentiation, but surprisingly, its expression is not widely used for this purpose. Our goals in this study were (1) to further confirm our microarray data, (2) to analize the value of P63 immunohistochemistry (IHC) in reducing the number of large cell carcinoma (LCC) diagnoses in surgical specimens, and (3) to investigate the potential of P63 IHC to minimize the proportion of "carcinoma NOS (not otherwise specified)" in a prospective series of small tumor samples. With these goals in mind, we studied (1) a tissue-microarray comprising 33 ACs and 99 SCCs on which we performed P63 IHC, (2) a series of 20 surgically resected LCCs studied for P63 and TTF-1 IHC, and (3) a prospective cohort of 66 small thoracic samples, including 32 carcinoma NOS, that were further classified by the result of P63 and TTF-1 IHC. The results in the three independent cohorts were as follows: (1) P63 IHC was differentially expressed in SCCs when compared to ACs (p<0.0001); (2) half of the 20 (50%) LCCs were positive for P63 and were reclassified as SCCs; and (3) all P63 positive cases (34%) were diagnosed as SCCs. P63 IHC is useful for the identification of lung SCCs.

  2. Supraorbital ethmoid cell: a consistent landmark for endoscopic identification of the anterior ethmoidal artery.

    PubMed

    Jang, David W; Lachanas, Vasileios A; White, Lauren C; Kountakis, Stilianos E

    2014-12-01

    To demonstrate that the supraorbital ethmoid cell (SOEC) is a consistent and reliable landmark in identification of the anterior ethmoidal artery (AEA). Retrospective radiographic study. Tertiary care rhinology practice. The computed tomography (CT) scans for 78 consecutive patients were evaluated for the presence of SOECs, degree of pneumatization, and location of the AEA in relation to fixed anatomic structures. Forty-one patients with normal SOECs were identified and compared with a group of 15 patients with pathological expansion of the SOEC secondary to inflammatory disease. The CT findings were correlated with endoscopic findings. The incidence of SOECs was 53%. Compared to normal SOECs, expanded SOECs had significantly greater pneumatization laterally (9.3 vs 18.5 mm, respectively; P < .0001) and AEAs that were significantly farther from the skull base (1.3 vs 6.6 mm, respectively; P < .0001). The distance between the AEA and the nasal beak was similar between the 2 groups (P = .1). More importantly, 68 of 68 sides with normal SOECs (100%) demonstrated the AEA within or in continuity with the posterior border of the SOEC opening. In patients with pathological expansion, the AEA remained within the posterior border of the SOEC opening in 19 of 19 sides (100%), despite significant expansion of the cell superolaterally. This is the first study to demonstrate a consistent landmark to identify the AEA even in cases of distorted anatomy of the frontal recess. Identifying the SOEC is a practical and reliable technique for minimizing the risk of injury to the AEA during frontal recess surgery. © American Academy of Otolaryngology—Head and Neck Surgery Foundation 2014.

  3. The Use of P63 Immunohistochemistry for the Identification of Squamous Cell Carcinoma of the Lung

    PubMed Central

    Conde, Esther; Angulo, Bárbara; Redondo, Pilar; Toldos, Oscar; García-García, Elena; Suárez-Gauthier, Ana; Rubio-Viqueira, Belén; Marrón, Carmen; García-Luján, Ricardo; Sánchez-Céspedes, Montse; López-Encuentra, Angel; Paz-Ares, Luis; López-Ríos, Fernando

    2010-01-01

    Introduction While some targeted agents should not be used in squamous cell carcinomas (SCCs), other agents might preferably target SCCs. In a previous microarray study, one of the top differentially expressed genes between adenocarcinomas (ACs) and SCCs is P63. It is a well-known marker of squamous differentiation, but surprisingly, its expression is not widely used for this purpose. Our goals in this study were (1) to further confirm our microarray data, (2) to analize the value of P63 immunohistochemistry (IHC) in reducing the number of large cell carcinoma (LCC) diagnoses in surgical specimens, and (3) to investigate the potential of P63 IHC to minimize the proportion of “carcinoma NOS (not otherwise specified)” in a prospective series of small tumor samples. Methods With these goals in mind, we studied (1) a tissue-microarray comprising 33 ACs and 99 SCCs on which we performed P63 IHC, (2) a series of 20 surgically resected LCCs studied for P63 and TTF-1 IHC, and (3) a prospective cohort of 66 small thoracic samples, including 32 carcinoma NOS, that were further classified by the result of P63 and TTF-1 IHC. Results The results in the three independent cohorts were as follows: (1) P63 IHC was differentially expressed in SCCs when compared to ACs (p<0.0001); (2) half of the 20 (50%) LCCs were positive for P63 and were reclassified as SCCs; and (3) all P63 positive cases (34%) were diagnosed as SCCs. Conclusions P63 IHC is useful for the identification of lung SCCs. PMID:20808915

  4. Identification and functional validation of HPV-mediated hypermethylation in head and neck squamous cell carcinoma.

    PubMed

    Lechner, Matthias; Fenton, Tim; West, James; Wilson, Gareth; Feber, Andrew; Henderson, Stephen; Thirlwell, Christina; Dibra, Harpreet K; Jay, Amrita; Butcher, Lee; Chakravarthy, Ankur R; Gratrix, Fiona; Patel, Nirali; Vaz, Francis; O'Flynn, Paul; Kalavrezos, Nicholas; Teschendorff, Andrew E; Boshoff, Chris; Beck, Stephan

    2013-01-01

    Human papillomavirus-positive (HPV+) head and neck squamous cell carcinoma (HNSCC) represents a distinct clinical and epidemiological condition compared with HPV-negative (HPV-) HNSCC. To test the possible involvement of epigenetic modulation by HPV in HNSCC, we conducted a genome-wide DNA-methylation analysis. Using laser-capture microdissection of 42 formalin-fixed paraffin wax-embedded (FFPE) HNSCCs, we generated DNA-methylation profiles of 18 HPV+ and 14 HPV- samples, using Infinium 450 k BeadArray technology. Methylation data were validated in two sets of independent HPV+/HPV- HNSCC samples (fresh-frozen samples and cell lines) using two independent methods (Infinium 450 k and whole-genome methylated DNA immunoprecipitation sequencing (MeDIP-seq)). For the functional analysis, an HPV- HNSCC cell line was transduced with lentiviral constructs containing the two HPV oncogenes (E6 and E7), and effects on methylation were assayed using the Infinium 450 k technology. Unsupervised clustering over the methylation variable positions (MVPs) with greatest variation showed that samples segregated in accordance with HPV status, but also that HPV+ tumors are heterogeneous. MVPs were significantly enriched at transcriptional start sites, leading to the identification of a candidate CpG island methylator phenotype in a sub-group of the HPV+ tumors. Supervised analysis identified a strong preponderance (87%) of MVPs towards hypermethylation in HPV+ HNSCC. Meta-analysis of our HNSCC and publicly available methylation data in cervical and lung cancers confirmed the observed DNA-methylation signature to be HPV-specific and tissue-independent. Grouping of MVPs into functionally more significant differentially methylated regions identified 43 hypermethylated promoter DMRs, including for three cadherins of the Polycomb group target genes. Integration with independent expression data showed strong negative correlation, especially for the cadherin gene-family members. Combinatorial

  5. Identification and functional validation of HPV-mediated hypermethylation in head and neck squamous cell carcinoma

    PubMed Central

    2013-01-01

    Background Human papillomavirus-positive (HPV+) head and neck squamous cell carcinoma (HNSCC) represents a distinct clinical and epidemiological condition compared with HPV-negative (HPV-) HNSCC. To test the possible involvement of epigenetic modulation by HPV in HNSCC, we conducted a genome-wide DNA-methylation analysis. Methods Using laser-capture microdissection of 42 formalin-fixed paraffin wax-embedded (FFPE) HNSCCs, we generated DNA-methylation profiles of 18 HPV+ and 14 HPV- samples, using Infinium 450 k BeadArray technology. Methylation data were validated in two sets of independent HPV+/HPV- HNSCC samples (fresh-frozen samples and cell lines) using two independent methods (Infinium 450 k and whole-genome methylated DNA immunoprecipitation sequencing (MeDIP-seq)). For the functional analysis, an HPV- HNSCC cell line was transduced with lentiviral constructs containing the two HPV oncogenes (E6 and E7), and effects on methylation were assayed using the Infinium 450 k technology. Results and discussion Unsupervised clustering over the methylation variable positions (MVPs) with greatest variation showed that samples segregated in accordance with HPV status, but also that HPV+ tumors are heterogeneous. MVPs were significantly enriched at transcriptional start sites, leading to the identification of a candidate CpG island methylator phenotype in a sub-group of the HPV+ tumors. Supervised analysis identified a strong preponderance (87%) of MVPs towards hypermethylation in HPV+ HNSCC. Meta-analysis of our HNSCC and publicly available methylation data in cervical and lung cancers confirmed the observed DNA-methylation signature to be HPV-specific and tissue-independent. Grouping of MVPs into functionally more significant differentially methylated regions identified 43 hypermethylated promoter DMRs, including for three cadherins of the Polycomb group target genes. Integration with independent expression data showed strong negative correlation, especially for the

  6. High Specificity in Circulating Tumor Cell Identification Is Required for Accurate Evaluation of Programmed Death-Ligand 1

    PubMed Central

    Schultz, Zachery D.; Warrick, Jay W.; Guckenberger, David J.; Pezzi, Hannah M.; Sperger, Jamie M.; Heninger, Erika; Saeed, Anwaar; Leal, Ticiana; Mattox, Kara; Traynor, Anne M.; Campbell, Toby C.; Berry, Scott M.; Beebe, David J.; Lang, Joshua M.

    2016-01-01

    Background Expression of programmed-death ligand 1 (PD-L1) in non-small cell lung cancer (NSCLC) is typically evaluated through invasive biopsies; however, recent advances in the identification of circulating tumor cells (CTCs) may be a less invasive method to assay tumor cells for these purposes. These liquid biopsies rely on accurate identification of CTCs from the diverse populations in the blood, where some tumor cells share characteristics with normal blood cells. While many blood cells can be excluded by their high expression of CD45, neutrophils and other immature myeloid subsets have low to absent expression of CD45 and also express PD-L1. Furthermore, cytokeratin is typically used to identify CTCs, but neutrophils may stain non-specifically for intracellular antibodies, including cytokeratin, thus preventing accurate evaluation of PD-L1 expression on tumor cells. This holds even greater significance when evaluating PD-L1 in epithelial cell adhesion molecule (EpCAM) positive and EpCAM negative CTCs (as in epithelial-mesenchymal transition (EMT)). Methods To evaluate the impact of CTC misidentification on PD-L1 evaluation, we utilized CD11b to identify myeloid cells. CTCs were isolated from patients with metastatic NSCLC using EpCAM, MUC1 or Vimentin capture antibodies and exclusion-based sample preparation (ESP) technology. Results Large populations of CD11b+CD45lo cells were identified in buffy coats and stained non-specifically for intracellular antibodies including cytokeratin. The amount of CD11b+ cells misidentified as CTCs varied among patients; accounting for 33–100% of traditionally identified CTCs. Cells captured with vimentin had a higher frequency of CD11b+ cells at 41%, compared to 20% and 18% with MUC1 or EpCAM, respectively. Cells misidentified as CTCs ultimately skewed PD-L1 expression to varying degrees across patient samples. Conclusions Interfering myeloid populations can be differentiated from true CTCs with additional staining criteria

  7. Identification and characterisation of citrullinated antigen-specific B cells in peripheral blood of patients with rheumatoid arthritis.

    PubMed

    Kerkman, Priscilla F; Fabre, Emeline; van der Voort, Ellen I H; Zaldumbide, Arnaud; Rombouts, Yoann; Rispens, Theo; Wolbink, Gertjan; Hoeben, Rob C; Spits, Hergen; Baeten, Dominique L P; Huizinga, Tom W J; Toes, René E M; Scherer, Hans U

    2016-06-01

    Immunity to citrullinated antigens is a hallmark of rheumatoid arthritis (RA). We set out to elucidate its biology by identifying and characterising citrullinated antigen-specific B cells in peripheral blood of patients with RA. Differentially labelled streptavidin and extravidin tetramers were conjugated to biotinylated CCP2 or control antigens and used in flow cytometry to identify citrullinated antigen-specific B cells in peripheral blood. Tetramer-positive and tetramer-negative B cells were isolated by fluorescence activated cell sorting (FACS) followed by in vitro culture and analysis of culture supernatants for the presence of antibodies against citrullinated protein antigens (ACPA) by ELISA. Cells were phenotypically characterised by flow cytometry. By combining differentially labelled CCP2 tetramers, we successfully separated citrullinated antigen-specific B cells from non-specific background signals. Isolated tetramer-positive B cells, but not tetramer-negative cells, produced large amounts of ACPA upon in vitro stimulation. Phenotypic analyses revealed that citrullinated antigen-specific B cells displayed markers of class-switched memory B cells and plasmablasts, whereas only few cells displayed a naïve phenotype. The frequency of tetramer-positive cells was high (up to 1/500 memory B cells with a median of 1/12 500 total B cells) and correlated with ACPA serum titres and spontaneous ACPA production in culture. We developed a technology to identify and isolate citrullinated antigen-specific B cells from peripheral blood of patients with RA. Most cells have a memory phenotype, express IgA or IgG and are present in relatively high frequencies. These data pave the path for a direct and detailed molecular characterisation of ACPA-expressing B cells and could lead to the identification of novel therapeutic targets. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://www.bmj.com/company/products-services/rights-and-licensing/

  8. Identification and quantitation of Vesivirus 2117 particles in bioreactor fluids from infected Chinese hamster ovary cell cultures.

    PubMed

    Qiu, Yongchang; Jones, Nathan; Busch, Michelle; Pan, Peng; Keegan, Jesse; Zhou, Weichang; Plavsic, Mark; Hayes, Michael; McPherson, John M; Edmunds, Tim; Zhang, Kate; Mattaliano, Robert J

    2013-05-01

    The prevention of adventitious agent contamination is a top priority throughout the entire biopharmaceutical production process. For example, although viral contamination of cell banks or cell cultures is rare, it can result in serious consequences (e.g., shutdown and decontamination of manufacturing facilities). To ensure virus free production, numerous in vivo and in vitro adventitious agent assays and biophysical characterizations such as electron microscopy are conducted on cell banks, raw materials, process materials, and drug substances throughout the manufacturing process. Molecular assays such as PCR and other nucleotide-based techniques are also routinely used for screening and identification of any viral agents. However, modern techniques in protein identification of complex protein mixtures have not yet been effectively integrated throughout the industry into current viral testing strategies. Here, we report the identification and quantitation of Vesivirus 2117 particles in bioreactor fluid from infected Chinese hamster ovary cell cultures by global protein sequencing using mass spectrometry in combination with multi-dimensional liquid-chromatography. Following mass spectrometric data acquisition and rigorous data analysis, six virus specific peptides were identified. These peptides were fragments of two structural proteins, capsid protein pre-cursor (four unique peptides) and small structural protein (two unique peptides), from the same species: Vesivirus 2117. Using stable heavy isotope-labeled peptides as internal standards, we also determined the absolute concentration of Vesivirus particles in the bioreactor fluid and the ratio of two capsid proteins (VP1:VP2) in the particles as approximately 9:1. The positive identification of Vesivirus 2117 was subsequently confirmed by RT-PCR. Copyright © 2012 Wiley Periodicals, Inc.

  9. Distributed parameter identification for a label-structured cell population dynamics model using CFSE histogram time-series data.

    PubMed

    Luzyanina, Tatyana; Roose, Dirk; Bocharov, Gennady

    2009-11-01

    In this work we address the problem of the robust identification of unknown parameters of a cell population dynamics model from experimental data on the kinetics of cells labelled with a fluorescence marker defining the division age of the cell. The model is formulated by a first order hyperbolic PDE for the distribution of cells with respect to the structure variable x (or z) being the intensity level (or the log(10)-transformed intensity level) of the marker. The parameters of the model are the rate functions of cell division, death, label decay and the label dilution factor. We develop a computational approach to the identification of the model parameters with a particular focus on the cell birth rate alpha(z) as a function of the marker intensity, assuming the other model parameters are scalars to be estimated. To solve the inverse problem numerically, we parameterize alpha(z) and apply a maximum likelihood approach. The parametrization is based on cubic Hermite splines defined on a coarse mesh with either equally spaced a priori fixed nodes or nodes to be determined in the parameter estimation procedure. Ill-posedness of the inverse problem is indicated by multiple minima. To treat the ill-posed problem, we apply Tikhonov regularization with the regularization parameter determined by the discrepancy principle. We show that the solution of the regularized parameter estimation problem is consistent with the data set with an accuracy within the noise level in the measurements.

  10. Identification of T-cell epitopes of Lol p 9, a major allergen of ryegrass (Lolium perenne) pollen.

    PubMed

    Blaher, B; Suphioglu, C; Knox, R B; Singh, M B; McCluskey, J; Rolland, J M

    1996-07-01

    T-cell recognition of Lol p 9, a major allergen of ryegrass pollen, was investigated by using a T-cell line and T-cell clones generated from the peripheral blood of an atopic donor. The T-cell line reacted with purified Lol p 9, as well as with crude ryegrass pollen extract, but failed to cross-react with Bermuda grass pollen extract. All of six T-cell clones generated from this line proliferated in response to Lol p 9. Epitope mapping was carried out with a panel of 34 overlapping synthetic peptides, which spanned the entire sequence of the Lol p 9 12R isoform. The T-cell line responded to two of the peptides, Lol p 9 (105-116) and Lol p 9 (193-204), whereas reactivity with one or other of these peptides was shown by five T-cell clones. These two peptides contained sequences consistent with motifs previously reported for major histocompatibility complex class II-restricted peptides. HLA antibody blocking studies showed that presentation of peptide Lol p 9 (105-116) to one T-cell clone was HLA-DR-restricted; this clone expressed a T helper cell phenotype (CD3+, CD4+) and the T-cell receptor alpha beta. The identification of immunodominant T-cell epitope(s) on allergens is essential for devising safer and more effective immunotherapy strategies, which can interrupt the chain of events leading to allergic disease.

  11. Identification of novel binding partners (annexins) for the cell death signal phosphatidylserine and definition of their recognition motif.

    PubMed

    Rosenbaum, Sabrina; Kreft, Sandra; Etich, Julia; Frie, Christian; Stermann, Jacek; Grskovic, Ivan; Frey, Benjamin; Mielenz, Dirk; Pöschl, Ernst; Gaipl, Udo; Paulsson, Mats; Brachvogel, Bent

    2011-02-18

    Identification and clearance of apoptotic cells prevents the release of harmful cell contents thereby suppressing inflammation and autoimmune reactions. Highly conserved annexins may modulate the phagocytic cell removal by acting as bridging molecules to phosphatidylserine, a characteristic phagocytosis signal of dying cells. In this study five members of the structurally and functionally related annexin family were characterized for their capacity to interact with phosphatidylserine and dying cells. The results showed that AnxA3, AnxA4, AnxA13, and the already described interaction partner AnxA5 can bind to phosphatidylserine and apoptotic cells, whereas AnxA8 lacks this ability. Sequence alignment experiments located the essential amino residues for the recognition of surface exposed phosphatidylserine within the calcium binding motifs common to all annexins. These amino acid residues were missing in the evolutionary young AnxA8 and when they were reintroduced by site directed mutagenesis AnxA8 gains the capability to interact with phosphatidylserine containing liposomes and apoptotic cells. By defining the evolutionary conserved amino acid residues mediating phosphatidylserine binding of annexins we show that the recognition of dying cells represent a common feature of most annexins. Hence, the individual annexin repertoire bound to the cell surface of dying cells may fulfil opsonin-like function in cell death recognition.

  12. Micrometer-Scale Magnetic-Resonance-Coupled Radio-Frequency Identification and Transceivers for Wireless Sensors in Cells

    NASA Astrophysics Data System (ADS)

    Hu, Xiaolin; Aggarwal, Kamal; Yang, Mimi X.; Parizi, Kokab B.; Xu, Xiaoqing; Akin, Demir; Poon, Ada S. Y.; Wong, H.-S. Philip

    2017-07-01

    We report the design, analysis, and characterization of a three-inductor radio-frequency identification (RFID) and transceiver system for potential applications in individual cell tracking and monitoring. The RFID diameter is 22 μ m and can be naturally internalized by living cells. Using magnetic resonance coupling, the system shows resonance shifts when the RFID is present and also when the RFID loading capacitance changes. It operates at 60 GHz with a high signal magnitude up to -50 dB and a sensitivity of 0.2. This miniaturized RFID with a high signal magnitude is a promising step toward continuous, real-time monitoring of activities at cellular levels.

  13. Identification and Characterization of Cell Type–Specific and Ubiquitous Chromatin Regulatory Structures in the Human Genome

    PubMed Central

    Xi, Hualin; Shulha, Hennady P; Lin, Jane M; Vales, Teresa R; Fu, Yutao; Bodine, David M; McKay, Ronald D. G; Chenoweth, Josh G; Tesar, Paul J; Furey, Terrence S; Ren, Bing; Weng, Zhiping; Crawford, Gregory E

    2007-01-01

    The identification of regulatory elements from different cell types is necessary for understanding the mechanisms controlling cell type–specific and housekeeping gene expression. Mapping DNaseI hypersensitive (HS) sites is an accurate method for identifying the location of functional regulatory elements. We used a high throughput method called DNase-chip to identify 3,904 DNaseI HS sites from six cell types across 1% of the human genome. A significant number (22%) of DNaseI HS sites from each cell type are ubiquitously present among all cell types studied. Surprisingly, nearly all of these ubiquitous DNaseI HS sites correspond to either promoters or insulator elements: 86% of them are located near annotated transcription start sites and 10% are bound by CTCF, a protein with known enhancer-blocking insulator activity. We also identified a large number of DNaseI HS sites that are cell type specific (only present in one cell type); these regions are enriched for enhancer elements and correlate with cell type–specific gene expression as well as cell type–specific histone modifications. Finally, we found that approximately 8% of the genome overlaps a DNaseI HS site in at least one the six cell lines studied, indicating that a significant percentage of the genome is potentially functional. PMID:17708682

  14. Characterization of the Transcriptome of Nascent Hair Cells and Identification of Direct Targets of the Atoh1 Transcription Factor

    PubMed Central

    Cai, Tiantian; Jen, Hsin-I; Kang, Hyojin; Klisch, Tiemo J.; Zoghbi, Huda Y.

    2015-01-01

    Hair cells are sensory receptors for the auditory and vestibular system in vertebrates. The transcription factor Atoh1 is both necessary and sufficient for the differentiation of hair cells, and is strongly upregulated during hair-cell regeneration in nonmammalian vertebrates. To identify genes involved in hair cell development and function, we performed RNA-seq profiling of purified Atoh1-expressing hair cells from the neonatal mouse cochlea. We identified >600 enriched transcripts in cochlear hair cells, of which 90% have not been previously shown to be expressed in hair cells. We identified 233 of these hair cell genes as candidates to be directly regulated by Atoh1 based on the presence of Atoh1 binding sites in their regulatory regions and by analyzing Atoh1 ChIP-seq datasets from the cerebellum and small intestine. We confirmed 10 of these genes as being direct Atoh1 targets in the cochlea by ChIP-PCR. The identification of candidate Atoh1 target genes is a first step in identifying gene regulatory networks for hair-cell development and may inform future studies on the potential role of Atoh1 in mammalian hair cell regeneration. PMID:25855195

  15. Quantitative multiplexed simulated-cell identification by SERS in microfluidic devices

    NASA Astrophysics Data System (ADS)

    Hoonejani, M. R.; Pallaoro, A.; Braun, G. B.; Moskovits, M.; Meinhart, C. D.

    2015-10-01

    A reliable identification of cells on the basis of their surface markers is of great interest for diagnostic and therapeutic applications. We present a multiplexed labeling and detection strategy that is applied to four microparticle populations, each mimicking cellular or bacterial samples with varying surface concentrations of up to four epitopes, using four distinct biotags that are meant to be used in conjunction with surface enhanced Raman spectroscopy (SERS) instead of fluorescence, together with microfluidics. Four populations of 6 μm polystyrene beads were incubated with different mixtures, ``cocktails'' of four SERS biotags (SBTs), simulating the approach that one would follow when seeking to identify multiple biomarkers encountered in biological applications. Populations were flowed in a microfluidic flow-focusing device and the SERS signal from individual beads was acquired during continuous flow. The spectrally rich SERS spectra enabled us to separate confidently the populations by utilizing principal component analysis (PCA). Also, using classical least squares (CLS), we were able to calculate the contributions of each SBT to the overall signal in each of the populations, and showed that the relative SBT contributions are consistent with the nominal percentage of each marker originally designed into that bead population, by functionalizing it with a given SBT cocktail. Our results demonstrate the multiplexing capability of SBTs in potential applications such as immunophenotyping.A reliable identification of cells on the basis of their surface markers is of great interest for diagnostic and therapeutic applications. We present a multiplexed labeling and detection strategy that is applied to four microparticle populations, each mimicking cellular or bacterial samples with varying surface concentrations of up to four epitopes, using four distinct biotags that are meant to be used in conjunction with surface enhanced Raman spectroscopy (SERS) instead of

  16. Quantitative image cytometry measurements of lipids, DNA, CD45 and cytokeratin for circulating tumor cell identification in a model system

    NASA Astrophysics Data System (ADS)

    Futia, Gregory L.; Qamar, Lubna; Behbakht, Kian; Gibson, Emily A.

    2016-04-01

    Circulating tumor cell (CTC) identification has applications in both early detection and monitoring of solid cancers. The rarity of CTCs, expected at ~1-50 CTCs per million nucleated blood cells (WBCs), requires identifying methods based on biomarkers with high sensitivity and specificity for accurate identification. Discovery of biomarkers with ever higher sensitivity and specificity to CTCs is always desirable to potentially find more CTCs in cancer patients thus increasing their clinical utility. Here, we investigate quantitative image cytometry measurements of lipids with the biomarker panel of DNA, Cytokeratin (CK), and CD45 commonly used to identify CTCs. We engineered a device for labeling suspended cell samples with fluorescent antibodies and dyes. We used it to prepare samples for 4 channel confocal laser scanning microscopy. The total data acquired at high resolution from one sample is ~ 1.3 GB. We developed software to perform the automated segmentation of these images into regions of interest (ROIs) containing individual cells. We quantified image features of total signal, spatial second moment, spatial frequency second moment, and their product for each ROI. We performed measurements on pure WBCs, cancer cell line MCF7 and mixed samples. Multivariable regressions and feature selection were used to determine combination features that are more sensitive and specific than any individual feature separately. We also demonstrate that computation of spatial characteristics provides higher sensitivity and specificity than intensity alone. Statistical models allowed quantification of the required sensitivity and specificity for detecting small levels of CTCs in a human blood sample.

  17. Identification of Receptor Ligands and Receptor Subtypes Using Antagonists in a Capillary Electrophoresis Single-Cell Biosensor Separation System

    NASA Astrophysics Data System (ADS)

    Fishman, Harvey A.; Orwar, Owe; Scheller, Richard H.; Zare, Richard N.

    1995-08-01

    A capillary electrophoresis system with single-cell biosensors as a detector has been used to separate and identify ligands in complex biological samples. The power of this procedure was significantly increased by introducing antagonists that inhibited the cellular response from selected ligand-receptor interactions. The single-cell biosensor was based on the ligand-receptor binding and G-protein-mediated signal transduction pathways in PC12 and NG108-15 cell lines. Receptor activation was measured as increases in cytosolic free calcium ion concentration by using fluorescence microscopy with the intracellular calcium ion indicator fluo-3 acetoxymethyl ester. Specifically, a mixture of bradykinin (BK) and acetylcholine (ACh) was fractionated and the components were identified by inhibiting the cellular response with icatibant (HOE 140), a selective antagonist to the BK B_2 receptor subtype (B_2BK), and atropine, an antagonist to muscarinic ACh receptor subtypes. Structurally related forms of BK were also identified based on inhibiting B_2BK receptors. Applications of this technique include identification of endogenous BK in a lysate of human hepatocellular carcinoma cells (Hep G2) and screening for bioactivity of BK degradation products in human blood plasma. The data demonstrate that the use of antagonists with a single-cell biosensor separation system aids identification of separated components and receptor subtypes.

  18. Immunoproteomic Identification of Human T Cell Antigens of Mycobacterium tuberculosis That Differentiate Healthy Contacts from Tuberculosis Patients*

    PubMed Central

    Deenadayalan, Anbarasu; Heaslip, Darragh; Rajendiran, Adhilakshmi Aavudaiyappan; Velayudham, Banurekha Vaithilingam; Frederick, Sheela; Yang, Hong-Liang; Dobos, Karen; Belisle, John T.; Raja, Alamelu

    2010-01-01

    Identification of Mycobacterium tuberculosis antigens inducing cellular immune responses is required to improve the diagnosis of and vaccine development against tuberculosis. To identify the antigens of M. tuberculosis that differentiated between tuberculosis (TB) patients and healthy contacts based on T cell reactivity, the culture filtrate of in vitro grown M. tuberculosis was fractionated by two-dimensional liquid phase electrophoresis and tested for the ability to stimulate T cells in a whole blood assay. This approach separated the culture filtrate into 350 fractions with sufficient protein quantity (at least 200 μg of protein) for mass spectrometry and immunological analyses. High levels of interferon-γ (IFN-γ) secretion were induced by 105 fractions in healthy contacts compared with TB patients (p < 0.05). Most interesting was the identification of 10 fractions that specifically induced strong IFN-γ production in the healthy contact population but not in TB patients. Other immunological measurements showed 42 fractions that induced significant lymphocyte proliferative responses in the healthy contact group compared with the TB patients. The tumor necrosis factor-α response for most of the fractions did not significantly differ in the tested groups, and the interleukin-4 response was below the detectable range for all fractions and both study groups. Proteomic characterization of the 105 fractions that induced a significant IFN-γ response in the healthy contacts compared with the TB patients led to the identification of 59 proteins of which 24 represented potentially novel T cell antigens. Likewise, the protein identification in the 10 healthy “contact-specific fractions” revealed 16 proteins that are key candidates as vaccine or diagnostic targets. PMID:20031926

  19. Identification of Primary Gene Targets of TFAP2C in Hormone Responsive Breast Carcinoma Cells

    PubMed Central

    Woodfield, George W.; Chen, Yizhen; Bair, Thomas B.; Domann, Frederick E.; Weigel, Ronald J.

    2010-01-01

    The TFAP2C transcription factor is involved in mammary development, differentiation and oncogenesis. Previous studies established a role for TFAP2C in the regulation of ESR1 (ERα) and ERBB2 (Her2) in breast carcinomas. However, the role of TFAP2C in different breast cancer phenotypes has not been examined in detail. To develop a more complete characterization of TFAP2C target genes, ChIP-seq with anti-TFAP2C antibody and expression arrays with TFAP2C knock down were analyzed in MCF-7 breast carcinoma cells. Genomic sequences common to the ChIP-seq data set defined the consensus sequence for TFAP2C chromatin binding as the nine base sequence SCCTSRGGS (S=G/C, R=A/G), which closely matches the previously defined optimal in vitro binding site. Comparing expression arrays before and after knock down of TFAP2C with ChIP-seq data demonstrated a conservative estimate that 8% of genes altered by TFAP2C expression are primary target genes and includes genes that are both induced and repressed by TFAP2C. A set of 447 primary target genes of TFAP2C was identified, which included ESR1 (ERα), FREM2, RET, FOXA1, WWOX, GREB1, MYC and members of the retinoic acid response pathway. The identification of ESR1, WWOX, GREB1 and FOXA1 as primary targets confirmed the role of TFAP2C in hormone response. TFAP2C plays a critical role in gene regulation in hormone responsive breast cancer and its target genes are different than for the Her2 breast cancer phenotype. PMID:20629094

  20. Characterization of tissue plasminogen activator binding proteins isolated from endothelial cells and other cell types

    SciTech Connect

    Beebe, D.P.; Wood, L.L.; Moos, M. )

    1990-07-15

    Human tissue plasminogen activator (t-PA) was shown to bind specifically to human osteosarcoma cells (HOS), and human epidermoid carcinoma cells (A-431 cells). Crosslinking studies with DTSSP demonstrated high molecular weight complexes (130,000) between {sup 125}I-t-PA and cell membrane protein on human umbilical vein endothelial cells (HUVEC), HOS, and A-431 cells. A 48-65,000 molecular weight complex was demonstrated after crosslinking t-PA peptide (res. 7-20) to cells. Ligand blotting of cell lysates which had been passed over a t-PA affinity column revealed binding of t-PA to 54,000 and 95,000 molecular weight proteins. Several t-PA binding proteins were identified in immunopurified cell lysates, including tubulin beta chain, plasminogen activator inhibitor type 1 and single chain urokinase.

  1. Overview of very small embryonic-like stem cells (VSELs) and methodology of their identification and isolation by flow cytometric methods.

    PubMed

    Zuba-Surma, Ewa K; Ratajczak, Mariusz Z

    2010-01-01

    The protocols presented here describe the procedures employed to identify and isolate very small embryonic-like stem cells (VSELs) using flow cytometric technologies including fluorescence-activated cell sorting (FACS). We describe the recommended steps in detail for their successful identification and isolation from adult tissues. These protocols were initially established to isolate such cells from murine bone marrow (BM) and human cord blood (CB) and may also be employed to isolate these primitive cells from other adult organs and embryonic tissues. Here, we focus on some critical parameters/key points required for the successful identification and purification of these rare cells by employing classical flow cytometry. In the last part of this unit, we also discuss a novel flow cytometric tool, ImageStream, an imaging flow cytometer, which allows better identification and morphological analysis of sorted cells.

  2. An UPLC-MS/MS method for the simultaneous identification and quantitation of cell wall phenolics in Brassica napus seeds.

    PubMed

    Frolov, Andrej; Henning, Anja; Böttcher, Christoph; Tissier, Alain; Strack, Dieter

    2013-02-13

    The seed residues left after pressing of rapeseed oil are rich in proteins and could be used for human nutrition and animal feeding. These press cakes contain, however, antinutritives, with fiber being the most abundant one. The analysis of fiber phenolic component (localized to seed coat cell walls) is, therefore, important in breeding and food quality control. However, correct structure and content assignments of cell wall-bound phenolics are challenging due to their low stability during sample preparation. Here, a novel LC-MS/MS-based method for the simultaneous identification and quantitation of 66 cell wall-bound phenolics and their derivatives is described. The method was internally standardized, corrected for degradation effects during sample preparation, and cross-validated with a well-established UV-based procedure. This approach was successfully applied to the analysis of cell wall phenolic patterns in different B. napus cultivars and proved to be suitable for marker compound search as well as assay development.

  3. Identification of different ALK mutations in a pair of neuroblastoma cell lines established at diagnosis and relapse

    PubMed Central

    Chen, Lindi; Humphreys, Angharad; Turnbull, Lisa; Bellini, Angela; Schleiermacher, Gudrun; Salwen, Helen; Cohn, Susan L.; Bown, Nick; Tweddle, Deborah A.

    2016-01-01

    Anaplastic Lymphoma Kinase (ALK) is a transmembrane receptor kinase that belongs to the insulin receptor superfamily and has previously been shown to play a role in cell proliferation, migration and invasion in neuroblastoma. Activating ALK mutations are reported in both hereditary and sporadic neuroblastoma tumours, and several ALK inhibitors are currently under clinical evaluation as novel treatments for neuroblastoma. Overall, mutations at codons F1174, R1275 and F1245 together account for ∼85% of reported ALK mutations in neuroblastoma. NBLW and NBLW-R are paired cell lines originally derived from an infant with metastatic MYCN amplified Stage IVS (Evans Criteria) neuroblastoma, at diagnosis and relapse, respectively. Using both Sanger and targeted deep sequencing, this study describes the identification of distinct ALK mutations in these paired cell lines, including the rare R1275L mutation, which has not previously been reported in a neuroblastoma cell line. Analysis of the sensitivity of NBLW and NBLW-R cells to a panel of ALK inhibitors (TAE-684, Crizotinib, Alectinib and Lorlatinib) revealed differences between the paired cell lines, and overall NBLW-R cells with the F1174L mutation were more resistant to ALK inhibitor induced apoptosis compared with NBLW cells. This pair of cell lines represents a valuable pre-clinical model of clonal evolution of ALK mutations associated with neuroblastoma progression. PMID:27888620

  4. The proper criteria for identification and sorting of very small embryonic-like stem cells, and some nomenclature issues.

    PubMed

    Suszynska, Malwina; Zuba-Surma, Ewa K; Maj, Magdalena; Mierzejewska, Kasia; Ratajczak, Janina; Kucia, Magda; Ratajczak, Mariusz Z

    2014-04-01

    Evidence has accumulated that both murine and human adult tissues contain early-development stem cells with a broader differentiation potential than other adult monopotent stem cells. These cells, being pluripotent or multipotent, exist at different levels of specification and most likely represent overlapping populations of cells that, depending on the isolation strategy, ex vivo expansion protocol, and markers employed for their identification, have been given different names. In this review, we will discuss a population of very small embryonic-like stem cells (VSELs) in the context of other stem cells that express pluripotent/multipotent markers isolated from adult tissues as well as review the most current, validated working criteria on how to properly identify and isolate these very rare cells. VSELs have been successfully purified in several laboratories; however, a few have failed to isolate them, which has raised some unnecessary controversy in the field. Therefore, in this short review, we will address the most important reasons that some investigators have experienced problems in isolating these very rare cells and discuss some still unresolved challenges which should be overcome before these cells can be widely employed in the clinic.

  5. Exploiting the power of LINE-1 retrotransposon mutagenesis for identification of genes involved in embryonic stem cell differentiation.

    PubMed

    Lenka, Nibedita; Krishnan, Shruthi; Board, Philip; Rangasamy, Danny

    2014-06-01

    Identifying the genes or epigenetic factors that control the self-renewal and differentiation of stem cells is critical to understanding the molecular basis of cell commitment. Although a number of insertional mutagenesis vectors have been developed for identifying gene functions in animal models, the L1 retrotransposition system offers additional advantages as a tool to disrupt genes in embryonic stem cells in order to identify their functions and the phenotypes associated with them. Recent advances in producing synthetic versions of L1 retrotransposon vector system and the optimization of techniques to accurately identify retrotransposon integration sites have increased their utility for gene discovery applications. We have developed a novel episomal, nonviral L1 retrotransposon vector using scaffold/matrix attachment regions that provides stable, sustained levels of retrotransposition in cell cultures without being affected by epigenetic silencing or from some of the common problems of vector integration. This modified vector contains a GFP marker whose expression occurs only after successful gene disruption events and thus the cells with disrupted genes can be easily picked for functional analysis. Here we present a method to disrupt gene function in embryonic stem cells that aid in the identification of genes involved in stem cell differentiation processes. The methods presented here can be easily adapted to the study of other types of cancer stem cells or induced pluripotent stem cells using the L1 retrotransposon as an insertional mutagen.

  6. Novel Approach of Using Near-Infrared Responsive PEGylated Gold Nanorod Coated Poly(l-lactide) Microneedles to Enhance the Antitumor Efficiency of Docetaxel-Loaded MPEG-PDLLA Micelles for Treating an A431 Tumor.

    PubMed

    Hao, Ying; Dong, MingLing; Zhang, TaoYe; Peng, JinRong; Jia, YanPeng; Cao, YiPing; Qian, ZhiYong

    2017-05-10

    The combination of chemotherapy and photothermal therapy (PTT) plays a significant role in synergistic tumor therapy. However, a high dosage of chemotherapy drugs or photothermal agents may cause series side effects. To overcome these challenges, we designed a near-infrared (NIR) responsive PEGylated gold nanorod (GNR-PEG) coated poly(l-lactide) microneedle (PLLA MN) system (GNR-PEG@MN) to enhance antitumor efficiency of docetaxel-loaded MPEG-PDLLA (MPEG-PDLLA-DTX) micelles for treating an A431 tumor. The as-made GNR-PEG@MNs contained only 31.83 ± 1.22 μg of GNR-PEG per patch and exhibited excellent heating efficacy both in vitro and in vivo. Meanwhile, GNR-PEG@MN with the height of 480 μm had good skin insertion ability and was harmless to the skin. On the other hand, GNR-PEG@MN had good heating transfer ability in vivo, and the tumor sites could reach 50 °C within 5 min. In comparison with chemotherapy and PTT alone, the combination of low dosage MPEG-PDLLA-DTX micelles (5 mg/kg) and GNR-PEG@MNs completely eradicated the A431 tumor without recurrence in vivo, demonstrating a remarkable synergetic effect. Hence, GNR-PEG@MN could be a promising carrier to enhance the antitumor effect of MPEG-PDLLA-DTX micelles for treating superficial tumors and is expected to have a great potential in clinical translation for human epidermoid cancer therapy.

  7. Involvement of aquaporin-3 in epidermal growth factor receptor signaling via hydrogen peroxide transport in cancer cells.

    PubMed

    Hara-Chikuma, Mariko; Watanabe, Sachiko; Satooka, Hiroki

    2016-03-18

    Aquaporin 3 (AQP3), a water/glycerol channel protein, is capable of transporting hydrogen peroxide (H2O2). Here, we show that AQP3-mediated intracellular H2O2 is involved in epidermal growth factor (EGF)-induced cell signaling and its dependent cell function in the EGF receptor (EGFR)-positive cancer cell lines A431 and H1666. AQP3 knockdown suppressed the transport into the cells of extracellular H2O2 produced in response to EGF in A431 and H1666 cells. EGF-induced Erk and Akt activation, which occurred through SHP2 and/or PTEN modulation, was impaired by AQP3 knockdown. Cell growth and migration induced by EGF stimulation were attenuated in AQP3 knockdown cells compared with those in control cells. Coincidentally, tumor growth of A431 cell xenografts in immunodeficient mice was decreased by AQP3 knockdown. Accordingly, a xenograft with AQP3 knockdown A431 cells significantly enhanced the survival of recipient mice compared with the transplantation with control cells. In addition, AQP3 associated with EGFR and NADPH oxidase 2, which we propose is linked to AQP3 producing a localized increase in intracellular H2O2 to function as a second messenger during EGFR cell signaling. Therefore, our findings suggest that AQP3 is required for EGF-EGFR cell signaling in cancer cells and is a therapeutic target for cancer progression.

  8. Extraction methods of red blood cell membrane proteins for Multidimensional Protein Identification Technology (MudPIT) analysis.

    PubMed

    De Palma, Antonella; Roveri, Antonella; Zaccarin, Mattia; Benazzi, Louise; Daminelli, Simone; Pantano, Giorgia; Buttarello, Mauro; Ursini, Fulvio; Gion, Massimo; Mauri, Pier Luigi

    2010-08-13

    Since red blood cells (RBCs) lack nuclei and organelles, cell membrane is their main load-bearing component and, according to a dynamic interaction with the cytoskeleton compartment, plays a pivotal role in their functioning. Even if erythrocyte membranes are available in large quantities, the low abundance and the hydrophobic nature of cell membrane proteins complicate their purification and detection by conventional 2D gel-based proteomic approaches. So, in order to increase the efficiency of RBC membrane proteome identification, here we took advantage of a simple and reproducible membrane sub-fractionation method coupled to Multidimensional Protein Identification Technology (MudPIT). In addition, the adoption of a stringent RBC filtration strategy from the whole blood, permitted to remove exhaustively contaminants, such as platelets and white blood cells, and to identify a total of 275 proteins in the three RBC membrane fractions collected and analysed. Finally, by means of software for the elaboration of the great quantity of data obtained and programs for statistical analysis and protein classification, it was possible to determine the validity of the entire system workflow and to assign the proper sub-cellular localization and function for the greatest number of the identified proteins.

  9. Identification of a surface glycoprotein on African green monkey kidney cells as a receptor for hepatitis A virus.

    PubMed Central

    Kaplan, G; Totsuka, A; Thompson, P; Akatsuka, T; Moritsugu, Y; Feinstone, S M

    1996-01-01

    Very little is known about the mechanism of cell entry of hepatitis A virus (HAV), and the identification of cellular receptors for this picornavirus has been elusive. Here we describe the molecular cloning of a cellular receptor for HAV using protective monoclonal antibodies raised against susceptible African green monkey kidney (AGMK) cells as probes. Monoclonal antibodies 190/4, 235/4 and 263/6, which reacted against similar epitopes, specifically protected AGMK cells against HAV infection by blocking the binding of HAV. Expression cloning and nucleotide sequence analysis of the cDNA coding for epitope 190/4 revealed a novel mucin-like class I integral membrane glycoprotein of 451 amino acids, the HAV cellular receptor 1 (HAVcr-1). Immunofluorescence analysis indicated that mouse Ltk- cells transfected with HAVcr-1 cDNA gained limited susceptibility to HAV infection, which was blocked by treatment with monoclonal antibody 190/4. Our results demonstrate that the HAVcr-1 polypeptide is an attachment receptor for HAV and strongly suggest that it is also a functional receptor which mediates HAV infection. This report constitutes the first identification of a cellular receptor for HAV. Images PMID:8861957

  10. Identification of a surface glycoprotein on African green monkey kidney cells as a receptor for hepatitis A virus.

    PubMed

    Kaplan, G; Totsuka, A; Thompson, P; Akatsuka, T; Moritsugu, Y; Feinstone, S M

    1996-08-15

    Very little is known about the mechanism of cell entry of hepatitis A virus (HAV), and the identification of cellular receptors for this picornavirus has been elusive. Here we describe the molecular cloning of a cellular receptor for HAV using protective monoclonal antibodies raised against susceptible African green monkey kidney (AGMK) cells as probes. Monoclonal antibodies 190/4, 235/4 and 263/6, which reacted against similar epitopes, specifically protected AGMK cells against HAV infection by blocking the binding of HAV. Expression cloning and nucleotide sequence analysis of the cDNA coding for epitope 190/4 revealed a novel mucin-like class I integral membrane glycoprotein of 451 amino acids, the HAV cellular receptor 1 (HAVcr-1). Immunofluorescence analysis indicated that mouse Ltk- cells transfected with HAVcr-1 cDNA gained limited susceptibility to HAV infection, which was blocked by treatment with monoclonal antibody 190/4. Our results demonstrate that the HAVcr-1 polypeptide is an attachment receptor for HAV and strongly suggest that it is also a functional receptor which mediates HAV infection. This report constitutes the first identification of a cellular receptor for HAV.

  11. Identification of an IRF-1 splicing tr