WorldWideScience

Sample records for cell polarity genes

  1. Apical Gene Transfer into Quiescent Human and Canine Polarized Intestinal Epithelial Cells by Lentivirus Vectors

    OpenAIRE

    Seppen, Jurgen; Barry, Simon C.; Klinkspoor, J. Henriette; Katen, Louis J.; Lee, Sum P; Garcia, J. Victor; Osborne, William R. A.

    2000-01-01

    Intestinal epithelial cells secrete a protective luminal mucus barrier inhibiting viral gene transfer. Quiescent, polarized monolayers of primary epithelial cells from dog gallbladder and human colon are efficiently transduced through the apical mucus side by lentivirus vectors, suggesting their application to intestinal gene therapy.

  2. Planar cell polarity effector gene Intu regulates cell fate-specific differentiation of keratinocytes through the primary cilia

    OpenAIRE

    Dai, D.; Li, L.; Huebner, A; H. Zeng; Guevara, E; Claypool, D J; Liu, A.; Chen, J.

    2012-01-01

    Genes involved in the planar cell polarity (PCP) signaling pathway are essential for a number of developmental processes in mammals, such as convergent extension and ciliogenesis. Tissue-specific PCP effector genes of the PCP signaling pathway are believed to mediate PCP signals in a tissue- and cell type-specific manner. However, how PCP signaling controls the morphogenesis of mammalian tissues remains unclear. In this study, we investigated the role of inturned (Intu), a tissue-specific PCP...

  3. Planar cell polarity effector gene Intu regulates cell fate-specific differentiation of keratinocytes through the primary cilia.

    Science.gov (United States)

    Dai, D; Li, L; Huebner, A; Zeng, H; Guevara, E; Claypool, D J; Liu, A; Chen, J

    2013-01-01

    Genes involved in the planar cell polarity (PCP) signaling pathway are essential for a number of developmental processes in mammals, such as convergent extension and ciliogenesis. Tissue-specific PCP effector genes of the PCP signaling pathway are believed to mediate PCP signals in a tissue- and cell type-specific manner. However, how PCP signaling controls the morphogenesis of mammalian tissues remains unclear. In this study, we investigated the role of inturned (Intu), a tissue-specific PCP effector gene, during hair follicle formation in mice. Tissue-specific disruption of Intu in embryonic epidermis resulted in hair follicle morphogenesis arrest because of the failure of follicular keratinocyte to differentiate. Targeting Intu in the epidermis resulted in almost complete loss of primary cilia in epidermal and follicular keratinocytes, and a suppressed hedgehog signaling pathway. Surprisingly, the epidermal stratification and differentiation programs and barrier function were not affected. These results demonstrate that tissue-specific PCP effector genes of the PCP signaling pathway control the differentiation of keratinocytes through the primary cilia in a cell fate- and context-dependent manner, which may be critical in orchestrating the propagation and interpretation of polarity signals established by the core PCP components. PMID:22935613

  4. Copy number variation analysis implicates the cell polarity gene glypican 5 as a human spina bifida candidate gene.

    Science.gov (United States)

    Bassuk, Alexander G; Muthuswamy, Lakshmi B; Boland, Riley; Smith, Tiffany L; Hulstrand, Alissa M; Northrup, Hope; Hakeman, Matthew; Dierdorff, Jason M; Yung, Christina K; Long, Abby; Brouillette, Rachel B; Au, Kit Sing; Gurnett, Christina; Houston, Douglas W; Cornell, Robert A; Manak, J Robert

    2013-03-15

    Neural tube defects (NTDs) are common birth defects of complex etiology. Family and population-based studies have confirmed a genetic component to NTDs. However, despite more than three decades of research, the genes involved in human NTDs remain largely unknown. We tested the hypothesis that rare copy number variants (CNVs), especially de novo germline CNVs, are a significant risk factor for NTDs. We used array-based comparative genomic hybridization (aCGH) to identify rare CNVs in 128 Caucasian and 61 Hispanic patients with non-syndromic lumbar-sacral myelomeningocele. We also performed aCGH analysis on the parents of affected individuals with rare CNVs where parental DNA was available (42 sets). Among the eight de novo CNVs that we identified, three generated copy number changes of entire genes. One large heterozygous deletion removed 27 genes, including PAX3, a known spina bifida-associated gene. A second CNV altered genes (PGPD8, ZC3H6) for which little is known regarding function or expression. A third heterozygous deletion removed GPC5 and part of GPC6, genes encoding glypicans. Glypicans are proteoglycans that modulate the activity of morphogens such as Sonic Hedgehog (SHH) and bone morphogenetic proteins (BMPs), both of which have been implicated in NTDs. Additionally, glypicans function in the planar cell polarity (PCP) pathway, and several PCP genes have been associated with NTDs. Here, we show that GPC5 orthologs are expressed in the neural tube, and that inhibiting their expression in frog and fish embryos results in NTDs. These results implicate GPC5 as a gene required for normal neural tube development. PMID:23223018

  5. Genetic interactions between planar cell polarity genes cause diverse neural tube defects in mice.

    Science.gov (United States)

    Murdoch, Jennifer N; Damrau, Christine; Paudyal, Anju; Bogani, Debora; Wells, Sara; Greene, Nicholas D E; Stanier, Philip; Copp, Andrew J

    2014-10-01

    Neural tube defects (NTDs) are among the commonest and most severe forms of developmental defect, characterized by disruption of the early embryonic events of central nervous system formation. NTDs have long been known to exhibit a strong genetic dependence, yet the identity of the genetic determinants remains largely undiscovered. Initiation of neural tube closure is disrupted in mice homozygous for mutations in planar cell polarity (PCP) pathway genes, providing a strong link between NTDs and PCP signaling. Recently, missense gene variants have been identified in PCP genes in humans with NTDs, although the range of phenotypes is greater than in the mouse mutants. In addition, the sequence variants detected in affected humans are heterozygous, and can often be detected in unaffected individuals. It has been suggested that interactions between multiple heterozygous gene mutations cause the NTDs in humans. To determine the phenotypes produced in double heterozygotes, we bred mice with all three pairwise combinations of Vangl2(Lp), Scrib(Crc) and Celsr1(Crsh) mutations, the most intensively studied PCP mutants. The majority of double-mutant embryos had open NTDs, with the range of phenotypes including anencephaly and spina bifida, therefore reflecting the defects observed in humans. Strikingly, even on a uniform genetic background, variability in the penetrance and severity of the mutant phenotypes was observed between the different double-heterozygote combinations. Phenotypically, Celsr1(Crsh);Vangl2(Lp);Scrib(Crc) triply heterozygous mutants were no more severe than doubly heterozygous or singly homozygous mutants. We propose that some of the variation between double-mutant phenotypes could be attributed to the nature of the protein disruption in each allele: whereas Scrib(Crc) is a null mutant and produces no Scrib protein, Celsr1(Crsh) and Vangl2(Lp) homozygotes both express mutant proteins, consistent with dominant effects. The variable outcomes of these genetic

  6. Planar cell polarity defects and defective Vangl2 trafficking in mutants for the COPII gene Sec24b

    NARCIS (Netherlands)

    Wansleeben, C.; Feitsma, H.; Montcouquiol, M.; Kroon, C.; Cuppen, E.; Meijlink, F.

    2010-01-01

    Among the cellular properties that are essential for the organization of tissues during animal development, the importance of cell polarity in the plane of epithelial sheets has become increasingly clear in the past decades. Planar cell polarity (PCP) signaling in vertebrates has indispensable roles

  7. The exon junction complex regulates the splicing of cell polarity gene dlg1 to control Wingless signaling in development

    Science.gov (United States)

    Liu, Min; Li, Yajuan; Liu, Aiguo; Li, Ruifeng; Su, Ying; Du, Juan; Li, Cheng; Zhu, Alan Jian

    2016-01-01

    Wingless (Wg)/Wnt signaling is conserved in all metazoan animals and plays critical roles in development. The Wg/Wnt morphogen reception is essential for signal activation, whose activity is mediated through the receptor complex and a scaffold protein Dishevelled (Dsh). We report here that the exon junction complex (EJC) activity is indispensable for Wg signaling by maintaining an appropriate level of Dsh protein for Wg ligand reception in Drosophila. Transcriptome analyses in Drosophila wing imaginal discs indicate that the EJC controls the splicing of the cell polarity gene discs large 1 (dlg1), whose coding protein directly interacts with Dsh. Genetic and biochemical experiments demonstrate that Dlg1 protein acts independently from its role in cell polarity to protect Dsh protein from lysosomal degradation. More importantly, human orthologous Dlg protein is sufficient to promote Dvl protein stabilization and Wnt signaling activity, thus revealing a conserved regulatory mechanism of Wg/Wnt signaling by Dlg and EJC. DOI: http://dx.doi.org/10.7554/eLife.17200.001 PMID:27536874

  8. Copy number variation analysis implicates the cell polarity gene glypican 5 as a human spina bifida candidate gene

    OpenAIRE

    Bassuk, Alexander G.; Muthuswamy, Lakshmi B.; Boland, Riley; Smith, Tiffany L.; Hulstrand, Alissa M.; Northrup, Hope; Hakeman, Matthew; Dierdorff, Jason M.; Yung, Christina K.; Long, Abby; Brouillette, Rachel B.; Au, Kit Sing; Gurnett, Christina; Houston, Douglas W.; Cornell, Robert A

    2012-01-01

    Neural tube defects (NTDs) are common birth defects of complex etiology. Family and population-based studies have confirmed a genetic component to NTDs. However, despite more than three decades of research, the genes involved in human NTDs remain largely unknown. We tested the hypothesis that rare copy number variants (CNVs), especially de novo germline CNVs, are a significant risk factor for NTDs. We used array-based comparative genomic hybridization (aCGH) to identify rare CNVs in 128 Cauca...

  9. The Wilms tumor gene, Wt1, is critical for mouse spermatogenesis via regulation of sertoli cell polarity and is associated with non-obstructive azoospermia in humans.

    Directory of Open Access Journals (Sweden)

    Xiao Na Wang

    Full Text Available Azoospermia is one of the major reproductive disorders which cause male infertility in humans; however, the etiology of this disease is largely unknown. In the present study, six missense mutations of WT1 gene were detected in 529 human patients with non-obstructive azoospermia (NOA, indicating a strong association between WT1 mutation and NOA. The Wilms tumor gene, Wt1, is specifically expressed in Sertoli cells (SCs which support spermatogenesis. To examine the functions of this gene in spermatogenesis, Wt1 was deleted in adult testis using Wt1(flox and Cre-ER(TM mice strains. We found that inactivation of Wt1 resulted in massive germ cell death and only SCs were present in most of the seminiferous tubules which was very similar to NOA in humans. In investigating the potential mechanism for this, histological studies revealed that the blood-testis barrier (BTB was disrupted in Wt1 deficient testes. In vitro studies demonstrated that Wt1 was essential for cell polarity maintenance in SCs. Further studies found that the expression of cell polarity associated genes (Par6b and E-cadherin and Wnt signaling genes (Wnt4, Wnt11 were downregulated in Wt1 deficient SCs, and that the expression of Par6b and E-cadherin was regulated by Wnt4. Our findings suggest that Wt1 is important in spermatogenesis by regulating the polarity of SCs via Wnt signaling pathway and that WT1 mutation is one of the genetic causes of NOA in humans.

  10. The predominant WT1 isoform (+KTS) encodes a DNA-binding protein targeting the planar cell polarity gene Scribble in renal podocytes.

    Science.gov (United States)

    Wells, Julie; Rivera, Miguel N; Kim, Woo Jae; Starbuck, Kristen; Haber, Daniel A

    2010-07-01

    WT1 encodes a tumor suppressor first identified by its inactivation in Wilms' Tumor. Although one WT1 splicing variant encodes a well-characterized zinc finger transcription factor, little is known about the function of the most prevalent WT1 isoform, whose DNA binding domain is disrupted by a three-amino acid (KTS) insertion. Using cells that conditionally express WT1(+KTS), we undertook a genome-wide chromatin immunoprecipitation and cloning analysis to identify candidate WT1(+KTS)-regulated promoters. We identified the planar cell polarity gene Scribble (SCRB) as the first WT1(+KTS) target gene in podocytes of the kidney. WT1 and SCRB expression patterns overlap precisely in developing renal glomeruli of mice, and WT1(+KTS) binds to a 33-nucleotide region within the Scribble promoter in mouse and human cell lines and kidneys. Together, our results support a role for the predominant WT1(+KTS) isoform in transcriptional regulation and suggest a link between the WT1-dependent tumor suppressor pathway and a key component of the planar cell polarity pathway. PMID:20571064

  11. The predominant WT1 isoform (+KTS) encodes a DNA binding protein targeting the planar cell polarity gene Scribble in renal podocytes

    Science.gov (United States)

    Wells, Julie; Rivera, Miguel N.; Kim, Woo Jae; Starbuck, Kristen; Haber, Daniel A.

    2010-01-01

    WT1 encodes a tumor suppressor, first identified by its inactivation in Wilms Tumor. While one WT1 splicing variant encodes a well-characterized zinc finger transcription factor, little is known about the function of the most prevalent WT1 isoform, whose DNA binding domain is disrupted by a three amino acid (KTS) insertion. Using cells which conditionally express WT1(+KTS), we undertook a genome-wide chromatin immunoprecipitation and cloning (ChIP-cloning) analysis to identify candidate WT1(+KTS) regulated promoters. We identified the planar cell polarity (PCP) gene Scribble (SCRB) as the first WT1(+KTS) target gene in podocytes of the kidney. WT1 and SCRB expression patterns overlap precisely in developing renal glomeruli of mice, and WT1(+KTS) binds to a 33 nucleotide region within the Scribble promoter in both mouse and human cell lines and kidneys. Together, our results support a role for the predominant WT1(+KTS) isoform in transcriptional regulation and suggest a link between the WT1-dependent tumor suppressor pathway and a key component of the planar cell polarity pathway. PMID:20571064

  12. Downhill exercise-induced changes in gene expression related with macrophage polarization and myogenic cells in the triceps long head of rats.

    Science.gov (United States)

    Minari, André Luis Araujo; Oyama, Lila Missae; Dos Santos, Ronaldo Vagner Thomatieli

    2015-02-01

    Macrophages are one of the most heterogenic immune cells involved in skeletal muscle regeneration. After skeletal muscle damage, M1 phenotypes exhibit pro-inflammatory reaction. In a later stage, they are converted to M2 phenotypes with anti-inflammatory properties. To study when gene expressions of macrophage polarization are changed after damage induced by downhill exercise to exhaustion is the objective of this paper. Before (CTRL) and 0 h (G0), 24 h (G24), 48 h (G48) and 72 h (G72) after 18 bouts of downhill exercise, the animals were euthanised, and the triceps were dissected. We measured gene expression of macrophages (CD68 and CD163), myogenic cells (MyoD and myogenin) and quantified cytokine secretion (interleukin (IL)-6, IL-10 and tumour necrosis factor alpha (TNF-α)). The CD68 expression was lower in G72 compared with G24 (P = 0.005) while CD163 was higher in G48 compared with G24 (P = 0.04). The MyoD expression was higher in G72 compared with G0 (P = 0.04). The myogenin expression was lower in G24 compared with CTRL (P = 0.01) and restored in G72 compared with G24 (P = 0.007). The TNF-α was significantly higher at all times after 24 h (all compared with CTRL, with P = 0.03). The CD68 and CD163 expressions behaved distinctly after exercise, which indicates macrophage polarization between 24 and 48 h. The distinct expression of myogenin, concomitantly with MyoD elevation in G72, indicates that myogenic cell differentiation and the significant change of TNF-α level show an important role of this cytokine in these processes. PMID:25249340

  13. Wnt-Dependent Control of Cell Polarity in Cultured Cells.

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    Runkle, Kristin B; Witze, Eric S

    2016-01-01

    The secreted ligand Wnt5a regulates cell polarity and polarized cell movement during development by signaling through the poorly defined noncanonical Wnt pathway. Cell polarity regulates most aspects of cell behavior including the organization of apical/basolateral membrane domains of epithelial cells, polarized cell divisions along a directional plane, and front rear polarity during cell migration. These characteristics of cell polarity allow coordinated cell movements required for tissue formation and organogenesis during embryonic development. Genetic model organisms have been used to identify multiple signaling pathways including Wnt5a that are required to establish cell polarity and regulate polarized cell behavior. However, the downstream signaling events that regulate these complex cellular processes are still poorly understood. The methods below describe assays to study Wnt5a-induced cell polarity in cultured cells, which may facilitate our understanding of these complex signaling pathways. PMID:27590152

  14. Coronavirus infection of polarized epithelial cells

    NARCIS (Netherlands)

    Rossen, J W; Horzinek, M C; Rottier, P J

    1995-01-01

    Epithelial cells are the first host cells to be infected by incoming c oronaviruses. Recent observations in vitro show that coronaviruses are released from a specific side of these polarized cells, and this polarized release might be important for the spread of the infection in vivo. Mechanisms for

  15. Membrane Organization and Dynamics in Cell Polarity

    OpenAIRE

    Orlando, Kelly; Guo, Wei

    2009-01-01

    The establishment and maintenance of cell polarity is important to a wide range of biological processes ranging from chemotaxis to embryogenesis. An essential feature of cell polarity is the asymmetric organization of proteins and lipids in the plasma membrane. In this article, we discuss how polarity regulators such as small GTP-binding proteins and phospholipids spatially and kinetically control vesicular trafficking and membrane organization. Conversely, we discuss how membrane trafficking...

  16. Profiling Signaling Polarity in Chemotactic Cells

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Yingchun; Ding, Shi-Jian; Wang, Wei; Jacobs, Jon M.; Qian, Weijun; Moore, Ronald J.; Yang, Feng; Camp, David G.; Smith, Richard D.; Klemke, Richard L.

    2007-05-15

    While directional movement requires morphological polarization characterized by formation of a leading pseudopodium at the front and a trailing rear at the back, little is known about how protein networks are spatially integrated to regulate this process. Here, we utilize a unique pseudopodial purification system and quantitative proteomics and phosphoproteomics to map the spatial relationship of 3509 proteins and 228 distinct sites of phosphorylation in polarized cells. Networks of signaling proteins, metabolic pathways, actin regulatory proteins, and kinase-substrate cascades were found to partition to different poles of the cell including components of the Ras/ERK pathway. Also, several novel proteins were found to be differentially phosphorylated at the front or rear of polarized cells and to localize to distinct subcellular structures. Our findings provide insight into the spatial organization of signaling networks that control cell movement and provide a comprehensive profile of proteins and their sites of phosphorylation that control cell polarization.

  17. Control of mammary epithelial differentiation: basement membrane induces tissue-specific gene expression in the absence of cell-cell interaction and morphological polarity

    OpenAIRE

    1991-01-01

    Functional differentiation in mammary epithelia requires specific hormones and local environmental signals. The latter are provided both by extracellular matrix and by communication with adjacent cells, their action being intricately connected in what appears to be a cascade of events leading to milk production. To distinguish between the influence of basement membrane and that of cell-cell contact in this process, we developed a novel suspension culture assay in which mammary epithelial cell...

  18. Polarized sorting and trafficking in epithelial cells

    Institute of Scientific and Technical Information of China (English)

    Xinwang Cao; Michal A Surma; Kai Simons

    2012-01-01

    The polarized distribution of proteins and lipids at the surface membrane of epithelial cells results in the formation of an apical and a basolateral domain,which are separated by tight junctions.The generation and maintenance of epithelial polarity require elaborate mechanisms that guarantee correct sorting and vectorial delivery of cargo molecules.This dynamic process involves the interaction of sorting signals with sorting machineries and the formation of transport carriers.Here we review the recent advances in the field of polarized sorting in epithelial cells.We especially highlight the role of lipid rafts in apical sorting.

  19. Appearance of differentiated cells derived from polar body nuclei in the silkworm, Bombyx mori

    Directory of Open Access Journals (Sweden)

    Hiroki eSakai

    2013-09-01

    Full Text Available AbstractIn Bombyx mori, polar body nuclei are observed until 9h after egg lying, however, the fate of polar body nuclei remains unclear. To examine the fate of polar body nuclei, we employed a mutation of serosal cell pigmentation, pink-eyed white egg (pe. The heterozygous pe/+pe females produced black serosal cells in white eggs, while pe/pe females did not produce black serosal cells in white eggs. These results suggest that the appearance of black serosal cells in white eggs depends on the genotype (pe/ +pe of the mother. Because the polar body nuclei had +pe genes in the white eggs laid by a pe/ +pe female, polar body nuclei participate in development and differentiate into functional cell (serosal cells. Analyses of serosal cells pigmentation indicated that approximately 30% of the eggs contained polar-body-nucleus-derived cells. These results demonstrate that polar-body-nucleus-derived cells appeared at a high frequency under natural conditions. Approximately 80% of polar-body-nucleus-derived cells appeared near the anterior pole and the dorsal side, which is opposite to where embryogenesis occurs. The number of cells derived from the polar body nuclei was very low. Approximately 26 % of these eggs contained only one black serosal cell. PCR-based analysis revealed that the polar-body-nucleus-derived cells disappeared in late embryonic stages (stage 25. Overall, polar-body-nuclei-derived cells were unlikely to contribute to embryos.

  20. Polarized Cell Division of Chlamydia trachomatis.

    Science.gov (United States)

    Abdelrahman, Yasser; Ouellette, Scot P; Belland, Robert J; Cox, John V

    2016-08-01

    Bacterial cell division predominantly occurs by a highly conserved process, termed binary fission, that requires the bacterial homologue of tubulin, FtsZ. Other mechanisms of bacterial cell division that are independent of FtsZ are rare. Although the obligate intracellular human pathogen Chlamydia trachomatis, the leading bacterial cause of sexually transmitted infections and trachoma, lacks FtsZ, it has been assumed to divide by binary fission. We show here that Chlamydia divides by a polarized cell division process similar to the budding process of a subset of the Planctomycetes that also lack FtsZ. Prior to cell division, the major outer-membrane protein of Chlamydia is restricted to one pole of the cell, and the nascent daughter cell emerges from this pole by an asymmetric expansion of the membrane. Components of the chlamydial cell division machinery accumulate at the site of polar growth prior to the initiation of asymmetric membrane expansion and inhibitors that disrupt the polarity of C. trachomatis prevent cell division. The polarized cell division of C. trachomatis is the result of the unipolar growth and FtsZ-independent fission of this coccoid organism. This mechanism of cell division has not been documented in other human bacterial pathogens suggesting the potential for developing Chlamydia-specific therapeutic treatments. PMID:27505160

  1. Coronaviruses in polarized epithelial cells

    NARCIS (Netherlands)

    Rossen, J W; Bekker, C P; Voorhout, W F; Horzinek, M C; Van der Ende, A; Strous, G J; Rottier, P J

    1995-01-01

    Coronaviruses have a marked tropism for epithelial cells. In this paper the interactions of the porcine transmissible gastroenteritis virus (TGEV) and mouse hepatitis virus (MHV-A59) with epithelial cells are compared. Porcine (LLC-PK1) and murine (mTAL) epithelial cells were grown on permeable supp

  2. Asymmetric protein localization in planar cell polarity

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    Peng, Ying; Axelrod, Jeffrey D.

    2016-01-01

    The polarization of epithelial cells along an axis orthogonal to their apical-basal axis is increasingly recognized for roles in a variety of developmental events and physiological functions. While now studied in many model organisms, mechanistic understanding is rooted in intensive investigations of Planar Cell Polarity (PCP) in Drosophila. Consensus has emerged that two molecular modules, referred to here as the global and core modules, operate upstream of effector proteins to produce morphological PCP. Proteins of the core module develop subcellular asymmetry, accumulating in two groups on opposite sides of cells, consistent with proposed functions in producing cell polarity and in communicating that polarity between neighboring cells. Less clear are the molecular and cell biological mechanisms underlying core module function in the generation and communication of subcellular asymmetry, and the relationship between the global and core modules. In this review, we discuss these two unresolved questions, highlighting important studies and potentially enlightening avenues for further investigation. It is likely that results from Drosophila will continue to inform our views of the growing list of examples of PCP in vertebrate systems. PMID:23140624

  3. Modeling the Control of Planar Cell Polarity

    Science.gov (United States)

    Axelrod, Jeffrey D.; Tomlin, Claire J.

    2016-01-01

    A growing list of medically important developmental defects and disease mechanisms can be traced to disruption of the Planar Cell Polarity (PCP) pathway. The PCP system polarizes cells in epithelial sheets along an axis orthogonal to their apical-basal axis. Studies in the fruitfly, Drosophila, have led to the concept of a modular system controlling PCP. The components of the PCP signaling modules, and the effector systems with which they interact, function together to produce emergent patterns. Experimental methods allow the manipulation of individual PCP signaling molecules in specified groups of cells; these interventions not only perturb the polarization of the targeted cells at a subcellular level, but also perturb patterns of polarity at the multicellular level, often affecting nearby cells in characteristic ways. These kinds of experiments should, in principle, allow one to infer the architecture within and between modules, but the relationships between molecular interactions and tissue-level pattern are sufficiently complex that they defy intuitive understanding. Mathematical modeling has been an important tool to address these problems. This review explores the emergence of a local signaling hypothesis, and describes how a local intercellular signal, coupled with a directional cue, can give rise to global pattern. We will discuss the critical role mathematical modeling has played in guiding and interpreting experimental results, and speculate about future roles for mathematical modeling of PCP. Mathematical models at varying levels of abstraction have and are expected to continue contributing in distinct ways to understanding the regulation of PCP signaling. PMID:21755606

  4. Gene transcription in polar bears (Ursus maritimus) from disparate populations

    Science.gov (United States)

    Bowen, Lizabeth; Miles, A. Keith; Waters, Shannon C.; Meyerson, Randi; Rode, Karyn D.; Atwood, Todd C.

    2015-01-01

    Polar bears in the Beaufort (SB) and Chukchi (CS) Seas experience different environments due primarily to a longer history of sea ice loss in the Beaufort Sea. Ecological differences have been identified as a possible reason for the generally poorer body condition and reproduction of Beaufort polar bears compared to those from the Chukchi, but the influence of exposure to other stressors remains unknown. We use molecular technology, quantitative PCR, to identify gene transcription differences among polar bears from the Beaufort and Chukchi Seas as well as captive healthy polar bears. We identified significant transcriptional differences among a priori groups (i.e., captive bears, SB 2012, SB 2013, CS 2013) for ten of the 14 genes of interest (i.e., CaM, HSP70, CCR3, TGFβ, COX2, THRα, T-bet, Gata3, CD69, and IL17); transcription levels of DRβ, IL1β, AHR, and Mx1 did not differ among groups. Multivariate analysis also demonstrated separation among the groups of polar bears. Specifically, we detected transcript profiles consistent with immune function impairment in polar bears from the Beaufort Sea, when compared with Chukchi and captive polar bears. Although there is no strong indication of differential exposure to contaminants or pathogens between CS and SB bears, there are clearly differences in important transcriptional responses between populations. Further investigation is warranted to refine interpretation of potential effects of described stress-related conditions for the SB population.

  5. Plant polar growth in tobacco disturbed by y-tubulin gene silencing

    Institute of Scientific and Technical Information of China (English)

    Shuang Zhao; Kun Yang; Qian Ma; Qi Wang; Xiaodan Wang; Yanhong Li

    2009-01-01

    To further understand the functions of y-tubulin in plant cells, we conducted a study in which the y-tubulin gene was down-regulated in tobacco plants (obtained by the Agrobacterium-mediated method). This involved transforming the target fragments, in which the sense and antisense partial y-tubulin cDNA fragments were ligated together, into Nicotiana tabacum var. Samsun NN. The y-tubulin down-regulated transformants developed multiple meristems or branches with trumpet-shaped leaves; their root generation also appeared abnormal, with the taproots undeveloped, whereas lateral roots were developed. In addition, the content of indole-3-acetic acid (IAA) and expression of polarity transportation vector PGPI were aberrant. These results suggest that y-tubulin gene silencing disturbed the polar growth of tobacco plants, and that this phenomenon was probably correlated with the IAA content and the polar transpor-tation process.

  6. A gas cell for stopping, storing and polarizing radioactive particles

    Science.gov (United States)

    Sytema, A.; van den Berg, J. E.; Böll, O.; Chernowitz, D.; Dijck, E. A.; Grasdijk, J. O.; Hoekstra, S.; Jungmann, K.; Mathavan, S. C.; Meinema, C.; Mohanty, A.; Müller, S. E.; Nuñez Portela, M.; Onderwater, C. J. G.; Pijpker, C.; Willmann, L.; Wilschut, H. W.

    2016-06-01

    A radioactive beam of 20Na is stopped in a gas cell filled with Ne gas. The stopped particles are polarized by optical pumping. The degree of polarization that can be achieved is studied. A maximum polarization of 50% was found. The dynamic processes in the cell are described with a phenomenological model.

  7. Auxin regulation of cell polarity in plants.

    Science.gov (United States)

    Pan, Xue; Chen, Jisheng; Yang, Zhenbiao

    2015-12-01

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

  8. American Society of Gene & Cell Therapy

    Science.gov (United States)

    ... Resources Clinical Trials Information Gene Therapy and Cell Therapy Terminology Gene Therapy & Cell Therapy Breakthroughs FAQs Gene Therapy and Cell Therapy Defined Gene Therapy and Cell Therapy for Diseases Sites of ...

  9. Mechanics and polarity in cell motility

    Science.gov (United States)

    Ambrosi, D.; Zanzottera, A.

    2016-09-01

    The motility of a fish keratocyte on a flat substrate exhibits two distinct regimes: the non-migrating and the migrating one. In both configurations the shape is fixed in time and, when the cell is moving, the velocity is constant in magnitude and direction. Transition from a stable configuration to the other one can be produced by a mechanical or chemotactic perturbation. In order to point out the mechanical nature of such a bistable behaviour, we focus on the actin dynamics inside the cell using a minimal mathematical model. While the protein diffusion, recruitment and segregation govern the polarization process, we show that the free actin mass balance, driven by diffusion, and the polymerized actin retrograde flow, regulated by the active stress, are sufficient ingredients to account for the motile bistability. The length and velocity of the cell are predicted on the basis of the parameters of the substrate and of the cell itself. The key physical ingredient of the theory is the exchange among actin phases at the edges of the cell, that plays a central role both in kinematics and in dynamics.

  10. Regulation of cell polarity determinants by the Retinoblastoma tumor suppressor protein.

    Science.gov (United States)

    Payankaulam, Sandhya; Yeung, Kelvin; McNeill, Helen; Henry, R William; Arnosti, David N

    2016-01-01

    In addition to their canonical roles in the cell cycle, RB family proteins regulate numerous developmental pathways, although the mechanisms remain obscure. We found that Drosophila Rbf1 associates with genes encoding components of the highly conserved apical-basal and planar cell polarity pathways, suggesting a possible regulatory role. Here, we show that depletion of Rbf1 in Drosophila tissues is indeed associated with polarity defects in the wing and eye. Key polarity genes aPKC, par6, vang, pk, and fmi are upregulated, and an aPKC mutation suppresses the Rbf1-induced phenotypes. RB control of cell polarity may be an evolutionarily conserved function, with important implications in cancer metastasis. PMID:26971715

  11. Loss of Cell Adhesion Increases Tumorigenic Potential of Polarity Deficient Scribble Mutant Cells.

    Directory of Open Access Journals (Sweden)

    Indrayani Waghmare

    Full Text Available Epithelial polarity genes are important for maintaining tissue architecture, and regulating growth. The Drosophila neoplastic tumor suppressor gene scribble (scrib belongs to the basolateral polarity complex. Loss of scrib results in disruption of its growth regulatory functions, and downregulation or mislocalization of Scrib is correlated to tumor growth. Somatic scribble mutant cells (scrib- surrounded by wild-type cells undergo apoptosis, which can be prevented by introduction of secondary mutations that provide a growth advantage. Using genetic tools in Drosophila, we analyzed the phenotypic effects of loss of scrib in different growth promoting backgrounds. We investigated if a central mechanism that regulates cell adhesion governs the growth and invasive potential of scrib mutant cells. Here we show that increased proliferation, and survival abilities of scrib- cells in different genetic backgrounds affect their differentiation, and intercellular adhesion. Further, loss of scrib is sufficient to cause reduced cell survival, activation of the JNK pathway and a mild reduction of cell adhesion. Our data show that for scrib cells to induce aggressive tumor growth characterized by loss of differentiation, cell adhesion, increased proliferation and invasion, cooperative interactions that derail signaling pathways play an essential role in the mechanisms leading to tumorigenesis. Thus, our study provides new insights on the effects of loss of scrib and the modification of these effects via cooperative interactions that enhance the overall tumorigenic potential of scrib deficient cells.

  12. Sterol-Rich Membrane Domains Define Fission Yeast Cell Polarity.

    Science.gov (United States)

    Makushok, Tatyana; Alves, Paulo; Huisman, Stephen Michiel; Kijowski, Adam Rafal; Brunner, Damian

    2016-05-19

    Cell polarization is crucial for the functioning of all organisms. The cytoskeleton is central to the process but its role in symmetry breaking is poorly understood. We study cell polarization when fission yeast cells exit starvation. We show that the basis of polarity generation is de novo sterol biosynthesis, cell surface delivery of sterols, and their recruitment to the cell poles. This involves four phases occurring independent of the polarity factor cdc42p. Initially, multiple, randomly distributed sterol-rich membrane (SRM) domains form at the plasma membrane, independent of the cytoskeleton and cell growth. These domains provide platforms on which the growth and polarity machinery assembles. SRM domains are then polarized by the microtubule-dependent polarity factor tea1p, which prepares for monopolar growth initiation and later switching to bipolar growth. SRM polarization requires F-actin but not the F-actin organizing polarity factors for3p and bud6p. We conclude that SRMs are key to cell polarization. PMID:27180904

  13. The predominant WT1 isoform (+KTS) encodes a DNA binding protein targeting the planar cell polarity gene Scribble in renal podocytes

    OpenAIRE

    Wells, Julie; Rivera, Miguel N.; Kim, Woo Jae; Starbuck, Kristen; Haber, Daniel A.

    2010-01-01

    WT1 encodes a tumor suppressor, first identified by its inactivation in Wilms Tumor. While one WT1 splicing variant encodes a well-characterized zinc finger transcription factor, little is known about the function of the most prevalent WT1 isoform, whose DNA binding domain is disrupted by a three amino acid (KTS) insertion. Using cells which conditionally express WT1(+KTS), we undertook a genome-wide chromatin immunoprecipitation and cloning (ChIP-cloning) analysis to identify candidate WT1(+...

  14. Shoot meristem function and leaf polarity: the role of class III HD-ZIP genes.

    Directory of Open Access Journals (Sweden)

    Mary E Byrne

    2006-06-01

    Full Text Available The shoot apical meristem comprises an organized cluster of cells with a central region population of self-maintaining stem cells providing peripheral region cells that are recruited to form differentiated lateral organs. Leaves, the principal lateral organ of the shoot, develop as polar structures typically with distinct dorsoventrality. Interdependent interactions between the meristem and developing leaf provide essential cues that serve both to maintain the meristem and to pattern dorsoventrality in the initiating leaf. A key component of both processes are the class III HD-ZIP genes. Current findings are defining the developmental role of members of this family and are identifying multiple mechanisms controlling expression of these genes.

  15. Polarizing intestinal epithelial cells electrically through Ror2

    OpenAIRE

    Cao, L; McCaig, CD; Scott, RH; Zhao, S.; G. Milne; Clevers, H; Zhao, M; Pu, J

    2014-01-01

    ABSTRACT The apicobasal polarity of enterocytes determines where the brush border membrane (apical membrane) will form, but how this apical membrane faces the lumen is not well understood. The electrical signal across the epithelium could serve as a coordinating cue, orienting and polarizing enterocytes. Here, we show that applying a physiological electric field to intestinal epithelial cells, to mimic the natural electric field created by the transepithelial potential difference, polarized p...

  16. Genomic evidence of geographically widespread effect of gene flow from polar bears into brown bears

    OpenAIRE

    Cahill, J.; Stirling, I.; Kistler, L.; Salamzade, R.; Ersmark, E.; Fulton, T.; Stiller, M.; Green, R.; Shapiro, B.

    2015-01-01

    Polar bears are an arctic, marine adapted species that is closely related to brown bears. Genome analyses have shown that polar bears are distinct and genetically homogeneous in comparison to brown bears. However, these analyses have also revealed a remarkable episode of polar bear gene flow into the population of brown bears that colonized the Admiralty, Baranof and Chichagof islands (ABC islands) of Alaska. Here, we present an analysis of data from a large panel of polar bear and brown bear...

  17. Polarity in Stem Cell Division: Asymmetric Stem Cell Division in Tissue Homeostasis

    OpenAIRE

    Yamashita, Yukiko M; Yuan, Hebao; Cheng, Jun; Hunt, Alan J.

    2010-01-01

    Many adult stem cells divide asymmetrically to balance self-renewal and differentiation, thereby maintaining tissue homeostasis. Asymmetric stem cell divisions depend on asymmetric cell architecture (i.e., cell polarity) within the cell and/or the cellular environment. In particular, as residents of the tissues they sustain, stem cells are inevitably placed in the context of the tissue architecture. Indeed, many stem cells are polarized within their microenvironment, or the stem cell niche, a...

  18. A Predictive Model for Yeast Cell Polarization in Pheromone Gradients

    Science.gov (United States)

    Calvez, Vincent; Voituriez, Raphaël; Gonçalves-Sá, Joana; Guo, Chin-Lin; Jiang, Xingyu; Murray, Andrew; Meunier, Nicolas

    2016-01-01

    Budding yeast cells exist in two mating types, a and α, which use peptide pheromones to communicate with each other during mating. Mating depends on the ability of cells to polarize up pheromone gradients, but cells also respond to spatially uniform fields of pheromone by polarizing along a single axis. We used quantitative measurements of the response of a cells to α-factor to produce a predictive model of yeast polarization towards a pheromone gradient. We found that cells make a sharp transition between budding cycles and mating induced polarization and that they detect pheromone gradients accurately only over a narrow range of pheromone concentrations corresponding to this transition. We fit all the parameters of the mathematical model by using quantitative data on spontaneous polarization in uniform pheromone concentration. Once these parameters have been computed, and without any further fit, our model quantitatively predicts the yeast cell response to pheromone gradient providing an important step toward understanding how cells communicate with each other. PMID:27077831

  19. A Predictive Model for Yeast Cell Polarization in Pheromone Gradients.

    Science.gov (United States)

    Muller, Nicolas; Piel, Matthieu; Calvez, Vincent; Voituriez, Raphaël; Gonçalves-Sá, Joana; Guo, Chin-Lin; Jiang, Xingyu; Murray, Andrew; Meunier, Nicolas

    2016-04-01

    Budding yeast cells exist in two mating types, a and α, which use peptide pheromones to communicate with each other during mating. Mating depends on the ability of cells to polarize up pheromone gradients, but cells also respond to spatially uniform fields of pheromone by polarizing along a single axis. We used quantitative measurements of the response of a cells to α-factor to produce a predictive model of yeast polarization towards a pheromone gradient. We found that cells make a sharp transition between budding cycles and mating induced polarization and that they detect pheromone gradients accurately only over a narrow range of pheromone concentrations corresponding to this transition. We fit all the parameters of the mathematical model by using quantitative data on spontaneous polarization in uniform pheromone concentration. Once these parameters have been computed, and without any further fit, our model quantitatively predicts the yeast cell response to pheromone gradient providing an important step toward understanding how cells communicate with each other. PMID:27077831

  20. Transcriptional activation of Arabidopsis axis patterning genes WOX8/9 links zygote polarity to embryo development.

    Science.gov (United States)

    Ueda, Minako; Zhang, Zhongjuan; Laux, Thomas

    2011-02-15

    In most flowering plants, the apical-basal body axis is established by an asymmetric division of the polarized zygote. In Arabidopsis, early embryo patterning is regulated by WOX homeobox genes, which are coexpressed in the zygote but become restricted to apical (WOX2) and basal (WOX8/9) cells. How the asymmetry of zygote division is regulated and connected to the daughter cell fates is largely unknown. Here, we show that expression of WOX8 is independent of the axis patterning signal auxin, but, together with the redundant gene WOX9, is activated in the zygote, its basal daughter cell, and the hypophysis by the zinc-finger transcription factor WRKY2. In wrky2 mutants, egg cells polarize normally but zygotes fail to reestablish polar organelle positioning from a transient symmetric state, resulting in equal cell division and distorted embryo development. Both defects are rescued by overexpressing WOX8, indicating that WRKY2-dependent WOX8 transcription links zygote polarization with embryo patterning. PMID:21316593

  1. Entry of genital Chlamydia trachomatis into polarized human epithelial cells.

    OpenAIRE

    Wyrick, P B; Choong, J; Davis, C H; Knight, S T; Royal, M O; Maslow, A S; Bagnell, C R

    1989-01-01

    To study the initial invasion process(es) of genital chlamydiae, a model system consisting of hormonally maintained primary cultures of human endometrial gland epithelial cells (HEGEC), grown in a polarized orientation on collagen-coated filters, was utilized. After Chlamydia trachomatis inoculation of the apical surface of polarized HEGEC, chlamydiae were readily visualized, by transmission electron microscopy, in coated pits and coated vesicles. This was true for HEGEC maintained in physiol...

  2. Expression of Opacity Proteins Interferes with the Transmigration of Neisseria gonorrhoeae across Polarized Epithelial Cells.

    Directory of Open Access Journals (Sweden)

    Daniel C Stein

    Full Text Available Neisseria gonorrhoeae (GC establishes infection at the mucosal surface of the human genital tract, most of which is lined with polarized epithelial cells. GC can cause localized as well as disseminated infections, leading to various complications. GC constantly change their surface structures via phase and antigenic variation, which has been implicated as a means for GC to establish infection at various anatomic locations of male and female genital tracks. However, the exact contribution of each surface molecule to bacterial infectivity remains elusive due to their phase variation. Using a GC derivative that is genetically devoid of all opa genes (MS11∆Opa, this study shows that Opa expression interferes with GC transmigration across polarized human epithelial cells. MS11∆Opa transmigrates across polarized epithelial cells much faster and to a greater extent than MS11Opa+, while adhering at a similar level as MS11Opa+. When MS11Opa+, able to phase vary Opa expression, was inoculated, only those bacteria that turn off Opa expression transmigrate across the polarized epithelial monolayer. Similar to bacteria alone or co-cultured with non-polarized epithelial cells, MS11∆Opa fails to form large microcolonies at the apical surface of polarized epithelial cells. Apical inoculation of MS11Opa+, but not MS11∆Opa, induces the recruitment of the Opa host-cell receptor carcinoembryonic antigen-related cell adhesion molecules (CEACAMs to the apical junction and the vicinity of bacterial adherent sites. Our results suggest that Opa expression limits gonococcal ability to invade into subepithelial tissues by forming tight interactions with neighboring bacteria and by inducing CEACAM redistribution to cell junctions.

  3. Apicobasal Polarity Controls Lymphocyte Adhesion to Hepatic Epithelial Cells

    Directory of Open Access Journals (Sweden)

    Natalia Reglero-Real

    2014-09-01

    Full Text Available Loss of apicobasal polarity is a hallmark of epithelial pathologies. Leukocyte infiltration and crosstalk with dysfunctional epithelial barriers are crucial for the inflammatory response. Here, we show that apicobasal architecture regulates the adhesion between hepatic epithelial cells and lymphocytes. Polarized hepatocytes and epithelium from bile ducts segregate the intercellular adhesion molecule 1 (ICAM-1 adhesion receptor onto their apical, microvilli-rich membranes, which are less accessible by circulating immune cells. Upon cell depolarization, hepatic ICAM-1 becomes exposed and increases lymphocyte binding. Polarized hepatic cells prevent ICAM-1 exposure to lymphocytes by redirecting basolateral ICAM-1 to apical domains. Loss of ICAM-1 polarity occurs in human inflammatory liver diseases and can be induced by the inflammatory cytokine tumor necrosis factor alpha (TNF-α. We propose that adhesion receptor polarization is a parenchymal immune checkpoint that allows functional epithelium to hamper leukocyte binding. This contributes to the haptotactic guidance of leukocytes toward neighboring damaged or chronically inflamed epithelial cells that expose their adhesion machinery.

  4. Polarized entry of canine parvovirus in an epithelial cell line.

    OpenAIRE

    Basak, S; Compans, R W

    1989-01-01

    The binding and uptake of canine parvovirus (CPV) in polarized epithelial cells were investigated by growing the cells on a permeable support and inoculating with the virus either from the apical or basolateral surface. Binding of radiolabeled CPV occurred preferentially on the basolateral surface. In contrast, when a similar experiment was carried out on nonpolarized A72 cells, virus binding occurred regardless of the direction of virus input. Binding appeared to be specific for CPV and coul...

  5. Winter diversity and expression of proteorhodopsin genes in a polar ocean.

    Science.gov (United States)

    Nguyen, Dan; Maranger, Roxane; Balagué, Vanessa; Coll-Lladó, Montserrat; Lovejoy, Connie; Pedrós-Alió, Carlos

    2015-08-01

    Mixotrophy is a valuable functional trait used by microbes when environmental conditions vary broadly or resources are limited. In the sunlit waters of the ocean, photoheterotrophy, a form of mixotrophy, is often mediated by proteorhodopsin (PR), a seven helices transmembrane protein binding the retinal chromophore. Altogether, they allow bacteria to capture photic energy for sensory and proton gradient formation cell functions. The seasonal occurrence and diversity of the gene coding for PR in cold oligotrophic polar oceans is not known and PR expression has not yet been reported. Here we show that PR is widely distributed among bacterial taxa, and that PR expression decreased markedly during the winter months in the Arctic Ocean. Gammaproteobacteria-like PR sequences were always dominant. However, within the second most common affiliation, there was a transition from Flavobacteria-like PR in early winter to Alphaproteobacteria-like PR in late winter. The phylogenetic shifts followed carbon dynamics, where patterns in expression were consistent with community succession, as identified by DNA community fingerprinting. Although genes for PR were always present, the trend in decreasing transcripts from January to February suggested reduced functional utility of PR during winter. Under winter darkness, sustained expression suggests that PR may continue to be useful for non-ATP forming functions, such as environmental sensing or small solute transport. The persistence of PR expression in winter among some bacterial groups may offer a competitive advantage, where its multifunctionality enhances microbial survival under harsh polar conditions. PMID:25700336

  6. Lipid polarity and sorting in epithelial cells

    OpenAIRE

    van Meer, G.; Simons, K.

    1988-01-01

    Apical and basolateral membrane domains of epithelial cell plasma membranes possess unique lipid compositions. The tight junction, the structure separating the two domains, forms a diffusion barrier for membrane components and thereby prevents intermixing of the two sets of lipids. The barrier apparently resides in the outer, exoplasmic leaflet of the plasma membrane bilayer. First data are now available on the generation of these differences in Madin-Darby canine kidney (MDCK) cells, grown o...

  7. Hybrid T-helper cells: stabilizing the moderate center in a polarized system.

    Directory of Open Access Journals (Sweden)

    Sui Huang

    Full Text Available Polarization of cell phenotypes, a common strategy to achieve cell type diversity in metazoa, results from binary cell-fate decisions in the branching pedigree of development. Such "either-or" fate decisions are controlled by two opposing cell fate-determining transcription factors. Each of the two distinct "master regulators" promotes differentiation of its respective sister lineage. But they also suppress one other, leading to their mutually exclusive expression in the two ensuing lineages. Thus, promiscuous coexistence of the antagonist regulators in the same cell, the hallmark of the common "undecided" progenitor of two sister lineages, is considered unstable. This antagonism ensures robust polarization into two discretely distinct cell types. But now the immune system's T-helper (Th cells and their two canonical subtypes, Th1 and Th2 cells, tell a different story, as revealed in three papers recently published in PLOS Biology. The intermediate state that co-expresses the two opposing master regulators of the Th1 and Th2 subtypes, T-bet and Gata3, is highly stable and is not necessarily an undecided precursor. Instead, the Th1/Th2 hybrid cell is a robust new type with properties of both Th1 and Th2 cells. These hybrid cells are functionally active and possess the benefit of moderation: self-limitation of effector T cell function to prevent excessive inflammation, a permanent risk in host defense that can cause tissue damage or autoimmunity. Gene regulatory network analysis suggests that stabilization of the intermediate center in a polarizing system can be achieved by minor tweaking of the architecture of the mutual suppression gene circuit, and thus is a design option readily available to evolution.

  8. Hybrid T-helper cells: stabilizing the moderate center in a polarized system.

    Science.gov (United States)

    Huang, Sui

    2013-01-01

    Polarization of cell phenotypes, a common strategy to achieve cell type diversity in metazoa, results from binary cell-fate decisions in the branching pedigree of development. Such "either-or" fate decisions are controlled by two opposing cell fate-determining transcription factors. Each of the two distinct "master regulators" promotes differentiation of its respective sister lineage. But they also suppress one other, leading to their mutually exclusive expression in the two ensuing lineages. Thus, promiscuous coexistence of the antagonist regulators in the same cell, the hallmark of the common "undecided" progenitor of two sister lineages, is considered unstable. This antagonism ensures robust polarization into two discretely distinct cell types. But now the immune system's T-helper (Th) cells and their two canonical subtypes, Th1 and Th2 cells, tell a different story, as revealed in three papers recently published in PLOS Biology. The intermediate state that co-expresses the two opposing master regulators of the Th1 and Th2 subtypes, T-bet and Gata3, is highly stable and is not necessarily an undecided precursor. Instead, the Th1/Th2 hybrid cell is a robust new type with properties of both Th1 and Th2 cells. These hybrid cells are functionally active and possess the benefit of moderation: self-limitation of effector T cell function to prevent excessive inflammation, a permanent risk in host defense that can cause tissue damage or autoimmunity. Gene regulatory network analysis suggests that stabilization of the intermediate center in a polarizing system can be achieved by minor tweaking of the architecture of the mutual suppression gene circuit, and thus is a design option readily available to evolution. PMID:23976879

  9. Deletion of the msdS/AfmsdC gene induces abnormal polarity and septation in Aspergillus fumigatus.

    Science.gov (United States)

    Li, Yanjie; Zhang, Lei; Wang, Depeng; Zhou, Hui; Ouyang, Haomiao; Ming, Jia; Jin, Cheng

    2008-07-01

    alpha-Mannosidases play an important role in the processing of mannose-containing glycans in eukaryotes. A deficiency in alpha-mannosidase is lethal in humans and cattle. In contrast to mammals, Saccharomyces cerevisiae does not require the endoplasmic reticulum alpha-mannosidase gene for growth. However, little is known of the consequence of loss of function of class I alpha-mannosidases in filamentous fungi. In this study, the msdS/AfmsdC gene was identified to encode 1,2-alpha-mannosidase MsdS in Aspergillus fumigatus. Soluble MsdS expressed in Escherichia coli was characterized as a typical class I alpha-mannosidase. The msdS gene was deleted by replacement of the msdS gene with a pyrG gene. Although the mutant showed a defect in N-glycan processing, as well as a reduction of cell wall components and a reduced ability of conidiation, it appeared that the rate of hyphal growth was not affected. Morphology analysis revealed abnormal polarity and septation at the stages of germination, hyphal growth and conidiation. Although the mechanism by which the N-glycan processing affects polarity and septation is unclear, our results show that msdS is involved in polarity and septation in A. fumigatus. PMID:18599824

  10. The Signaling Mechanisms Underlying Cell Polarity and Chemotaxis

    OpenAIRE

    Wang, Fei

    2009-01-01

    Chemotaxis—the directed movement of cells in a gradient of chemoattractant—is essential for neutrophils to crawl to sites of inflammation and infection and for Dictyostelium discoideum (D. discoideum) to aggregate during morphogenesis. Chemoattractant-induced activation of spatially localized cellular signals causes cells to polarize and move toward the highest concentration of the chemoattractant. Extensive studies have been devoted to achieving a better understanding of the mechanism(s) use...

  11. Vectorial secretion of proteoglycans by polarized rat uterine epithelial cells

    OpenAIRE

    1988-01-01

    We have studied proteoglycan secretion using a recently developed system for the preparing of polarized primary cultures of rat uterine epithelial cells. To mimic their native environment better and provide a system for discriminating apical from basolateral compartments, we cultured cells on semipermeable supports impregnated with biomatrix. Keratan sulfate proteoglycans (KSPG) as well as heparan sulfate- containing molecules (HS[PG]) were the major sulfated products synthesized and secreted...

  12. Dressed Spin of Polarized 3He in a Cell

    CERN Document Server

    Chu, P H; Peng, J C; Beck, D H; Chandler, D E; Clayton, S; Hu, B Z; Ngan, S Y; Sham, C H; So, L H; Williamson, S; Yoder, J

    2010-01-01

    We report a measurement of the modification of the effective precession frequency of polarized 3He atoms in response to a dressing field in a room temperature cell. The 3He atoms were polarized using the metastability spin-exchange method. An oscillating dressing field is then applied perpendicular to the constant magnetic field. Modification of the 3He effective precession frequency was observed over a broad range of the amplitude and frequency of the dressing field. The observed effects are compared with calculations based on quantum optics formalism.

  13. Lis1 mediates planar polarity of auditory hair cells through regulation of microtubule organization

    OpenAIRE

    Sipe, Conor W.; Liu, Lixia; Lee, Jianyi; Grimsley-Myers, Cynthia; Lu, Xiaowei

    2013-01-01

    The V-shaped hair bundles atop auditory hair cells and their uniform orientation are manifestations of epithelial planar cell polarity (PCP) required for proper perception of sound. PCP is regulated at the tissue level by a conserved core Wnt/PCP pathway. However, the hair cell-intrinsic polarity machinery is poorly understood. Recent findings implicate hair cell microtubules in planar polarization of hair cells. To elucidate the microtubule-mediated polarity pathway, we analyzed Lis1 functio...

  14. Satellite Cells in Muscular Dystrophy - Lost in Polarity.

    Science.gov (United States)

    Chang, Natasha C; Chevalier, Fabien P; Rudnicki, Michael A

    2016-06-01

    Recent findings employing the mdx mouse model for Duchenne muscular dystrophy (DMD) have revealed that muscle satellite stem cells play a direct role in contributing to disease etiology and progression of DMD, the most common and severe form of muscular dystrophy. Lack of dystrophin expression in DMD has critical consequences in satellite cells including an inability to establish cell polarity, abrogation of asymmetric satellite stem-cell divisions, and failure to enter the myogenic program. Thus, muscle wasting in dystrophic mice is not only caused by myofiber fragility but is exacerbated by intrinsic satellite cell dysfunction leading to impaired regeneration. Despite intense research and clinical efforts, there is still no effective cure for DMD. In this review we highlight recent research advances in DMD and discuss the current state of treatment and, importantly, how we can incorporate satellite cell-targeted therapeutic strategies to correct satellite cell dysfunction in DMD. PMID:27161598

  15. Genomic evidence of geographically widespread effect of gene flow from polar bears into brown bears.

    Science.gov (United States)

    Cahill, James A; Stirling, Ian; Kistler, Logan; Salamzade, Rauf; Ersmark, Erik; Fulton, Tara L; Stiller, Mathias; Green, Richard E; Shapiro, Beth

    2015-03-01

    Polar bears are an arctic, marine adapted species that is closely related to brown bears. Genome analyses have shown that polar bears are distinct and genetically homogeneous in comparison to brown bears. However, these analyses have also revealed a remarkable episode of polar bear gene flow into the population of brown bears that colonized the Admiralty, Baranof and Chichagof islands (ABC islands) of Alaska. Here, we present an analysis of data from a large panel of polar bear and brown bear genomes that includes brown bears from the ABC islands, the Alaskan mainland and Europe. Our results provide clear evidence that gene flow between the two species had a geographically wide impact, with polar bear DNA found within the genomes of brown bears living both on the ABC islands and in the Alaskan mainland. Intriguingly, while brown bear genomes contain up to 8.8% polar bear ancestry, polar bear genomes appear to be devoid of brown bear ancestry, suggesting the presence of a barrier to gene flow in that direction. PMID:25490862

  16. Minimal model for spontaneous cell polarization and edge activity in oscillating, rotating and migrating cells

    Science.gov (United States)

    Raynaud, Franck; Ambühl, Mark E.; Gabella, Chiara; Bornert, Alicia; Sbalzarini, Ivo F.; Meister, Jean-Jacques; Verkhovsky, Alexander B.

    2016-04-01

    How cells break symmetry and organize activity at their edges to move directionally is a fundamental question in cell biology. Physical models of cell motility commonly incorporate gradients of regulatory proteins and/or feedback from the motion itself to describe the polarization of this edge activity. These approaches, however, fail to explain cell behaviour before the onset of polarization. We use polarizing and moving fish epidermal cells as a model system to bridge the gap between cell behaviours before and after polarization. Our analysis suggests a novel and simple principle of self-organizing cell activity, in which local cell-edge dynamics depends on the distance from the cell centre, but not on the orientation with respect to the front-back axis. We validate this principle with a stochastic model that faithfully reproduces a range of cell-migration behaviours. Our findings indicate that spontaneous polarization, persistent motion and cell shape are emergent properties of the local cell-edge dynamics controlled by the distance from the cell centre.

  17. A Three-Dimensional Cell Culture Model To Study Enterovirus Infection of Polarized Intestinal Epithelial Cells.

    Science.gov (United States)

    Drummond, Coyne G; Nickerson, Cheryl A; Coyne, Carolyn B

    2016-01-01

    Despite serving as the primary entry portal for coxsackievirus B (CVB), little is known about CVB infection of the intestinal epithelium, owing at least in part to the lack of suitable in vivo models and the inability of cultured cells to recapitulate the complexity and structure associated with the gastrointestinal (GI) tract. Here, we report on the development of a three-dimensional (3-D) organotypic cell culture model of Caco-2 cells to model CVB infection of the gastrointestinal epithelium. We show that Caco-2 cells grown in 3-D using the rotating wall vessel (RWV) bioreactor recapitulate many of the properties of the intestinal epithelium, including the formation of well-developed tight junctions, apical-basolateral polarity, brush borders, and multicellular complexity. In addition, transcriptome analyses using transcriptome sequencing (RNA-Seq) revealed the induction of a number of genes associated with intestinal epithelial differentiation and/or intestinal processes in vivo when Caco-2 cells were cultured in 3-D. Applying this model to CVB infection, we found that although the levels of intracellular virus production were similar in two-dimensional (2-D) and 3-D Caco-2 cell cultures, the release of infectious CVB was enhanced in 3-D cultures at early stages of infection. Unlike CVB, the replication of poliovirus (PV) was significantly reduced in 3-D Caco-2 cell cultures. Collectively, our studies show that Caco-2 cells grown in 3-D using the RWV bioreactor provide a cell culture model that structurally and transcriptionally represents key aspects of cells in the human GI tract and can thus be used to expand our understanding of enterovirus-host interactions in intestinal epithelial cells. IMPORTANCE Coxsackievirus B (CVB), a member of the enterovirus family of RNA viruses, is associated with meningitis, pericarditis, diabetes, dilated cardiomyopathy, and myocarditis, among other pathologies. CVB is transmitted via the fecal-oral route and encounters the

  18. A Three-Dimensional Cell Culture Model To Study Enterovirus Infection of Polarized Intestinal Epithelial Cells

    Science.gov (United States)

    Drummond, Coyne G.

    2015-01-01

    ABSTRACT Despite serving as the primary entry portal for coxsackievirus B (CVB), little is known about CVB infection of the intestinal epithelium, owing at least in part to the lack of suitable in vivo models and the inability of cultured cells to recapitulate the complexity and structure associated with the gastrointestinal (GI) tract. Here, we report on the development of a three-dimensional (3-D) organotypic cell culture model of Caco-2 cells to model CVB infection of the gastrointestinal epithelium. We show that Caco-2 cells grown in 3-D using the rotating wall vessel (RWV) bioreactor recapitulate many of the properties of the intestinal epithelium, including the formation of well-developed tight junctions, apical-basolateral polarity, brush borders, and multicellular complexity. In addition, transcriptome analyses using transcriptome sequencing (RNA-Seq) revealed the induction of a number of genes associated with intestinal epithelial differentiation and/or intestinal processes in vivo when Caco-2 cells were cultured in 3-D. Applying this model to CVB infection, we found that although the levels of intracellular virus production were similar in two-dimensional (2-D) and 3-D Caco-2 cell cultures, the release of infectious CVB was enhanced in 3-D cultures at early stages of infection. Unlike CVB, the replication of poliovirus (PV) was significantly reduced in 3-D Caco-2 cell cultures. Collectively, our studies show that Caco-2 cells grown in 3-D using the RWV bioreactor provide a cell culture model that structurally and transcriptionally represents key aspects of cells in the human GI tract and can thus be used to expand our understanding of enterovirus-host interactions in intestinal epithelial cells. IMPORTANCE Coxsackievirus B (CVB), a member of the enterovirus family of RNA viruses, is associated with meningitis, pericarditis, diabetes, dilated cardiomyopathy, and myocarditis, among other pathologies. CVB is transmitted via the fecal-oral route and

  19. [Polar coordinates representation based leukocyte segmentation of microscopic cell images].

    Science.gov (United States)

    Gu, Guanghua; Cui, Dong; Hao, Lianwang

    2010-12-01

    We propose an algorithm for segmentation of the overlapped leukocyte in the microscopic cell image. The histogram of the saturation channel in the cell image is smoothed to obtain the meaningful global valley point by the fingerprint smoothing method, and then the nucleus can be segmented. A circular region, containing the entire regions of the leukocyte, is marked off according to the equivalent sectional radius of the nucleus. Then, the edge of the overlapped leukocyte is represented by polar coordinates. The overlapped region by the change of the polar angle of the edge pixels is determined, and the closed edge of the leukocyte integrating the gradient information of the overlapped region is reconstructed. Finally, the leukocyte is exactly extracted. The experimental results show that our method has good performance in terms of recall ratio, precision ratio and pixel error ratio. PMID:21374971

  20. Centrosome polarization in T cells: a task for formins

    Directory of Open Access Journals (Sweden)

    Laura eAndrés-Delgado

    2013-07-01

    Full Text Available T-cell antigen receptor (TCR engagement triggers the rapid reorientation of the centrosome, which is associated with the secretory machinery, towards the immunological synapse (IS for polarized protein trafficking. Recent evidence indicates that upon TCR triggering the INF2 formin, together with the formins DIA1 and FMNL1, promotes the formation of a specialized array of stable detyrosinated MTs that breaks the symmetrical organization of the T-cell microtubule (MT cytoskeleton. The detyrosinated MT array and TCR-induced tyrosine phosphorylation should coincide for centrosome polarization. We propose that the pushing forces produced by the detyrosinated MT array, which modify the position of the centrosome, in concert with Src kinase dependent TCR signaling, which provide the reference frame with respect to which the centrosome reorients, result in the repositioning of the centrosome to the IS.

  1. Polarization and reprogramming of myeloid-derived suppressor cells

    OpenAIRE

    Yang, Wen-Chin; Ma, Ge; Chen, Shu-hsia; Pan, Ping-Ying

    2013-01-01

    Myeloid-derived suppressor cells (MDSC) have recently emerged as one of the central regulators of the immune system. In recent years, interest in understanding MDSC biology and applying MDSC for therapeutic purpose has exploded exponentially. Despite recent progress in MDSC biology, the mechanisms underlying MDSC development from expansion and activation to polarization in different diseases remain poorly understood. More recent studies have demonstrated that two MDSC subsets, M (monocytic)-M...

  2. X11/Mint genes control polarized localization of axonal membrane proteins in vivo.

    Science.gov (United States)

    Gross, Garrett G; Lone, G Mohiddin; Leung, Lok Kwan; Hartenstein, Volker; Guo, Ming

    2013-05-01

    Mislocalization of axonal proteins can result in misassembly and/or miswiring of neural circuits, causing disease. To date, only a handful of genes that control polarized localization of axonal membrane proteins have been identified. Here we report that Drosophila X11/Mint proteins are required for targeting several proteins, including human amyloid precursor protein (APP) and Drosophila APP-like protein (APPL), to axonal membranes and for their exclusion from dendrites of the mushroom body in Drosophila, a brain structure involved in learning and memory. Axonal localization of APP is mediated by an endocytic motif, and loss of X11/Mint results in a dramatic increase in cell-surface levels of APPL, especially on dendrites. Mutations in genes required for endocytosis show similar mislocalization of these proteins to dendrites, and strongly enhance defects seen in X11/Mint mutants. These results suggest that X11/Mint-dependent endocytosis in dendrites may serve to promote the axonal localization of membrane proteins. Since X11/Mint binds to APP, and abnormal trafficking of APP contributes to Alzheimer's disease, deregulation of X11/Mint may be important for Alzheimer's disease pathogenesis. PMID:23658195

  3. MMP28 promotes macrophage polarization toward M2 cells and augments pulmonary fibrosis

    Science.gov (United States)

    Gharib, Sina A.; Johnston, Laura K.; Huizar, Isham; Birkland, Timothy P.; Hanson, Josiah; Wang, Ying; Parks, William C.; Manicone, Anne M.

    2014-01-01

    Members of the MMP family function in various processes of innate immunity, particularly in controlling important steps in leukocyte trafficking and activation. MMP28 (epilysin) is a member of this family of proteinases, and we have found that MMP28 is expressed by macrophages and regulates their recruitment to the lung. We hypothesized that MMP28 regulates other key macrophage responses, such as macrophage polarization. Furthermore, we hypothesized that these MMP28-dependent changes in macrophage polarization would alter fibrotic responses in the lung. We examined the gene expression changes in WT and Mmp28−/− BMDMs, stimulated with LPS or IL-4/IL-13 to promote M1 and M2 cells, respectively. We also collected macrophages from the lungs of Pseudomonas aeruginosa-exposed WT and Mmp28−/− mice to evaluate changes in macrophage polarization. Lastly, we evaluated the macrophage polarization phenotypes during bleomycin-induced pulmonary fibrosis in WT and Mmp28−/− mice and assessed mice for differences in weight loss and total collagen levels. We found that MMP28 dampens proinflammatory macrophage function and promots M2 programming. In both in vivo models, we found deficits in M2 polarization in Mmp28−/− mice. In bleomycin-induced lung injury, these changes were associated with reduced fibrosis. MMP28 is an important regulator of macrophage polarization, promoting M2 function. Loss of MMP28 results in reduced M2 polarization and protection from bleomycin-induced fibrosis. These findings highlight a novel role for MMP28 in macrophage biology and pulmonary disease. PMID:23964118

  4. Efficient Gene Transfer in Bacterial Cell Chains

    OpenAIRE

    Babic, Ana; Berkmen, Melanie B.; Lee, Catherine A.; Grossman, Alan D.

    2011-01-01

    Horizontal gene transfer contributes to evolution and the acquisition of new traits. In bacteria, horizontal gene transfer is often mediated by conjugative genetic elements that transfer directly from cell to cell. Integrative and conjugative elements (ICEs; also known as conjugative transposons) are mobile genetic elements that reside within a host genome but can excise to form a circle and transfer by conjugation to recipient cells. ICEs contribute to the spread of genes involved in pathoge...

  5. Inter-tissue mechanical stress affects Frizzled-mediated planar cell polarity in the Drosophila notum epidermis

    OpenAIRE

    Olguín, Patricio; Glavic, Alvaro; Mlodzik, Marek

    2011-01-01

    Frizzled/Planar Cell Polarity (Fz/PCP) signaling controls the orientation of sensory bristles and cellular hairs (trichomes) along the antero-posterior axis of the Drosophila thorax (notum) [1–4]. A subset of the trichome-producing notum cells differentiate as “tendon cells”, serving as attachment sites for the indirect flight muscles (IFMs) to the exoskeleton [5]. Through the analysis of chascon (chas), a gene identified by its ability to disrupt Fz/PCP signaling under overexpression conditi...

  6. Planar Cell Polarity Controls Pancreatic Beta Cell Differentiation and Glucose Homeostasis

    DEFF Research Database (Denmark)

    Cortijo, Cedric; Gouzi, Mathieu; Tissir, Fadel;

    2012-01-01

    .5 synchronously to apicobasal polarization of pancreas progenitors. Loss of function of the two PCP core components Celsr2 and Celsr3 shows that they control the differentiation of endocrine cells from polarized progenitors, with a prevalent effect on insulin-producing beta cells. This results in a decreased...... glucose clearance. Loss of Celsr2 and 3 leads to a reduction of Jun phosphorylation in progenitors, which, in turn, reduces beta cell differentiation from endocrine progenitors. These results highlight the importance of the PCP pathway in cell differentiation in vertebrates. In addition, they reveal that...

  7. The arabidopsis thaliana AGRAVITROPIC 1 gene encodes a component of the polar-auxin-transport efflux carrier

    Science.gov (United States)

    Chen, R.; Hilson, P.; Sedbrook, J.; Rosen, E.; Caspar, T.; Masson, P. H.

    1998-01-01

    Auxins are plant hormones that mediate many aspects of plant growth and development. In higher plants, auxins are polarly transported from sites of synthesis in the shoot apex to their sites of action in the basal regions of shoots and in roots. Polar auxin transport is an important aspect of auxin functions and is mediated by cellular influx and efflux carriers. Little is known about the molecular identity of its regulatory component, the efflux carrier [Estelle, M. (1996) Current Biol. 6, 1589-1591]. Here we show that mutations in the Arabidopsis thaliana AGRAVITROPIC 1 (AGR1) gene involved in root gravitropism confer increased root-growth sensitivity to auxin and decreased sensitivity to ethylene and an auxin transport inhibitor, and cause retention of exogenously added auxin in root tip cells. We used positional cloning to show that AGR1 encodes a putative transmembrane protein whose amino acid sequence shares homologies with bacterial transporters. When expressed in Saccharomyces cerevisiae, AGR1 promotes an increased efflux of radiolabeled IAA from the cells and confers increased resistance to fluoro-IAA, a toxic IAA-derived compound. AGR1 transcripts were localized to the root distal elongation zone, a region undergoing a curvature response upon gravistimulation. We have identified several AGR1-related genes in Arabidopsis, suggesting a global role of this gene family in the control of auxin-regulated growth and developmental processes.

  8. Characterization of a pancreatic islet cell tumor in a polar bear (Ursus maritimus).

    Science.gov (United States)

    Fortin, Jessica S; Benoit-Biancamano, Marie-Odile

    2014-01-01

    Herein, we report a 25-year-old male polar bear suffering from a pancreatic islet cell tumor. The aim of this report is to present a case of this rare tumor in a captive polar bear. The implication of potential risk factors such as high carbohydrate diet or the presence of amyloid fibril deposits was assessed. Necropsy examination revealed several other changes, including nodules observed in the liver, spleen, pancreas, intestine, and thyroid glands that were submitted for histopathologic analysis. Interestingly, the multiple neoplastic nodules were unrelated and included a pancreatic islet cell tumor. Immunohistochemistry of the pancreas confirmed the presence of insulin and islet amyloid polypeptide (IAPP) within the pancreatic islet cells. The IAPP gene was extracted from the paraffin-embedded liver tissue and sequenced. IAPP cDNA from the polar bear exhibits some differences as compared to the sequence published for several other species. Different factors responsible for neoplasms in bears such as diet, infectious agents, and industrial chemical exposure are reviewed. This case report raised several issues that further studies may address by evaluating the prevalence of cancers in captive or wild animals. PMID:25273481

  9. Entry of genital Chlamydia trachomatis into polarized human epithelial cells.

    Science.gov (United States)

    Wyrick, P B; Choong, J; Davis, C H; Knight, S T; Royal, M O; Maslow, A S; Bagnell, C R

    1989-01-01

    To study the initial invasion process(es) of genital chlamydiae, a model system consisting of hormonally maintained primary cultures of human endometrial gland epithelial cells (HEGEC), grown in a polarized orientation on collagen-coated filters, was utilized. After Chlamydia trachomatis inoculation of the apical surface of polarized HEGEC, chlamydiae were readily visualized, by transmission electron microscopy, in coated pits and coated vesicles. This was true for HEGEC maintained in physiologic concentrations of estrogen (proliferative phase) and of estrogen plus progesterone (secretory phase), despite the finding that association of chlamydiae with secretory-phase HEGEC is significantly reduced (P = 0.025; A.S. Maslow, C.H. Davis, J. Choong, and P.B. Wyrick, Am. J. Obstet. Gynecol. 159:1006-1014, 1988). In contrast, chlamydiae were rarely observed in the clathrin-associated structures if the HEGEC were cultured on plastic surfaces. The same pattern of coated pit versus noncoated pit entry was reproducible in HeLa cells. The quantity of coated pits associated with isolated membrane sheets derived from HeLa cells, grown on poly-L-lysine-coated cover slips in medium containing the female hormones, was not significantly different as monitored by radiolabeling studies and by laser scanning microscopy. These data suggest that culture conditions which mimic in vivo cellular organization may enhance entry into coated pits for some obligate intracellular pathogens. Images PMID:2744852

  10. Challenge for lowering concentration polarization in solid oxide fuel cells

    Science.gov (United States)

    Shimada, Hiroyuki; Suzuki, Toshio; Yamaguchi, Toshiaki; Sumi, Hirofumi; Hamamoto, Koichi; Fujishiro, Yoshinobu

    2016-01-01

    In the scope of electrochemical phenomena, concentration polarization at electrodes is theoretically inevitable, and lowering the concentration overpotential to improve the performance of electrochemical cells has been a continuing challenge. Electrodes with highly controlled microstructure, i.e., high porosity and uniform large pores are therefore essential to achieve high performance electrochemical cells. In this study, state-of-the-art technology for controlling the microstructure of electrodes has been developed for realizing high performance support electrodes of solid oxide fuel cells (SOFCs). The key is controlling the porosity and pore size distribution to improve gas diffusion, while maintaining the integrity of the electrolyte and the structural strength of actual sized electrode supports needed for the target application. Planar anode-supported SOFCs developed in this study realize 5 μm thick dense electrolyte (yttria-stabilized zirconia: YSZ) and the anode substrate (Ni-YSZ) of 53.6 vol.% porosity with a large median pore diameter of 0.911 μm. Electrochemical measurements reveal that the performance of the anode-supported SOFCs improves with increasing anode porosity. This Ni-YSZ anode minimizes the concentration polarization, resulting in a maximum power density of 3.09 W cm-2 at 800 °C using humidified hydrogen fuel without any electrode functional layers.

  11. Hybrid cell lines constitute a potential reservoir of polarized cells: isolation and study of highly differentiated hepatoma-derived hybrid cells able to form functional bile canaliculi in vitro

    OpenAIRE

    1991-01-01

    A large number of hepatoma cell lines has been used to study expression and regulation of liver-specific function. However these cells, even the most differentiated, are morphologically far from hepatocytes. In no case is the typical hepatocyte cell polarity well maintained. Cell hybridization has been used as a potential means for turning on specific genes. From hybrids between well differentiated Fao rat hepatoma cells and WI 38 human fibroblasts, we have attempted to isolate segregated cel...

  12. Airway epithelial homeostasis and planar cell polarity signaling depend on multiciliated cell differentiation

    Science.gov (United States)

    Vladar, Eszter K.; Nayak, Jayakar V.; Milla, Carlos E.; Axelrod, Jeffrey D.

    2016-01-01

    Motile airway cilia that propel contaminants out of the lung are oriented in a common direction by planar cell polarity (PCP) signaling, which localizes PCP protein complexes to opposite cell sides throughout the epithelium to orient cytoskeletal remodeling. In airway epithelia, PCP is determined in a 2-phase process. First, cell-cell communication via PCP complexes polarizes all cells with respect to the proximal-distal tissue axis. Second, during ciliogenesis, multiciliated cells (MCCs) undergo cytoskeletal remodeling to orient their cilia in the proximal direction. The second phase not only directs cilium polarization, but also consolidates polarization across the epithelium. Here, we demonstrate that in airway epithelia, PCP depends on MCC differentiation. PCP mutant epithelia have misaligned cilia, and also display defective barrier function and regeneration, indicating that PCP regulates multiple aspects of airway epithelial homeostasis. In humans, MCCs are often sparse in chronic inflammatory diseases, and these airways exhibit PCP dysfunction. The presence of insufficient MCCs impairs mucociliary clearance in part by disrupting PCP-driven polarization of the epithelium. Consistent with defective PCP, barrier function and regeneration are also disrupted. Pharmacological stimulation of MCC differentiation restores PCP and reverses these defects, suggesting its potential for broad therapeutic benefit in chronic inflammatory disease.

  13. Intertissue mechanical stress affects Frizzled-mediated planar cell polarity in the Drosophila notum epidermis.

    Science.gov (United States)

    Olguín, Patricio; Glavic, Alvaro; Mlodzik, Marek

    2011-02-01

    Frizzled/planar cell polarity (Fz/PCP) signaling controls the orientation of sensory bristles and cellular hairs (trichomes) along the anteroposterior axis of the Drosophila thorax (notum). A subset of the trichome-producing notum cells differentiate as "tendon cells," serving as attachment sites for the indirect flight muscles (IFMs) to the exoskeleton. Through the analysis of chascon (chas), a gene identified by its ability to disrupt Fz/PCP signaling under overexpression conditions, and jitterbug (jbug)/filamin, we show that maintenance of anteroposterior planar polarization requires the notum epithelia to balance mechanical stress generated by the attachment of the IFMs. chas is expressed in notum tendon cells, and its loss of function disturbs cellular orientation at and near the regions where IFMs attach to the epidermis. This effect is independent of the Fz/PCP and fat/dachsous systems. The chas phenotype arises during normal shortening of the IFMs and is suppressed by genetic ablation of the IFMs. chas acts through jbug/filamin and cooperates with MyosinII to modulate the mechanoresponse of notum tendon cells. These observations support the notion that the ability of epithelia to respond to mechanical stress generated by one or more interactions with other tissues during development and organogenesis influences the maintenance of its shape and PCP features. PMID:21276726

  14. Mycobacterium tuberculosislpdC, Rv0462, induces dendritic cell maturation and Th1 polarization

    Energy Technology Data Exchange (ETDEWEB)

    Heo, Deok Rim [Department of Microbiology and Immunology, School of Medicine, Pusan National University, Beom-eo Ri, Mulgum Eop, Yangsan, Gyeongsangnam-do 626-770 (Korea, Republic of); Shin, Sung Jae; Kim, Woo Sik [Department of Microbiology, College of Medicine, Chungnam National University, Munwha-Dong, Jung-Ku, Daejeon 301-747 (Korea, Republic of); Noh, Kyung Tae; Park, Jin Wook; Son, Kwang Hee [Department of Microbiology and Immunology, School of Medicine, Pusan National University, Beom-eo Ri, Mulgum Eop, Yangsan, Gyeongsangnam-do 626-770 (Korea, Republic of); Park, Won Sun [Department of Physiology, Kangwon National University, School of Medicine, Chuncheon 200-701 (Korea, Republic of); Lee, Min-Goo [Department of Physiology, Korea University, College of Medicine, Anam-dong, Sungbuk-Gu, Seoul 136-705 (Korea, Republic of); Kim, Daejin [Department of Anatomy, Chung-Ang University, College of Medicine, 221 Heuksuk-Dong, Dongjak-Ku, Seoul 156-756 (Korea, Republic of); Shin, Yong Kyoo [Department of Pharmacology, Chung-Ang University, College of Medicine, 221 Heuksuk-Dong, Dongjak-Ku, Seoul 156-756 (Korea, Republic of); Jung, In Duk, E-mail: jungid@pusan.ac.kr [Department of Microbiology and Immunology, School of Medicine, Pusan National University, Beom-eo Ri, Mulgum Eop, Yangsan, Gyeongsangnam-do 626-770 (Korea, Republic of); Research Institute of Convergence of Biomedical Science and Technology, Pusan National University Yangsan Hospital, Beom-eo Ri, Mulgum Eop, Yangsan, Gyeongsangnam-do 626-770 (Korea, Republic of); Park, Yeong-Min, E-mail: immunpym@pusan.ac.kr [Department of Microbiology and Immunology, School of Medicine, Pusan National University, Beom-eo Ri, Mulgum Eop, Yangsan, Gyeongsangnam-do 626-770 (Korea, Republic of); Research Institute of Convergence of Biomedical Science and Technology, Pusan National University Yangsan Hospital, Beom-eo Ri, Mulgum Eop, Yangsan, Gyeongsangnam-do 626-770 (Korea, Republic of)

    2011-08-05

    Highlights: {yields} Treatment with Rv0462 induces the expression of surface molecules and the production of cytokines in DCs. {yields} Rv0462 induces the activation of MAPKs. {yields} Rv0462-treated DCs enhances the proliferation of CD4{sup +} T cells. -- Abstract: Mycobacterium tuberculosis, the etiological factor of pulmonary tuberculosis, causes significant morbidity and mortality worldwide. Activation of host immune responses for containment of mycobacterial infections involves participation of innate immune cells, such as dendritic cells (DCs). In this study, we demonstrated that the gene encoding lipoamide dehydrogenase C (lpdC) from M. tuberculosis, Rv0462, induce maturation and activation of DCs involved in the MAPKs signaling pathway. Moreover, Rv0462-treated DCs activated naive T cells, polarized CD4{sup +} and CD8{sup +} T cells to secrete IFN-{gamma} in syngeneic mixed lymphocyte reactions, which would be expected to contribute to Th1 polarization of the immune response. Our results suggest that Rv0462 can contribute to the innate and adaptive immune responses during tuberculosis infection, and thus modulate the clinical course of tuberculosis.

  15. Mycobacterium tuberculosislpdC, Rv0462, induces dendritic cell maturation and Th1 polarization

    International Nuclear Information System (INIS)

    Highlights: → Treatment with Rv0462 induces the expression of surface molecules and the production of cytokines in DCs. → Rv0462 induces the activation of MAPKs. → Rv0462-treated DCs enhances the proliferation of CD4+ T cells. -- Abstract: Mycobacterium tuberculosis, the etiological factor of pulmonary tuberculosis, causes significant morbidity and mortality worldwide. Activation of host immune responses for containment of mycobacterial infections involves participation of innate immune cells, such as dendritic cells (DCs). In this study, we demonstrated that the gene encoding lipoamide dehydrogenase C (lpdC) from M. tuberculosis, Rv0462, induce maturation and activation of DCs involved in the MAPKs signaling pathway. Moreover, Rv0462-treated DCs activated naive T cells, polarized CD4+ and CD8+ T cells to secrete IFN-γ in syngeneic mixed lymphocyte reactions, which would be expected to contribute to Th1 polarization of the immune response. Our results suggest that Rv0462 can contribute to the innate and adaptive immune responses during tuberculosis infection, and thus modulate the clinical course of tuberculosis.

  16. Planar cell polarity enables posterior localization of nodal cilia and left-right axis determination during mouse and Xenopus embryogenesis.

    Directory of Open Access Journals (Sweden)

    Dragana Antic

    Full Text Available Left-right asymmetry in vertebrates is initiated in an early embryonic structure called the ventral node in human and mouse, and the gastrocoel roof plate (GRP in the frog. Within these structures, each epithelial cell bears a single motile cilium, and the concerted beating of these cilia produces a leftward fluid flow that is required to initiate left-right asymmetric gene expression. The leftward fluid flow is thought to result from the posterior tilt of the cilia, which protrude from near the posterior portion of each cell's apical surface. The cells, therefore, display a morphological planar polarization. Planar cell polarity (PCP is manifested as the coordinated, polarized orientation of cells within epithelial sheets, or as directional cell migration and intercalation during convergent extension. A set of evolutionarily conserved proteins regulates PCP. Here, we provide evidence that vertebrate PCP proteins regulate planar polarity in the mouse ventral node and in the Xenopus gastrocoel roof plate. Asymmetric anterior localization of VANGL1 and PRICKLE2 (PK2 in mouse ventral node cells indicates that these cells are planar polarized by a conserved molecular mechanism. A weakly penetrant Vangl1 mutant phenotype suggests that compromised Vangl1 function may be associated with left-right laterality defects. Stronger functional evidence comes from the Xenopus GRP, where we show that perturbation of VANGL2 protein function disrupts the posterior localization of motile cilia that is required for leftward fluid flow, and causes aberrant expression of the left side-specific gene Nodal. The observation of anterior-posterior PCP in the mouse and in Xenopus embryonic organizers reflects a strong evolutionary conservation of this mechanism that is important for body plan determination.

  17. Pluripotent Stem Cells and Gene Therapy

    OpenAIRE

    Simara, Pavel; Motl, Jason A.; Kaufman, Dan S.

    2013-01-01

    Human pluripotent stem cells represent an accessible cell source for novel cell-based clinical research and therapies. With the realization of induced pluripotent stem cells (iPSCs), it is possible to produce almost any desired cell type from any patient's cells. Current developments in gene modification methods have opened the possibility for creating genetically corrected human iPSCs for certain genetic diseases that could be used later in autologous transplantation. Promising preclinical s...

  18. Differentiation of embryonic stem cells transfected by ibeB gene

    Institute of Scientific and Technical Information of China (English)

    SHANG Deshu; FANG Wengang; CHEN Yuhua

    2005-01-01

    We have previously identified an E. coli deter- minant, ibeB gene locus contributing to invasion of human brain microvascular endothelial cells. In the present study, we established embryonic stem (ES) cell lines overexpressing IbeB and found that exogenic ibeB gene could start-up expression of a neural stem cell specific marker, nestin, and give rise to polar changes. In analysis of IbeB location, it was found that GFP-IbeB fusion protein targeted at the ES cell nucleus. These data suggests that ibeB gene may play an important role in the regulation of nestin expression.

  19. Shoot Meristem Function and Leaf Polarity: The Role of Class III HD–ZIP Genes

    OpenAIRE

    Byrne, Mary E

    2006-01-01

    The shoot apical meristem comprises an organized cluster of cells with a central region population of self-maintaining stem cells providing peripheral region cells that are recruited to form differentiated lateral organs. Leaves, the principal lateral organ of the shoot, develop as polar structures typically with distinct dorsoventrality. Interdependent interactions between the meristem and developing leaf provide essential cues that serve both to maintain the meristem and to pattern dorsoven...

  20. Muscle Stem Cell Fate Is Controlled by the Cell-Polarity Protein Scrib

    Directory of Open Access Journals (Sweden)

    Yusuke Ono

    2015-02-01

    Full Text Available Satellite cells are resident skeletal muscle stem cells that supply myonuclei for homeostasis, hypertrophy, and repair in adult muscle. Scrib is one of the major cell-polarity proteins, acting as a potent tumor suppressor in epithelial cells. Here, we show that Scrib also controls satellite-cell-fate decisions in adult mice. Scrib is undetectable in quiescent cells but becomes expressed during activation. Scrib is asymmetrically distributed in dividing daughter cells, with robust accumulation in cells committed to myogenic differentiation. Low Scrib expression is associated with the proliferative state and preventing self-renewal, whereas high Scrib levels reduce satellite cell proliferation. Satellite-cell-specific knockout of Scrib in mice causes a drastic and insurmountable defect in muscle regeneration. Thus, Scrib is a regulator of tissue stem cells, controlling population expansion and self-renewal with Scrib expression dynamics directing satellite cell fate.

  1. Interferon-γ and Tumor Necrosis Factor-α Polarize Bone Marrow Stromal Cells Uniformly to a Th1 Phenotype.

    Science.gov (United States)

    Jin, Ping; Zhao, Yuanlong; Liu, Hui; Chen, Jinguo; Ren, Jiaqiang; Jin, Jianjian; Bedognetti, Davide; Liu, Shutong; Wang, Ena; Marincola, Francesco; Stroncek, David

    2016-01-01

    Activated T cells polarize mesenchymal stromal cells (MSCs) to a proinflammatory Th1 phenotype which likely has an important role in amplifying the immune response in the tumor microenvironment. We investigated the role of interferon gamma (IFN-γ) and tumor necrosis factor alpha (TNF-α), two factors produced by activated T cells, in MSC polarization. Gene expression and culture supernatant analysis showed that TNF-α and IFN-γ stimulated MSCs expressed distinct sets of proinflammatory factors. The combination of IFN-γ and TNF-α was synergistic and induced a transcriptome most similar to that found in MSCs stimulated with activated T cells and similar to that found in the inflamed tumor microenvironment; a Th1 phenotype with the expression of the immunosuppressive factors IL-4, IL-10, CD274/PD-L1 and indoleamine 2,3 dioxygenase (IDO). Single cell qRT-PCR analysis showed that the combination of IFN-γ and TNF-α polarized uniformly to this phenotype. The combination of IFN-γ and TNF-α results in the synergist uniform polarization of MSCs toward a primarily Th1 phenotype. The stimulation of MSCs by IFN-γ and TNF-α released from activated tumor infiltrating T cells is likely responsible for the production of many factors that characterize the tumor microenvironment. PMID:27211104

  2. Endothelial Cell Migration and Vascular Endothelial Growth Factor Expression Are the Result of Loss of Breast Tissue Polarity

    Energy Technology Data Exchange (ETDEWEB)

    Chen, Amy; Cuevas, Ileana; Kenny, Paraic A; Miyake, Hiroshi; Mace, Kimberley; Ghajar, Cyrus; Boudreau, Aaron; Bissell, Mina; Boudreau, Nancy

    2009-05-26

    Recruiting a new blood supply is a rate-limiting step in tumor progression. In a three-dimensional model of breast carcinogenesis, disorganized, proliferative transformed breast epithelial cells express significantly higher expression of angiogenic genes compared with their polarized, growth-arrested nonmalignant counterparts. Elevated vascular endothelial growth factor (VEGF) secretion by malignant cells enhanced recruitment of endothelial cells (EC) in heterotypic cocultures. Significantly, phenotypic reversion of malignant cells via reexpression of HoxD10, which is lost in malignant progression, significantly attenuated VEGF expression in a hypoxia-inducible factor 1{alpha}-independent fashion and reduced EC migration. This was due primarily to restoring polarity: forced proliferation of polarized, nonmalignant cells did not induce VEGF expression and EC recruitment, whereas disrupting the architecture of growth-arrested, reverted cells did. These data show that disrupting cytostructure activates the angiogenic switch even in the absence of proliferation and/or hypoxia and restoring organization of malignant clusters reduces VEGF expression and EC activation to levels found in quiescent nonmalignant epithelium. These data confirm the importance of tissue architecture and polarity in malignant progression.

  3. Epithelial cell polarity and tumorigenesis: new perspectives for cancer detection and treatment

    Institute of Scientific and Technical Information of China (English)

    Danila CORADINI; Claudia CASARSA; Saro ORIANA

    2011-01-01

    Loss of cell-cell adhesion and cell polarity is commonly observed in tumors of epithelial origin and correlates with their invasion into adjacent tissues and formation of metastases. Growing evidence indicates that loss of cell polarity and cell-cell adhesion may also be important in early stage of cancer. In first part of this review, we delineate the current understanding of the mechanisms that establish and maintain the polarity of epithelial tissues and discuss the involvement of cell polarity and apical junctional complex components in tumor pathogenesis. In the second part we address the clinical significance of cell polarity and junctional complex components in can- cer diagnosis and prognosis. Finally, we explore their potential use as therapeutic targets in the treatment of cancer.

  4. Circumpolar Genetic Structure and Recent Gene Flow of Polar Bears: A Reanalysis.

    Directory of Open Access Journals (Sweden)

    René M Malenfant

    Full Text Available Recently, an extensive study of 2,748 polar bears (Ursus maritimus from across their circumpolar range was published in PLOS ONE, which used microsatellites and mitochondrial haplotypes to apparently show altered population structure and a dramatic change in directional gene flow towards the Canadian Archipelago-an area believed to be a future refugium for polar bears as their southernmost habitats decline under climate change. Although this study represents a major international collaborative effort and promised to be a baseline for future genetics work, methodological shortcomings and errors of interpretation undermine some of the study's main conclusions. Here, we present a reanalysis of this data in which we address some of these issues, including: (1 highly unbalanced sample sizes and large amounts of systematically missing data; (2 incorrect calculation of FST and of significance levels; (3 misleading estimates of recent gene flow resulting from non-convergence of the program BayesAss. In contrast to the original findings, in our reanalysis we find six genetic clusters of polar bears worldwide: the Hudson Bay Complex, the Western and Eastern Canadian Arctic Archipelago, the Western and Eastern Polar Basin, and-importantly-we reconfirm the presence of a unique and possibly endangered cluster of bears in Norwegian Bay near Canada's expected last sea-ice refugium. Although polar bears' abundance, distribution, and population structure will certainly be negatively affected by ongoing-and increasingly rapid-loss of Arctic sea ice, these genetic data provide no evidence of strong directional gene flow in response to recent climate change.

  5. Circumpolar Genetic Structure and Recent Gene Flow of Polar Bears: A Reanalysis.

    Science.gov (United States)

    Malenfant, René M; Davis, Corey S; Cullingham, Catherine I; Coltman, David W

    2016-01-01

    Recently, an extensive study of 2,748 polar bears (Ursus maritimus) from across their circumpolar range was published in PLOS ONE, which used microsatellites and mitochondrial haplotypes to apparently show altered population structure and a dramatic change in directional gene flow towards the Canadian Archipelago-an area believed to be a future refugium for polar bears as their southernmost habitats decline under climate change. Although this study represents a major international collaborative effort and promised to be a baseline for future genetics work, methodological shortcomings and errors of interpretation undermine some of the study's main conclusions. Here, we present a reanalysis of this data in which we address some of these issues, including: (1) highly unbalanced sample sizes and large amounts of systematically missing data; (2) incorrect calculation of FST and of significance levels; (3) misleading estimates of recent gene flow resulting from non-convergence of the program BayesAss. In contrast to the original findings, in our reanalysis we find six genetic clusters of polar bears worldwide: the Hudson Bay Complex, the Western and Eastern Canadian Arctic Archipelago, the Western and Eastern Polar Basin, and-importantly-we reconfirm the presence of a unique and possibly endangered cluster of bears in Norwegian Bay near Canada's expected last sea-ice refugium. Although polar bears' abundance, distribution, and population structure will certainly be negatively affected by ongoing-and increasingly rapid-loss of Arctic sea ice, these genetic data provide no evidence of strong directional gene flow in response to recent climate change. PMID:26974333

  6. Asymmetric division triggers cell-specific gene expression through coupled capture and stabilization of a phosphatase

    OpenAIRE

    Bradshaw, Niels; Losick, Richard

    2015-01-01

    Formation of a division septum near a randomly chosen pole during sporulation in Bacillus subtilis creates unequal sized daughter cells with dissimilar programs of gene expression. An unanswered question is how polar septation activates a transcription factor (σF) selectively in the small cell. We present evidence that the upstream regulator of σF, the phosphatase SpoIIE, is compartmentalized in the small cell by transfer from the polar septum to the adjacent cell pole where SpoIIE is protect...

  7. Polarized Trafficking of the Sorting Receptor SorLA in Neurons and MDCK Cells

    DEFF Research Database (Denmark)

    Klinger, Stine C; Højland, Anne; Jain, Shweta;

    2016-01-01

    The sorting receptor SorLA is highly expressed in neurons and is also found in other polarized cells. The receptor has been reported to participate in the trafficking of several ligands, some of which are linked to human diseases, including the amyloid precursor protein, TrkB and lipoprotein lipase...... (LpL). Despite this, only the trafficking in non-polarized cells has been described so far. Due to the many differences between polarized and non-polarized cells, we examined the localization and trafficking of SorLA in epithelial Madin-Darby canine kidney (MDCK) cells and rat hippocampal neurons. We...

  8. Gene sensitizes cancer cells to chemotherapy drugs

    Science.gov (United States)

    NCI scientists have found that a gene, Schlafen-11 (SLFN11), sensitizes cells to substances known to cause irreparable damage to DNA.  As part of their study, the researchers used a repository of 60 cell types to identify predictors of cancer cell respons

  9. Rho GTPases and regulation of cell migration and polarization in human corneal epithelial cells.

    Directory of Open Access Journals (Sweden)

    Aihua Hou

    Full Text Available PURPOSE: Epithelial cell migration is required for regeneration of tissues and can be defective in a number of ocular surface diseases. This study aimed to determine the expression pattern of Rho family small G-proteins in human corneal epithelial cells to test their requirement in directional cell migration. METHODS: Rho family small G-protein expression was assessed by reverse transcription-polymerase chain reaction. Dominant-inhibitory constructs encoding Rho proteins or Rho protein targeting small interfering RNA were transfected into human corneal epithelial large T antigen cells, and wound closure rate were evaluated by scratch wounding assay, and a complementary non-traumatic cell migration assay. Immunofluorescence staining was performed to study cell polarization and to assess Cdc42 downstream effector. RESULTS: Cdc42, Chp, Rac1, RhoA, TC10 and TCL were expressed in human corneal epithelial cells. Among them, Cdc42 and TCL were found to significantly affect cell migration in monolayer scratch assays. These results were confirmed through the use of validated siRNAs directed to Cdc42 and TCL. Scramble siRNA transfected cells had high percentage of polarized cells than Cdc42 or TCL siRNA transfected cells at the wound edge. We showed that the Cdc42-specific effector p21-activated kinase 4 localized predominantly to cell-cell junctions in cell monolayers, but failed to translocate to the leading edge in Cdc42 siRNA transfected cells after monolayer wounding. CONCLUSION: Rho proteins expressed in cultured human corneal epithelial cells, and Cdc42, TCL facilitate two-dimensional cell migration in-vitro. Although silencing of Cdc42 and TCL did not noticeably affect the appearance of cell adhesions at the leading edge, the slower migration of these cells indicates both GTP-binding proteins play important roles in promoting cell movement of human corneal epithelial cells.

  10. X11/Mint Genes Control Polarized Localization of Axonal Membrane Proteins in Vivo

    OpenAIRE

    Garrett G Gross; Lone, G. Mohiddin; Leung, Lok Kwan; Hartenstein, Volker; Guo, Ming

    2013-01-01

    Mislocalization of axonal proteins can result in misassembly and/or miswiring of neural circuits, causing disease. To date, only a handful of genes that control polarized localization of axonal membrane proteins have been identified. Here we report that Drosophila X11/Mint proteins are required for targeting several proteins, including human amyloid precursor protein (APP) and Drosophila APP-like protein (APPL), to axonal membranes and for their exclusion from dendrites of the mushroom body i...

  11. Patterns of polarity in the Escherichia coli car AB gene cluster.

    OpenAIRE

    Gigot, D; Crabeel, M; Feller, A; Charlier, D; Lissens, W; Glansdorff, N.; Piérard, A

    1980-01-01

    The direction of transcription of the carAB gene cluster, which codes for Escherichia coli carbamoylphosphate synthase, was deduced from the effects of phage Mu-1 insertions in each of the two genes and from the results of ribonucleic acid-deoxyribonucleic acid hybridization experiments relating the quantity of car messenger ribonucleic acid to the location of various car mutations. The car locus appears to constitute an operon polarized from carA to carB. The levels of carA and carB products...

  12. Dishevelled is essential for neural connectivity and planar cell polarity in planarians.

    Science.gov (United States)

    Almuedo-Castillo, Maria; Saló, Emili; Adell, Teresa

    2011-02-15

    The Wingless/Integrated (Wnt) signaling pathway controls multiple events during development and homeostasis. It comprises multiple branches, mainly classified according to their dependence on β-catenin activation. The Wnt/β-catenin branch is essential for the establishment of the embryonic anteroposterior (AP) body axis throughout the phylogenetic tree. It is also required for AP axis establishment during planarian regeneration. Wnt/β-catenin-independent signaling encompasses several different pathways, of which the most extensively studied is the planar cell polarity (PCP) pathway, which is responsible for planar polarization of cell structures within an epithelial sheet. Dishevelled (Dvl) is the hub of Wnt signaling because it regulates and channels the Wnt signal into every branch. Here, we analyze the role of Schmidtea mediterranea Dvl homologs (Smed-dvl-1 and Smed-dvl-2) using gene silencing. We demonstrate that in addition to a role in AP axis specification, planarian Dvls are involved in at least two different β-catenin-independent processes. First, they are essential for neural connectivity through Smed-wnt5 signaling. Second, Smed-dvl-2, together with the S. mediterranea homologs of Van-Gogh (Vang) and Diversin (Div), is required for apical positioning of the basal bodies of epithelial cells. These data represent evidence not only of the function of the PCP network in lophotrocozoans but of the involvement of the PCP core elements Vang and Div in apical positioning of the cilia. PMID:21282632

  13. Kif3a regulates planar polarization of auditory hair cells through both ciliary and non-ciliary mechanisms

    OpenAIRE

    Sipe, Conor W.; Lu, Xiaowei

    2011-01-01

    Auditory hair cells represent one of the most prominent examples of epithelial planar polarity. In the auditory sensory epithelium, planar polarity of individual hair cells is defined by their V-shaped hair bundle, the mechanotransduction organelle located on the apical surface. At the tissue level, all hair cells display uniform planar polarity across the epithelium. Although it is known that tissue planar polarity is controlled by non-canonical Wnt/planar cell polarity (PCP) signaling, the ...

  14. Pluripotent Stem Cells and Gene Therapy

    Science.gov (United States)

    Simara, Pavel; Motl, Jason A.; Kaufman, Dan S.

    2013-01-01

    Human pluripotent stem cells represent an accessible cell source for novel cell-based clinical research and therapies. With the realization of induced pluripotent stem cells (iPSCs), it is possible to produce almost any desired cell type from any patient's cells. Current developments in gene modification methods have opened the possibility for creating genetically corrected human iPSCs for certain genetic diseases that could be used later in autologous transplantation. Promising preclinical studies have demonstrated correction of disease-causing mutations in a number of hematological, neuronal and muscular disorders. This review aims to summarize these recent advances with a focus on iPSC generation techniques, as well as gene modification methods. We will then further discuss some of the main obstacles remaining to be overcome before successful application of human pluripotent stem cell-based therapy arrives in the clinic and what the future of stem cell research may look like. PMID:23353080

  15. Mechanistic Framework for Establishment, Maintenance, and Alteration of Cell Polarity in Plants

    Directory of Open Access Journals (Sweden)

    Pankaj Dhonukshe

    2012-01-01

    Full Text Available Cell polarity establishment, maintenance, and alteration are central to the developmental and response programs of nearly all organisms and are often implicated in abnormalities ranging from patterning defects to cancer. By residing at the distinct plasma membrane domains polar cargoes mark the identities of those domains, and execute localized functions. Polar cargoes are recruited to the specialized membrane domains by directional secretion and/or directional endocytic recycling. In plants, auxin efflux carrier PIN proteins display polar localizations in various cell types and play major roles in directional cell-to-cell transport of signaling molecule auxin that is vital for plant patterning and response programs. Recent advanced microscopy studies applied to single cells in intact plants reveal subcellular PIN dynamics. They uncover the PIN polarity generation mechanism and identified important roles of AGC kinases for polar PIN localization. AGC kinase family members PINOID, WAG1, and WAG2, belonging to the AGC-3 subclass predominantly influence the polar localization of PINs. The emerging mechanism for AGC-3 kinases action suggests that kinases phosphorylate PINs mainly at the plasma membrane after initial symmetric PIN secretion for eventual PIN internalization and PIN sorting into distinct ARF-GEF-regulated polar recycling pathways. Thus phosphorylation status directs PIN translocation to different cell sides. Based on these findings a mechanistic framework evolves that suggests existence of cell side-specific recycling pathways in plants and implicates AGC3 kinases for differential PIN recruitment among them for eventual PIN polarity establishment, maintenance, and alteration.

  16. Brown and polar bear Y chromosomes reveal extensive male-biased gene flow within brother lineages.

    Science.gov (United States)

    Bidon, Tobias; Janke, Axel; Fain, Steven R; Eiken, Hans Geir; Hagen, Snorre B; Saarma, Urmas; Hallström, Björn M; Lecomte, Nicolas; Hailer, Frank

    2014-06-01

    Brown and polar bears have become prominent examples in phylogeography, but previous phylogeographic studies relied largely on maternally inherited mitochondrial DNA (mtDNA) or were geographically restricted. The male-specific Y chromosome, a natural counterpart to mtDNA, has remained underexplored. Although this paternally inherited chromosome is indispensable for comprehensive analyses of phylogeographic patterns, technical difficulties and low variability have hampered its application in most mammals. We developed 13 novel Y-chromosomal sequence and microsatellite markers from the polar bear genome and screened these in a broad geographic sample of 130 brown and polar bears. We also analyzed a 390-kb-long Y-chromosomal scaffold using sequencing data from published male ursine genomes. Y chromosome evidence support the emerging understanding that brown and polar bears started to diverge no later than the Middle Pleistocene. Contrary to mtDNA patterns, we found 1) brown and polar bears to be reciprocally monophyletic sister (or rather brother) lineages, without signals of introgression, 2) male-biased gene flow across continents and on phylogeographic time scales, and 3) male dispersal that links the Alaskan ABC islands population to mainland brown bears. Due to female philopatry, mtDNA provides a highly structured estimate of population differentiation, while male-biased gene flow is a homogenizing force for nuclear genetic variation. Our findings highlight the importance of analyzing both maternally and paternally inherited loci for a comprehensive view of phylogeographic history, and that mtDNA-based phylogeographic studies of many mammals should be reevaluated. Recent advances in sequencing technology render the analysis of Y-chromosomal variation feasible, even in nonmodel organisms. PMID:24667925

  17. Sequential development of apical-basal and planar polarities in aggregating epitheliomuscular cells of Hydra.

    Science.gov (United States)

    Seybold, Anna; Salvenmoser, Willi; Hobmayer, Bert

    2016-04-01

    Apical-basal and planar cell polarities are hallmarks of metazoan epithelia required to separate internal and external environments and to regulate trans- and intracellular transport, cytoskeletal organization, and morphogenesis. Mechanisms of cell polarization have been intensively studied in bilaterian model organisms, particularly in early embryos and cultured cells, while cell polarity in pre-bilaterian tissues is poorly understood. Here, we have studied apical-basal and planar polarization in regenerating (aggregating) clusters of epitheliomuscular cells of Hydra, a simple representative of the ancestral, pre-bilaterian phylum Cnidaria. Immediately after dissociation, single epitheliomuscular cells do not exhibit cellular polarity, but they polarize de novo during aggregation. Reestablishment of the Hydra-specific epithelial bilayer is a result of short-range cell sorting. In the early phase of aggregation, apical-basal polarization starts with an enlargement of the epithelial apical-basal diameter and by the development of belt-like apical septate junctions. Specification of the basal pole of epithelial cells occurs shortly later and is linked to synthesis of mesoglea, development of hemidesmosome-like junctions, and formation of desmosome-like junctions connecting the basal myonemes of neighbouring cells. Planar polarization starts, while apical-basal polarization is already ongoing. It is executed gradually starting with cell-autonomous formation, parallelization, and condensation of myonemes at the basal end of each epithelial cell and continuing with a final planar alignment of epitheliomuscular cells at the tissue level. Our findings reveal that epithelial polarization in Hydra aggregates occurs in defined steps well accessible by histological and ultrastructural techniques and they will provide a basis for future molecular studies. PMID:26921448

  18. Regulation of differentiation and polarized secretion in mammary epithelial cells maintained in culture: extracellular matrix and membrane polarity influences

    OpenAIRE

    1987-01-01

    Several previous studies have demonstrated that mammary epithelial cells from pregnant mice retain their differentiated characteristics and their secretory potential in culture only when maintained on stromal collagen gels floated in the culture medium. The cellular basis for these culture requirements was investigated by the monitoring of milk protein synthesis and polarized secretion from the mouse mammary epithelial cell line, COMMA-1-D. Experiments were directed towards gaining an underst...

  19. Competition of two distinct actin networks for actin defines a bistable switch for cell polarization

    Science.gov (United States)

    Lomakin, Alexis J.; Lee, Kun-Chun; Han, Sangyoon J.; Bui, D A.; Davidson, Michael; Mogilner, Alex; Danuser, Gaudenz

    2015-01-01

    Symmetry-breaking polarization enables functional plasticity of cells and tissues and is yet not well understood. Here we show that epithelial cells, hard-wired to maintain a static morphology and to preserve tissue organization, can spontaneously switch to a migratory polarized phenotype upon relaxation of the actomyosin cytoskeleton. We find that myosin-II engages actin in the formation of cortical actomyosin bundles and thus makes it unavailable for deployment in the process of dendritic growth normally driving cell motility. At low contractility regimes epithelial cells polarize in a front-back manner due to emergence of actin retrograde flows powered by dendritic polymerization of actin. Coupled to cell movement, the flows transport myosin-II from the front to the back of the cell, where the motor locally “locks” actin in contractile bundles. This polarization mechanism could be employed by embryonic and cancer epithelial cells in microenvironments where high contractility-driven cell motion is inefficient. PMID:26414403

  20. Heat induces gene amplification in cancer cells

    International Nuclear Information System (INIS)

    Highlights: ► This study discovered that heat exposure (hyperthermia) results in gene amplification in cancer cells. ► Hyperthermia induces DNA double strand breaks. ► DNA double strand breaks are considered to be required for the initiation of gene amplification. ► The underlying mechanism of heat-induced gene amplification is generation of DNA double strand breaks. -- Abstract: Background: Hyperthermia plays an important role in cancer therapy. However, as with radiation, it can cause DNA damage and therefore genetic instability. We studied whether hyperthermia can induce gene amplification in cancer cells and explored potential underlying molecular mechanisms. Materials and methods: (1) Hyperthermia: HCT116 colon cancer cells received water-submerged heating treatment at 42 or 44 °C for 30 min; (2) gene amplification assay using N-(phosphoacetyl)-L-aspartate (PALA) selection of cabamyl-P-synthetase, aspartate transcarbarmylase, dihydro-orotase (cad) gene amplified cells; (3) southern blotting for confirmation of increased cad gene copies in PALA-resistant cells; (4) γH2AX immunostaining to detect γH2AX foci as an indication for DNA double strand breaks. Results: (1) Heat exposure at 42 or 44 °C for 30 min induces gene amplification. The frequency of cad gene amplification increased by 2.8 and 6.5 folds respectively; (2) heat exposure at both 42 and 44 °C for 30 min induces DNA double strand breaks in HCT116 cells as shown by γH2AX immunostaining. Conclusion: This study shows that heat exposure can induce gene amplification in cancer cells, likely through the generation of DNA double strand breaks, which are believed to be required for the initiation of gene amplification. This process may be promoted by heat when cellular proteins that are responsible for checkpoints, DNA replication, DNA repair and telomere functions are denatured. To our knowledge, this is the first study to provide direct evidence of hyperthermia induced gene amplification.

  1. Heat induces gene amplification in cancer cells

    Energy Technology Data Exchange (ETDEWEB)

    Yan, Bin, E-mail: yanbin@mercyhealth.com [Department of Radiation Oncology, University of Mississippi Medical Center, Jackson, MS 39213 (United States); Mercy Cancer Center, Mercy Medical Center-North Iowa, Mason City, IA 50401 (United States); Ouyang, Ruoyun [Department of Respiratory Medicine, The Second Xiangya Hospital, Xinagya School of Medicine, Central South University, Changsha 410011 (China); Huang, Chenghui [Department of Radiation Oncology, University of Mississippi Medical Center, Jackson, MS 39213 (United States); Department of Oncology, The Third Xiangya Hospital, Xinagya School of Medicine, Central South University, Changsha 410013 (China); Liu, Franklin [Department of Radiation Oncology, Duke University Medical Center, Durham, NC 27710 (United States); Neill, Daniel [Department of Radiation Oncology, University of Mississippi Medical Center, Jackson, MS 39213 (United States); Li, Chuanyuan [Dermatology, Duke University Medical Center, Durham, NC 27710 (United States); Dewhirst, Mark [Department of Radiation Oncology, Duke University Medical Center, Durham, NC 27710 (United States)

    2012-10-26

    Highlights: Black-Right-Pointing-Pointer This study discovered that heat exposure (hyperthermia) results in gene amplification in cancer cells. Black-Right-Pointing-Pointer Hyperthermia induces DNA double strand breaks. Black-Right-Pointing-Pointer DNA double strand breaks are considered to be required for the initiation of gene amplification. Black-Right-Pointing-Pointer The underlying mechanism of heat-induced gene amplification is generation of DNA double strand breaks. -- Abstract: Background: Hyperthermia plays an important role in cancer therapy. However, as with radiation, it can cause DNA damage and therefore genetic instability. We studied whether hyperthermia can induce gene amplification in cancer cells and explored potential underlying molecular mechanisms. Materials and methods: (1) Hyperthermia: HCT116 colon cancer cells received water-submerged heating treatment at 42 or 44 Degree-Sign C for 30 min; (2) gene amplification assay using N-(phosphoacetyl)-L-aspartate (PALA) selection of cabamyl-P-synthetase, aspartate transcarbarmylase, dihydro-orotase (cad) gene amplified cells; (3) southern blotting for confirmation of increased cad gene copies in PALA-resistant cells; (4) {gamma}H2AX immunostaining to detect {gamma}H2AX foci as an indication for DNA double strand breaks. Results: (1) Heat exposure at 42 or 44 Degree-Sign C for 30 min induces gene amplification. The frequency of cad gene amplification increased by 2.8 and 6.5 folds respectively; (2) heat exposure at both 42 and 44 Degree-Sign C for 30 min induces DNA double strand breaks in HCT116 cells as shown by {gamma}H2AX immunostaining. Conclusion: This study shows that heat exposure can induce gene amplification in cancer cells, likely through the generation of DNA double strand breaks, which are believed to be required for the initiation of gene amplification. This process may be promoted by heat when cellular proteins that are responsible for checkpoints, DNA replication, DNA repair and

  2. Positioning of polarity formation by extracellular signaling during asymmetric cell division.

    Science.gov (United States)

    Seirin Lee, Sungrim

    2016-07-01

    Anterior-posterior (AP) polarity formation of cell membrane proteins plays a crucial role in determining cell asymmetry, which ultimately generates cell diversity. In Caenorhabditis elegans, a single fertilized egg cell (P0), its daughter cell (P1), and the germline precursors (P2 and P3 cells) form two exclusive domains of different PAR proteins on the membrane along the anterior-posterior axis. However, the phenomenon of polarity reversal has been observed in which the axis of asymmetric cell division of the P2 and P3 cells is formed in an opposite manner to that of the P0 and P1 cells. The extracellular signal MES-1/SRC-1 has been shown to induce polarity reversal, but the detailed mechanism remains elusive. Here, using a mathematical model, I explore the mechanism by which MES-1/SRC-1 signaling can induce polarity reversal and ultimately affect the process of polarity formation. I show that a positive correlation between SRC-1 and the on-rate of PAR-2 is the essential mechanism underlying polarity reversal, providing a mathematical basis for the orientation of cell polarity patterns. PMID:27086039

  3. A polarized cell model for Chikungunya virus infection: entry and egress of virus occurs at the apical domain of polarized cells.

    Directory of Open Access Journals (Sweden)

    Pei Jin Lim

    2014-02-01

    Full Text Available Chikungunya virus (CHIKV has resulted in several outbreaks in the past six decades. The clinical symptoms of Chikungunya infection include fever, skin rash, arthralgia, and an increasing incidence of encephalitis. The re-emergence of CHIKV with more severe pathogenesis highlights its potential threat on our human health. In this study, polarized HBMEC, polarized Vero C1008 and non-polarized Vero cells grown on cell culture inserts were infected with CHIKV apically or basolaterally. Plaque assays, viral binding assays and immunofluorescence assays demonstrated apical entry and release of CHIKV in polarized HBMEC and Vero C1008. Drug treatment studies were performed to elucidate both host cell and viral factors involved in the sorting and release of CHIKV at the apical domain of polarized cells. Disruption of host cell myosin II, microtubule and microfilament networks did not disrupt the polarized release of CHIKV. However, treatment with tunicamycin resulted in a bi-directional release of CHIKV, suggesting that N-glycans of CHIKV envelope glycoproteins could serve as apical sorting signals.

  4. EGFR signaling promotes self-renewal through the establishment of cell polarity in Drosophila follicle stem cells.

    Science.gov (United States)

    Castanieto, Angela; Johnston, Michael J; Nystul, Todd G

    2014-01-01

    Epithelial stem cells divide asymmetrically, such that one daughter replenishes the stem cell pool and the other differentiates. We found that, in the epithelial follicle stem cell (FSC) lineage of the Drosophila ovary, epidermal growth factor receptor (EGFR) signaling functions specifically in the FSCs to promote the unique partially polarized state of the FSC, establish apical-basal polarity throughout the lineage, and promote FSC maintenance in the niche. In addition, we identified a novel connection between EGFR signaling and the cell-polarity regulator liver kinase B1 (LKB1), which indicates that EGFR signals through both the Ras-Raf-MEK-Erk pathway and through the LKB1-AMPK pathway to suppress apical identity. The development of apical-basal polarity is the earliest visible difference between FSCs and their daughters, and our findings demonstrate that the EGFR-mediated regulation of apical-basal polarity is essential for the segregation of stem cell and daughter cell fates. PMID:25437306

  5. Improved Gene Targeting through Cell Cycle Synchronization.

    Directory of Open Access Journals (Sweden)

    Vasiliki Tsakraklides

    Full Text Available Gene targeting is a challenge in organisms where non-homologous end-joining is the predominant form of recombination. We show that cell division cycle synchronization can be applied to significantly increase the rate of homologous recombination during transformation. Using hydroxyurea-mediated cell cycle arrest, we obtained improved gene targeting rates in Yarrowia lipolytica, Arxula adeninivorans, Saccharomyces cerevisiae, Kluyveromyces lactis and Pichia pastoris demonstrating the broad applicability of the method. Hydroxyurea treatment enriches for S-phase cells that are active in homologous recombination and enables previously unattainable genomic modifications.

  6. Gene expressions changes in bronchial epithelial cells

    DEFF Research Database (Denmark)

    Remy, S.; Verstraelen, S.; Van Den Heuvel, R.;

    2014-01-01

    cells were exposed during 6, 10, and 24 h to 4 respiratory sensitizers and 6 non-respiratory sensitizers (3 skin sensitizers and 3 respiratory irritants) at a concentration inducing 20% cell viability loss after 24 h. Changes in gene expression were evaluated using Agilent Whole Human Genome 4 x 44 K...... differentially expressed compared to vehicle control for each chemical. The results show that the NRF2-mediated oxidative stress response is activated in the cell line after stimulation with all of the chemicals that were selected in our study, and that - at the level of gene expression - this pathway shows no...

  7. Role of flagella in adherence, internalization, and translocation of Campylobacter jejuni in nonpolarized and polarized epithelial cell cultures.

    Science.gov (United States)

    Grant, C C; Konkel, M E; Cieplak, W; Tompkins, L S

    1993-01-01

    Previous studies of Campylobacter jejuni have suggested that flagellin is an adhesin for epithelial cells and that motility is a virulence factor of this bacterium. The role of flagella in the interactions of C. jejuni with nonpolarized and polarized epithelial cells was examined with flagellar mutants. Flagellated, nonmotile (flaA flaB+ Mot-) and nonflagellated, nonmotile (flaA flaB Mot-) mutants of C. jejuni were constructed by in vivo homologous recombination and gene replacement techniques. Both classes of mutants were found to adhere to cells of human epithelial origin (INT 407) equally well; however, on the basis of the percentage of the inoculum internalized, internalization of the flaA flaB Mot- mutants was decreased by factors ranging from approximately 30 to 40 compared with the parent. The flaA flaB+ Mot- mutant was internalized by the INT 407 cells at levels six- to sevenfold higher than the flaA flaB Mot- mutants. Both classes of mutants, unlike the parent, were unable to translocate across polarized Caco-2 monolayers. These results indicate that flagella are not involved in C. jejuni adherence to epithelial cells but that they do play a role in internalization. Furthermore, the results suggest that either the motility of C. jejuni or the product of flaA is essential for the bacterium to cross polarized epithelial cell monolayers. Images PMID:8478066

  8. Flotillins are involved in the polarization of primitive and mature hematopoietic cells.

    Directory of Open Access Journals (Sweden)

    Lawrence Rajendran

    Full Text Available BACKGROUND: Migration of mature and immature leukocytes in response to chemokines is not only essential during inflammation and host defense, but also during development of the hematopoietic system. Many molecules implicated in migratory polarity show uniform cellular distribution under non-activated conditions, but acquire a polarized localization upon exposure to migratory cues. METHODOLOGY/PRINCIPAL FINDINGS: Here, we present evidence that raft-associated endocytic proteins (flotillins are pre-assembled in lymphoid, myeloid and primitive hematopoietic cells and accumulate in the uropod during migration. Furthermore, flotillins display a polarized distribution during immunological synapse formation. Employing the membrane lipid-order sensitive probe Laurdan, we show that flotillin accumulation in the immunological synapse is concomittant with membrane ordering in these regions. CONCLUSIONS: Together with the observation that flotillin polarization does not occur in other polarized cell types such as polarized epithelial cells, our results suggest a specific role for flotillins in hematopoietic cell polarization. Based on our results, we propose that in hematopoietic cells, flotillins provide intrinsic cues that govern segregation of certain microdomain-associated molecules during immune cell polarization.

  9. The RPG gene of Medicago truncatula controls Rhizobium-directed polar growth during infection

    OpenAIRE

    Arrighi, Jean-François; Godfroy, Olivier; de Billy, Françoise; Saurat, Olivier; Jauneau, Alain; Gough, Clare

    2008-01-01

    Rhizobia can infect roots of host legume plants and induce new organs called nodules, in which they fix atmospheric nitrogen. Infection generally starts with root hair curling, then proceeds inside newly formed, intracellular tubular structures called infection threads. A successful symbiotic interaction relies on infection threads advancing rapidly at their tips by polar growth through successive cell layers of the root toward developing nodule primordia. To identify a plant component that c...

  10. Dystroglycan is required for polarizing the epithelial cells and the oocyte in Drosophila

    DEFF Research Database (Denmark)

    Deng, Wu-Min; Schneider, Martina; Frock, Richard;

    2003-01-01

    The transmembrane protein Dystroglycan is a central element of the dystrophin-associated glycoprotein complex, which is involved in the pathogenesis of many forms of muscular dystrophy. Dystroglycan is a receptor for multiple extracellular matrix (ECM) molecules such as Laminin, agrin and perlecan......-autonomously for cellular polarity in two different cell types, the epithelial cells (apicobasal polarity) and the oocyte (anteroposterior polarity). Loss of Dystroglycan function in follicle and disc epithelia results in expansion of apical markers to the basal side of cells and overexpression results in a...... reduced apical localization of these same markers. In Dystroglycan germline clones early oocyte polarity markers fail to be localized to the posterior, and oocyte cortical F-actin organization is abnormal. Dystroglycan is also required non-cell-autonomously to organize the planar polarity of basal actin...

  11. Capture and sorting of multiple cells by polarization-controlled three-beam interference

    Science.gov (United States)

    Hou, Yu; Wang, Zuobin; Hu, Yaowei; Li, Dayou; Qiu, Renxi

    2016-03-01

    For the capture and sorting of multiple cells, a sensitive and highly efficient polarization-controlled three-beam interference set-up has been developed. With the theory of superposition of three beams, simulations on the influence of polarization angle upon the intensity distribution and the laser gradient force change with different polarization angles have been carried out. By controlling the polarization angle of the beams, various intensity distributions and different sizes of dots are obtained. We have experimentally observed multiple optical tweezers and the sorting of cells with different polarization angles, which are in accordance with the theoretical analysis. The experimental results have shown that the polarization angle affects the shapes and feature sizes of the interference patterns and the trapping force.

  12. A two-step mechanism underlies the planar polarization of regenerating sensory hair cells

    OpenAIRE

    López-Schier, Hernán; Hudspeth, A. J.

    2006-01-01

    The restoration of planar cell polarity is an essential but poorly understood step toward physiological recovery during sensory-organ regeneration. Investigating this issue in the lateral line of the zebrafish, we found that hair cells regenerate in pairs along a single axis established by the restricted localization and oriented division of their progenitors. By analyzing mutants lacking the planar-polarity determinant Vangl2, we ascertained that the uniaxial production of hair cells and the...

  13. Advantages and mechanisms of polarity and cell shape determination in Caulobacter crescentus

    OpenAIRE

    Lawler, Melanie L.; Brun, Yves V.

    2007-01-01

    The tremendous diversity of bacterial cell shapes and the targeting of proteins and macromolecular complexes to specific subcellular sites strongly suggest that cellular organization provides important advantages for bacteria in their environment. Key advances have been made in the understanding of the mechanism and function of polarity and cell shape by studying the aquatic bacterium Caulobacter crescentus, whose cell cycle progression involves the ordered synthesis of different polar struct...

  14. The Rho GTPase Cdc42 regulates hair cell planar polarity and cellular patterning in the developing cochlea

    OpenAIRE

    Anna Kirjavainen; Maarja Laos; Tommi Anttonen; Ulla Pirvola

    2015-01-01

    Hair cells of the organ of Corti (OC) of the cochlea exhibit distinct planar polarity, both at the tissue and cellular level. Planar polarity at tissue level is manifested as uniform orientation of the hair cell stereociliary bundles. Hair cell intrinsic polarity is defined as structural hair bundle asymmetry; positioning of the kinocilium/basal body complex at the vertex of the V-shaped bundle. Consistent with strong apical polarity, the hair cell apex displays prominent actin and microtubul...

  15. Directional memory arises from long-lived cytoskeletal asymmetries in polarized chemotactic cells.

    Science.gov (United States)

    Prentice-Mott, Harrison V; Meroz, Yasmine; Carlson, Andreas; Levine, Michael A; Davidson, Michael W; Irimia, Daniel; Charras, Guillaume T; Mahadevan, L; Shah, Jagesh V

    2016-02-01

    Chemotaxis, the directional migration of cells in a chemical gradient, is robust to fluctuations associated with low chemical concentrations and dynamically changing gradients as well as high saturating chemical concentrations. Although a number of reports have identified cellular behavior consistent with a directional memory that could account for behavior in these complex environments, the quantitative and molecular details of such a memory process remain unknown. Using microfluidics to confine cellular motion to a 1D channel and control chemoattractant exposure, we observed directional memory in chemotactic neutrophil-like cells. We modeled this directional memory as a long-lived intracellular asymmetry that decays slower than observed membrane phospholipid signaling. Measurements of intracellular dynamics revealed that moesin at the cell rear is a long-lived element that when inhibited, results in a reduction of memory. Inhibition of ROCK (Rho-associated protein kinase), downstream of RhoA (Ras homolog gene family, member A), stabilized moesin and directional memory while depolymerization of microtubules (MTs) disoriented moesin deposition and also reduced directional memory. Our study reveals that long-lived polarized cytoskeletal structures, specifically moesin, actomyosin, and MTs, provide a directional memory in neutrophil-like cells even as they respond on short time scales to external chemical cues. PMID:26764383

  16. Differential expression of cell adhesion genes

    DEFF Research Database (Denmark)

    Stein, Wilfred D; Litman, Thomas; Fojo, Tito;

    2005-01-01

    It is well known that tumors arising from tissues such as kidney, pancreas, liver and stomach are particularly refractory to treatment. Searching for new anticancer drugs using cells in culture has yielded some effective therapies, but these refractory tumors remain intractable. Studies that...... survival might, therefore, act through such a matrix-to-cell suppression of apoptosis. Indeed, correlative mining of gene expression and patient survival databases suggests that poor survival in patients with metastatic cancer correlates highly with tumor expression of a common theme: the genes involved in...

  17. Dkk-1 Inhibits Intestinal Epithelial Cell Migration by Attenuating Directional Polarization of Leading Edge Cells

    Science.gov (United States)

    Koch, Stefan; Capaldo, Christopher T.; Samarin, Stanislav; Nava, Porfirio; Neumaier, Irmgard; Skerra, Arne; Sacks, David B.; Parkos, Charles A.

    2009-01-01

    Wnt signaling pathways regulate proliferation, motility, and survival in a variety of human cell types. Dickkopf-1 (Dkk-1) is a secreted Wnt antagonist that has been proposed to regulate tissue homeostasis in the intestine. In this report, we show that Dkk-1 is secreted by intestinal epithelial cells after wounding and that it inhibits cell migration by attenuating the directional orientation of migrating epithelial cells. Dkk-1 exposure induced mislocalized activation of Cdc42 in migrating cells, which coincided with a displacement of the polarity protein Par6 from the leading edge. Consequently, the relocation of the microtubule organizing center and the Golgi apparatus in the direction of migration was significantly and persistently inhibited in the presence of Dkk-1. Small interfering RNA-induced down-regulation of Dkk-1 confirmed that extracellular exposure to Dkk-1 was required for this effect. Together, these data demonstrate a novel role of Dkk-1 in the regulation of directional polarization of migrating intestinal epithelial cells, which contributes to the effect of Dkk-1 on wound closure in vivo. PMID:19776352

  18. Polarized neural stem cells derived from adult bone marrow stromal cells develop a rosette-like structure.

    Science.gov (United States)

    Darabi, Shahram; Tiraihi, Taki; Ruintan, Atefeh; Abbaszadeh, Hojatt Allah; Delshad, AliReza; Taheri, Taher

    2013-09-01

    Bone marrow stromal cells (BMSCs) were reported to form floating aggregation of cells with expression of nestin, a marker for neural stem cells (NSCs). The purpose of this investigation is to evaluate the morphology and the molecular markers expressed by NSCs derived from these neurospheres. The BMSCs were isolated from Sprague Dawley rats and evaluated for osteogenesis, lipogenesis, and expression of fibronectin, CD90, CD106, CD31, and Oct4. The BMSCs were cultured with Dulbecco's modified Eagle's medium (DMEM)/F12 containing 15% fetal bovine serum, then with DMEM/F12 containing 2% B27, basic fibroblast growth factor, and epidermal growth factor. The cell aggregates or spheres were stained with acridine orange, which showed that the neurospheres comprised aggregated cells at either premitotic/postsynthetic (PS), postmitotic/presynthetic (PM) phases of cell cycle, or a mixture of both. The NSCs harvested from the neurospheres were polar with eccentric nucleus, and at either a PS or a PM cell cycle phases, some cells at the latter phase tended to form rosette-like structures. The cells were immunostained for molecular markers such as nestin, neurofilament 68 (NF68), NF160, and NF200 and glial fibrillary acidic protein (GFAP). Myelin basic protein (MBP), the pluripotency (Oct4, Nanog, and SOX2), and the differentiation genes (NeuroD1, Tubb4, and Musashi I) were also evaluated using reverse transcription polymerase chain reaction (RT-PCR). Nestin, NF68, NF160, NF200, GFAP, O4, and N-cadherin were expressed in the NSCs. The percentage of immunoreactive cells to nestin was significantly higher than that of the other neuronal markers. MBP was not expressed in BMSCs, neurospheres, and NSCs. The neurospheres were immunoreactive to GFAP. RT-PCR showed the expression of NeuroD1 and Musashi I. The pluripotency gene (SOX2) was expressed in NSCs. Oct4 and Nanog were expressed in BMSCs, while Oct4 and SOX2 were expressed in the neurosphere. This indicates that a pluripotency

  19. Cell shape, spreading symmetry, and the polarization of stress-fibers in cells

    Energy Technology Data Exchange (ETDEWEB)

    Zemel, A [Institute of Dental Sciences, Faculty of Dental Medicine, and the Fritz Haber Center for Molecular Dynamics, Hebrew University-Hadassah Medical Center, Jerusalem, 91120 (Israel); Rehfeldt, F [III. Physikalisches Institut, Georg-August-Universitaet, 37077 Goettingen (Germany); Brown, A E X [Department of Physics and Astronomy, University of Pennsylvania, Philadelphia, PA 19104 (United States); Discher, D E [Graduate Group of Physics and Astronomy, University of Pennsylvania, Philadelphia, PA 19104 (United States); Safran, S A [Department of Materials and Interfaces, Weizmann Institute of Science, Rehovot 76100 (Israel)

    2010-05-19

    The active regulation of cellular forces during cell adhesion plays an important role in the determination of cell size, shape, and internal structure. While on flat, homogeneous and isotropic substrates some cells spread isotropically, others spread anisotropically and assume elongated structures. In addition, in their native environment as well as in vitro experiments, the cell shape and spreading asymmetry can be modulated by the local distribution of adhesive molecules and topography of the environment. We present a simple elastic model and experiments on stem cells to explain the variation of cell size with the matrix rigidity. In addition, we predict the experimental consequences of two mechanisms of acto-myosin polarization and focus here on the effect of the cell spreading asymmetry on the regulation of the stress-fiber alignment in the cytoskeleton. We show that when cell spreading is sufficiently asymmetric the alignment of acto-myosin forces in the cell increases monotonically with the matrix rigidity; however, in general this alignment is non-monotonic, as shown previously. These results highlight the importance of the symmetry characteristics of cell spreading in the regulation of cytoskeleton structure and suggest a mechanism by which different cell types may acquire different morphologies and internal structures in different mechanical environments.

  20. Cell shape, spreading symmetry, and the polarization of stress-fibers in cells

    International Nuclear Information System (INIS)

    The active regulation of cellular forces during cell adhesion plays an important role in the determination of cell size, shape, and internal structure. While on flat, homogeneous and isotropic substrates some cells spread isotropically, others spread anisotropically and assume elongated structures. In addition, in their native environment as well as in vitro experiments, the cell shape and spreading asymmetry can be modulated by the local distribution of adhesive molecules and topography of the environment. We present a simple elastic model and experiments on stem cells to explain the variation of cell size with the matrix rigidity. In addition, we predict the experimental consequences of two mechanisms of acto-myosin polarization and focus here on the effect of the cell spreading asymmetry on the regulation of the stress-fiber alignment in the cytoskeleton. We show that when cell spreading is sufficiently asymmetric the alignment of acto-myosin forces in the cell increases monotonically with the matrix rigidity; however, in general this alignment is non-monotonic, as shown previously. These results highlight the importance of the symmetry characteristics of cell spreading in the regulation of cytoskeleton structure and suggest a mechanism by which different cell types may acquire different morphologies and internal structures in different mechanical environments.

  1. Polar release of pathogenic Old World hantaviruses from renal tubular epithelial cells

    Directory of Open Access Journals (Sweden)

    Krautkrämer Ellen

    2012-11-01

    Full Text Available Abstract Background Epithelio- and endotheliotropic viruses often exert polarized entry and release that may be responsible for viral spread and dissemination. Hantaviruses, mostly rodent-borne members of the Bunyaviridae family infect epithelial and endothelial cells of different organs leading to organ dysfunction or even failure. Endothelial and renal epithelial cells belong to the target cells of Old World hantavirus. Therefore, we examined the release of hantaviruses in several renal epithelial cell culture models. We used Vero cells that are commonly used in hantavirus studies and primary human renal epithelial cells (HREpC. In addition, we analyzed MDCKII cells, an epithelial cell line of a dog kidney, which represents a widely accepted in vitro model of polarized monolayers for their permissiveness for hantavirus infection. Results Vero C1008 and primary HREpCs were grown on porous-support filter inserts for polarization. Monolayers were infected with hantavirus Hantaan (HTNV and Puumala (PUUV virus. Supernatants from the apical and basolateral chamber of infected cells were analyzed for the presence of infectious particles by re-infection of Vero cells. Viral antigen and infectious particles of HTNV and PUUV were exclusively detected in supernatants collected from the apical chamber of infected Vero C1008 cells and HREpCs. MDCKII cells were permissive for hantavirus infection and polarized MDCKII cells released infectious hantaviral particles from the apical surface corresponding to the results of Vero and primary human epithelial cells. Conclusions Pathogenic Old World hantaviruses are released from the apical surface of different polarized renal epithelial cells. We characterized MDCKII cells as a suitable polarized cell culture model for hantavirus infection studies.

  2. A comparison of mathematical models for polarization of single eukaryotic cells in response to guided cues.

    Directory of Open Access Journals (Sweden)

    Alexandra Jilkine

    2011-04-01

    Full Text Available Polarization, a primary step in the response of an individual eukaryotic cell to a spatial stimulus, has attracted numerous theoretical treatments complementing experimental studies in a variety of cell types. While the phenomenon itself is universal, details differ across cell types, and across classes of models that have been proposed. Most models address how symmetry breaking leads to polarization, some in abstract settings, others based on specific biochemistry. Here, we compare polarization in response to a stimulus (e.g., a chemoattractant in cells typically used in experiments (yeast, amoebae, leukocytes, keratocytes, fibroblasts, and neurons, and, in parallel, responses of several prototypical models to typical stimulation protocols. We find that the diversity of cell behaviors is reflected by a diversity of models, and that some, but not all models, can account for amplification of stimulus, maintenance of polarity, adaptation, sensitivity to new signals, and robustness.

  3. The role of polar pili in the adherence of Pseudomonas aeruginosa to injured canine tracheal cells: a semiquantitative morphologic study.

    Science.gov (United States)

    Zoutman, D E; Hulbert, W C; Pasloske, B L; Joffe, A M; Volpel, K; Trebilcock, M K; Paranchych, W

    1991-03-01

    Pseudomonas aeruginosa adheres to respiratory epithelial cells in a highly specific fashion. In order to study the role of P. aeruginosa polar pili in the adherence process we conducted a quantitative morphological electron microscopic examination of P. aeruginosa adherence to SO2 injured canine tracheal cells in vitro. A pilin lacking background strain of P. aeruginosa PAK (BLP2) was constructed using a gene replacement and it in turn was engineered to express either the pilin gene of P. aeruginosa PAO, PAK, or no pilin gene. After 30 minutes incubation of these bacterial strains with injured canine tracheal rings the P. aeruginosa strains expressing pili adhered quantitatively more to the injured tracheal cells than did the pili lacking strains. PAO bearing strains adhered in greater numbers than PAK bearing strains. Healthy tracheal cells did not have any bacteria bound to their surfaces. The bacteria bound to the cilia and lateral edge of the exfoliating tracheal cells. Invasion of tracheal cells by piliated P. aeruginosa bacteria and penetration into the submucosa was also demonstrated. These data confirm the role of pili as important adhesins to injured tracheal cells. The difference in the adherence characteristics of pilin types PAK versus PAO may relate to the differences in the primary structure of these two pilin molecules. PMID:1675811

  4. Vectorial insertion of apical and basolateral membrane proteins in polarized epithelial cells revealed by quantitative 3D live cell imaging

    OpenAIRE

    Hua, Wei; Sheff, David; Toomre, Derek; Mellman, Ira

    2006-01-01

    Although epithelial cells are known to exhibit a polarized distribution of membrane components, the pathways responsible for delivering membrane proteins to their appropriate domains remain unclear. Using an optimized approach to three-dimensional live cell imaging, we have visualized the transport of newly synthesized apical and basolateral membrane proteins in fully polarized filter-grown Madin–Darby canine kidney cells. We performed a detailed quantitative kinetic analysis of trans-Golgi n...

  5. Gene expression profiles in irradiated cancer cells

    International Nuclear Information System (INIS)

    Knowledge of the molecular and genetic mechanisms underlying cellular response to radiation may provide new avenues to develop innovative predictive tests of radiosensitivity of tumours and normal tissues and to improve individual therapy. Nowadays very few studies describe molecular changes induced by hadrontherapy treatments, therefore this field has to be explored and clarified. High-throughput methodologies, such as DNA microarray, allow us to analyse mRNA expression of thousands of genes simultaneously in order to discover new genes and pathways as targets of response to hadrontherapy. Our aim is to elucidate the molecular networks involved in the sensitivity/resistance of cancer cell lines subjected to hadrontherapy treatments with a genomewide approach by using cDNA microarray technology to identify gene expression profiles and candidate genes responsible of differential cellular responses

  6. Induced differentiation of cancer cells: second generation potent hybrid polar compounds target cell cycle regulators

    International Nuclear Information System (INIS)

    Hybrid polar compounds are potent inducers of differentiation of a wide variety of cancer transformed cells. Hexamethylene bisacetamide (HMBA) has been used as a prototype of these compounds to investigate their mechanism of action. Employing murine erythroleukemia (MEL) cells as a model, three characteristics of inducer-mediated commitment to terminal differentiation were demonstrated: (I) induced commitment was stochastic, requiring up to 5 cell cycles to recruit essentially all cells to commit to growth arrest in G1; (II) inducers caused a prolongation of the initial G1; and (III) the hybrid polar compounds induced a wide variety of transformed cells to terminal differentiation. These findings suggested that the rate limiting factor or factors for induction by these agents may be at the level of protein(s) regulating G1-to-S progression, which are common to most eukaryotic cells. It was found that HMBA induced a profound suppression of cyclin dependent kinase, cdk4, which reflected a marked decrease in stability of the protein, and is a critical change in the pathway of induced differentiation. HMBA also induced an increase in pRB and in the active, underphosphorylated form of this protein, an increase in the pRB related protein, p107, and an increase in the cyclin dependent kinase inhibitor, p21. Further, the free form of the transcription factor, E2F, was markedly decreased within hours of exposure of transformed cells to HMBA and found to complex with p107 and cdk 2. A phase II clinical trial was conducted using HMBA to treat patients with myelodysplastic syndrome (MDS) or acute myelogenous leukemia. Of 28 patients, 9 patients achieved a complete or partial remission lasting from 1 to 16 months. These clinical studies also provided direct evidence that HMBA induces differentiation of transformed cells in patients. In four separate courses of treatment with HMBA, a patient with MDS and the monosomy 7 karyotype marking the malignant clone of bone marrow blast

  7. Long-term trends of the Polar and Arctic cells influencing the Arctic climate since 1989

    Science.gov (United States)

    Qian, Weihong; Wu, Kaijun; Leung, Jeremy Cheuk-Hin; Shi, Jian

    2016-03-01

    The strengthening and broadening trends of the Hadley cell have been revealed, while the existence of the Arctic cell has also been confirmed in previous studies. This study extends previous strengthening trend analyses of the Hadley cell to the Polar and Arctic cells in the Northern Hemisphere and explores their climate influences. Results show that the Polar cell experienced an abrupt change from a slow to a rapid strengthening trend in 1989, while the Arctic cell showed an insignificant strengthening trend and a significant weakening trend successively. The strengthening subsidence flow associated with the Polar and Arctic cells can partly explain the warming surface air temperature and declining sea ice concentration through the increasing tropospheric height and temperature trends. These results provide new insights for understanding the interdecadal relationship between atmospheric circulation and climate change in the Arctic region.

  8. The young and happy marriage of membrane traffic and cell polarity

    OpenAIRE

    Thompson, Barry J; Perez, Franck; Vaccari, Thomas

    2012-01-01

    The ESF–EMBO Meeting on ‘Cell polarity and Membrane Traffic' took place in April 2012. It brought together scientists from two once very separate fields and highlighted the emerging interdependence between them.

  9. Polarization of the epithelial layer and apical localization of integrins are required for engulfment of apoptotic cells in the Drosophila ovary

    Directory of Open Access Journals (Sweden)

    Tracy L. Meehan

    2015-12-01

    Full Text Available Inefficient clearance of dead cells or debris by epithelial cells can lead to or exacerbate debilitating conditions such as retinitis pigmentosa, macular degeneration, chronic obstructive pulmonary disease and asthma. Despite the importance of engulfment by epithelial cells, little is known about the molecular changes that are required within these cells. The misregulation of integrins has previously been associated with disease states, suggesting that a better understanding of the regulation of receptor trafficking could be key to treating diseases caused by defects in phagocytosis. Here, we demonstrate that the integrin heterodimer αPS3/βPS becomes apically enriched and is required for engulfment by the epithelial follicle cells of the Drosophila ovary. We found that integrin heterodimer localization and function is largely directed by the α-subunit. Moreover, proper cell polarity promotes asymmetric integrin enrichment, suggesting that αPS3/βPS trafficking occurs in a polarized fashion. We show that several genes previously known for their roles in trafficking and cell migration are also required for engulfment. Moreover, as in mammals, the same α-integrin subunit is required by professional and non-professional phagocytes and migrating cells in Drosophila. Our findings suggest that migrating and engulfing cells use common machinery, and demonstrate a crucial role for integrin function and polarized trafficking of integrin subunits during engulfment. This study also establishes the epithelial follicle cells of the Drosophila ovary as a powerful model for understanding the molecular changes required for engulfment by a polarized epithelium.

  10. Genes involved in cell division in mycoplasmas

    Directory of Open Access Journals (Sweden)

    Frank Alarcón

    2007-01-01

    Full Text Available Bacterial cell division has been studied mainly in model systems such as Escherichia coli and Bacillus subtilis, where it is described as a complex process with the participation of a group of proteins which assemble into a multiprotein complex called the septal ring. Mycoplasmas are cell wall-less bacteria presenting a reduced genome. Thus, it was important to compare their genomes to analyze putative genes involved in cell division processes. The division and cell wall (dcw cluster, which in E. coli and B. subtilis is composed of 16 and 17 genes, respectively, is represented by only three to four genes in mycoplasmas. Even the most conserved protein, FtsZ, is not present in all mycoplasma genomes analyzed so far. A model for the FtsZ protein from Mycoplasma hyopneumoniae and Mycoplasma synoviae has been constructed. The conserved residues, essential for GTP/GDP binding, are present in FtsZ from both species. A strong conservation of hydrophobic amino acid patterns is observed, and is probably necessary for the structural stability of the protein when active. M. synoviae FtsZ presents an extended amino acid sequence at the C-terminal portion of the protein, which may participate in interactions with other still unknown proteins crucial for the cell division process.

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

    OpenAIRE

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

    1987-01-01

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

  12. Cdc42 and noncanonical Wnt signal transduction pathways cooperate to promote cell polarity

    OpenAIRE

    Schlessinger, Karni; McManus, Edward J.; Hall, Alan

    2007-01-01

    Scratch-induced disruption of cultured monolayers induces polarity in front row cells that can be visualized by spatially localized polymerization of actin at the front of the cell and reorientation of the centrosome/Golgi to face the leading edge. We previously reported that centrosomal reorientation and microtubule polarization depend on a Cdc42-regulated signal transduction pathway involving activation of the Par6/aPKC complex followed by inhibition of GSK-3β and accumulation of the adenom...

  13. Local Pheromone Release from Dynamic Polarity Sites Underlies Cell-Cell Pairing during Yeast Mating.

    Science.gov (United States)

    Merlini, Laura; Khalili, Bita; Bendezú, Felipe O; Hurwitz, Daniel; Vincenzetti, Vincent; Vavylonis, Dimitrios; Martin, Sophie G

    2016-04-25

    Cell pairing is central for many processes, including immune defense, neuronal connection, hyphal fusion, and sexual reproduction. How does a cell orient toward a partner, especially when faced with multiple choices? Fission yeast Schizosaccharomyces pombe P and M cells, which respectively express P and M factor pheromones [1, 2], pair during the mating process induced by nitrogen starvation. Engagement of pheromone receptors Map3 and Mam2 [3, 4] with their cognate pheromone ligands leads to activation of the Gα protein Gpa1 to signal sexual differentiation [3, 5, 6]. Prior to cell pairing, the Cdc42 GTPase, a central regulator of cell polarization, forms dynamic zones of activity at the cell periphery at distinct locations over time [7]. Here we show that Cdc42-GTP polarization sites contain the M factor transporter Mam1, the general secretion machinery, which underlies P factor secretion, and Gpa1, suggesting that these are sub-cellular zones of pheromone secretion and signaling. Zone lifetimes scale with pheromone concentration. Computational simulations of pair formation through a fluctuating zone show that the combination of local pheromone release and sensing, short pheromone decay length, and pheromone-dependent zone stabilization leads to efficient pair formation. Consistently, pairing efficiency is reduced in the absence of the P factor protease. Similarly, zone stabilization at reduced pheromone levels, which occurs in the absence of the predicted GTPase-activating protein for Ras, leads to reduction in pairing efficiency. We propose that efficient cell pairing relies on fluctuating local signal emission and perception, which become locked into place through stimulation. PMID:27020743

  14. Myosin Id is required for planar cell polarity in ciliated tracheal and ependymal epithelial cells.

    Science.gov (United States)

    Hegan, Peter S; Ostertag, Eric; Geurts, Aron M; Mooseker, Mark S

    2015-10-01

    In wild type (WT) tracheal epithelial cells, ciliary basal bodies are oriented such that all cilia on the cell surface beat in the same upward direction. This precise alignment of basal bodies and, as a result, the ciliary axoneme, is termed rotational planar cell polarity (PCP). Rotational PCP in the multi-ciliated epithelial cells of the trachea is perturbed in rats lacking myosin Id (Myo1d). Myo1d is localized in the F-actin and basal body rich subapical cortex of the ciliated tracheal epithelial cell. Scanning and transmission electron microscopy of Myo1d knock out (KO) trachea revealed that the unidirectional bending pattern is disrupted. Instead, cilia splay out in a disordered, often radial pattern. Measurement of the alignment axis of the central pair axonemal microtubules was much more variable in the KO, another indicator that rotational PCP is perturbed. The asymmetric localization of the PCP core protein Vangl1 is lost. Both the velocity and linearity of cilia-driven movement of beads above the tracheal mucosal surface was impaired in the Myo1d KO. Multi-ciliated brain ependymal epithelial cells exhibit a second form of PCP termed translational PCP in which basal bodies and attached cilia are clustered at the anterior side of the cell. The precise asymmetric clustering of cilia is disrupted in the ependymal cells of the Myo1d KO rat. While basal body clustering is maintained, left-right positioning of the clusters is lost. PMID:26446290

  15. Porcine aminopeptidase N mediated polarized infection by porcine epidemic diarrhea virus in target cells

    International Nuclear Information System (INIS)

    Infection of polarized intestinal epithelial cells by porcine epidemic diarrhea virus (PEDV) was characterized. Indirect immunofluorescence assay, real-time PCR, and transmission electron microscopy confirmed PEDV can be successfully propagated in immortalized swine small intestine epithelial cells (IECs). Infection involved porcine aminpeptidase N (pAPN), a reported cellular receptor for PEDV, transient expression of pAPN and siRNA targeted pAPN increased and decreased the infectivity of PEDV in IECs, respectively. Subsequently, polarized entry into and release from both Vero E6 and IECs was analyzed. PEDV entry into polarized cells and pAPN grown on membrane inserts occurs via apical membrane. The progeny virus released into the medium was also quantified which demonstrated that PEDV is preferentially released from the apical membrane. Collectively, our data demonstrate that pAPN, the cellular receptor for PEDV, mediates polarized PEDV infection. These results imply the possibility that PEDV infection may proceed by lateral spread of virus in intestinal epithelial cells. - Highlights: • PEDV infection of polarized intestinal epithelial cells (IECs) was characterized. • Porcine aminpeptidase N (pAPN) facilitated PEDV infection in IECs. • PEDV entry into and release from polarized cell via its apical membrane. • PEDV infection may proceed by lateral spread of virus in IECs

  16. Porcine aminopeptidase N mediated polarized infection by porcine epidemic diarrhea virus in target cells

    Energy Technology Data Exchange (ETDEWEB)

    Cong, Yingying; Li, Xiaoxue; Bai, Yunyun [College of Veterinary Medicine, Northeast Agricultural University, Harbin 150030 (China); Lv, Xiaonan [College of Veterinary Medicine, Northeast Agricultural University, Harbin 150030 (China); CAS Key Lab for Biomedical Effects of Nanomaterials and Nanosafety, National Center for Nanoscience & Technology of China, Beijing 100090 (China); Herrler, Georg [Institute for Virology, University of Veterinary Medicine, Hannover D-30559 (Germany); Enjuanes, Luis [Department of Molecular and Cell Biology, Centro Nacional de Biotecnología (CNB-CSIC), Campus Universidad Autónoma de Madrid, Cantoblanco, Madrid (Spain); Zhou, Xingdong [College of Veterinary Medicine, Northeast Agricultural University, Harbin 150030 (China); Qu, Bo [Faculty of Life Sciences, Northeast Agricultural University, Harbin 150030 (China); Meng, Fandan [Institute for Virology, University of Veterinary Medicine, Hannover D-30559 (Germany); Cong, Chengcheng [College Animal Husbandry and Veterinary Medicine, Shenyang Agricultural University, Shenyang 110161 (China); Ren, Xiaofeng; Li, Guangxing [College of Veterinary Medicine, Northeast Agricultural University, Harbin 150030 (China)

    2015-04-15

    Infection of polarized intestinal epithelial cells by porcine epidemic diarrhea virus (PEDV) was characterized. Indirect immunofluorescence assay, real-time PCR, and transmission electron microscopy confirmed PEDV can be successfully propagated in immortalized swine small intestine epithelial cells (IECs). Infection involved porcine aminpeptidase N (pAPN), a reported cellular receptor for PEDV, transient expression of pAPN and siRNA targeted pAPN increased and decreased the infectivity of PEDV in IECs, respectively. Subsequently, polarized entry into and release from both Vero E6 and IECs was analyzed. PEDV entry into polarized cells and pAPN grown on membrane inserts occurs via apical membrane. The progeny virus released into the medium was also quantified which demonstrated that PEDV is preferentially released from the apical membrane. Collectively, our data demonstrate that pAPN, the cellular receptor for PEDV, mediates polarized PEDV infection. These results imply the possibility that PEDV infection may proceed by lateral spread of virus in intestinal epithelial cells. - Highlights: • PEDV infection of polarized intestinal epithelial cells (IECs) was characterized. • Porcine aminpeptidase N (pAPN) facilitated PEDV infection in IECs. • PEDV entry into and release from polarized cell via its apical membrane. • PEDV infection may proceed by lateral spread of virus in IECs.

  17. The viral spike protein is not involved in the polarized sorting of coronaviruses in epithelial cells

    NARCIS (Netherlands)

    Rossen, J W; de Beer, R; Godeke, G J; Raamsman, M J; Horzinek, M C; Vennema, H; Rottier, P J

    1998-01-01

    Coronaviruses are assembled by budding into a pre-Golgi compartment from which they are transported along the secretory pathway to leave the cell. In cultured epithelial cells, they are released in a polarized fashion; depending on the virus and cell type, they are sorted preferentially either to th

  18. Polarization of cells and soft objects driven by mechanical interactions: Consequences for migration and chemotaxis

    Science.gov (United States)

    Leoni, M.; Sens, P.

    2015-02-01

    We study a generic model for the polarization and motility of self-propelled soft objects, biological cells, or biomimetic systems, interacting with a viscous substrate. The active forces generated by the cell on the substrate are modeled by means of oscillating force multipoles at the cell-substrate interface. Symmetry breaking and cell polarization for a range of cell sizes naturally "emerge" from long range mechanical interactions between oscillating units, mediated both by the intracellular medium and the substrate. However, the harnessing of cell polarization for motility requires substrate-mediated interactions. Motility can be optimized by adapting the oscillation frequency to the relaxation time of the system or when the substrate and cell viscosities match. Cellular noise can destroy mechanical coordination between force-generating elements within the cell, resulting in sudden changes of polarization. The persistence of the cell's motion is found to depend on the cell size and the substrate viscosity. Within such a model, chemotactic guidance of cell motion is obtained by directionally modulating the persistence of motion, rather than by modulating the instantaneous cell velocity, in a way that resembles the run and tumble chemotaxis of bacteria.

  19. The Balance Between the Novel Protein Target of Wingless and the Drosophila Rho-Associated Kinase Pathway Regulates Planar Cell Polarity in the Drosophila Wing

    OpenAIRE

    Chung, SeYeon; Kim, Sangjoon; Yoon, Jeongsook; Adler, Paul N.; Yim, Jeongbin

    2007-01-01

    Planar cell polarity (PCP) signaling is mediated by the serpentine receptor Frizzled (Fz) and transduced by Dishevelled (Dsh). Wingless (Wg) signaling utilizes Drosophila Frizzled 2 (DFz2) as a receptor and also requires Dsh for transducing signals to regulate cell proliferation and differentiation in many developmental contexts. Distinct pathways are activated downstream of Dsh in Wg- and Fz-signaling pathways. Recently, a number of genes, which have essential roles as downstream components ...

  20. Genes of pyelonephritogenic E. coli required for digalactoside-specific agglutination of human cells.

    OpenAIRE

    Lindberg, F P; Lund, B; Normark, S

    1984-01-01

    Most pyelonephritic Escherichia coli strains bind to digalactoside-containing glycolipids on uroepithelial cells. Purified Pap pili (pili associated with pyelonephritis) show the same binding specificity. A non-polar mutation early in the papA pilin gene abolishes formation of Pap pili but does not affect the degree of digalactoside-specific hemagglutination. Three novel pap genes, papE , papF and papG are defined in this report. The papF and papG gene products are both required for digalacto...

  1. Cholinergic regulation of VIP gene expression in human neuroblastoma cells

    DEFF Research Database (Denmark)

    Kristensen, Bo; Georg, Birgitte; Fahrenkrug, Jan

    Vasoactive intestinal polypeptide, muscarinic receptor, neuroblastoma cell, mRNA, gene expression, peptide processing......Vasoactive intestinal polypeptide, muscarinic receptor, neuroblastoma cell, mRNA, gene expression, peptide processing...

  2. The final cut: cell polarity meets cytokinesis at the bud neck in S. cerevisiae.

    Science.gov (United States)

    Juanes, Maria Angeles; Piatti, Simonetta

    2016-08-01

    Cell division is a fundamental but complex process that gives rise to two daughter cells. It includes an ordered set of events, altogether called "the cell cycle", that culminate with cytokinesis, the final stage of mitosis leading to the physical separation of the two daughter cells. Symmetric cell division equally partitions cellular components between the two daughter cells, which are therefore identical to one another and often share the same fate. In many cases, however, cell division is asymmetrical and generates two daughter cells that differ in specific protein inheritance, cell size, or developmental potential. The budding yeast Saccharomyces cerevisiae has proven to be an excellent system to investigate the molecular mechanisms governing asymmetric cell division and cytokinesis. Budding yeast is highly polarized during the cell cycle and divides asymmetrically, producing two cells with distinct sizes and fates. Many components of the machinery establishing cell polarization during budding are relocalized to the division site (i.e., the bud neck) for cytokinesis. In this review we recapitulate how budding yeast cells undergo polarized processes at the bud neck for cell division. PMID:27085703

  3. Differential effects of Mycobacterium bovis - derived polar and apolar lipid fractions on bovine innate immune cells

    Directory of Open Access Journals (Sweden)

    Pirson Chris

    2012-06-01

    Full Text Available Abstract Mycobacterial lipids have long been known to modulate the function of a variety of cells of the innate immune system. Here, we report the extraction and characterisation of polar and apolar free lipids from Mycobacterium bovis AF 2122/97 and identify the major lipids present in these fractions. Lipids found included trehalose dimycolate (TDM and trehalose monomycolate (TMM, the apolar phthiocerol dimycocersates (PDIMs, triacyl glycerol (TAG, pentacyl trehalose (PAT, phenolic glycolipid (PGL, and mono-mycolyl glycerol (MMG. Polar lipids identified included glucose monomycolate (GMM, diphosphatidyl glycerol (DPG, phenylethanolamine (PE and a range of mono- and di-acylated phosphatidyl inositol mannosides (PIMs. These lipid fractions are capable of altering the cytokine profile produced by fresh and cultured bovine monocytes as well as monocyte derived dendritic cells. Significant increases in the production of IL-10, IL-12, MIP-1β, TNFα and IL-6 were seen after exposure of antigen presenting cells to the polar lipid fraction. Phenotypic characterisation of the cells was performed by flow cytometry and significant decreases in the expression of MHCII, CD86 and CD1b were found after exposure to the polar lipid fraction. Polar lipids also significantly increased the levels of CD40 expressed by monocytes and cultured monocytes but no effect was seen on the constitutively high expression of CD40 on MDDC or on the levels of CD80 expressed by any of the cells. Finally, the capacity of polar fraction treated cells to stimulate alloreactive lymphocytes was assessed. Significant reduction in proliferative activity was seen after stimulation of PBMC by polar fraction treated cultured monocytes whilst no effect was seen after lipid treatment of MDDC. These data demonstrate that pathogenic mycobacterial polar lipids may significantly hamper the ability of the host APCs to induce an appropriate immune response to an invading pathogen.

  4. Gene and cell therapy for muscle regeneration

    OpenAIRE

    Stilhano, Roberta Sessa; Martins, Leonardo; Ingham, Sheila Jean McNeill; Pesquero, João Bosco; Huard, Johnny

    2015-01-01

    Skeletal muscle injury and healing are multifactorial processes, involving three steps of healing: (1) degeneration and inflammation, (2) regeneration, and (3) fibrosis. Fibrous tissue hinders the muscle’s complete recovery and current therapies fail in achieving total muscle recovery. Gene and cell therapy (or both) are potential future treatments for severe muscular injuries. Stem cells’ properties associated with growth factors or/and cytokines can improve muscle healing and permit long-te...

  5. Mammalian aPKC/Par polarity complex mediated regulation of epithelial division orientation and cell fate

    International Nuclear Information System (INIS)

    Oriented cell division is a key regulator of tissue architecture and crucial for morphogenesis and homeostasis. Balanced regulation of proliferation and differentiation is an essential property of tissues not only to drive morphogenesis but also to maintain and restore homeostasis. In many tissues orientation of cell division is coupled to the regulation of differentiation producing daughters with similar (symmetric cell division, SCD) or differential fate (asymmetric cell division, ACD). This allows the organism to generate cell lineage diversity from a small pool of stem and progenitor cells. Division orientation and/or the ratio of ACD/SCD need to be tightly controlled. Loss of orientation or an altered ratio can promote overgrowth, alter tissue architecture and induce aberrant differentiation, and have been linked to morphogenetic diseases, cancer and aging. A key requirement for oriented division is the presence of a polarity axis, which can be established through cell intrinsic and/or extrinsic signals. Polarity proteins translate such internal and external cues to drive polarization. In this review we will focus on the role of the polarity complex aPKC/Par3/Par6 in the regulation of division orientation and cell fate in different mammalian epithelia. We will compare the conserved function of this complex in mitotic spindle orientation and distribution of cell fate determinants and highlight common and differential mechanisms in which this complex is used by tissues to adapt division orientation and cell fate to the specific properties of the epithelium

  6. Mammalian aPKC/Par polarity complex mediated regulation of epithelial division orientation and cell fate

    Energy Technology Data Exchange (ETDEWEB)

    Vorhagen, Susanne; Niessen, Carien M., E-mail: carien.niessen@uni-koeln.de

    2014-11-01

    Oriented cell division is a key regulator of tissue architecture and crucial for morphogenesis and homeostasis. Balanced regulation of proliferation and differentiation is an essential property of tissues not only to drive morphogenesis but also to maintain and restore homeostasis. In many tissues orientation of cell division is coupled to the regulation of differentiation producing daughters with similar (symmetric cell division, SCD) or differential fate (asymmetric cell division, ACD). This allows the organism to generate cell lineage diversity from a small pool of stem and progenitor cells. Division orientation and/or the ratio of ACD/SCD need to be tightly controlled. Loss of orientation or an altered ratio can promote overgrowth, alter tissue architecture and induce aberrant differentiation, and have been linked to morphogenetic diseases, cancer and aging. A key requirement for oriented division is the presence of a polarity axis, which can be established through cell intrinsic and/or extrinsic signals. Polarity proteins translate such internal and external cues to drive polarization. In this review we will focus on the role of the polarity complex aPKC/Par3/Par6 in the regulation of division orientation and cell fate in different mammalian epithelia. We will compare the conserved function of this complex in mitotic spindle orientation and distribution of cell fate determinants and highlight common and differential mechanisms in which this complex is used by tissues to adapt division orientation and cell fate to the specific properties of the epithelium.

  7. Optimizing autologous cell grafts to improve stem cell gene therapy.

    Science.gov (United States)

    Psatha, Nikoletta; Karponi, Garyfalia; Yannaki, Evangelia

    2016-07-01

    Over the past decade, stem cell gene therapy has achieved unprecedented curative outcomes for several genetic disorders. Despite the unequivocal success, clinical gene therapy still faces challenges. Genetically engineered hematopoietic stem cells are particularly vulnerable to attenuation of their repopulating capacity once exposed to culture conditions, ultimately leading to low engraftment levels posttransplant. This becomes of particular importance when transduction rates are low or/and competitive transplant conditions are generated by reduced-intensity conditioning in the absence of a selective advantage of the transduced over the unmodified cells. These limitations could partially be overcome by introducing megadoses of genetically modified CD34(+) cells into conditioned patients or by transplanting hematopoietic stem cells hematopoietic stem cells with high engrafting and repopulating potential. On the basis of the lessons gained from cord blood transplantation, we summarize the most promising approaches to date of increasing either the numbers of hematopoietic stem cells for transplantation or/and their engraftability, as a platform toward the optimization of engineered stem cell grafts. PMID:27106799

  8. Protocadherin FAT1 binds Ena/VASP proteins and is necessary for actin dynamics and cell polarization

    OpenAIRE

    Moeller, Marcus J.; Soofi, Abdulsalam; Braun, Gerald S; Li, Xiaodong; Watzl, Carsten; Kriz, Wilhelm; Holzman, Lawrence B.

    2004-01-01

    Cell migration requires integration of cellular processes resulting in cell polarization and actin dynamics. Previous work using tools of Drosophila genetics suggested that protocadherin fat serves in a pathway necessary for determining cell polarity in the plane of a tissue. Here we identify mammalian FAT1 as a proximal element of a signaling pathway that determines both cellular polarity in the plane of the monolayer and directed actin-dependent cell motility. FAT1 is localized to the leadi...

  9. Calponin 2 Acts As an Effector of Noncanonical Wnt-Mediated Cell Polarization during Neural Crest Cell Migration

    OpenAIRE

    Bärbel Ulmer; Cathrin Hagenlocher; Silke Schmalholz; Sabrina Kurz; Axel Schweickert; Ayelet Kohl; Lee Roth; Dalit Sela-Donenfeld; Martin Blum

    2013-01-01

    Neural crest cells (NCCs) migrate throughout the embryo to differentiate into cell types of all germ layers. Initial directed NCC emigration relies on planar cell polarity (PCP), which through the activity of the small GTPases RhoA and Rac governs the actin-driven formation of polarized cell protrusions. We found that the actin binding protein calponin 2 (Cnn2) was expressed in protrusions at the leading edge of migratory NCCs in chicks and frogs. Cnn2 knockdown resulted in NCC migration defe...

  10. Specific polar subpopulations of astral microtubules control spindle orientation and symmetric neural stem cell division.

    Science.gov (United States)

    Mora-Bermúdez, Felipe; Matsuzaki, Fumio; Huttner, Wieland B

    2014-01-01

    Mitotic spindle orientation is crucial for symmetric vs asymmetric cell division and depends on astral microtubules. Here, we show that distinct subpopulations of astral microtubules exist, which have differential functions in regulating spindle orientation and division symmetry. Specifically, in polarized stem cells of developing mouse neocortex, astral microtubules reaching the apical and basal cell cortex, but not those reaching the central cell cortex, are more abundant in symmetrically than asymmetrically dividing cells and reduce spindle orientation variability. This promotes symmetric divisions by maintaining an apico-basal cleavage plane. The greater abundance of apical/basal astrals depends on a higher concentration, at the basal cell cortex, of LGN, a known spindle-cell cortex linker. Furthermore, newly developed specific microtubule perturbations that selectively decrease apical/basal astrals recapitulate the symmetric-to-asymmetric division switch and suffice to increase neurogenesis in vivo. Thus, our study identifies a novel link between cell polarity, astral microtubules, and spindle orientation in morphogenesis. PMID:24996848

  11. Climatic anomalous patterns associated with the Arctic and Polar cell strength variations

    Science.gov (United States)

    Qian, Weihong; Wu, Kaijun; Leung, Jeremy Cheuk-Hin

    2016-03-01

    The Arctic cell as a reversed and closed loop next to the Polar cell has been recently revealed in the Northern Hemisphere (NH). In this paper, we study the interannual variability of the Arctic and Polar cell strengths during 1979-2012, and their influence on surface air temperature (SAT), precipitation, and sea-ice concentration (SIC) at mid- and high-latitudes of the NH. We show that there is a significant negative correlation between the Arctic and Polar cell strengths. Both the Arctic and Polar cell strengths can well indicate the recurring climatic anomalies of SAT, precipitation, and SIC in their extreme winters. The surface large-scale cold-warm and dry-wet anomalous patterns in these extreme winters are directly linked with the vertical structure of height and temperature anomalies in the troposphere. Results suggest that the past climatic anomalies are better indicated by the strength anomalies of the Polar and Arctic cells than the traditional indices of mid-high latitude pattern such as the Arctic Oscillation and North Atlantic Oscillation. This study illustrates a three-dimensional picture of atmospheric variable anomalies in the troposphere that result in surface climatic anomalies.

  12. The tumor suppressor Apc controls planar cell polarities central to gut homeostasis

    OpenAIRE

    Bellis, Julien; Duluc, Isabelle; Romagnolo, Béatrice; Perret, Christine; Faux, Maree C.; Dujardin, Denis; Formstone, Caroline; Lightowler, Sally; Ramsay, Robert G.; Freund, Jean-Noël; De Mey, Jan R.

    2012-01-01

    The stem cells (SCs) at the bottom of intestinal crypts tightly contact niche-supporting cells and fuel the extraordinary tissue renewal of intestinal epithelia. Their fate is regulated stochastically by populational asymmetry, yet whether asymmetrical fate as a mode of SC division is relevant and whether the SC niche contains committed progenitors of the specialized cell types are under debate. We demonstrate spindle alignments and planar cell polarities, which form a novel functional unit t...

  13. Genome-Wide Gene Expression Profiling Revealed a Critical Role for GATA3 in the Maintenance of the Th2 Cell Identity

    OpenAIRE

    Sasaki, Tetsuya; Onodera, Atsushi; Hosokawa, Hiroyuki; Watanabe, Yukiko; Horiuchi, Shu; Yamashita, Junji; Tanaka, Hitoshi; Ogawa, Yasumasa; Suzuki, Yutaka; Nakayama, Toshinori

    2013-01-01

    Functionally polarized CD4+ T helper (Th) cells such as Th1, Th2 and Th17 cells are central to the regulation of acquired immunity. However, the molecular mechanisms governing the maintenance of the polarized functions of Th cells remain unclear. GATA3, a master regulator of Th2 cell differentiation, initiates the expressions of Th2 cytokine genes and other Th2-specific genes. GATA3 also plays important roles in maintaining Th2 cell function and in continuous chromatin remodeling of Th2 cytok...

  14. Polarization Affects Airway Epithelial Conditioning of Monocyte-Derived Dendritic Cells

    DEFF Research Database (Denmark)

    Papazian, Dick; Chhoden, Tashi; Arge, Maria;

    2015-01-01

    Airway epithelial cells (AECs) form polarized barriers that interact with inhaled allergens and are involved in immune homeostasis. We examined how monocyte-derived dendritic cells (MDDCs) are affected by contact with the airway epithelium. In traditional setups, bronchial epithelial cell lines...... were allowed to polarize on filter inserts, and MDDCs were allowed to adhere to the epithelial basal side. In an optimized setup, the cell application was reversed, and the culture conditions were modified to preserve cellular polarization and integrity. These two parameters were crucial for the MDDCs....... In conclusion, we determined that AEC conditioning favoring cellular integrity leads to a tolerogenic MDDC phenotype, which is likely to be important in regulating immune responses against commonly inhaled allergens....

  15. Roles of imprinted genes in neural stem cells.

    Science.gov (United States)

    Hoffmann, Anke; Daniel, Guillaume; Schmidt-Edelkraut, Udo; Spengler, Dietmar

    2014-01-01

    Imprinted genes and neural stem cells (NSC) play an important role in the developing and mature brain. A central theme of imprinted gene function in NSCs is cell survival and G1 arrest to control cell division, cell-cycle exit, migration and differentiation. Moreover, genomic imprinting can be epigenetically switched off at some genes to ensure stem cell quiescence and differentiation. At the genome scale, imprinted genes are organized in dynamic networks formed by interchromosomal interactions and transcriptional coregulation of imprinted and nonimprinted genes. Such multilayered networks may synchronize NSC activity with the demand from the niche resembling their roles in adjusting fetal size. PMID:25431944

  16. Evidence for Nuclear Tensor Polarization of Deuterium Molecules in Storage Cells

    International Nuclear Information System (INIS)

    Deuterium molecules were obtained by recombination, on a copper surface, of deuterium atoms prepared in specific hyperfine states. The molecules were stored for about 5ms in an open-ended cylindrical cell, placed in a 23mT magnetic field, and their tensor polarization was measured by elastic scattering of 704MeV electrons. The results of the measurements are consistent with the deuterium molecules retaining the tensor polarization of the initial atoms. copyright 1997 The American Physical Society

  17. Cell Targeting in Anti-Cancer Gene Therapy

    OpenAIRE

    Lila, Mohd Azmi Mohd; Siew, John Shia Kwong; Zakaria, Hayati; Saad, Suria Mohd; Ni, Lim Shen; Abdullah, Jafri Malin

    2004-01-01

    Gene therapy is a promising approach towards cancer treatment. The main aim of the therapy is to destroy cancer cells, usually by apoptotic mechanisms, and preserving others. However, its application has been hindered by many factors including poor cellular uptake, non-specific cell targeting and undesirable interferences with other genes or gene products. A variety of strategies exist to improve cellular uptake efficiency of gene-based therapies. This paper highlights advancements in gene th...

  18. Iron repletion relocalizes hephaestin to a proximal basolateral compartment in polarized MDCK and Caco2 cells

    Energy Technology Data Exchange (ETDEWEB)

    Lee, Seung-Min [Department of Biological Sciences, University of Columbia, NY (United States); Department of Nutritional Science and Toxicology, University of California, Berkeley, CA (United States); Attieh, Zouhair K. [Department of Laboratory Science and Technology, American University of Science and Technology, Ashrafieh (Lebanon); Department of Nutritional Science and Toxicology, University of California, Berkeley, CA (United States); Son, Hee Sook [Department of Food Science and Human Nutrition, College of Human Ecology, Chonbuk National University (Korea, Republic of); Department of Nutritional Science and Toxicology, University of California, Berkeley, CA (United States); Chen, Huijun [Medical School, Nanjing University, Nanjing 210008, Jiangsu Province (China); Department of Nutritional Science and Toxicology, University of California, Berkeley, CA (United States); Bacouri-Haidar, Mhenia [Department of Biology, Faculty of Sciences (I), Lebanese University, Hadath (Lebanon); Department of Nutritional Science and Toxicology, University of California, Berkeley, CA (United States); Vulpe, Chris D., E-mail: vulpe@berkeley.edu [Department of Nutritional Science and Toxicology, University of California, Berkeley, CA (United States)

    2012-05-11

    Highlights: Black-Right-Pointing-Pointer Hephaestin localizes in the perinuclear space in non-polarized cells. Black-Right-Pointing-Pointer Hephaestin localizes in the perinuclear space in iron deficient and polarized cells. Black-Right-Pointing-Pointer Hephaestin with apical iron moves near to basolateral membrane of polarized cells. Black-Right-Pointing-Pointer Peri-basolateral location of hephaestin is accessible to the extracellular space. Black-Right-Pointing-Pointer Hephaestin is involved in iron mobilization from the intestine to circulation. -- Abstract: While intestinal cellular iron entry in vertebrates employs multiple routes including heme and non-heme routes, iron egress from these cells is exclusively channeled through the only known transporter, ferroportin. Reduced intestinal iron export in sex-linked anemia mice implicates hephaestin, a ferroxidase, in this process. Polarized cells are exposed to two distinct environments. Enterocytes contact the gut lumen via the apical surface of the cell, and through the basolateral surface, to the body. Previous studies indicate both local and systemic control of iron uptake. We hypothesized that differences in iron availability at the apical and/or basolateral surface may modulate iron uptake via cellular localization of hephaestin. We therefore characterized the localization of hephaestin in two models of polarized epithelial cell lines, MDCK and Caco2, with varying iron availability at the apical and basolateral surfaces. Our results indicate that hephaestin is expressed in a supra-nuclear compartment in non-polarized cells regardless of the iron status of the cells and in iron deficient and polarized cells. In polarized cells, we found that both apical (as FeSO{sub 4}) and basolateral iron (as the ratio of apo-transferrin to holo-transferrin) affect mobilization of hephaestin from the supra-nuclear compartment. We find that the presence of apical iron is essential for relocalization of hephaestin to a

  19. Kruppel-like factor 4 regulates intestinal epithelial cell morphology and polarity.

    Directory of Open Access Journals (Sweden)

    Tianxin Yu

    Full Text Available Krüppel-like factor 4 (KLF4 is a zinc finger transcription factor that plays a vital role in regulating cell lineage differentiation during development and maintaining epithelial homeostasis in the intestine. In normal intestine, KLF4 is predominantly expressed in the differentiated epithelial cells. It has been identified as a tumor suppressor in colorectal cancer. KLF4 knockout mice demonstrated a decrease in number of goblet cells in the colon, and conditional ablation of KLF4 from the intestinal epithelium led to altered epithelial homeostasis. However, the role of KLF4 in differentiated intestinal cells and colon cancer cells, as well as the mechanism by which it regulates homeostasis and represses tumorigenesis in the intestine is not well understood. In our study, KLF4 was partially depleted in the differentiated intestinal epithelial cells by a tamoxifen-inducible Cre recombinase. We found a significant increase in the number of goblet cells in the KLF4-deleted small intestine, suggesting that KLF4 is not only required for goblet cell differentiation, but also required for maintaining goblet cell numbers through its function in inhibiting cell proliferation. The number and position of Paneth cells also changed. This is consistent with the KLF4 knockout study using villin-Cre [1]. Through immunohistochemistry (IHC staining and statistical analysis, we found that a stem cell and/or tuft cell marker, DCAMKL1, and a proliferation marker, Ki67, are affected by KLF4 depletion, while an enteroendocrine cell marker, neurotensin (NT, was not affected. In addition, we found KLF4 depletion altered the morphology and polarity of the intestinal epithelial cells. Using a three-dimensional (3D intestinal epithelial cyst formation assay, we found that KLF4 is essential for cell polarity and crypt-cyst formation in human colon cancer cells. These findings suggest that, as a tumor suppressor in colorectal cancer, KLF4 affects intestinal epithelial cell

  20. Inter-tissue mechanical stress affects Frizzled-mediated planar cell polarity in the Drosophila notum epidermis

    Science.gov (United States)

    Olguín, Patricio; Glavic, Alvaro; Mlodzik, Marek

    2011-01-01

    Summary Frizzled/Planar Cell Polarity (Fz/PCP) signaling controls the orientation of sensory bristles and cellular hairs (trichomes) along the antero-posterior axis of the Drosophila thorax (notum) [1–4]. A subset of the trichome-producing notum cells differentiate as “tendon cells”, serving as attachment sites for the indirect flight muscles (IFMs) to the exoskeleton [5]. Through the analysis of chascon (chas), a gene identified by its ability to disrupt Fz/PCP signaling under overexpression conditions, and jitterbug (jbug)/filamin [6], we show that maintenance of antero-posterior planar polarization requires the notum epithelia to balance mechanical stress generated by the attachment of the IFMs. chas is expressed in notum tendon cells and its loss-of-function disturbs cellular orientation at and near the regions where IFMs attach to the epidermis. This effect is independent of the Fz/PCP and fat (ft)/dachsous (ds) systems [7]. The chas phenotype arises during normal shortening of the IFMs [8] and is suppressed by genetic ablation of the IFMs. chas acts through jbug/filamin and cooperates with MyosinII to modulate the mechano-response of notum tendon cells. These observations support the notion that the ability of epithelia to respond to mechanical stress generated by interaction(s) with other tissues during development/organogenesis influences the maintenance of its shape and PCP features. PMID:21276726

  1. Direct cell lysis for single-cell gene expression profiling

    Directory of Open Access Journals (Sweden)

    DavidSvec

    2013-11-01

    Full Text Available The interest to analyze single and few cell samples is rapidly increasing. Numerous extraction protocols to purify nucleic acids are available, but most of them compromise severely on yield to remove contaminants and are therefore not suitable for the analysis of samples containing small numbers of transcripts only. Here, we evaluate 17 direct cell lysis protocols for transcript yield and compatibility with downstream reverse transcription quantitative real-time PCR. Four endogenously expressed genes are assayed together with RNA and DNA spikes in the samples. We found bovine serum albumin (BSA to be the best lysis agent, resulting in efficient cell lysis, high RNA stability and enhanced reverse transcription efficiency. Furthermore, we found direct cell lysis with BSA superior to standard column based extraction methods, when analyzing from 1 up to 512 mammalian cells. In conclusion, direct cell lysis protocols based on BSA can be applied with most cell collection methods and are compatible with most analytical workflows to analyze single cells as well as samples composed of small numbers of cells.

  2. Itinerant exosomes: emerging roles in cell and tissue polarity

    OpenAIRE

    Lakkaraju, Aparna; Rodriguez-Boulan, Enrique

    2008-01-01

    Cells use secreted signals (e.g. chemokines and growth factors) and sophisticated vehicles such as argosomes, cytonemes, tunneling nanotubes and exosomes to relay important information to other cells, often over large distances. Exosomes, 30–100-nm intraluminal vesicles of multivesicular bodies (MVB) released upon exocytic fusion of the MVB with the plasma membrane, are increasingly recognized as a novel mode of cell-independent communication. Exosomes have been shown to function in antigen p...

  3. THE GENE EXPRESSION PROFILE OF HIGHLY METASTATIC HUMAN OVARIAN CANCER CELL LINE BY GENE CHIP

    Institute of Scientific and Technical Information of China (English)

    吕桂泉; 许沈华; 牟瀚舟; 朱赤红; 羊正炎; 高永良; 楼洪坤; 刘祥麟; 杨文; 程勇

    2001-01-01

    To study the gene expression of high metastatic human ovarian carcinoma cell line (HO-8910PM) and to screen for novel metastasis- associated genes by cDNA microarray. Methods: The cDNA was retro-transcribed from equal quantity mRNA derived from tissues of highly metastatic ovarian carcinoma cell line and normal ovarian, and was labeled with Cy5 and Cy3 fluorescence as probes. The mixed probes were hybridized with BioDoor 4096 double dot human whole gene chip. The chip was scanned by scanArray 3000 laser scanner. The acquired image was analyzed by ImaGene 3.0 software. Results: By applying the cDNA microarray we found: A total of 323 genes whose expression level were 3 times higher or lower in HO-8910PM cell than normal ovarian epithelium cell were screened out, with 71 higher and 252 lower respectively. Among these 10 were new genes. 67 genes showed expression difference bigger than 6 times between HO-8910PM cell and normal ovarian epithelium cell, among these genes 12 were higher, 55 lower, and two new genes were found. Conclusion: cDNA microarray technique is effective in screening the differentially expressed genes between human ovarian cancer cell line (HO-8910PM) and normal ovarian epithelium cell. Using the cDNA microarray to analyze of human ovarian cancer cell line gene expression profile difference will help the gene diagnosis, treatment and protection.

  4. [Adipose-derived stem cells promote the polarization from M1 macrophages to M2 macrophages].

    Science.gov (United States)

    Yin, Xuehong; Pang, Chunyan; Bai, Li; Zhang, Ying; Geng, Lixia

    2016-03-01

    Objective To investigate the effects of adipose-derived stem cells (ADSCs) on M1/M2 macrophages and whether ADSCs are able to promote the polarization from M1 macrophages to M2 macrophages. Methods M1 macrophages were induced from J774.1 macrophages by 24-hour stimulation of lipopolysaccharide (LPS) and interferon γ (IFN-γ), and M2 macrophages were induced from J774.1 macrophages by interleukin 4 (IL-4) for another 24 hours. Then M1/M2 macrophages were separately cultured in the presence of ADSCs for 24 hours. The M1/M2 macrophages and their corresponding supernatants were collected for further analysis. The expressions of IL-6, tumor necrosis factor α (TNF-α), inducible nitric oxide synthase (iNOS), CC chemokine ligand 2 (CCL2), CD86, arginase 1 (Arg1), mannose receptors/CD206 (MR/CD206), IL-10, found in inflammatory zone 1 (FIZZ1), chitinase 3-like 3 (Ym-1) were detected by real-time PCR and ELISA. Results ADSCs significantly decreased the levels of IL-6, TNF-α, iNOS, CCL2 and CD86, and increased the levels of Arg1, CD206 and IL-10 in M1 macrophages. In the supernatant of M1 macrophages, the expressions of IL-6 and TNF-α were reduced, while those of CD206 were enhanced. In M2 macrophages, ADSCs resulted in down-regulation of IL-6, TNF-α, iNOS, CD86 and up-regulation of Arg1, CD206, FIZZ-1, Ym-1 and IL-10. In the supernatant of M2 macrophages, the expression levels of IL-6 and TNF-α were down-regulated and those of CD206 were up-regulated. Conclusion ADSCs can inhibit the gene expression of M1 macrophages and promote the gene expression of M2 macrophages, as well as mediate the polarization from M1 macrophages to M2 macrophages. PMID:26927552

  5. Functional analysis of Peutz-Jeghers mutations reveals that the LKB1 C-terminal region exerts a crucial role in regulating both the AMPK pathway and the cell polarity.

    NARCIS (Netherlands)

    Forcet, C.; Etienne-Manneville, S.; Gaude, H.; Fournier, L.; Debilly, S.; Salmi, M.; Baas, A.; Olschwang, S.; Clevers, J.C.; Billaud, M.

    2005-01-01

    Germline mutations of the LKB1 gene are responsible for the cancer-prone Peutz-Jeghers syndrome (PJS). LKB1 encodes a serine-threonine kinase that acts as a regulator of cell cycle, metabolism and cell polarity. The majority of PJS missense mutations abolish LKB1 enzymatic activity and thereby impai

  6. Targeted gene conversion induced by triplex-directed psoralen interstrand crosslinks in mammalian cells.

    Science.gov (United States)

    Liu, Yaobin; Nairn, Rodney S; Vasquez, Karen M

    2009-10-01

    Correction of a defective gene is a promising approach for both basic research and clinical gene therapy. However, the absence of site-specific targeting and the low efficiency of homologous recombination in human cells present barriers to successful gene targeting. In an effort to overcome these barriers, we utilized triplex-forming oligonucleotides (TFOs) conjugated to a DNA interstrand crosslinking (ICL) agent, psoralen (pTFO-ICLs), to improve the gene targeting efficiency at a specific site in DNA. Gene targeting events were monitored by the correction of a deletion on a recipient plasmid with the homologous sequence from a donor plasmid in human cells. The mechanism underlying this event is stimulation of homologous recombination by the pTFO-ICL. We found that pTFO-ICLs are efficient in inducing targeted gene conversion (GC) events in human cells. The deletion size in the recipient plasmid influenced both the recombination frequency and spectrum of recombinants; i.e. plasmids with smaller deletions had a higher frequency and proportion of GC events. The polarity of the pTFO-ICL also had a prominent effect on recombination. Our results suggest that pTFO-ICL induced intermolecular recombination provides an efficient method for targeted gene correction in mammalian cells. PMID:19726585

  7. Basolateral Invasion and Trafficking of Campylobacter jejuni in Polarized Epithelial Cells

    OpenAIRE

    Bouwman, L.I.; Niewold, P.; van Putten, J.P.M.

    2013-01-01

    Campylobacter jejuni is a major cause of bacterial diarrheal disease. Most enteropathogenic bacteria including C. jejuni can invade cultured eukaryotic cells via an actin- and/or microtubule-dependent and an energy-consuming uptake process. Recently, we identified a novel highly efficient C. jejuni invasion pathway that involves bacterial migration into the subcellular space of non-polarized epithelial cells (termed subvasion) followed by invasion from the cell basis. Here we report cellular ...

  8. Tumor cell-activated CARD9 signaling contributes to metastasis-associated macrophage polarization

    OpenAIRE

    Yang, M; Shao, J-H; Miao, Y-J; Cui, W.; Qi, Y-F; Han, J-H; Lin, X.; J. Du

    2014-01-01

    Macrophages are critical immune effector cells of the tumor microenvironment that promote seeding, extravasation and persistent growth of tumor cells in primary tumors and metastatic sites. Tumor progression and metastasis are affected by dynamic changes in the specific phenotypes of macrophage subpopulations; however, the mechanisms by which tumor cells modulate macrophage polarization remain incompletely understood. Caspase recruitment domain-containing protein 9 (CARD9) is a central adapto...

  9. A Catalytic Role for Mod5 in the Formation of the Tea1 Cell Polarity Landmark

    OpenAIRE

    Bicho, Claudia C.; Kelly, David A.; Snaith, Hilary A.; Goryachev, Andrew B.; Sawin, Kenneth E.

    2010-01-01

    Summary Many systems regulating cell polarity involve stable landmarks defined by internal cues [1–5]. In the rod-shaped fission yeast Schizosaccharomyces pombe, microtubules regulate polarized vegetative growth via a landmark involving the protein Tea1 [6–9]. Tea1 is delivered to cell tips as packets of molecules associated with growing microtubule ends [10] and anchored at the plasma membrane via a mechanism involving interaction with the membrane protein Mod5 [11, 12]. Tea1 and Mod5 are hi...

  10. Essential function for PDLIM2 in cell polarization in three-dimensional cultures by feedback regulation of the β1-integrin-RhoA signaling axis.

    Science.gov (United States)

    Deevi, Ravi Kiran; Cox, Orla T; O'Connor, Rosemary

    2014-05-01

    PDLIM2 is a cytoskeletal and nuclear PDZ-LIM domain protein that regulates the stability of Nuclear Factor kappa-B (NFκB) and other transcription factors, and is required for polarized cell migration. PDLIM2 expression is suppressed by methylation in different cancers, but is strongly expressed in invasive breast cancer cells that have undergone an Epithelial Mesenchymal Transition (EMT). PDLIM2 is also expressed in non-transformed breast myoepithelial MCF10A cells and here we asked whether it is important for maintaining the polarized, epithelial phenotype of these cells. Suppression of PDLIM2 in MCF10A cells was sufficient to disrupt cell polarization and acini formation with increased proliferation and reduced apoptosis in the luminal space compared to control acini with hollow lumina. Spheroids with suppressed PDLIM2 exhibited increased expression of cell-cell and cell-matrix adhesion proteins including beta 1 (β1) integrin. Interestingly, levels of the Insulin-like growth factor 1 receptor (IGF-1 R) and Receptor of activated protein kinase C 1 (RACK1), which scaffolds IGF-1R to β1 integrin, were also increased, indicating a transformed phenotype. Focal Adhesion Kinase (FAK) and cofilin phosphorylation, and RhoA Guanosine Triphosphatase (GTPase) activity were all enhanced in these spheroids compared to control acini. Importantly, inhibition of either FAK or Rho Kinase (ROCK) was sufficient to rescue the polarity defect. We conclude that PDLIM2 expression is essential for feedback regulation of the β1-integrin-RhoA signalling axis and integration of cellular microenvironment signals with gene expression to control the polarity of breast epithelial acini structures. This is a mechanism by which PDLIM2 could mediate tumour suppression in breast epithelium. PMID:24863845

  11. Essential Function for PDLIM2 in Cell Polarization in Three-Dimensional Cultures by Feedback Regulation of the β1-Integrin–RhoA Signaling Axis

    Directory of Open Access Journals (Sweden)

    Ravi Kiran Deevi

    2014-05-01

    Full Text Available PDLIM2 is a cytoskeletal and nuclear PDZ-LIM domain protein that regulates the stability of Nuclear Factor kappa-B (NFκB and other transcription factors, and is required for polarized cell migration. PDLIM2 expression is suppressed by methylation in different cancers, but is strongly expressed in invasive breast cancer cells that have undergone an Epithelial Mesenchymal Transition (EMT. PDLIM2 is also expressed in non-transformed breast myoepithelial MCF10A cells and here we asked whether it is important for maintaining the polarized, epithelial phenotype of these cells. Suppression of PDLIM2 in MCF10A cells was sufficient to disrupt cell polarization and acini formation with increased proliferation and reduced apoptosis in the luminal space compared to control acini with hollow lumina. Spheroids with suppressed PDLIM2 exhibited increased expression of cell-cell and cell-matrix adhesion proteins including beta 1 (β1 integrin. Interestingly, levels of the Insulin-like growth factor 1 receptor (IGF-1 R and Receptor of activated protein kinase C 1 (RACK1, which scaffolds IGF-1R to β1 integrin, were also increased, indicating a transformed phenotype. Focal Adhesion Kinase (FAK and cofilin phosphorylation, and RhoA Guanosine Triphosphatase (GTPase activity were all enhanced in these spheroids compared to control acini. Importantly, inhibition of either FAK or Rho Kinase (ROCK was sufficient to rescue the polarity defect. We conclude that PDLIM2 expression is essential for feedback regulation of the β1-integrin-RhoA signalling axis and integration of cellular microenvironment signals with gene expression to control the polarity of breast epithelial acini structures. This is a mechanism by which PDLIM2 could mediate tumour suppression in breast epithelium.

  12. Rap1 integrates tissue polarity, lumen formation, and tumorigenicpotential in human breast epithelial cells

    Energy Technology Data Exchange (ETDEWEB)

    Itoh, Masahiko; Nelson, Celeste M.; Myers, Connie A.; Bissell,Mina J.

    2006-09-29

    Maintenance of apico-basal polarity in normal breast epithelial acini requires a balance between cell proliferation, cell death, and proper cell-cell and cell-extracellular matrix signaling. Aberrations in any of these processes can disrupt tissue architecture and initiate tumor formation. Here we show that the small GTPase Rap1 is a crucial element in organizing acinar structure and inducing lumen formation. Rap1 activity in malignant HMT-3522 T4-2 cells is appreciably higher than in S1 cells, their non-malignant counterparts. Expression of dominant-negative Rap1 resulted in phenotypic reversion of T4-2 cells, led to formation of acinar structures with correct apico-basal polarity, and dramatically reduced tumor incidence despite the persistence of genomic abnormalities. The resulting acini contained prominent central lumina not observed when other reverting agents were used. Conversely, expression of dominant-active Rap1 in T4-2 cells inhibited phenotypic reversion and led to increased invasiveness and tumorigenicity. Thus, Rap1 acts as a central regulator of breast architecture, with normal levels of activation instructing apical polarity during acinar morphogenesis, and increased activation inducing tumor formation and progression to malignancy.

  13. Activation of JNK signaling links IgI mutations to disruption of the cell polarity and epithelial organization in Drosophila imaginal discs

    Institute of Scientific and Technical Information of China (English)

    Ming-wei Zhu; Tian-chi Xin; Shun-yan Weng; Yin Gao; Ying-jie Zhang; Qi Li; Ming-fa Li

    2010-01-01

    Dear Editor, Identification of Drosophila melanogaster as a model organism for cancer research has facilitated the exploration of human tumor malignancy. In Drosophila, lossof-function mutations in the neoplastic tumor suppressor genes (nTSGs) lethal(2)giant larvae (lgl), discs large (dlg) or scribble (scrib) cause a malignant tumor-like phenotype characteristic of disrupted cell polarity and overgrowth in epithelial tissues such as imaginal discs [1].

  14. HIGH EFFICIENCY RETROVIRUS-MEDIATED GENE TRANSFER TO LEUKEMIA CELLS

    Institute of Scientific and Technical Information of China (English)

    FU Jian-xin; CHEN Zi-xing; CEN Jian-nong; WANG Wei; RUAN Chang-geng

    1999-01-01

    Objective: To establish an efficient and safe gene transfer system mediated by retrovirus for gene marking and gene therapy of human leukemia. Method: The retroviral vector LXSN, containing the neomycin resistance (NeoR) gene, was transferred into amphotropic packaging cells GP+envAm12 by liposome transfection or by ecotropic retrovirus transduction. Amphotropic retrovirus in supernatants with higher titer was used to infect human leukemic cell lines NB4, U937, and THP-1.The efficiency of gene transfer was assayed on colonies formed by transduced K562 cells. Results: The titer of DOSPER directly transfected GP+envAm12 cells determined on NIH3T3 cells was 8.0×105 CFU/ml, while that of producer infected with retrovirus was 1.6×107CFU/ml. Integration of NeoR gene into all leukemia cells was confirmed by polymerase chain reaction (PCR).Absence of replication-competent virus was proved by both nested PCR for env gene and marker gene rescue assay. Gene transfer with the efficiency as high as 93.3 to 100% in K562 cells was verified by seminested PCR for integrated NeoR gene on colonies after 7 days' culture.Conclusion: The efficiency and safety of retrovirus mediated gene transfer system might provide an optimal system in gene therapy for leukemia or genetic diseases.

  15. Gene expression profiling of dendritic cells reveals important mechanisms associated with predisposition to Staphylococcus infections.

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    Mehdi Toufeer

    Full Text Available BACKGROUND: Staphylococcus aureus is a major pathogen of humans and animals and emerging antibiotic-resistant strains have further increased the concern of this health issue. Host genetics influence susceptibility to S. aureus infections, and the genes determining the outcome of infections should be identified to find alternative therapies to treatment with antibiotics. Here, we used outbred animals from a divergent selection based on susceptibility towards Staphylococcus infection to explore host immunogenetics. METHODOLOGY/PRINCIPAL FINDINGS: We investigated how dendritic cells respond to heat-inactivated S. aureus and whether dendritic cells from animals showing different degrees of susceptibility had distinct gene expression profiles. We measured gene expression levels of in vitro S. aureus-stimulated bone marrow-derived dendritic cells at three different time points (0, 3 and 8 hrs by using 15 k ovine Agilent microarrays. Furthermore, differential expression of a selected number of genes was confirmed by RT-qPCR. Gene signatures of stimulated DCs were obtained and showed that genes involved in the inflammatory process and T helper cell polarization were highly up-regulated upon stimulation. Moreover, a set of 204 genes were statistically differentially expressed between susceptible and resistant animals, and grouped them according to their predisposition to staphylococcal infection. Interestingly, over-expression of the C1q and Ido1 genes was observed in the resistant line and suggested a role of classical pathway of complement and early regulation of inflammation pathways, respectively. On the contrary, over expression of genes involved in the IL1R pathway was observed in susceptible animals. Furthermore, the leucocyte extravasation pathway was also found to be dominant in the susceptible line. CONCLUSION/SIGNIFICANCE: We successfully obtained Staphylococcus aureus associated gene expression of ovine BM-DC in an 8-hour kinetics experiment

  16. Gene Expression Profiling of Dendritic Cells Reveals Important Mechanisms Associated with Predisposition to Staphylococcus Infections

    Science.gov (United States)

    Toufeer, Mehdi; Bonnefont, Cécile M. D.; Foulon, Eliane; Caubet, Cécile; Tasca, Christian; Aurel, Marie-Rose; Robert-Granié, Christèle; Rupp, Rachel; Foucras, Gilles

    2011-01-01

    Background Staphylococcus aureus is a major pathogen of humans and animals and emerging antibiotic-resistant strains have further increased the concern of this health issue. Host genetics influence susceptibility to S. aureus infections, and the genes determining the outcome of infections should be identified to find alternative therapies to treatment with antibiotics. Here, we used outbred animals from a divergent selection based on susceptibility towards Staphylococcus infection to explore host immunogenetics. Methodology/Principal Findings We investigated how dendritic cells respond to heat-inactivated S. aureus and whether dendritic cells from animals showing different degrees of susceptibility had distinct gene expression profiles. We measured gene expression levels of in vitro S. aureus-stimulated bone marrow-derived dendritic cells at three different time points (0, 3 and 8 hrs) by using 15 k ovine Agilent microarrays. Furthermore, differential expression of a selected number of genes was confirmed by RT-qPCR. Gene signatures of stimulated DCs were obtained and showed that genes involved in the inflammatory process and T helper cell polarization were highly up-regulated upon stimulation. Moreover, a set of 204 genes were statistically differentially expressed between susceptible and resistant animals, and grouped them according to their predisposition to staphylococcal infection. Interestingly, over-expression of the C1q and Ido1 genes was observed in the resistant line and suggested a role of classical pathway of complement and early regulation of inflammation pathways, respectively. On the contrary, over expression of genes involved in the IL1R pathway was observed in susceptible animals. Furthermore, the leucocyte extravasation pathway was also found to be dominant in the susceptible line. Conclusion/Significance We successfully obtained Staphylococcus aureus associated gene expression of ovine BM-DC in an 8-hour kinetics experiment. The distinct

  17. Polarized THG microscopy identifies compositionally different lipid droplets in mammalian cells.

    Science.gov (United States)

    Bautista, Godofredo; Pfisterer, Simon G; Huttunen, Mikko J; Ranjan, Sanjeev; Kanerva, Kristiina; Ikonen, Elina; Kauranen, Martti

    2014-11-18

    Cells store excess lipids as two major compounds, triacylglycerols (TAGs) and cholesteryl esters (CEs), inside lipid droplets (LDs). The degree of lipid ordering is considered to play a major role in the mobility and enzymatic processing of lipids in LDs. Here, we provide evidence that polarized third-harmonic generation (THG) microscopy distinguishes between native TAG- and CE-enriched LDs in cells due to the different ordering of the two lipid species. We first demonstrate that the responses from synthetic TAG- and CE-enriched LDs using THG microscopy with linear and circular polarizations differ according to their different intrinsic ordering. We then employ simulations to dissect how polarization effects influence the THG from an isotropic LD. Finally, we induce TAG- and CE-enriched LDs in murine macrophages and demonstrate that polarized THG responses increase in a nonlinear fashion with increasing CE/TAG ratio. This suggests that with an increasing CE content, there is a rather sharp transition toward increased LD ordering. Our results demonstrate that polarized THG microscopy enables label-free quantitative analysis of LD ordering and discriminates between compositionally different LDs in intact mammalian cells. PMID:25418291

  18. Requirement for Dlgh-1 in planar cell polarity and skeletogenesis during vertebrate development.

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    Charlene Rivera

    Full Text Available The development of specialized organs is tightly linked to the regulation of cell growth, orientation, migration and adhesion during embryogenesis. In addition, the directed movements of cells and their orientation within the plane of a tissue, termed planar cell polarity (PCP, appear to be crucial for the proper formation of the body plan. In Drosophila embryogenesis, Discs large (dlg plays a critical role in apical-basal cell polarity, cell adhesion and cell proliferation. Craniofacial defects in mice carrying an insertional mutation in Dlgh-1 suggest that Dlgh-1 is required for vertebrate development. To determine what roles Dlgh-1 plays in vertebrate development, we generated mice carrying a null mutation in Dlgh-1. We found that deletion of Dlgh-1 caused open eyelids, open neural tube, and misorientation of cochlear hair cell stereociliary bundles, indicative of defects in planar cell polarity (PCP. Deletion of Dlgh-1 also caused skeletal defects throughout the embryo. These findings identify novel roles for Dlgh-1 in vertebrates that differ from its well-characterized roles in invertebrates and suggest that the Dlgh-1 null mouse may be a useful animal model to study certain human congenital birth defects.

  19. Polarization measurements on single-step co-fired solid oxide fuel cells (SOFCs)

    Energy Technology Data Exchange (ETDEWEB)

    Yoon, Kyung Joong; Zink, Peter; Gopalan, Srikanth; Pal, Uday B. [Department of Manufacturing Engineering, Boston University, Boston, MA 02215 (United States)

    2007-10-11

    Anode-supported planar solid oxide fuel cells (SOFC) were successfully fabricated by a single step co-firing process. The cells comprised of a Ni + yttria-stabilized zirconia (YSZ) anode, a YSZ or scandia-stabilized zirconia (ScSZ) electrolyte, a (La{sub 0.85}Ca{sub 0.15}){sub 0.97}MnO{sub 3} (LCM) + YSZ cathode active layer, and an LCM cathode current collector layer. The fabrication process involved tape casting of the anode, screen printing of the electrolyte and the cathode, and single step co-firing of the green-state cells in the temperature range of 1300-1330 C for 2 h. Cells were tested in the temperature range of 700-800 C with humidified hydrogen as fuel and air as oxidant. Cell test results and polarization modeling showed that the polarization losses were dominated by the ohmic loss associated with the electrodes and the activation polarization of the cathode, and that the ohmic loss due to the ionic resistance of the electrolyte and the activation polarization of the anode were relatively insignificant. Ohmic resistance associated with the electrode was lowered by improving the electrical contact between the electrode and the current collector. Activation polarization of the cathode was reduced by the improvement of the microstructure of the cathode active layer and lowering the cell sintering temperature. The cell performance was further improved by increasing the porosity in the anode. As a result, the maximum power density of 1.5 W cm{sup -2} was achieved at 800 C with humidified hydrogen and air. (author)

  20. Multisite phosphorylation of the guanine nucleotide exchange factor Cdc24 during yeast cell polarization.

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    Stephanie C Wai

    Full Text Available BACKGROUND: Cell polarization is essential for processes such as cell migration and asymmetric cell division. A common regulator of cell polarization in most eukaryotic cells is the conserved Rho GTPase, Cdc42. In budding yeast, Cdc42 is activated by a single guanine nucleotide exchange factor, Cdc24. The mechanistic details of Cdc24 activation at the onset of yeast cell polarization are unclear. Previous studies have suggested an important role for phosphorylation of Cdc24, which may regulate activity or function of the protein, representing a key step in the symmetry breaking process. METHODOLOGY/PRINCIPAL FINDINGS: Here, we directly ask whether multisite phosphorylation of Cdc24 plays a role in its regulation. We identify through mass spectrometry analysis over thirty putative in vivo phosphorylation sites. We first focus on sites matching consensus sequences for cyclin-dependent and p21-activated kinases, two kinase families that have been previously shown to phosphorylate Cdc24. Through site-directed mutagenesis, yeast genetics, and light and fluorescence microscopy, we show that nonphosphorylatable mutations of these consensus sites do not lead to any detectable consequences on growth rate, morphology, kinetics of polarization, or localization of the mutant protein. We do, however, observe a change in the mobility shift of mutant Cdc24 proteins on SDS-PAGE, suggesting that we have indeed perturbed its phosphorylation. Finally, we show that mutation of all identified phosphorylation sites does not cause observable defects in growth rate or morphology. CONCLUSIONS/SIGNIFICANCE: We conclude that lack of phosphorylation on Cdc24 has no overt functional consequences in budding yeast. Yeast cell polarization may be more tightly regulated by inactivation of Cdc42 by GTPase activating proteins or by alternative methods of Cdc24 regulation, such as conformational changes or oligomerization.

  1. Complement protein C1q directs macrophage polarization and limits inflammasome activity during the uptake of apoptotic cells

    Science.gov (United States)

    Benoit, Marie E.; Clarke, Elizabeth V.; Morgado, Pedro; Fraser, Deborah A.; Tenner, Andrea J.

    2012-01-01

    Deficiency in C1q, the recognition component of the classical complement cascade and a pattern recognition receptor involved in apoptotic cell clearance, leads to lupus-like auto-immune diseases characterized by auto-antibodies to self proteins and aberrant innate immune cell activation likely due to impaired clearance of apoptotic cells. Here, we developed an autologous system using primary human lymphocytes and monocyte-derived macrophages (HMDMs) to characterize the effect of C1q on macrophage gene expression profiles during the uptake of apoptotic cells. C1q bound to autologous apoptotic lymphocytes modulated expression of genes associated with JAK/STAT signaling, chemotaxis, immunoregulation and NLRP3 inflammasome activation in LPS-stimulated HMDMs. Specifically, C1q sequentially induced type I interferons (IFNs), IL-27 and IL-10 in LPS-stimulated HMDMs and IL-27 in HMDMs when incubated with AL conditioned media. Co-incubation with C1q tails prevented the induction of type I IFNs and IL-27 in a dose dependent manner and neutralization of type I IFNs partially prevented IL-27 induction by C1q. Finally, C1q decreased procaspase-1 cleavage and caspase-1 dependent cleavage of IL-1β suggesting potent inhibitory effect of C1q on inflammasome activation. These results identify specific molecular pathways induced by C1q to suppress macrophage inflammation providing potential therapeutic targets to control macrophage polarization, and thus inflammation and autoimmunity. PMID:22523386

  2. Expression of recombination-activating genes and T cell receptor gene recombination in the human T cell leukemia cell line

    Institute of Scientific and Technical Information of China (English)

    ZOU Hong-yun; MA Li; MENG Min-jie; YAO Xin-sheng; LIN Ying; WU Zhen-qiang; HE Xiao-wei; WANG Ju-fang; WANG Xiao-ning

    2007-01-01

    Background Recent studies have suggested that mature T cells can change their specificity through reexpression of recombination-activating genes (RAG) and RAG-mediated V(D)J recombination. This process is named receptor revision and has been observed in mature peripheral T cells from transgenic mice and human donors. However, whether the receptor revision in mature T cells is a random or orientated process remains poorly understood. Here we used the Jurkat human T cell line, which represents a mature stage of T cell development, as a model to investigate the regulation of T cell receptor (TCR) gene recombination.Methods TCR Dβ-Jβ signal joint T cell receptor excision DNA circles (sjTRECs) were determined by nested and seminested PCR. Double-strand DNA breaks at recombination signal sequences (RSSs) in the TCRVβ chain locus were detected by ligation-mediated-PCR. Further analysis of the complementarity-determining region 3 (CDR3) size of the TCRVβ chain was examined by the TCR GeneScan technique.Results RAG1, RAG2, and three crucial components of the nonhomologous DNA end-joining (NHEJ) pathway were readily detected in Jurkat. Characteristics of junctional diversity of Dβ2-Jβ2 signal joints and ds RSS breaks associated with the Dβ25' and Dβ 23' sites were detected in DNA from Jurkat cells. CDR3 size and the gene sequences of the TCRVβ chain did not change during cell proliferation.Conclusions RAG1 and RAG2 and ongoing TCR gene recombination are coexpressed in Jurkat cells, but the ongoing recombination process may not play a role in modification of the TCR repertoire. However, the results suggest that Jurkat could be used as a model for studying the regulation of RAGs and V(D)J recombination and as a "special" model of the coexistence of TCR gene rearrangements and "negative" receptor revision.

  3. Aptamer-guided gene targeting in yeast and human cells

    OpenAIRE

    Ruff, Patrick; Koh, Kyung Duk; Keskin, Havva; Pai, Rekha B.; Storici, Francesca

    2014-01-01

    Gene targeting is a genetic technique to modify an endogenous DNA sequence in its genomic location via homologous recombination (HR) and is useful both for functional analysis and gene therapy applications. HR is inefficient in most organisms and cell types, including mammalian cells, often limiting the effectiveness of gene targeting. Therefore, increasing HR efficiency remains a major challenge to DNA editing. Here, we present a new concept for gene correction based on the development of DN...

  4. Transcriptome network analysis reveals candidate genes for renal cell carcinoma

    OpenAIRE

    Wei Zhai; Yun-Fei Xu; Min Liu; Jun-Hua Zheng

    2012-01-01

    Context: Renal cell carcinoma (RCC) is a kidney cancer that originates in renal parenchyma and it is the most common type of kidney cancer with approximately 80% lethal cases. Aims: To interpret the mechanism, explore the regulation of TF-target genes and TF-pathway, and identify the potential key genes of renal cell carcinoma. Settings and Design: After constructing a regulation network from differently expressed genes and transcription factors, pathway regulation network and gene onto...

  5. Distinct Gene Regulatory Pathways for Human Innate versus Adaptive Lymphoid Cells.

    Science.gov (United States)

    Koues, Olivia I; Collins, Patrick L; Cella, Marina; Robinette, Michelle L; Porter, Sofia I; Pyfrom, Sarah C; Payton, Jacqueline E; Colonna, Marco; Oltz, Eugene M

    2016-05-19

    Innate lymphoid cells (ILCs) serve as sentinels in mucosal tissues, sensing release of soluble inflammatory mediators, rapidly communicating danger via cytokine secretion, and functioning as guardians of tissue homeostasis. Although ILCs have been extensively studied in model organisms, little is known about these "first responders" in humans, especially their lineage and functional kinships to cytokine-secreting T helper (Th) cell counterparts. Here, we report gene regulatory circuitries for four human ILC-Th counterparts derived from mucosal environments, revealing that each ILC subset diverges as a distinct lineage from Th and circulating natural killer cells but shares circuitry devoted to functional polarization with their Th counterparts. Super-enhancers demarcate cohorts of cell-identity genes in each lineage, uncovering new modes of regulation for signature cytokines, new molecules that likely impart important functions to ILCs, and potential mechanisms for autoimmune disease SNP associations within ILC-Th subsets. PMID:27156452

  6. Nonviral Gene Delivery to Mesenchymal Stem Cells Using Cationic Liposomes for Gene and Cell Therapy

    Directory of Open Access Journals (Sweden)

    C. Madeira

    2010-01-01

    Full Text Available Mesenchymal stem cells (MSCs hold a great promise for application in several therapies due to their unique biological characteristics. In order to harness their full potential in cell-or gene-based therapies it might be advantageous to enhance some of their features through gene delivery strategies. Accordingly, we are interested in developing an efficient and safe methodology to genetically engineer human bone marrow MSC (BM MSC, enhancing their therapeutic efficacy in Regenerative Medicine. The plasmid DNA delivery was optimized using a cationic liposome-based reagent. Transfection efficiencies ranged from ~2% to ~35%, resulting from using a Lipid/DNA ratio of 1.25 with a transgene expression of 7 days. Importantly, the number of plasmid copies in different cell passages was quantified for the first time and ~20,000 plasmid copies/cell were obtained independently of cell passage. As transfected MSC have shown high viabilities (>90% and recoveries (>52% while maintaining their multipotency, this might be an advantageous transfection strategy when the goal is to express a therapeutic gene in a safe and transient way.

  7. The Rho GTPase Cdc42 regulates hair cell planar polarity and cellular patterning in the developing cochlea

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    Anna Kirjavainen

    2015-03-01

    Full Text Available Hair cells of the organ of Corti (OC of the cochlea exhibit distinct planar polarity, both at the tissue and cellular level. Planar polarity at tissue level is manifested as uniform orientation of the hair cell stereociliary bundles. Hair cell intrinsic polarity is defined as structural hair bundle asymmetry; positioning of the kinocilium/basal body complex at the vertex of the V-shaped bundle. Consistent with strong apical polarity, the hair cell apex displays prominent actin and microtubule cytoskeletons. The Rho GTPase Cdc42 regulates cytoskeletal dynamics and polarization of various cell types, and, thus, serves as a candidate regulator of hair cell polarity. We have here induced Cdc42 inactivation in the late-embryonic OC. We show the role of Cdc42 in the establishment of planar polarity of hair cells and in cellular patterning. Abnormal planar polarity was displayed as disturbances in hair bundle orientation and morphology and in kinocilium/basal body positioning. These defects were accompanied by a disorganized cell-surface microtubule network. Atypical protein kinase C (aPKC, a putative Cdc42 effector, colocalized with Cdc42 at the hair cell apex, and aPKC expression was altered upon Cdc42 depletion. Our data suggest that Cdc42 together with aPKC is part of the machinery establishing hair cell planar polarity and that Cdc42 acts on polarity through the cell-surface microtubule network. The data also suggest that defects in apical polarization are influenced by disturbed cellular patterning in the OC. In addition, our data demonstrates that Cdc42 is required for stereociliogenesis in the immature cochlea.

  8. Regulated genes in mesenchymal stem cells and gastriccancer

    Institute of Scientific and Technical Information of China (English)

    Shihori Tanabe; Kazuhiko Aoyagi; Hiroshi Yokozaki; Hiroki Sasaki

    2015-01-01

    AIM To investigate the genes regulated in mesenchymalstem cells (MSCs) and diffuse-type gastric cancer (GC),gene expression was analyzed.METHODS: Gene expression of MSCs and diffuse-typeGC cells were analyzed by microarray. Genes relatedto stem cells, cancer and the epithelial-mesenchymaltransition (EMT) were extracted from human genelists using Gene Ontology and reference information.Gene panels were generated, and messenger RNAgene expression in MSCs and diffuse-type GC cells wasanalyzed. Cluster analysis was performed using the NCSSsoftware.RESULTS: The gene expression of regulator of G-proteinsignaling 1 (RGS1) was up-regulated in diffuse-type GCcells compared with MSCs. A panel of stem-cell relatedgenes and genes involved in cancer or the EMT wereexamined. Stem-cell related genes, such as growtharrest-specific 6, musashi RNA-binding protein 2 andhairy and enhancer of split 1 (Drosophila), NOTCHfamily genes and Notch ligands, such as delta-like 1(Drosophila) and Jagged 2, were regulated.CONCLUSION: Expression of RGS1 is up-regulated,and genes related to stem cells and NOTCH signalingare altered in diffuse-type GC compared with MSCs.

  9. Simple system for evaluating retardation of liquid crystal cells using grating type liquid crystal polarization splitters

    Science.gov (United States)

    Honma, Michinori; Nose, Toshiaki

    2016-04-01

    We propose a unique optical system for measuring the retardation of birefringent films using a pair of liquid crystal (LC) gratings; that is, the examined birefringent films are inserted between two LC gratings. Because the LC grating functions as a polarization beam splitter for circularly polarized light, the proposed system is optically equivalent to the measurement system using a pair of two circular polarizers. First, the polarization splitting performance of the LC grating is discussed. It is found that a sufficiently high voltage (such that the retardation is less than a half wavelength) has to be applied for the almost pure circularly polarized diffracted light. Next, the measurement of the retardation of a homogeneous LC cell as an examined birefringent film was demonstrated using the proposed method. The proposed method is revealed to have the same measurement performance as that of the conventional method using a pair of linear polarizers and has an advantage that there is no need for the optic axis of the test birefringent specimen to be set at a specific angle.

  10. Polarized exocyst-mediated vesicle fusion directs intracellular lumenogenesis within the C. elegans excretory cell.

    Science.gov (United States)

    Armenti, Stephen T; Chan, Emily; Nance, Jeremy

    2014-10-01

    Lumenogenesis of small seamless tubes occurs through intracellular membrane growth and directed vesicle fusion events. Within the Caenorhabditis elegans excretory cell, which forms seamless intracellular tubes (canals) that mediate osmoregulation, lumens grow in length and diameter when vesicles fuse with the expanding lumenal surface. Here, we show that lumenal vesicle fusion depends on the small GTPase RAL-1, which localizes to vesicles and acts through the exocyst vesicle-tethering complex. Loss of either the exocyst or RAL-1 prevents excretory canal lumen extension. Within the excretory canal and other polarized cells, the exocyst co-localizes with the PAR polarity proteins PAR-3, PAR-6 and PKC-3. Using early embryonic cells to determine the functional relationships between the exocyst and PAR proteins, we show that RAL-1 recruits the exocyst to the membrane, while PAR proteins concentrate membrane-localized exocyst proteins to a polarized domain. These findings reveal that RAL-1 and the exocyst direct the polarized vesicle fusion events required for intracellular lumenogenesis of the excretory cell, suggesting mechanistic similarities in the formation of topologically distinct multicellular and intracellular lumens. PMID:25102190

  11. Kv7.1 surface expression is regulated by epithelial cell polarization

    DEFF Research Database (Denmark)

    Andersen, Martin N; Olesen, Søren-Peter; Rasmussen, Hanne Borger

    2011-01-01

    deafness, and several mutations have been described as trafficking mutations. To learn more about the basic mechanisms that regulate K(V)7.1 surface expression, we have investigated the trafficking of K(V)7.1 during the polarization process of the epithelial cell line Madin-Darby Canine Kidney (MDCK) using...

  12. On temperature variations during 3He Polarization experiments in Pomeranchuk cells

    DEFF Research Database (Denmark)

    Geng, Q.; Rasmussen, Finn Berg

    1984-01-01

    Simple model calculations have been performed in relation to temperature changes in decompression experiments with Pomeranchuk cells, aiming at the production of spin polarized liquid **3He. Comparison with reported experiments indicates that thermal contact with the surroundings is too strong...

  13. Comparison study of distinguishing cancerous and normal prostate epithelial cells by confocal and polarization diffraction imaging.

    Science.gov (United States)

    Jiang, Wenhuan; Lu, Jun Qing; Yang, Li V; Sa, Yu; Feng, Yuanming; Ding, Junhua; Hu, Xin-Hua

    2016-07-01

    Accurate classification of malignant cells from benign ones can significantly enhance cancer diagnosis and prognosis by detection of circulating tumor cells (CTCs). We have investigated two approaches of quantitative morphology and polarization diffraction imaging on two prostate cell types to evaluate their feasibility as single-cell assay methods toward CTC detection after cell enrichment. The two cell types have been measured by a confocal imaging method to obtain their three-dimensional morphology parameters and by a polarization diffraction imaging flow cytometry (p-DIFC) method to obtain image texture parameters. The support vector machine algorithm was applied to examine the accuracy of cell classification with the morphology and diffraction image parameters. Despite larger mean values of cell and nuclear sizes of the cancerous prostate cells than the normal ones, it has been shown that the morphologic parameters cannot serve as effective classifiers. In contrast, accurate classification of the two prostate cell types can be achieved with high classification accuracies on measured data acquired separately in three measurements. These results provide strong evidence that the p-DIFC method has the potential to yield morphology-related “fingerprints” for accurate and label-free classification of the two prostate cell types. PMID:26616011

  14. Influenza H5N1 virus infection of polarized human alveolar epithelial cells and lung microvascular endothelial cells

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    Yuen Kit M

    2009-10-01

    Full Text Available Abstract Background Highly pathogenic avian influenza (HPAI H5N1 virus is entrenched in poultry in Asia and Africa and continues to infect humans zoonotically causing acute respiratory disease syndrome and death. There is evidence that the virus may sometimes spread beyond respiratory tract to cause disseminated infection. The primary target cell for HPAI H5N1 virus in human lung is the alveolar epithelial cell. Alveolar epithelium and its adjacent lung microvascular endothelium form host barriers to the initiation of infection and dissemination of influenza H5N1 infection in humans. These are polarized cells and the polarity of influenza virus entry and egress as well as the secretion of cytokines and chemokines from the virus infected cells are likely to be central to the pathogenesis of human H5N1 disease. Aim To study influenza A (H5N1 virus replication and host innate immune responses in polarized primary human alveolar epithelial cells and lung microvascular endothelial cells and its relevance to the pathogenesis of human H5N1 disease. Methods We use an in vitro model of polarized primary human alveolar epithelial cells and lung microvascular endothelial cells grown in transwell culture inserts to compare infection with influenza A subtype H1N1 and H5N1 viruses via the apical or basolateral surfaces. Results We demonstrate that both influenza H1N1 and H5N1 viruses efficiently infect alveolar epithelial cells from both apical and basolateral surface of the epithelium but release of newly formed virus is mainly from the apical side of the epithelium. In contrast, influenza H5N1 virus, but not H1N1 virus, efficiently infected polarized microvascular endothelial cells from both apical and basolateral aspects. This provides a mechanistic explanation for how H5N1 virus may infect the lung from systemic circulation. Epidemiological evidence has implicated ingestion of virus-contaminated foods as the source of infection in some instances and our

  15. DNA-mediated gene transfer into ataxia-telangiectasia cells

    International Nuclear Information System (INIS)

    The complete description of the genetic lesion(s) underlying the AT mutation might, therefore, highlight not only a DNA-repair pathwa, but also an important aspect of the physiology of lymphocytes. DNA-mediated gene transfer into eukaryotic cells has proved a powerful tool for the molecular cloning of certain mammalian genes. The possibility to clone a given gene using this technology depends, basically, on the availability of a selectable marker associated with the expression of the transfected gene in the recipient cell. Recently, a human DNA repair gene has been cloned in CHO mutant cells by taking advantage of the increased resistance to ultraviolet radiation of the transformants. As a preliminary step toward the molecular cloning of the AT gene(s), the authors have attempted to confer radioresistance to AT cells by transfection with normal human DNA

  16. Gene expression profile of renal cell carcinoma clear cell type

    Directory of Open Access Journals (Sweden)

    Marcos F. Dall’Oglio

    2010-08-01

    Full Text Available PURPOSE: The determination of prognosis in patients with renal cell carcinoma (RCC is based, classically, on stage and histopathological aspects. The metastatic disease develops in one third of patients after surgery, even in localized tumors. There are few options for treating those patients, and even the new target designed drugs have shown low rates of success in controlling disease progression. Few studies used high throughput genomic analysis in renal cell carcinoma for determination of prognosis. This study is focused on the identification of gene expression signatures in tissues of low-risk, high-risk and metastatic RCC clear cell type (RCC-CCT. MATERIALS AND METHODS: We analyzed the expression of approximately 55,000 distinct transcripts using the Whole Genome microarray platform hybridized with RNA extracted from 19 patients submitted to surgery to treat RCC-CCT with different clinical outcomes. They were divided into three groups (1 low risk, characterized by pT1, Fuhrman grade 1 or 2, no microvascular invasion RCC; (2 high risk, pT2-3, Fuhrman grade 3 or 4 with, necrosis and microvascular invasion present and (3 metastatic RCC-CCT. Normal renal tissue was used as control. RESULTS: After comparison of differentially expressed genes among low-risk, high-risk and metastatic groups, we identified a group of common genes characterizing metastatic disease. Among them Interleukin-8 and Heat shock protein 70 were over-expressed in metastasis and validated by real-time polymerase chain reaction. CONCLUSION: These findings can be used as a starting point to generate molecular markers of RCC-CCT as well as a target for the development of innovative therapies.

  17. Can Ferroelectric Polarization Explain the High Performance of Hybrid Halide Perovskite Solar Cells?

    OpenAIRE

    Sherkar, Tejas; Koster, L Jan Anton

    2016-01-01

    The power conversion efficiency of photovoltaic cells based on the use of hybrid halide perovskites, CH3NH3PbX3 (X = Cl, Br, I), now exceeds 20%. Recently, it was suggested that this high performance originates from the presence of ferroelectricity in the perovskite, which is hypothesized to lower charge recombination in the device. Here, we investigate and quantify the influence of mesoscale ferroelectric polarization on the device performance of perovskite solar cells. We implement a 3D dri...

  18. Dchs1–Fat4 regulation of polarized cell behaviours during skeletal morphogenesis

    OpenAIRE

    Mao, Yaopan; Kuta, Anna; Crespo-Enriquez, Ivan; Whiting, Danielle; Martin, Tina; Mulvaney, Joanna; Irvine, Kenneth D.; Francis-West, Philippa

    2016-01-01

    Skeletal shape varies widely across species as adaptation to specialized modes of feeding and locomotion, but how skeletal shape is established is unknown. An example of extreme diversity in the shape of a skeletal structure can be seen in the sternum, which varies considerably across species. Here we show that the Dchs1-Fat4 planar cell polarity pathway controls cell orientation in the early skeletal condensation to define the shape and relative dimensions of the mouse sternum. These changes...

  19. Basolateral invasion and trafficking of Campylobacter jejuni in polarized epithelial cells.

    Directory of Open Access Journals (Sweden)

    Lieneke I Bouwman

    Full Text Available Campylobacter jejuni is a major cause of bacterial diarrheal disease. Most enteropathogenic bacteria including C. jejuni can invade cultured eukaryotic cells via an actin- and/or microtubule-dependent and an energy-consuming uptake process. Recently, we identified a novel highly efficient C. jejuni invasion pathway that involves bacterial migration into the subcellular space of non-polarized epithelial cells (termed subvasion followed by invasion from the cell basis. Here we report cellular requirements of this entry mechanism and the subsequent intracellular trafficking route of C. jejuni in polarized islands of Caco-2 intestinal epithelial cells. Advanced microscopy on infected cells revealed that C. jejuni invades the polarized intestinal cells via the subcellular invasion pathway. Remarkably, invasion was not blocked by the inhibitors of microtubule dynamics colchicine or paclitaxel, and was even enhanced after disruption of host cell actin filaments by cytochalasin D. Invasion also continued after dinitrophenol-induced cellular depletion of ATP, whereas this compound effectively inhibited the uptake of invasive Escherichia coli. Confocal microscopy demonstrated that intracellular C. jejuni resided in membrane-bound CD63-positive cellular compartments for up to 24 h. Establishment of a novel luciferase reporter-based bacterial viability assay, developed to overcome the limitations of the classical bacterial recovery assay, demonstrated that a subset of C. jejuni survived intracellularly for up to 48 h. Taken together, our results indicate that C. jejuni is able to actively invade polarized intestinal epithelial cells via a novel actin- and microtubule-independent mechanism and remains metabolically active in the intracellular niche for up to 48 hours.

  20. Bifidobacterium bifidum Actively Changes the Gene Expression Profile Induced by Lactobacillus acidophilus in Murine Dendritic Cells

    DEFF Research Database (Denmark)

    Weiss, Gudrun Margarethe; Rasmussen, Simon; Fink, Lisbeth Nielsen; Jarmer, Hanne Østergaard; Nielsen, Birgit Margrethe Nøhr; Frøkiær, Hanne

    2010-01-01

    cytokine IL-12 in DC, whereas bifidobacteria do not induce IL-12 but inhibit the IL-12 production induced by lactobacilli. In the present study, genome-wide microarrays were used to investigate the gene expression pattern of murine DC stimulated with Lactobacillus acidophilus NCFM and Bifidobacterium......Dendritic cells (DC) play a pivotal regulatory role in activation of both the innate as well as the adaptive immune system by responding to environmental microorganisms. We have previously shown that Lactobacillus acidophilus induces a strong production of the pro-inflammatory and Th1 polarizing...... bifidum Z9. L. acidophilus NCFM strongly induced expression of interferon (IFN)-beta, other virus defence genes, and cytokine and chemokine genes related to the innate and the adaptive immune response. By contrast, B. bifidum Z9 up-regulated genes encoding cytokines and chemokines related to the innate...

  1. Solid-State NMR on bacterial cells: selective cell wall signal enhancement and resolution improvement using dynamic nuclear polarization

    International Nuclear Information System (INIS)

    Dynamic nuclear polarization (DNP) enhanced solid-state nuclear magnetic resonance (NMR) has recently emerged as a powerful technique for the study of material surfaces. In this study, we demonstrate its potential to investigate cell surface in intact cells. Using Bacillus subtilis bacterial cells as an example, it is shown that the polarizing agent 1-(TEMPO-4-oxy)-3-(TEMPO-4-amino)propan-2-ol (TOTAPOL) has a strong binding affinity to cell wall polymers (peptidoglycan). This particular interaction is thoroughly investigated with a systematic study on extracted cell wall materials, disrupted cells, and entire cells, which proved that TOTAPOL is mainly accumulating in the cell wall. This property is used on one hand to selectively enhance or suppress cell wall signals by controlling radical concentrations and on the other hand to improve spectral resolution by means of a difference spectrum. Comparing DNP-enhanced and conventional solid-state NMR, an absolute sensitivity ratio of 24 was obtained on the entire cell sample. This important increase in sensitivity together with the possibility of enhancing specifically cell wall signals and improving resolution really opens new avenues for the use of DNP-enhanced solid-state NMR as an on-cell investigation tool. (authors)

  2. Progress in gene transfer by germ cells in mammals

    Institute of Scientific and Technical Information of China (English)

    2008-01-01

    Use of germ cells as vectors for transgenesis in mammals has been well developed and offers exciting prospects for experimental and applied biology, agricultural and medical sciences.Such approach is referred to as either male germ cell mediated gene transfer (MGCMGT)or female germ cell mediated gene transfer(FGCMGT)technique.Sperm-mediated gene transfer (SMGT),including its alternative method,testis-mediated gene transfer(TMGT),becomes an established and reliable method for transgenesis.They have been extensively used for producing transgenic animals.The newly developed approach of FGCMGT,ovary-mediated gene transfer(OMGT) is also a novel and useful tool for efficient transgenesis.This review highlights an overview of the recent progress in germ cell mediated gene transfer techniques,methods developed and mechanisms of nucleic acid uptake by germ cells.

  3. Polarized trafficking of the sorting receptor SorLA in neurons and MDCK cells.

    Science.gov (United States)

    Klinger, Stine C; Højland, Anne; Jain, Shweta; Kjolby, Mads; Madsen, Peder; Svendsen, Anna Dorst; Olivecrona, Gunilla; Bonifacino, Juan S; Nielsen, Morten S

    2016-07-01

    The sorting receptor SorLA is highly expressed in neurons and is also found in other polarized cells. The receptor has been reported to participate in the trafficking of several ligands, some of which are linked to human diseases, including the amyloid precursor protein, TrkB, and Lipoprotein Lipase (LpL). Despite this, only the trafficking in nonpolarized cells has been described so far. Due to the many differences between polarized and nonpolarized cells, we examined the localization and trafficking of SorLA in epithelial Madin-Darby canine kidney (MDCK) cells and rat hippocampal neurons. We show that SorLA is mainly found in sorting endosomes and on the basolateral surface of MDCK cells and in the somatodendritic domain of neurons. This polarized distribution of SorLA respectively depends on an acidic cluster and an extended version of this cluster and involves the cellular adaptor complex AP-1. Furthermore, we show that SorLA can mediate transcytosis across a tight cell layer. PMID:27192064

  4. Macrophage polarization reflects T cell composition of tumor microenvironment in pediatric classical Hodgkin lymphoma and has impact on survival.

    Directory of Open Access Journals (Sweden)

    Mário H M Barros

    Full Text Available Macrophages have been implicated in the pathogenesis of classical Hodgkin lymphoma (cHL and have been suggested to have a negative impact on outcome. Most studies addressing the role of macrophages in cHL have relied on identification of macrophages by generic macrophage antigens, e.g., CD68. We have therefore conducted an in situ analysis of macrophage polarization in a series of 100 pediatric cHL (pcHL cases using double staining immunohistochemistry, combining CD68 or CD163 with pSTAT1 (M1-like or CMAF (M2-like. M1- or M2-polarised microenvironment was defined by an excess of one population over the other (>1.5. Expression of STAT1 and LYZ genes was also evaluated by RT-qPCR. Patients 1.5 was associated with higher numbers of CD68+pSTAT1+ (P=0.025 and CD163+pSTAT1+ macrophages (P 1.5 was associated with better OS (P= 0.037. In conclusion, macrophage polarization in pcHL correlates with prevalent local T cell response and may be influenced by the EBV-status of neoplastic cells. Besides, M1-like and M2-like macrophages displayed differential effects on outcome in pcHL.

  5. Gene expression profiles of hepatic cell-type specific marker genes in progression of liver fibrosis

    Institute of Scientific and Technical Information of China (English)

    Yoshiyuki Takahara; Mitsuo Takahashi; Hiroki Wagatsuma; Fumihiko Yokoya; Qing-Wei Zhang; Mutsuyo Yamaguchi; Hiroyuki Aburatani; Norifumi Kawada

    2006-01-01

    AIM: To determine the gene expression profile data for the whole liver during development of dimethylnitrosamine (DMN)-induced hepatic fibrosis.METHODS: Marker genes were identified for different types of hepatic cells, including hepatic stellate cells (HSCs), Kupffer cells (including other inflammatory cells),and hepatocytes, using independent temporal DNA microarray data obtained from isolated hepatic cells.RESULTS: The cell-type analysis of gene expression gave several key results and led to formation of three hypotheses: (1) changes in the expression of HSCspecific marker genes during fibrosis were similar to gene expression data in in vitro cultured HSCs, suggesting a major role of the self-activating characteristics of HSCs in formation of fibrosis; (2) expression of mast cell-specific marker genes reached a peak during liver fibrosis,suggesting a possible role of mast cells in formation of fibrosis; and (3) abnormal expression of hepatocytespecific marker genes was found across several metabolic pathways during fibrosis, including sulfur-containing amino acid metabolism, fatty acid metabolism, and drug metabolism, suggesting a mechanistic relationship between these abnormalities and symptoms of liver fibrosis.CONCLUSION: Analysis of marker genes for specific hepatic cell types can identify the key aspects of fibrogenesis. Sequential activation of inflammatory cells and the self-supporting properties of HSCs play an important role in development of fibrosis.

  6. Participation of the cell polarity protein PALS1 to T-cell receptor-mediated NF-κB activation.

    Directory of Open Access Journals (Sweden)

    Gabrielle Carvalho

    Full Text Available BACKGROUND: Beside their established function in shaping cell architecture, some cell polarity proteins were proposed to participate to lymphocyte migration, homing, scanning, as well as activation following antigen receptor stimulation. Although PALS1 is a central component of the cell polarity network, its expression and function in lymphocytes remains unknown. Here we investigated whether PALS1 is present in T cells and whether it contributes to T Cell-Receptor (TCR-mediated activation. METHODOLOGY/PRINCIPAL FINDINGS: By combining RT-PCR and immunoblot assays, we found that PALS1 is constitutively expressed in human T lymphocytes as well as in Jurkat T cells. siRNA-based knockdown of PALS1 hampered TCR-induced activation and optimal proliferation of lymphocyte. We further provide evidence that PALS1 depletion selectively hindered TCR-driven activation of the transcription factor NF-κB. CONCLUSIONS: The cell polarity protein PALS1 is expressed in T lymphocytes and participates to the optimal activation of NF-κB following TCR stimulation.

  7. Constrained Nonlinear and Mixed Effects Differential Equation Models for Dynamic Cell Polarity Signaling

    OpenAIRE

    Xiao, Zhen; Brunel, Nicolas; Yang, Zhenbiao; Cui, Xinping

    2016-01-01

    The key of tip growth in eukaryotes is the polarized distribution on plasma membrane of a particle named ROP1. This distribution is the result of a positive feedback loop, whose mechanism can be described by a Differential Equation parametrized by two meaningful parameters kpf and knf . We introduce a mechanistic Integro-Differential Equation (IDE) derived from a spatiotemporal model of cell polarity and we show how this model can be fitted to real data, i.e., ROP1 intensities measured on pol...

  8. ERG gene rearrangements are common in prostatic small cell carcinomas

    OpenAIRE

    Lotan, Tamara L.; Gupta, Nilesh S; Wang, Wenle; Toubaji, Antoun; Haffner, Michael C; Chaux, Alcides; Hicks, Jessica L.; Meeker, Alan K.; Bieberich, Charles J.; De Marzo, Angelo M.; Epstein, Jonathan I; Netto, George J.

    2011-01-01

    Small cell carcinoma of the prostate is a rare subtype with an aggressive clinical course. Despite the frequent occurrence of ERG gene rearrangements in acinar carcinoma, the incidence of these rearrangements in prostatic small cell carcinoma is unclear. In addition, molecular markers to distinguish prostatic small cell carcinomas from lung and bladder small cell carcinomas may be clinically useful. We examined the occurrence of ERG gene rearrangements by fluorescence in situ hybridization in...

  9. Trafficking through COPII stabilises cell polarity and drives secretion during Drosophila epidermal differentiation.

    Directory of Open Access Journals (Sweden)

    Michaela Norum

    Full Text Available BACKGROUND: The differentiation of an extracellular matrix (ECM at the apical side of epithelial cells implies massive polarised secretion and membrane trafficking. An epithelial cell is hence engaged in coordinating secretion and cell polarity for a correct and efficient ECM formation. PRINCIPAL FINDINGS: We are studying the molecular mechanisms that Drosophila tracheal and epidermal cells deploy to form their specific apical ECM during differentiation. In this work we demonstrate that the two genetically identified factors haunted and ghost are essential for polarity maintenance, membrane topology as well as for secretion of the tracheal luminal matrix and the cuticle. We show that they code for the Drosophila COPII vesicle-coating components Sec23 and Sec24, respectively, that organise vesicle transport from the ER to the Golgi apparatus. CONCLUSION: Taken together, epithelial differentiation during Drosophila embryogenesis is a concerted action of ECM formation, plasma membrane remodelling and maintenance of cell polarity that all three rely mainly, if not absolutely, on the canonical secretory pathway from the ER over the Golgi apparatus to the plasma membrane. Our results indicate that COPII vesicles constitute a central hub for these processes.

  10. Recording of polarization holograms in a liquid crystal cell with a photosensitive chalcogenide orientation layer [Invited].

    Science.gov (United States)

    Sheremet, Nina; Kurioz, Yuriy; Slyusarenko, Kostyantyn; Trunov, Michael; Reznikov, Yuriy

    2013-08-01

    Polarization gratings have been recorded in a combined liquid crystal (LC) cell made of a substrate covered with a photosensitive chalcogenide orientation layer and a reference substrate covered with a rubbed polyimide film. The gratings are formed due to the spatially modulated light-induced easy orientation axis on the chalcogenide surface recorded by two beams with opposite circular polarizations. The gratings are permanent, but they can be erased by one of the recording beams and re-recorded. The diffraction intensity of the circularly polarized light is achromatic and does not depend on the birefringence of the LC. The diffraction efficiency of the grating is of the order of a few percents. Application of an ac field causes a strong increase of the diffraction efficiency up to 45%. PMID:23913086

  11. Cell polarity and patterning by PIN trafficking through early endosomal compartments in Arabidopsis thaliana.

    Directory of Open Access Journals (Sweden)

    Hirokazu Tanaka

    2013-05-01

    Full Text Available PIN-FORMED (PIN proteins localize asymmetrically at the plasma membrane and mediate intercellular polar transport of the plant hormone auxin that is crucial for a multitude of developmental processes in plants. PIN localization is under extensive control by environmental or developmental cues, but mechanisms regulating PIN localization are not fully understood. Here we show that early endosomal components ARF GEF BEN1 and newly identified Sec1/Munc18 family protein BEN2 are involved in distinct steps of early endosomal trafficking. BEN1 and BEN2 are collectively required for polar PIN localization, for their dynamic repolarization, and consequently for auxin activity gradient formation and auxin-related developmental processes including embryonic patterning, organogenesis, and vasculature venation patterning. These results show that early endosomal trafficking is crucial for cell polarity and auxin-dependent regulation of plant architecture.

  12. Microarray gene expression profiling and analysis in renal cell carcinoma

    Directory of Open Access Journals (Sweden)

    Sadhukhan Provash

    2004-06-01

    Full Text Available Abstract Background Renal cell carcinoma (RCC is the most common cancer in adult kidney. The accuracy of current diagnosis and prognosis of the disease and the effectiveness of the treatment for the disease are limited by the poor understanding of the disease at the molecular level. To better understand the genetics and biology of RCC, we profiled the expression of 7,129 genes in both clear cell RCC tissue and cell lines using oligonucleotide arrays. Methods Total RNAs isolated from renal cell tumors, adjacent normal tissue and metastatic RCC cell lines were hybridized to affymatrix HuFL oligonucleotide arrays. Genes were categorized into different functional groups based on the description of the Gene Ontology Consortium and analyzed based on the gene expression levels. Gene expression profiles of the tissue and cell line samples were visualized and classified by singular value decomposition. Reverse transcription polymerase chain reaction was performed to confirm the expression alterations of selected genes in RCC. Results Selected genes were annotated based on biological processes and clustered into functional groups. The expression levels of genes in each group were also analyzed. Seventy-four commonly differentially expressed genes with more than five-fold changes in RCC tissues were identified. The expression alterations of selected genes from these seventy-four genes were further verified using reverse transcription polymerase chain reaction (RT-PCR. Detailed comparison of gene expression patterns in RCC tissue and RCC cell lines shows significant differences between the two types of samples, but many important expression patterns were preserved. Conclusions This is one of the initial studies that examine the functional ontology of a large number of genes in RCC. Extensive annotation, clustering and analysis of a large number of genes based on the gene functional ontology revealed many interesting gene expression patterns in RCC. Most

  13. Polarization of migrating monocytic cells is independent of PI 3-kinase activity.

    Directory of Open Access Journals (Sweden)

    Silvia Volpe

    Full Text Available BACKGROUND: Migration of mammalian cells is a complex cell type and environment specific process. Migrating hematopoietic cells assume a rapid amoeboid like movement when exposed to gradients of chemoattractants. The underlying signaling mechanisms remain controversial with respect to localization and distribution of chemotactic receptors within the plasma membrane and the role of PI 3-kinase activity in cell polarization. METHODOLOGY/PRINCIPAL FINDINGS: We present a novel model for the investigation of human leukocyte migration. Monocytic THP-1 cells transfected with the alpha(2A-adrenoceptor (alpha(2AAR display comparable signal transduction responses, such as calcium mobilization, MAP-kinase activation and chemotaxis, to the noradrenaline homologue UK 14'304 as when stimulated with CCL2, which binds to the endogenous chemokine receptor CCR2. Time-lapse video microscopy reveals that chemotactic receptors remain evenly distributed over the plasma membrane and that their internalization is not required for migration. Measurements of intramolecular fluorescence resonance energy transfer (FRET of alpha(2AAR-YFP/CFP suggest a uniform activation of the receptors over the entire plasma membrane. Nevertheless, PI 3-kinase activation is confined to the leading edge. When reverting the gradient of chemoattractant by moving the dispensing micropipette, polarized monocytes--in contrast to neutrophils--rapidly flip their polarization axis by developing a new leading edge at the previous posterior side. Flipping of the polarization axis is accompanied by re-localization of PI-3-kinase activity to the new leading edge. However, reversal of the polarization axis occurs in the absence of PI 3-kinase activation. CONCLUSIONS/SIGNIFICANCE: Accumulation and internalization of chemotactic receptors at the leading edge is dispensable for cell migration. Furthermore, uniformly distributed receptors allow the cells to rapidly reorient and adapt to changes in the

  14. Genes that confer the identity of the renin cell.

    Science.gov (United States)

    Brunskill, Eric W; Sequeira-Lopez, Maria Luisa S; Pentz, Ellen S; Lin, Eugene; Yu, Jing; Aronow, Bruce J; Potter, S Steven; Gomez, R Ariel

    2011-12-01

    Renin-expressing cells modulate BP, fluid-electrolyte homeostasis, and kidney development, but remarkably little is known regarding the genetic regulatory network that governs the identity of these cells. Here we compared the gene expression profiles of renin cells with most cells in the kidney at various stages of development as well as after a physiologic challenge known to induce the transformation of arteriolar smooth muscle cells into renin-expressing cells. At all stages, renin cells expressed a distinct set of genes characteristic of the renin phenotype, which was vastly different from other cell types in the kidney. For example, cells programmed to exhibit the renin phenotype expressed Akr1b7, and maturing cells expressed angiogenic factors necessary for the development of the kidney vasculature and RGS (regulator of G-protein signaling) genes, suggesting a potential relationship between renin cells and pericytes. Contrary to the plasticity of arteriolar smooth muscle cells upstream from the glomerulus, which can transiently acquire the embryonic phenotype in the adult under physiologic stress, the adult juxtaglomerular cell always possessed characteristics of both smooth muscle and renin cells. Taken together, these results identify the gene expression profile of renin-expressing cells at various stages of maturity, and suggest that juxtaglomerular cells maintain properties of both smooth muscle and renin-expressing cells, likely to allow the rapid control of body fluids and BP through both contractile and endocrine functions. PMID:22034642

  15. Genetic Variation and Divergence of Genes Involved in Leaf Adaxial-Abaxial Polarity Establishment in Brassica rapa.

    Science.gov (United States)

    Liang, Jianli; Liu, Bo; Wu, Jian; Cheng, Feng; Wang, Xiaowu

    2016-01-01

    Alterations in leaf adaxial-abaxial (ad-ab) polarity are one of the main factors that influence leaf curvature. In Chinese cabbage, leaf incurvature is an essential prerequisite to the formation of a leafy head. Identifying ad-ab patterning genes and investigating their genetic variation may facilitate elucidation of the mechanisms underlying leaf incurvature during head formation. Comparative genomic analysis of 45 leaf ad-ab patterning genes in Brassica rapa based on 26 homologs of Arabidopsis thaliana indicated that these genes underwent expansion and were retained after whole genome triplication (WGT). We also assessed the nucleotide diversity and selection footprints of these 45 genes in a collection of 94 Brassica rapa accessions that were composed of heading and non-heading morphotypes. Six of the 45 genes showed significant negative Tajima's D indices and nucleotide diversity reduction in heading accessions compared to those in non-heading accessions, indicating that they underwent purifying selection. Further testing of the BrARF3.1 gene, which was one of the selection signals from a larger collection, confirmed that purifying selection did occur. Our results provide genetic evidence that ad-ab patterning genes are involved in leaf incurvature, which is associated with formation of a leafy head, as well as promote an understanding of the genetic mechanism underlying leafy head formation in Chinese cabbage. PMID:26904064

  16. Genetic Variation and Divergence of Genes Involved in Leaf Adaxial-abaxial Polarity Establishment in Brassica rapa

    Directory of Open Access Journals (Sweden)

    Jianli eLiang

    2016-02-01

    Full Text Available Alterations in leaf adaxial–abaxial (ad-ab polarity are one of the main factors that are responsible for leaf curvature. In Chinese cabbage, to form a leafy head, leaf incurvature is an essential prerequisite. Identifying ad-ab patterning genes and investigating its genetic variations will facilitate in elucidating the mechanism underlying leaf incurvature during head formation. In the present study we conducted comparative genomic analysis of the identification of 45 leaf ad-ab patterning genes in Brassica rapa based on 26 homologs in Arabidopsis thaliana, indicating that these genes underwent expansion and were retained after whole genome triplication (WGT. We also assessed the nucleotide diversity and selection footprints of these 45 genes in a collection of 94 Brassica rapa accessions that were composed of heading and non-heading morphotypes. Six of the 45 genes showed significant negative Tajima’s D indices and nucleotide diversity reduction in heading accessions compared to that in non-heading accessions, indicating that these underwent purifying selection. Further testing of the BrARF3.1 gene, which was one of the selection signals from a larger collection, confirmed that purifying selection did occur. Our results provide genetic evidence that ad-ab patterning genes are involved in leaf incurvature that is associated in the formation of a leafy head, as well as promote an understanding of the genetic mechanism underlying leafy head formation in Chinese cabbage.

  17. Integrative characterization of germ cell-specific genes from mouse spermatocyte UniGene library

    Directory of Open Access Journals (Sweden)

    Eddy Edward M

    2007-07-01

    Full Text Available Abstract Background The primary regulator of spermatogenesis, a highly ordered and tightly regulated developmental process, is an intrinsic genetic program involving male germ cell-specific genes. Results We analyzed the mouse spermatocyte UniGene library containing 2155 gene-oriented transcript clusters. We predict that 11% of these genes are testis-specific and systematically identified 24 authentic genes specifically and abundantly expressed in the testis via in silico and in vitro approaches. Northern blot analysis disclosed various transcript characteristics, such as expression level, size and the presence of isoform. Expression analysis revealed developmentally regulated and stage-specific expression patterns in all of the genes. We further analyzed the genes at the protein and cellular levels. Transfection assays performed using GC-2 cells provided information on the cellular characteristics of the gene products. In addition, antibodies were generated against proteins encoded by some of the genes to facilitate their identification and characterization in spermatogenic cells and sperm. Our data suggest that a number of the gene products are implicated in transcriptional regulation, nuclear integrity, sperm structure and motility, and fertilization. In particular, we found for the first time that Mm.333010, predicted to contain a trypsin-like serine protease domain, is a sperm acrosomal protein. Conclusion We identify 24 authentic genes with spermatogenic cell-specific expression, and provide comprehensive information about the genes. Our findings establish a new basis for future investigation into molecular mechanisms underlying male reproduction.

  18. Restoration of cell polarity and bile excretion function of hepatocytes in sandwich-culture

    Institute of Scientific and Technical Information of China (English)

    ZHANG Xian-jie; WANG Ying; SUN Jia-bang; SONG Mao-min; QIAO Xin

    2007-01-01

    Objective:To investigate the nature of the restoration of cell polarity and bile excretion function in Sandwich-cultured hepatocytes.Methods:Freshly isolated hepatocytes from male Sprague-Dawley rats were cultured in a double layer collagen gel Sandwich configuration.Morphological changes were observed under a inverted microscope.The domain specific membrane associated protein DPP Ⅳ was tested by immunofluorescenee,and the bile excretion function was determined by using fluorescein diacetate.Hepatocytes cultured on a single layer of collagen gel were taken as control.Results:Adult rat hepatocytes cultured in a double layer collagen gel sandwich configuration regained its morphological and functional polarity and maintained polygonal morphology for at least 4 weeks.Immunofluorescence studies USing antibodies against DPP Ⅳ showed polarity restoration as early as 48 h.After cultured in the double layer collagen gel Sandwich configuration for 96 h the hepatocytes began to excrete bile;while hepatocytes cultured on a single layer collagen gel had no bile excretion.Conclusion:Hepatocytes cultured in a double layer collagen gel Sandwich configuration are able to regain their morphological and functional polarity givan certain conditions.Hepaotcyte culture is a useful tool for the study of polarity restoration.

  19. Mesenchymal stem cells alleviate experimental asthma by inducing polarization of alveolar macrophages.

    Science.gov (United States)

    Song, Xiaolian; Xie, Shuanshuan; Lu, Kun; Wang, Changhui

    2015-04-01

    The reparative and immunoregulatory properties of mesenchymal stromal cells (MSCs) have made them attractive candidates for cellular therapy. However, the underlying mechanism of the effects of transplanted MSCs on allergic asthma remains elusive. Here, we show that administration of MSCs isolated from human bone marrow provoked a pronounced polarization in alveolar macrophages to M2 subtypes, rather than induced an increase in the total macrophage number, and efficiently inhibited hallmark features of asthma, including airway hyperresponsiveness and eosinophilic accumulation. Moreover, transforming growth factor beta (TGF-β) signaling pathway appeared to mediate the effects of MSCs on macrophage polarization and subsequently the inhibition of hallmark features of asthma. Inhibition of TGF-β signaling was sufficient to inhibit the macrophage polarization in response to MSCs and consequently reserved the inhibitory effects of macrophage polarization on hallmark features of asthma. Collectively, our data demonstrate that human MSCs have immunosuppressive activity on asthma, which is mediated by TGF-β-signaling-dependent alveolar macrophage polarization. PMID:24958014

  20. Bifidobacterium bifidum actively changes the gene expression profile induced by Lactobacillus acidophilus in murine dendritic cells.

    Science.gov (United States)

    Weiss, Gudrun; Rasmussen, Simon; Nielsen Fink, Lisbeth; Jarmer, Hanne; Nøhr Nielsen, Birgit; Frøkiaer, Hanne

    2010-01-01

    Dendritic cells (DC) play a pivotal regulatory role in activation of both the innate as well as the adaptive immune system by responding to environmental microorganisms. We have previously shown that Lactobacillus acidophilus induces a strong production of the pro-inflammatory and Th1 polarizing cytokine IL-12 in DC, whereas bifidobacteria do not induce IL-12 but inhibit the IL-12 production induced by lactobacilli. In the present study, genome-wide microarrays were used to investigate the gene expression pattern of murine DC stimulated with Lactobacillus acidophilus NCFM and Bifidobacterium bifidum Z9. L. acidophilus NCFM strongly induced expression of interferon (IFN)-beta, other virus defence genes, and cytokine and chemokine genes related to the innate and the adaptive immune response. By contrast, B. bifidum Z9 up-regulated genes encoding cytokines and chemokines related to the innate immune response. Moreover, B. bifidum Z9 inhibited the expression of the Th1-promoting genes induced by L. acidophilus NCFM and had an additive effect on genes of the innate immune response and Th2 skewing genes. The gene encoding Jun dimerization protein 2 (JDP2), a transcription factor regulating the activation of JNK, was one of the few genes only induced by B. bifidum Z9. Neutralization of IFN-beta abrogated L. acidophilus NCFM-induced expression of Th1-skewing genes, and blocking of the JNK pathway completely inhibited the expression of IFN-beta. Our results indicate that B. bifidum Z9 actively inhibits the expression of genes related to the adaptive immune system in murine dendritic cells and that JPD2 via blocking of IFN-beta plays a central role in this regulatory mechanism. PMID:20548777

  1. Polarization independent beam fanning using a multi-domain liquid crystal cell.

    Science.gov (United States)

    Ren, Hongwen; Wu, Shin-Tson

    2009-07-01

    Polarization independent beam fanning using a multi-domain liquid crystal (LC) cell is demonstrated experimentally. In the neighboring domains, the LC directors are aligned in orthogonal directions. To prove concepts, two hybrid-aligned LC cells with four and six domains were fabricated. Applying a voltage across the LC layer will change the phase difference between the neighboring domains. When the phase difference is 2mpi (m is an integer), the LC cell will not disturb the incident beam. However, if the phase shift is (2m + 1)pi, the outgoing beam will fan out into several beams; the number of fanout beams is equal to the domain number. PMID:19582068

  2. Double suicide genes selectively kill human umbilical vein endothelial cells

    OpenAIRE

    Liu Lunxu; Wang Yanping; Mei Longyong; Jia Weiguo; Che Guowei

    2011-01-01

    Abstract Background To construct a recombinant adenovirus containing CDglyTK double suicide genes and evaluate the killing effect of the double suicide genes driven by kinase domain insert containing receptor (KDR) promoter on human umbilical vein endothelial cells. Methods Human KDR promoter, Escherichia coli (E. coli) cytosine deaminase (CD) gene and the herpes simplex virus-thymidine kinase (TK) gene were cloned using polymerase chain reaction (PCR). Plasmid pKDR-CDglyTK was constructed wi...

  3. T cells stimulate catabolic gene expression by the stromal cells from giant cell tumor of bone

    Energy Technology Data Exchange (ETDEWEB)

    Cowan, Robert W. [Department of Pathology and Molecular Medicine, McMaster University, 1280 Main St. W., Hamilton, ON, Canada L8S 4L8 (Canada); Juravinski Cancer Centre, 699 Concession St., Hamilton, ON, Canada L8V 5C2 (Canada); Ghert, Michelle [Juravinski Cancer Centre, 699 Concession St., Hamilton, ON, Canada L8V 5C2 (Canada); Department of Surgery, McMaster University, 1280 Main St. W., Hamilton, ON, Canada L8S 4L8 (Canada); Singh, Gurmit, E-mail: gurmit.singh@jcc.hhsc.ca [Department of Pathology and Molecular Medicine, McMaster University, 1280 Main St. W., Hamilton, ON, Canada L8S 4L8 (Canada); Juravinski Cancer Centre, 699 Concession St., Hamilton, ON, Canada L8V 5C2 (Canada)

    2012-03-23

    Highlights: Black-Right-Pointing-Pointer Two T cell lines stimulate PTHrP, RANKL, MMP13 gene expression in GCT cell cultures. Black-Right-Pointing-Pointer CD40 expressed by stromal cells; CD40L detected in whole tumor but not cultures. Black-Right-Pointing-Pointer Effect of CD40L treatment on GCT cells increased PTHrP and MMP13 gene expression. Black-Right-Pointing-Pointer PTHrP treatment increased MMP13 expression, while inhibition decreased expression. Black-Right-Pointing-Pointer T cells may stimulate GCT stromal cells and promote the osteolysis of the tumor. -- Abstract: The factors that promote the localized bone resorption by giant cell tumor of bone (GCT) are not fully understood. We investigated whether T cells could contribute to bone resorption by stimulating expression of genes for parathyroid hormone-related protein (PTHrP), matrix metalloproteinase (MMP)-13, and the receptor activator of nuclear-factor {kappa}B ligand (RANKL). Two cell lines, Jurkat clone E6-1 and D1.1, were co-cultured with isolated GCT stromal cells. Real-time PCR analyses demonstrated a significant increase of all three genes following 48 h incubation, and PTHrP and MMP-13 gene expression was also increased at 24 h. Further, we examined the expression of CD40 ligand (CD40L), a protein expressed by activated T cells, and its receptor, CD40, in GCT. Immunohistochemistry results revealed expression of the CD40 receptor in both the stromal cells and giant cells of the tumor. RNA collected from whole GCT tissues showed expression of CD40LG, which was absent in cultured stromal cells, and suggests that CD40L is expressed within GCT. Stimulation of GCT stromal cells with CD40L significantly increased expression of the PTHrP and MMP-13 genes. Moreover, we show that inhibition of PTHrP with neutralizing antibodies significantly decreased MMP13 expression by the stromal cells compared to IgG-matched controls, whereas stimulation with PTHrP (1-34) increased MMP-13 gene expression. These

  4. Trafficking of epidermal growth factor receptor ligands in polarized epithelial cells.

    Science.gov (United States)

    Singh, Bhuminder; Coffey, Robert J

    2014-01-01

    A largely unilamellar epithelial layer lines body cavities and organ ducts such as the digestive tract and kidney tubules. This polarized epithelium is composed of biochemically and functionally separate apical and basolateral surfaces. The epidermal growth factor receptor (EGFR) signaling pathway is a critical regulator of epithelial homeostasis and is perturbed in a number of epithelial disorders. It is underappreciated that in vivo EGFR signaling is most often initiated by cell-surface delivery and processing of one of seven transmembrane ligands, resulting in release of the soluble form that binds EGFR. In polarized epithelial cells, EGFR is restricted largely to the basolateral surface, and apical or basolateral ligand delivery therefore has important biological consequences. In vitro approaches have been used to study the biosynthesis, cell-surface delivery, proteolytic processing, and release of soluble EGFR ligands in polarized epithelial cells. We review these results, discuss their relevance to normal physiology, and demonstrate the pathophysiological consequences of aberrant trafficking. These studies have uncovered a rich diversity of apico-basolateral trafficking mechanisms among the EGFR ligands, provided insights into the pathogenesis of an inherited magnesium-wasting disorder of the kidney (isolated renal hypomagnesemia), and identified a new mode of EGFR ligand signaling via exosomes. PMID:24215440

  5. Lethal(2)giant larvae is required in the follicle cells for formation of the initial AP asymmetry and the oocyte polarity during Drosophila oogenesis

    Institute of Scientific and Technical Information of China (English)

    Qi Li; Tianchi Xin; Wenlian Chen; Mingwei Zhu; Mingfa Li

    2008-01-01

    The intricately regulated differentiation of the somatic follicle cell lineages into distinct subpopulations with specific functions plays an essential role in Drosophila egg development. At early oogenesis, induction of the stalk cells generates the first anteroposterior (AP) asymmetry in the egg chamber by inducing the posterior localization of the oocyte. Later, the properly specified posterior follicle cells signal to polarize the oocyte along the AP and dorsoventral (DV) axes at mid-oogenesis. Here, we show that lethal(2)giant larvae (Igt), a Drosophila tumor suppressor gene, is required in the follicle cells for the differentiation of both stalk cells and posterior follicle cells. Loss-of-function mutations in Igl cause oocyte mispositioning in the younger one of the fused chambers, due to lack of the stalk. Removal of Igl function from the posterior follicle cells using the FLP/FRT system results in loss of the oocyte polarity that is elicited by the failure of those posterior cells to differentiate normally. Thus, we provide the first demonstration that Igl is implicated in the formation of the initial AP asymmetry and the patterning of the AP and DV axes in the oocyte by acting in the specification of a subset of somatic follicle cells.

  6. Antitumor effect of murine dendritic and tumor cells transduced with IL-2 gene Antitumor effect of murine dendritic and tumor cells transduced with IL-2 gene

    Directory of Open Access Journals (Sweden)

    Justyna Wojas-Turek

    2012-10-01

    Full Text Available Interleukin (IL- 2 acts on a number of types of immune cells promoting their effector functions. To replace
    systemic administration of recombinant form of this cytokine, various genetically modified cells have been used in
    different preclinical models for tumor growth inhibition. In this study, dendritic or tumor cells transduced with retroviral
    vector carrying IL-2 gene (JAWS II/IL-2, X63/IL-2, MC38/IL-2 cells alone or combined with tumor antigenstimulated
    dendritic cells (JAWS II/TAg were exploited to treat colon carcinoma MC38-bearing mice. After the
    peritumoral injection of vaccine cells, the tumor growth delay and the increase in the number of tumor infiltrating
    CD4+ and CD8+ T lymphocytes were noted. A considerable increase in CD4+ cell influx into tumor tissue was observed
    when JAWS II/IL-2 cells or JAWS II/TAg with syngeneic MC38/IL-2 cells were applied. The increase in
    intensity of CD8+ cell infiltration was associated with immune reaction triggered by the same combination of applied
    cells or JAWS II/TAg with allogeneic X63/IL-2 cells. The effect observed in vivo was accompanied by MC38/0 cell
    specific cytotoxic activity of spleen cells in vitro. Thus, the application of vaccines, including IL-2-secreting cells of
    various origins, was able to induce different antitumor responses polarized by exogenous IL-2 and the encountered
    tumor antigen.Interleukin (IL- 2 acts on a number of types of immune cells promoting their effector functions. To replace
    systemic administration of recombinant form of this cytokine, various genetically modified cells have been used in
    different preclinical models for tumor growth inhibition. In this study, dendritic or tumor cells transduced with retroviral
    vector carrying IL-2 gene (JAWS II/IL-2, X63/IL-2, MC38/IL-2 cells alone or combined with tumor antigenstimulated
    dendritic cells (JAWS II/TAg were exploited to treat colon

  7. Indexing TNF-α gene expression using a gene-targeted reporter cell line

    Directory of Open Access Journals (Sweden)

    Engelhardt John F

    2009-02-01

    Full Text Available Abstract Background Current cell-based drug screening technologies utilize randomly integrated reporter genes to index transcriptional activity of an endogenous gene of interest. In this context, reporter expression is controlled by known genetic elements that may only partially capture gene regulation and by unknown features of chromatin specific to the integration site. As an alternative technology, we applied highly efficient gene-targeting with recombinant adeno-associated virus to precisely integrate a luciferase reporter gene into exon 1 of the HeLa cell tumor necrosis factor-alpha (TNF-α gene. Drugs known to induce TNF-α expression were then used to compare the authenticity of gene-targeted and randomly integrated transcriptional reporters. Results TNF-α-targeted reporter activity reflected endogenous TNF-α mRNA expression, whereas randomly integrated TNF-α reporter lines gave variable expression in response to transcriptional and epigenetic regulators. 5,6-Dimethylxanthenone-4-acetic acid (DMXAA, currently used in cancer clinical trials to induce TNF-α gene transcription, was only effective at inducing reporter expression from TNF-α gene-targeted cells. Conclusion We conclude that gene-targeted reporter cell lines provide predictive indexing of gene transcription for drug discovery.

  8. PINCH1 regulates cell-matrix and cell-cell adhesions, cell polarity and cell survival during the peri-implantation stage

    DEFF Research Database (Denmark)

    Li, Shaohua; Bordoy, Randi; Stanchi, Fabio;

    2005-01-01

    integrin or Ilk, loss of PINCH1 arrested development at the peri-implantation stage. In contrast to beta1 integrin or Ilk mutants, however, disruption of the PINCH1 gene produced implantation chambers with visible cell clumps even at embryonic day 9.5. In order to define the phenotype leading to the peri...

  9. Genes and Gene Networks Involved in Sodium Fluoride-Elicited Cell Death Accompanying Endoplasmic Reticulum Stress in Oral Epithelial Cells

    Directory of Open Access Journals (Sweden)

    Yoshiaki Tabuchi

    2014-05-01

    Full Text Available Here, to understand the molecular mechanisms underlying cell death induced by sodium fluoride (NaF, we analyzed gene expression patterns in rat oral epithelial ROE2 cells exposed to NaF using global-scale microarrays and bioinformatics tools. A relatively high concentration of NaF (2 mM induced cell death concomitant with decreases in mitochondrial membrane potential, chromatin condensation and caspase-3 activation. Using 980 probe sets, we identified 432 up-regulated and 548 down-regulated genes, that were differentially expressed by >2.5-fold in the cells treated with 2 mM of NaF and categorized them into 4 groups by K-means clustering. Ingenuity® pathway analysis revealed several gene networks from gene clusters. The gene networks Up-I and Up-II included many up-regulated genes that were mainly associated with the biological function of induction or prevention of cell death, respectively, such as Atf3, Ddit3 and Fos (for Up-I and Atf4 and Hspa5 (for Up-II. Interestingly, knockdown of Ddit3 and Hspa5 significantly increased and decreased the number of viable cells, respectively. Moreover, several endoplasmic reticulum (ER stress-related genes including, Ddit3, Atf4 and Hapa5, were observed in these gene networks. These findings will provide further insight into the molecular mechanisms of NaF-induced cell death accompanying ER stress in oral epithelial cells.

  10. CAMSAP3 orients the apical-to-basal polarity of microtubule arrays in epithelial cells.

    Science.gov (United States)

    Toya, Mika; Kobayashi, Saeko; Kawasaki, Miwa; Shioi, Go; Kaneko, Mari; Ishiuchi, Takashi; Misaki, Kazuyo; Meng, Wenxiang; Takeichi, Masatoshi

    2016-01-12

    Polarized epithelial cells exhibit a characteristic array of microtubules that are oriented along the apicobasal axis of the cells. The minus-ends of these microtubules face apically, and the plus-ends face toward the basal side. The mechanisms underlying this epithelial-specific microtubule assembly remain unresolved, however. Here, using mouse intestinal cells and human Caco-2 cells, we show that the microtubule minus-end binding protein CAMSAP3 (calmodulin-regulated-spectrin-associated protein 3) plays a pivotal role in orienting the apical-to-basal polarity of microtubules in epithelial cells. In these cells, CAMSAP3 accumulated at the apical cortices, and tethered the longitudinal microtubules to these sites. Camsap3 mutation or depletion resulted in a random orientation of these microtubules; concomitantly, the stereotypic positioning of the nucleus and Golgi apparatus was perturbed. In contrast, the integrity of the plasma membrane was hardly affected, although its structural stability was decreased. Further analysis revealed that the CC1 domain of CAMSAP3 is crucial for its apical localization, and that forced mislocalization of CAMSAP3 disturbs the epithelial architecture. These findings demonstrate that apically localized CAMSAP3 determines the proper orientation of microtubules, and in turn that of organelles, in mature mammalian epithelial cells. PMID:26715742

  11. A polarized gas internal target using a storage cell in an electron storage ring

    International Nuclear Information System (INIS)

    The first experiment using a storage cell to increase the thickness of an internal polarized gas target in an electron beam storage ring was performed at the VEPP-3 facility. We describe the storage cell technique as applied in this measurement of elastic and inelastic electron scattering from tensor polarized deuterium. An analysis of electron-beam-induced depolarization of the target was performed and experimental tests were carried out which verify the effect. Other effects causing depolarization of the target are discussed as well as the means by which they are overcome. The effective pzz of the target, shown to be stable over 8 months, was 0.57±0.05; the total target thickness was increased over that of a jet target by a factor of fifteen. (orig.)

  12. Gene expression profiles of human bone marrow derived mesenchymal stem cells and tendon cells

    Institute of Scientific and Technical Information of China (English)

    胡庆柳; 朴英杰; 邹飞

    2003-01-01

    Objective To study the gene expression profiles of human bone marrow derived mesenchymal stem cells and tendon cells.Methods Total RNA extracted from human bone marrow derived mesenchymal stem cells and tendon cells underwent reverse transcription, and the products were labeled with α-32P dCTP. The cDNA probes of total RNA were hybridized to cDNA microarray with 1176 genes, and then the signals were analyzed by AtlasImage analysis software Version 1.01a.Results Fifteen genes associated with cell proliferation and signal transduction were up-regulated, and one gene that takes part in cell-to-cell adhesion was down-regulated in tendon cells.Conclusion The 15 up-regulated and one down-regulated genes may be beneficial to the orientational differentiation of mesenchymal stem cells into tendon cells.

  13. Geometry of the Gene Expression Space of Individual Cells.

    Directory of Open Access Journals (Sweden)

    Yael Korem

    2015-07-01

    Full Text Available There is a revolution in the ability to analyze gene expression of single cells in a tissue. To understand this data we must comprehend how cells are distributed in a high-dimensional gene expression space. One open question is whether cell types form discrete clusters or whether gene expression forms a continuum of states. If such a continuum exists, what is its geometry? Recent theory on evolutionary trade-offs suggests that cells that need to perform multiple tasks are arranged in a polygon or polyhedron (line, triangle, tetrahedron and so on, generally called polytopes in gene expression space, whose vertices are the expression profiles optimal for each task. Here, we analyze single-cell data from human and mouse tissues profiled using a variety of single-cell technologies. We fit the data to shapes with different numbers of vertices, compute their statistical significance, and infer their tasks. We find cases in which single cells fill out a continuum of expression states within a polyhedron. This occurs in intestinal progenitor cells, which fill out a tetrahedron in gene expression space. The four vertices of this tetrahedron are each enriched with genes for a specific task related to stemness and early differentiation. A polyhedral continuum of states is also found in spleen dendritic cells, known to perform multiple immune tasks: cells fill out a tetrahedron whose vertices correspond to key tasks related to maturation, pathogen sensing and communication with lymphocytes. A mixture of continuum-like distributions and discrete clusters is found in other cell types, including bone marrow and differentiated intestinal crypt cells. This approach can be used to understand the geometry and biological tasks of a wide range of single-cell datasets. The present results suggest that the concept of cell type may be expanded. In addition to discreet clusters in gene-expression space, we suggest a new possibility: a continuum of states within a

  14. Gene manipulated peritoneal cell patch repairs infarcted myocardium

    OpenAIRE

    Huang, Wei; Zhang, Dongsheng; Millard, Ronald W.; Wang, Tao; Zhao, Tiemin; Fan, Guo-Chang; Ashraf, Atif; Xu, Meifeng; ASHRAF, Muhammad; Wang, Yigang

    2009-01-01

    A gene manipulated cell patch using a homologous peritoneum substrate was developed and applied after myocardial infarction to repair scarred myocardium. We genetically engineered male rat mesenchymal stem cells (MSC) using adenoviral transduction to over-express CXCR4/green fluorescent protein (GFP) (MSCCXCR4) or MSCNull or siRNA targeting CXCR4 (MSCsiRNA). Gene expression was studied by real-time quantitative PCR (qPCR) and enzyme-linked immunosorbent assay (ELISA). Cells were cultured on e...

  15. Use of genes and cells in regenerative medicine

    OpenAIRE

    Simonson, Oscar

    2015-01-01

    Regenerative medicine is a discipline that aims to achieve regeneration of cells, tissue or organs in order to restore or establish normal functions. There are several strategies that can be used to achieve this goal. Many of the strategies are based on use of genes or cells to regenerate organ functions. The present thesis aim to investigate different gene and cell based methods for the use in regenerative medicine. In paper I a novel peptide conjugate is described for the...

  16. Polycystin-1 is a microtubule-driven desmosome-associated component in polarized epithelial cells

    Energy Technology Data Exchange (ETDEWEB)

    Basora, Nuria, E-mail: Nuria.Basora@USherbrooke.ca [Department of Anatomy and Cell Biology, Faculty of Medicine and Health Sciences, Universite de Sherbrooke, Sherbrooke, Quebec, Canada J1N 5N4 (Canada); Tetreault, Marie-Pier; Boucher, Marie-Pierre; Herring, Elizabeth; Beaulieu, Jean-Francois [Department of Anatomy and Cell Biology, Faculty of Medicine and Health Sciences, Universite de Sherbrooke, Sherbrooke, Quebec, Canada J1N 5N4 (Canada)

    2010-05-15

    In this study, we have analyzed the expression and localization of polycystin-1 in intestinal epithelial cells, a system lacking primary cilia. Polycystin-1 was found to be expressed in the epithelium of the small intestine during development and levels remained elevated in the adult. Dual-labelling indirect immunofluorescence revealed polycystin-1 at sites of cell-cell contact co-localizing with the desmosomes both in situ as well as in polarized Caco-2/15 cells. In unpolarized cultures of Caco-2/15 cells, polycystin-1 was recruited to the cell surface early during initiation of cell junction assembly. In isolated Caco-2/15 cells and HIEC-6 cell cultures, where junctional complexes are absent, polycystin-1 was found predominantly associated with the cytoskeletal elements of the intermediate filaments and microtubule networks. More precisely, polycystin-1 was seen as brightly labelled puncta decorating the keratin-18 positive filaments as well as the {beta}-tubulin positive microtubules, which was particularly obvious in the lamellipodia. Treatment with the microtubule-disrupting agent, nocodazole, eliminated the microtubule association of polycystin-1 but did not seem to affect its association with keratin or the desmosomes. Taken together these data suggest that polycystin-1 is involved with the establishment of cell-cell junctions in absorptive intestinal epithelial cells and exploits the microtubule-based machinery in order to be transported to the plasma membrane.

  17. Polycystin-1 is a microtubule-driven desmosome-associated component in polarized epithelial cells

    International Nuclear Information System (INIS)

    In this study, we have analyzed the expression and localization of polycystin-1 in intestinal epithelial cells, a system lacking primary cilia. Polycystin-1 was found to be expressed in the epithelium of the small intestine during development and levels remained elevated in the adult. Dual-labelling indirect immunofluorescence revealed polycystin-1 at sites of cell-cell contact co-localizing with the desmosomes both in situ as well as in polarized Caco-2/15 cells. In unpolarized cultures of Caco-2/15 cells, polycystin-1 was recruited to the cell surface early during initiation of cell junction assembly. In isolated Caco-2/15 cells and HIEC-6 cell cultures, where junctional complexes are absent, polycystin-1 was found predominantly associated with the cytoskeletal elements of the intermediate filaments and microtubule networks. More precisely, polycystin-1 was seen as brightly labelled puncta decorating the keratin-18 positive filaments as well as the β-tubulin positive microtubules, which was particularly obvious in the lamellipodia. Treatment with the microtubule-disrupting agent, nocodazole, eliminated the microtubule association of polycystin-1 but did not seem to affect its association with keratin or the desmosomes. Taken together these data suggest that polycystin-1 is involved with the establishment of cell-cell junctions in absorptive intestinal epithelial cells and exploits the microtubule-based machinery in order to be transported to the plasma membrane.

  18. A glycophospholipid membrane anchor acts as an apical targeting signal in polarized epithelial cells

    OpenAIRE

    1989-01-01

    Glycosyl-phosphatidylinositol- (GPI) anchored proteins contain a large extracellular protein domain that is linked to the membrane via a glycosylated form of phosphatidylinositol. We recently reported the polarized apical distribution of all endogenous GPI-anchored proteins in the MDCK cell line (Lisanti, M. P., M. Sargiacomo, L. Graeve, A. R. Saltiel, and E. Rodriguez-Boulan. 1988. Proc. Natl. Acad. Sci. USA. 85:9557-9561). To study the role of this mechanism of membrane anchoring in targeti...

  19. Single-cell PCR profiling of gene expression in hematopoiesis.

    Science.gov (United States)

    Teles, José; Enver, Tariq; Pina, Cristina

    2014-01-01

    Single-cell analysis of gene expression offers the possibility of exploring cellular and molecular heterogeneity in stem and developmental cell systems, including cancer, to infer routes of cellular specification and their respective gene regulatory modules. PCR-based technologies, although limited to the analysis of a predefined set of genes, afford a cost-effective balance of throughput and biological information and have become a method of choice in stem cell laboratories. Here we describe an experimental and analytical protocol based on the Fluidigm microfluidics platform for the simultaneous expression analysis of 48 or 96 genes in multiples of 48 or 96 cells. We detail wet laboratory procedures and describe clustering, principal component analysis, correlation, and classification tools for the inference of cellular pathways and gene networks. PMID:25062620

  20. Polarity of fatty acid uptake and metabolism in a human intestinal cell line (CACO-2)

    International Nuclear Information System (INIS)

    Free fatty acids (ffa) can enter the intestinal cell via the apical (AP) or basolateral (BL) membrane. The authors are using the Caco-2 intestinal cell line to examine the polarity of ffa uptake and metabolism in the enterocyte. Cells are grown on permeable polycarbonate Transwell filters in order to obtain access to both AP and BL compartments. Differentiated Caco-2 cells form tight polarized monolayers which express small intestine-specific enzymes and are impermeable to the fluid phase marker Lucifer Yellow. Submicellar concentrations of 3H-palmitic acid (2uM) were added to AP or BL sides of Caco-2 monolayers at 37 degrees C and cells were incubated for various times between 2 and 120 minutes. Total AP and BL uptake is similar; however, when relative membrane surface areas are accounted for, AP uptake is about 2-fold higher. The metabolism of AP and BL ffa is not significantly different: triacylglycerol and phosphatidylcholine account for most of the metabolites (32±4 and 24±2% respectively at 5 minutes). Little ffa oxidation is observed. Preincubation with albumin-bound 2-monoolein (100uM) and palmitate (50uM) increases the level of TG metabolites. The results suggest that in this cell line the uptake of AP ffa may be greater than BL ffa, but that AP (dietary) ffa and BL (plasma) ffa are metabolized similarly

  1. Polarity of fatty acid uptake and metabolism in a human intestinal cell line (CACO-2)

    Energy Technology Data Exchange (ETDEWEB)

    Trotter, P.J.; Storch, J. (Harvard School of Public Health, Boston, MA (United States))

    1990-02-26

    Free fatty acids (ffa) can enter the intestinal cell via the apical (AP) or basolateral (BL) membrane. The authors are using the Caco-2 intestinal cell line to examine the polarity of ffa uptake and metabolism in the enterocyte. Cells are grown on permeable polycarbonate Transwell filters in order to obtain access to both AP and BL compartments. Differentiated Caco-2 cells form tight polarized monolayers which express small intestine-specific enzymes and are impermeable to the fluid phase marker Lucifer Yellow. Submicellar concentrations of {sup 3}H-palmitic acid (2uM) were added to AP or BL sides of Caco-2 monolayers at 37{degrees}C and cells were incubated for various times between 2 and 120 minutes. Total AP and BL uptake is similar; however, when relative membrane surface areas are accounted for, AP uptake is about 2-fold higher. The metabolism of AP and BL ffa is not significantly different: triacylglycerol and phosphatidylcholine account for most of the metabolites (32{plus minus}4 and 24{plus minus}2% respectively at 5 minutes). Little ffa oxidation is observed. Preincubation with albumin-bound 2-monoolein (100uM) and palmitate (50uM) increases the level of TG metabolites. The results suggest that in this cell line the uptake of AP ffa may be greater than BL ffa, but that AP (dietary) ffa and BL (plasma) ffa are metabolized similarly.

  2. Tumor cell-activated CARD9 signaling contributes to metastasis-associated macrophage polarization.

    Science.gov (United States)

    Yang, M; Shao, J-H; Miao, Y-J; Cui, W; Qi, Y-F; Han, J-H; Lin, X; Du, J

    2014-08-01

    Macrophages are critical immune effector cells of the tumor microenvironment that promote seeding, extravasation and persistent growth of tumor cells in primary tumors and metastatic sites. Tumor progression and metastasis are affected by dynamic changes in the specific phenotypes of macrophage subpopulations; however, the mechanisms by which tumor cells modulate macrophage polarization remain incompletely understood. Caspase recruitment domain-containing protein 9 (CARD9) is a central adaptor protein of innate immune responses to extracellular pathogens. We report that increased CARD9 expression is primarily localized in infiltrated macrophages and significantly associated with advanced histopathologic stage and the presence of metastasis. Using CARD9-deficient (CARD9(-/-)) mice, we show that bone marrow-derived CARD9 promotes liver metastasis of colon carcinoma cells. Mechanistic studies reveal that CARD9 contributes to tumor metastasis by promoting metastasis-associated macrophage polarization through activation of the nuclear factor-kappa B signaling pathway. We further demonstrate that tumor cell-secreted vascular endothelial growth factor facilitates spleen tyrosine kinase activation in macrophages, which is necessary for formation of the CARD9-B-cell lymphoma/leukemia 10-mucosa-associated lymphoid tissue lymphoma translocation protein 1 complex. Taken together, our results indicating that CARD9 is a regulator of metastasis-associated macrophages will lead to new insights into evolution of the microenvironments supporting tumor metastasis, thereby providing targets for anticancer therapies. PMID:24722209

  3. Co-regulation of cell polarization and migration by caveolar proteins PTRF/Cavin-1 and caveolin-1.

    Directory of Open Access Journals (Sweden)

    Michelle M Hill

    Full Text Available Caveolin-1 and caveolae are differentially polarized in migrating cells in various models, and caveolin-1 expression has been shown to quantitatively modulate cell migration. PTRF/cavin-1 is a cytoplasmic protein now established to be also necessary for caveola formation. Here we tested the effect of PTRF expression on cell migration. Using fluorescence imaging, quantitative proteomics, and cell migration assays we show that PTRF/cavin-1 modulates cellular polarization, and the subcellular localization of Rac1 and caveolin-1 in migrating cells as well as PKCα caveola recruitment. PTRF/cavin-1 quantitatively reduced cell migration, and induced mesenchymal epithelial reversion. Similar to caveolin-1, the polarization of PTRF/cavin-1 was dependent on the migration mode. By selectively manipulating PTRF/cavin-1 and caveolin-1 expression (and therefore caveola formation in multiple cell systems, we unveil caveola-independent functions for both proteins in cell migration.

  4. Patterns of expression of cell wall related genes in sugarcane

    Directory of Open Access Journals (Sweden)

    Lima D.U.

    2001-01-01

    Full Text Available Our search for genes related to cell wall metabolism in the sugarcane expressed sequence tag (SUCEST database (http://sucest.lbi.dcc.unicamp.br resulted in 3,283 reads (1% of the total reads which were grouped into 459 clusters (potential genes with an average of 7.1 reads per cluster. To more clearly display our correlation coefficients, we constructed surface maps which we used to investigate the relationship between cell wall genes and the sugarcane tissues libraries from which they came. The only significant correlations that we found between cell wall genes and/or their expression within particular libraries were neutral or synergetic. Genes related to cellulose biosynthesis were from the CesA family, and were found to be the most abundant cell wall related genes in the SUCEST database. We found that the highest number of CesA reads came from the root and stem libraries. The genes with the greatest number of reads were those involved in cell wall hydrolases (e.g. beta-1,3-glucanases, xyloglucan endo-beta-transglycosylase, beta-glucosidase and endo-beta-mannanase. Correlation analyses by surface mapping revealed that the expression of genes related to biosynthesis seems to be associated with the hydrolysis of hemicelluloses, pectin hydrolases being mainly associated with xyloglucan hydrolases. The patterns of cell wall related gene expression in sugarcane based on the number of reads per cluster reflected quite well the expected physiological characteristics of the tissues. This is the first work to provide a general view on plant cell wall metabolism through the expression of related genes in almost all the tissues of a plant at the same time. For example, developing flowers behaved similarly to both meristematic tissues and leaf-root transition zone tissues. Besides providing a basis for future research on the mechanisms of plant development which involve the cell wall, our findings will provide valuable tools for plant engineering in the

  5. Optically-driven red blood cell rotor in linearly polarized laser tweezers

    Indian Academy of Sciences (India)

    Manas Khan; Samarendra K Mohanty; A K Sood

    2005-11-01

    We have constructed a dual trap optical tweezers set-up around an inverted microscope where both the traps can be independently controlled and manipulated in all the three dimensions. Here we report our observations on rotation of red blood cells (RBCs) in a linearly polarized optical trap. Red blood cells deform and become twisted in hypertonic phosphate buffer saline and when trapped, experience an unbalanced radiation pressure force. The torque generated from the unbalanced force causes the trapped RBC to rotate. Addition of Ca++ ions in the solution, keeping the osmolarity same, makes the cell membranes stiffer and the cells deform less. Thus the speed of rotation of the red blood cells can be controlled, as less deformation and in turn less asymmetry in shape produces less torque under the radiation pressure resulting in slower rotation at the same laser power.

  6. Non-invasive imaging of cellulose microfibril orientation within plant cell walls by polarized Raman microspectroscopy.

    Science.gov (United States)

    Sun, Lan; Singh, Seema; Joo, Michael; Vega-Sanchez, Miguel; Ronald, Pamela; Simmons, Blake A; Adams, Paul; Auer, Manfred

    2016-01-01

    Cellulose microfibrils represent the major scaffold of plant cell walls. Different packing and orientation of the microfibrils at the microscopic scale determines the macroscopic properties of cell walls and thus affect their functions with a profound effect on plant survival. We developed a polarized Raman microspectroscopic method to determine cellulose microfibril orientation within rice plant cell walls. Employing an array of point measurements as well as area imaging and subsequent Matlab-assisted data processing, we were able to characterize the distribution of cellulose microfibril orientation in terms of director angle and anisotropy magnitude. Using this approach we detected differences between wild type rice plants and the rice brittle culm mutant, which shows a more disordered cellulose microfibril arrangement, and differences between different tissues of a wild type rice plant. This novel non-invasive Raman imaging approach allows for quantitative assessment of cellulose fiber orientation in cell walls of herbaceous plants, an important advancement in cell wall characterization. PMID:26137889

  7. Patterns of cell division revealed by transcriptional regulation of genes during the cell cycle in plants.

    OpenAIRE

    Fobert, P R; Coen, E S; Murphy, G. J.; Doonan, J H

    1994-01-01

    Transcripts from five cell cycle related genes accumulate in isolated cells dispersed throughout the actively dividing regions of plant meristems. We propose that this pattern reflects gene expression during particular phases of the cell division cycle. The high proportion of isolated cells suggests that synchrony between daughter cells is rapidly lost following mitosis. This is the first time that such a cell specific expression pattern has been described in a higher organism. Counterstainin...

  8. Cell Pluripotency Levels Associated with Imprinted Genes in Human

    Directory of Open Access Journals (Sweden)

    Liyun Yuan

    2015-01-01

    Full Text Available Pluripotent stem cells are exhibited similarly in the morphology, gene expression, growth properties, and epigenetic modification with embryonic stem cells (ESCs. However, it is still controversial that the pluripotency of induced pluripotent stem cell (iPSC is much inferior to ESC, and the differentiation capacity of iPSC and ESC can also be separated by transcriptome and epigenetics. miRNAs, which act in posttranscriptional regulation of gene expression and are involved in many basic cellular processes, may reveal the answer. In this paper, we focused on identifying the hidden relationship between miRNAs and imprinted genes in cell pluripotency. Total miRNA expression patterns in iPSC and ES cells were comprehensively analysed and linked with human imprinted genes, which show a global picture of their potential function in pluripotent level. A new CPA4-KLF14 region which locates in chromosomal homologous segments (CHSs within mammals and include both imprinted genes and significantly expressed miRNAs was first identified. Molecular network analysis showed genes interacted with imprinted genes closely and enriched in modules such as cancer, cell death and survival, and tumor morphology. This imprinted region may provide a new look for those who are interested in cell pluripotency of hiPSCs and hESCs.

  9. Cell Pluripotency Levels Associated with Imprinted Genes in Human.

    Science.gov (United States)

    Yuan, Liyun; Tang, Xiaoyan; Zhang, Binyan; Ding, Guohui

    2015-01-01

    Pluripotent stem cells are exhibited similarly in the morphology, gene expression, growth properties, and epigenetic modification with embryonic stem cells (ESCs). However, it is still controversial that the pluripotency of induced pluripotent stem cell (iPSC) is much inferior to ESC, and the differentiation capacity of iPSC and ESC can also be separated by transcriptome and epigenetics. miRNAs, which act in posttranscriptional regulation of gene expression and are involved in many basic cellular processes, may reveal the answer. In this paper, we focused on identifying the hidden relationship between miRNAs and imprinted genes in cell pluripotency. Total miRNA expression patterns in iPSC and ES cells were comprehensively analysed and linked with human imprinted genes, which show a global picture of their potential function in pluripotent level. A new CPA4-KLF14 region which locates in chromosomal homologous segments (CHSs) within mammals and include both imprinted genes and significantly expressed miRNAs was first identified. Molecular network analysis showed genes interacted with imprinted genes closely and enriched in modules such as cancer, cell death and survival, and tumor morphology. This imprinted region may provide a new look for those who are interested in cell pluripotency of hiPSCs and hESCs. PMID:26504487

  10. Chimera proteins with affinity for membranes and microtubule tips polarize in the membrane of fission yeast cells.

    Science.gov (United States)

    Recouvreux, Pierre; Sokolowski, Thomas R; Grammoustianou, Aristea; Ten Wolde, Pieter Rein; Dogterom, Marileen

    2016-02-16

    Cell polarity refers to a functional spatial organization of proteins that is crucial for the control of essential cellular processes such as growth and division. To establish polarity, cells rely on elaborate regulation networks that control the distribution of proteins at the cell membrane. In fission yeast cells, a microtubule-dependent network has been identified that polarizes the distribution of signaling proteins that restricts growth to cell ends and targets the cytokinetic machinery to the middle of the cell. Although many molecular components have been shown to play a role in this network, it remains unknown which molecular functionalities are minimally required to establish a polarized protein distribution in this system. Here we show that a membrane-binding protein fragment, which distributes homogeneously in wild-type fission yeast cells, can be made to concentrate at cell ends by attaching it to a cytoplasmic microtubule end-binding protein. This concentration results in a polarized pattern of chimera proteins with a spatial extension that is very reminiscent of natural polarity patterns in fission yeast. However, chimera levels fluctuate in response to microtubule dynamics, and disruption of microtubules leads to disappearance of the pattern. Numerical simulations confirm that the combined functionality of membrane anchoring and microtubule tip affinity is in principle sufficient to create polarized patterns. Our chimera protein may thus represent a simple molecular functionality that is able to polarize the membrane, onto which additional layers of molecular complexity may be built to provide the temporal robustness that is typical of natural polarity patterns. PMID:26831106

  11. Effect of toxic components on microbial fuel cell-polarization curves and estimation of the type of toxic inhibition

    NARCIS (Netherlands)

    Stein, N.E.; Hamelers, H.V.M.; Straten, G. van; Keesman, K.J.

    2012-01-01

    Polarization curves are of paramount importance for the detection of toxic components in microbial fuel cell (MFC) based biosensors. In this study, polarization curves were made under non-toxic conditions and under toxic conditions after the addition of various concentrations of nickel, bentazon, so

  12. NKp46 clusters at the immune synapse and regulates NK cell polarization

    Directory of Open Access Journals (Sweden)

    Uzi eHadad

    2015-09-01

    Full Text Available Natural killer cells play an important role in first-line defense against tumor and virus-infected cells. The activity of NK cells is tightly regulated by a repertoire of cell-surface expressed inhibitory and activating receptors. NKp46 is a major NK cell activating receptor that is involved in the elimination of target cells. NK cells form different types of synapses that result in distinct functional outcomes: cytotoxic, inhibitory, and regulatory. Recent studies revealed that complex integration of NK receptor signaling controls cytoskeletal rearrangement and other immune synapse-related events. However the distinct nature by which NKp46 participates in NK immunological synapse formation and function remains unknown. In this study we determined that NKp46 forms microclusters structures at the immune synapse between NK cells and target cells. Over-expression of human NKp46 is correlated with increased accumulation of F-actin mesh at the immune synapse. Concordantly, knock-down of NKp46 in primary human NK cells decreased recruitment of F-actin to the synapse. Live cell imaging experiments showed a linear correlation between NKp46 expression and lytic granules polarization to the immune synapse. Taken together, our data suggest that NKp46 signaling directly regulates the NK lytic immune synapse from early formation to late function.

  13. The cell cycle-regulated genes of Schizosaccharomyces pombe.

    Directory of Open Access Journals (Sweden)

    Anna Oliva

    2005-07-01

    Full Text Available Many genes are regulated as an innate part of the eukaryotic cell cycle, and a complex transcriptional network helps enable the cyclic behavior of dividing cells. This transcriptional network has been studied in Saccharomyces cerevisiae (budding yeast and elsewhere. To provide more perspective on these regulatory mechanisms, we have used microarrays to measure gene expression through the cell cycle of Schizosaccharomyces pombe (fission yeast. The 750 genes with the most significant oscillations were identified and analyzed. There were two broad waves of cell cycle transcription, one in early/mid G2 phase, and the other near the G2/M transition. The early/mid G2 wave included many genes involved in ribosome biogenesis, possibly explaining the cell cycle oscillation in protein synthesis in S. pombe. The G2/M wave included at least three distinctly regulated clusters of genes: one large cluster including mitosis, mitotic exit, and cell separation functions, one small cluster dedicated to DNA replication, and another small cluster dedicated to cytokinesis and division. S. pombe cell cycle genes have relatively long, complex promoters containing groups of multiple DNA sequence motifs, often of two, three, or more different kinds. Many of the genes, transcription factors, and regulatory mechanisms are conserved between S. pombe and S. cerevisiae. Finally, we found preliminary evidence for a nearly genome-wide oscillation in gene expression: 2,000 or more genes undergo slight oscillations in expression as a function of the cell cycle, although whether this is adaptive, or incidental to other events in the cell, such as chromatin condensation, we do not know.

  14. In vivo determination of fibril orientation in plant cell walls with polarization CSLM

    International Nuclear Information System (INIS)

    Congo Red fluorescence is used to detect cellulose in the wall of plant cells. The orientation of the cellulose fibrils is determined by using polarized light for excitation. The absorption characteristics of Congo Red make this approach a method of choice for applications with any standard confocal scanning laser microscope (CSLM). The semiquantitative character of CSLM observations combined with the non-toxicity of the stain allow a very fast and reliable assessment of cellulose orientation in the wall of living plant cells. (author)

  15. The HP0256 gene product is involved in motility and cell envelope architecture of Helicobacter pylori

    LENUS (Irish Health Repository)

    Douillard, Francois P

    2010-04-08

    Abstract Background Helicobacter pylori is the causative agent for gastritis, and peptic and duodenal ulcers. The bacterium displays 5-6 polar sheathed flagella that are essential for colonisation and persistence in the gastric mucosa. The biochemistry and genetics of flagellar biogenesis in H. pylori has not been fully elucidated. Bioinformatics analysis suggested that the gene HP0256, annotated as hypothetical, was a FliJ homologue. In Salmonella, FliJ is a chaperone escort protein for FlgN and FliT, two proteins that themselves display chaperone activity for components of the hook, the rod and the filament. Results Ablation of the HP0256 gene in H. pylori significantly reduced motility. However, flagellin and hook protein synthesis was not affected in the HP0256 mutant. Transmission electron transmission microscopy revealed that the HP0256 mutant cells displayed a normal flagellum configuration, suggesting that HP0256 was not essential for assembly and polar localisation of the flagella in the cell. Interestingly, whole genome microarrays of an HP0256 mutant revealed transcriptional changes in a number of genes associated with the flagellar regulon and the cell envelope, such as outer membrane proteins and adhesins. Consistent with the array data, lack of the HP0256 gene significantly reduced adhesion and the inflammatory response in host cells. Conclusions We conclude that HP0256 is not a functional counterpart of FliJ in H. pylori. However, it is required for full motility and it is involved, possibly indirectly, in expression of outer membrane proteins and adhesins involved in pathogenesis and adhesion.

  16. A noninvasive transfer system for polarized renal tubule epithelial cell sheets using temperature-responsive culture dishes

    Directory of Open Access Journals (Sweden)

    Kushida A.

    2005-08-01

    Full Text Available We used temperature-responsive culture dishes onto which the temperature-responsive polymer, poly(Nisopropylacrylamide, was covalently grafted for tissue engineering. Confluent cells harvested as intact sheets from these surfaces by simple temperature reduction can be transferred to various surfaces including additional culture dishes, other cell sheets, and tissues. In order to examine the maintenance of cell polarity, Madin-Darby canine kidney cells and human primary renal proximal tubule epithelial cells which had developed apical-basal cell polarity in culture, were subjected to cell sheet transfer. This functional and structural cell polarity, which is susceptible to treatment with trypsin, was examined by immunohistochemistry and transmission electron microscopy. Using our cell-sheet method, the noninvasive transfer of these cell sheets retaining typical distributions of Na+/K+-ATPase, GLUT-1, SGLT-1, aquaporin-1, neutral endopeptidase and dipeptidylendopeptidase IV, could be achieved. The transferred cell sheets also developed numerous microvilli and tight junctions at the apical and lateral membranes, respectively. For biochemical analysis, immunoblotting of occludin, a transmembrane protein that composes tight junctions, was conducted and results confirmed that occludin remained intact after cell sheet transfer. This two-dimensional cell sheet manipulation method promises to be useful for tissue engineering as well as in the investigation of epithelial cell polarity.

  17. Mutations in exocyst complex subunit SEC6 gene impaired polar auxin transport and PIN protein recycling in Arabidopsis primary root.

    Science.gov (United States)

    Tan, Xiaoyun; Feng, Yihong; Liu, Yulong; Bao, Yiqun

    2016-09-01

    Polar auxin transport, which is critical for land plant pattern formation and directional growth, is largely depended on asymmetric distribution of PIN proteins at the plasma membrane (PM). Endocytosis and recycling processes play important roles in regulating PIN protein distribution and abundance at the PM. Two subunits (SEC8, EXO70A1) of exocyst, an octameric vesicle-tethering complex, have been reported to be involved in PIN protein recycling in Arabidopsis. However, the function of exocyst complex in PIN protein recycling and polar auxin transport remains incompletely understood. In this study, we utilized two SEC6 down-regulation mutants (PRsec6-1 and PRsec6-2) to investigate the role of exocyst subunit SEC6 in the primary root development, polar auxin transport and PIN proteins recycling. We found that in PRsec6 mutants: 1. Primary root growth was retarded, and lateral root initiation were compromised. 2. Primary roots were sensitive to exogenous auxin 1-napthalene acetic acid (NAA) but not 2,4-dichlorophenoxy (2.4-D). 3. Recycling of PIN1 and PIN2 proteins from the Brefeldin A (BFA) compartment to the PM was delayed. 4. Vesicles accumulated in the primary root tip cells, especially accumulated in the cytosol closed to the PM. These results further demonstrated that the exocyst complex plays an important role in PIN protein recycling and polar auxin transport in Arabidopsis primary root. PMID:27457987

  18. Dystroglycan loss disrupts polarity and beta-casein induction inmammary epithelial cells by perturbing laminin anchoring

    Energy Technology Data Exchange (ETDEWEB)

    Weir, M. Lynn; Oppizzi, Maria Luisa; Henry, Michael D.; Onishi,Akiko; Campbell, Kevin P.; Bissell, Mina J.; Muschler, John L.

    2006-02-17

    Precise contact between epithelial cells and their underlying basement membrane is critical to the maintenance of tissue architecture and function. To understand the role that the laminin receptor dystroglycan (DG) plays in these processes, we assayed cell responses to laminin-111 following conditional ablation of DG expression in cultured mammary epithelial cells (MECs). Strikingly, DG loss disrupted laminin-111-induced polarity and {beta}-casein production, and abolished laminin assembly at the step of laminin binding to the cell surface. DG re-expression restored these deficiencies. Investigations of mechanism revealed that DG cytoplasmic sequences were not necessary for laminin assembly and signaling, and only when the entire mucin domain of extracellular DG was deleted did laminin assembly not occur. These results demonstrate that DG is essential as a laminin-111 co-receptor in MECs that functions by mediating laminin anchoring to the cell surface, a process that allows laminin polymerization, tissue polarity, and {beta}-casein induction. The observed loss of laminin-111 assembly and signaling in DG-/-MECs provides insights into the signaling changes occurring in breast carcinomas and other cancers, where DG's laminin-binding function is frequently defective.

  19. T cell receptor reversed polarity recognition of a self-antigen major histocompatibility complex.

    Science.gov (United States)

    Beringer, Dennis X; Kleijwegt, Fleur S; Wiede, Florian; van der Slik, Arno R; Loh, Khai Lee; Petersen, Jan; Dudek, Nadine L; Duinkerken, Gaby; Laban, Sandra; Joosten, Antoinette; Vivian, Julian P; Chen, Zhenjun; Uldrich, Adam P; Godfrey, Dale I; McCluskey, James; Price, David A; Radford, Kristen J; Purcell, Anthony W; Nikolic, Tatjana; Reid, Hugh H; Tiganis, Tony; Roep, Bart O; Rossjohn, Jamie

    2015-11-01

    Central to adaptive immunity is the interaction between the αβ T cell receptor (TCR) and peptide presented by the major histocompatibility complex (MHC) molecule. Presumably reflecting TCR-MHC bias and T cell signaling constraints, the TCR universally adopts a canonical polarity atop the MHC. We report the structures of two TCRs, derived from human induced T regulatory (iT(reg)) cells, complexed to an MHC class II molecule presenting a proinsulin-derived peptide. The ternary complexes revealed a 180° polarity reversal compared to all other TCR-peptide-MHC complex structures. Namely, the iT(reg) TCR α-chain and β-chain are overlaid with the α-chain and β-chain of MHC class II, respectively. Nevertheless, this TCR interaction elicited a peptide-reactive, MHC-restricted T cell signal. Thus TCRs are not 'hardwired' to interact with MHC molecules in a stereotypic manner to elicit a T cell signal, a finding that fundamentally challenges our understanding of TCR recognition. PMID:26437244

  20. [Targeting of type IV carbonic anhydrases in Capan-1 human pancreatic duct cells is concomitant of the polarization].

    Science.gov (United States)

    Mairal, A; Fanjul, M; Hollande, E

    1996-01-01

    Carbonic anhydrases II and IV play an essential role in the synthesis and secretion of HCO3- ions in pancreatic duct cells. Secretion of these ions is regulated by the CFTR (cystic fibrosis transmembrane conductance regulator) chloride channel. In the present study, the expression of carbonic anhydrases IV and their targeting to plasma membranes were examined during the growth of human pancreatic duct cells in vitro. Human cancerous pancreatic duct cells of Capan-1 cell line which polarize during their growth were used. We show that: a) these cells express carbonic anhydrases IV continuously during growth in culture, and the expression depends on the stage of growth and the conformation of the cells; b) carbonic anhydrases IV are seen in the cytoplasm in non-polarized cells, but become progressively anchored to plasma membranes as the cells polarize, being targeted to the apical membranes of polarized cells; c) the subcellular distribution of carbonic anhydrases IV indicates that these enzymes are synthetized in rough endoplasmic reticulum and then transported towards the plasma membrane using the classical secretory pathway through the Golgi apparatus. The results indicated that targeting of carbonic anhydrases IV in Capan-1 cells is linked to cellular polarization. PMID:8881572

  1. Mast cells dysregulate apoptotic and cell cycle genes in mucosal squamous cell carcinoma

    Directory of Open Access Journals (Sweden)

    Davis Paul

    2006-12-01

    Full Text Available Abstract Background Mucosal squamous cell carcinoma of the head and neck is a disease of high mortality and morbidity. Interactions between the squamous cell carcinoma and the host's local immunity, and how the latter contributes to the biological behavior of the tumor are unclear. In vivo studies have demonstrated sequential mast cell infiltration and degranulation during squamous cell carcinogenesis. The degree of mast cell activation correlates closely with distinct phases of hyperkeratosis, dysplasia, carcinoma in-situ and invasive carcinoma. However, the role of mast cells in carcinogenesis is unclear. Aim This study explores the effects of mast cells on the proliferation and gene expression profile of mucosal squamous cell carcinoma using human mast cell line (HMC-1 and human glossal squamous cell carcinoma cell line (SCC25. Methods HMC-1 and SCC25 were co-cultured in a two-compartment chamber, separated by a polycarbonate membrane. HMC-1 was stimulated to degranulate with calcium ionophore A23187. The experiments were done in quadruplicate. Negative controls were established where SCC25 were cultured alone without HMC-1. At 12, 24, 48 and 72 hours, proliferation and viability of SCC25 were assessed with MTT colorimetric assay. cDNA microarray was employed to study differential gene expression between co-cultured and control SCC25. Results HMC-1/SCC25 co-culture resulted in suppression of growth rate for SCC-25 (34% compared with 110% for the control by 72 hours, p Conclusion We show that mast cells have a direct inhibitory effect on the proliferation of mucosal squamous cell carcinoma in vitro by dysregulating key genes in apoptosis and cell cycle control.

  2. Regulation of cell-to-cell variability in divergent gene expression

    Science.gov (United States)

    Yan, Chao; Wu, Shuyang; Pocetti, Christopher; Bai, Lu

    2016-03-01

    Cell-to-cell variability (noise) is an important feature of gene expression that impacts cell fitness and development. The regulatory mechanism of this variability is not fully understood. Here we investigate the effect on gene expression noise in divergent gene pairs (DGPs). We generated reporters driven by divergent promoters, rearranged their gene order, and probed their expressions using time-lapse fluorescence microscopy and single-molecule fluorescence in situ hybridization (smFISH). We show that two genes in a co-regulated DGP have higher expression covariance compared with the separate, tandem and convergent configurations, and this higher covariance is caused by more synchronized firing of the divergent transcriptions. For differentially regulated DGPs, the regulatory signal of one gene can stochastically `leak' to the other, causing increased gene expression noise. We propose that the DGPs' function in limiting or promoting gene expression noise may enhance or compromise cell fitness, providing an explanation for the conservation pattern of DGPs.

  3. Bone cell-material interactions on metal-ion doped polarized hydroxyapatite

    International Nuclear Information System (INIS)

    The objective of this work is to study the influence of Mg2+ and Sr2+ dopants on in vitro bone cell-material interactions of electrically polarized hydroxyapatite [HAp, Ca10(PO4)6(OH)2] ceramics with an aim to achieve additional advantage of matching bone chemistry along with the original benefits of electrical polarization treatment relevant to biomedical applications. To achieve our research objective, commercial phase pure HAp has been doped with MgO, and SrO in single, and binary compositions. All samples have been sintered at 1200 deg. C for 2 h and subsequently polarized using an external d.c. field (2.0 kV/cm) at 400 deg. C for 1 h. Combined addition of 1 wt.% MgO/1 wt.% SrO in HAp has been most beneficial in enhancing the polarizability in which stored charge was 4.19 μC/cm2 compared to pure HAp of 2.23 μC/cm2. Bone cell-material interaction has been studied by culturing with human fetal osteoblast cells (hFOB) for a maximum of 7 days. Scanning electron microscope (SEM) images of cell morphology reveal that favorable surface properties and dopant chemistry lead to good cellular adherence and spreading on negatively charged surfaces of both Sr2+ and Mg2+ doped HAp samples over undoped HAp. MTT assay results at 7 days show the highest viable cell densities on the negatively charged surfaces of binary doped HAp samples, while positive charged doped HAp surfaces exhibit limited cellular growth in comparison to neutral surfaces.

  4. Gene Therapy In Squamous Cell Carcinoma – A Short Review

    Directory of Open Access Journals (Sweden)

    Soma Susan Varghese

    2011-07-01

    Full Text Available Oral cancer remains one of the leading causes of death world wide. Various means to destroy tumor cells preferentially have been developed; gene therapy is one among them with less treatment morbidity. Gene therapy involves the transfer of therapeutic or working copy of genes into a specific cell of an individual in order to repair a faulty copy of gene. The alteration can be accomplished by repairing or replacing the damaged DNA by various strategies and vectors. To date genetically altered viruses are commonly used as gene delivery vehicle (vector which has an advantage of evolutionary selection of host-virus relation. Non viral vectors which include the physical transfection of genes can be accomplished by electrophoration, microinjection, or use of ballistic particles and chemical transfection by forming liposomes.

  5. Comparison of clinical grade type 1 polarized and standard matured dendritic cells for cancer immunotherapy

    DEFF Research Database (Denmark)

    Hansen, Morten; Hjortø, Gertrud Malene; Donia, Marco;

    2013-01-01

    induction of type 1 effector T cells. Standard matured clinical grade DCs “sDCs” were compared with DCs matured with either of two type 1 polarizing maturation cocktails; the alpha-type-1 DCs “αDC1s” (TNF-α, IL-1β, IFN-γ, IFN-α, Poly(I:C)) and “mDCs” (monophosphoryl lipid A (MPL), IFN-γ) or a mixed cocktail...... – “mpDCs”, containing MPL, IFN-γ and PGE2. αDC1s and mDCs secreted IL-12 directly and following re-stimulation with CD40L-expressing cells and they mainly secreted the T effector cell attracting chemokines CXCL10 and CCL5 as opposed to sDCs that mainly secreted CCL22, known to attract regulatory T cells...

  6. Cancer-associated fibroblasts as another polarized cell type of the tumor microenvironment

    Directory of Open Access Journals (Sweden)

    MartinAugsten

    2014-03-01

    Full Text Available Tumor- or cancer-associated fibroblasts (CAFs are one of the most abundant stromal cell types in different carcinomas and comprise a heterogeneous cell population. Classically, CAFs are assigned with pro-tumorigenic effects stimulating tumor growth and progression. More recent studies demonstrated also tumor-inhibitory effects of CAFs suggesting that tumor-residing fibroblasts exhibit a similar degree of plasticity as other stromal cell types. Reciprocal interactions with the tumor milieu and different sources of origin are emerging as two important factors underlying CAF heterogeneity. This review highlights recent advances in our understanding of CAF biology and proposes to expand the term of cellular ´polarization´, previously introduced to describe different activation states of various immune cells, onto CAFs to reflect their phenotypic diversity.

  7. Coupling of cytoplasm and adhesion dynamics determines cell polarization and locomotion

    CERN Document Server

    Bock, Martin; Möhl, Christoph

    2009-01-01

    Observations of single epidermal cells on flat adhesive substrates have revealed two distinct morphological and functional states, namely a non-migrating symmetric unpolarized state and a migrating asymmetric polarized state. These states are characterized by different spatial distributions and dynamics of important biochemical cell components: F-actin and myosin-II form the contractile part of the cytoskeleton, and integrin receptors in the plasma membrane connect F-actin filaments to the substratum. In this way, focal adhesion complexes are assembled, which determine cytoskeletal force transduction and subsequent cell locomotion. So far, physical models have reduced this phenomenon either to gradients in regulatory control molecules or to different mechanics of the actin filament system in different regions of the cell. Here we offer an alternative and self-organizational model incorporating polymerization, pushing and sliding of filaments, as well as formation of adhesion sites and their force dependent ki...

  8. Gene Transfer between Salmonella enterica Serovar Typhimurium inside Epithelial Cells

    OpenAIRE

    Ferguson, Gayle C.; Heinemann, Jack A.; Kennedy, Martin A

    2002-01-01

    Virulence and antibiotic resistance genes transfer between bacteria by bacterial conjugation. Conjugation also mediates gene transfer from bacteria to eukaryotic organisms, including yeast and human cells. Predicting when and where genes transfer by conjugation could enhance our understanding of the risks involved in the release of genetically modified organisms, including those being developed for use as vaccines. We report here that Salmonella enterica serovar Typhimurium conjugated inside ...

  9. Expression and subcellular localization of aquaporin water channels in the polarized hepatocyte cell line, WIF-B

    OpenAIRE

    Marinelli Raúl A; Splinter Patrick L; Tietz Pamela S; Gradilone Sergio A; LaRusso Nicholas F

    2005-01-01

    Abstract Background Recent data suggest that canalicular bile secretion involves selective expression and coordinated regulation of aquaporins (AQPs), a family of water channels proteins. In order to further characterize the role of AQPs in this process, an in vitro cell system with retained polarity and expression of AQPs and relevant solute transporters involved in bile formation is highly desirable. The WIF-B cell line is a highly differentiated and polarized rat hepatoma/human fibroblast ...

  10. Genome-Wide Gene Expression Profiling Revealed a Critical Role for GATA3 in the Maintenance of the Th2 Cell Identity.

    Directory of Open Access Journals (Sweden)

    Tetsuya Sasaki

    Full Text Available Functionally polarized CD4+ T helper (Th cells such as Th1, Th2 and Th17 cells are central to the regulation of acquired immunity. However, the molecular mechanisms governing the maintenance of the polarized functions of Th cells remain unclear. GATA3, a master regulator of Th2 cell differentiation, initiates the expressions of Th2 cytokine genes and other Th2-specific genes. GATA3 also plays important roles in maintaining Th2 cell function and in continuous chromatin remodeling of Th2 cytokine gene loci. However, it is unclear whether continuous expression of GATA3 is required to maintain the expression of various other Th2-specific genes. In this report, genome-wide DNA gene expression profiling revealed that GATA3 expression is critical for the expression of a certain set of Th2-specific genes. We demonstrated that GATA3 dependency is reduced for some Th2-specific genes in fully developed Th2 cells compared to that observed in effector Th2 cells, whereas it is unchanged for other genes. Moreover, effects of a loss of GATA3 expression in Th2 cells on the expression of cytokine and cytokine receptor genes were examined in detail. A critical role of GATA3 in the regulation of Th2-specific gene expression is confirmed in in vivo generated antigen-specific memory Th2 cells. Therefore, GATA3 is required for the continuous expression of the majority of Th2-specific genes involved in maintaining the Th2 cell identity.

  11. The blood-brain barrier induces differentiation of migrating monocytes into Th17-polarizing dendritic cells.

    Science.gov (United States)

    Ifergan, Igal; Kébir, Hania; Bernard, Monique; Wosik, Karolina; Dodelet-Devillers, Aurore; Cayrol, Romain; Arbour, Nathalie; Prat, Alexandre

    2008-03-01

    Trafficking of antigen-presenting cells into the CNS is essential for lymphocyte reactivation within the CNS compartment. Although perivascular dendritic cells found in inflammatory lesions are reported to polarize naive CD4+ T lymphocytes into interleukin-17-secreting-cells, the origin of those antigen-presenting cells remains controversial. We demonstrate that a subset of CD14+ monocytes migrate across the inflamed human blood-brain barrier (BBB) and differentiate into CD83+CD209+ dendritic cells under the influence of BBB-secreted transforming growth factor-beta and granulocyte-macrophage colony-stimulating factor. We also demonstrate that these dendritic cells secrete interleukin-12p70, transforming growth factor-beta and interleukin-6 and promote the proliferation and expansion of distinct populations of interferon-gamma-secreting Th1 and interleukin-17-secreting Th17 CD4+ T lymphocytes. We further confirmed the abundance of such dendritic cells in situ, closely associated with microvascular BBB-endothelial cells within acute multiple sclerosis lesions, as well as a significant number of CD4+ interleukin-17+ T lymphocytes in the perivascular infiltrate. Our data support the notion that functional perivascular myeloid CNS dendritic cells arise as a consequence of migration of CD14+ monocytes across the human BBB, through the concerted actions of BBB-secreted transforming growth factor-beta and granulocyte-macrophage colony-stimulating factor. PMID:18156156

  12. Developmental regulation of planar cell polarity and hair-bundle morphogenesis in auditory hair cells: lessons from human and mouse genetics.

    Science.gov (United States)

    Lu, Xiaowei; Sipe, Conor W

    2016-01-01

    Hearing loss is the most common and costly sensory defect in humans and genetic causes underlie a significant proportion of affected individuals. In mammals, sound is detected by hair cells (HCs) housed in the cochlea of the inner ear, whose function depends on a highly specialized mechanotransduction organelle, the hair bundle. Understanding the factors that regulate the development and functional maturation of the hair bundle is crucial for understanding the pathophysiology of human deafness. Genetic analysis of deafness genes in animal models, together with complementary forward genetic screens and conditional knock-out mutations in essential genes, have provided great insights into the molecular machinery underpinning hair-bundle development and function. In this review, we highlight recent advances in our understanding of hair-bundle morphogenesis, with an emphasis on the molecular pathways governing hair-bundle polarity and orientation. We next discuss the proteins and structural elements important for hair-cell mechanotransduction as well as hair-bundle cohesion and maintenance. In addition, developmental signals thought to regulate tonotopic features of HCs are introduced. Finally, novel approaches that complement classic genetics for studying the molecular etiology of human deafness are presented. WIREs Dev Biol 2016, 5:85-101. doi: 10.1002/wdev.202 For further resources related to this article, please visit the WIREs website. PMID:26265594

  13. Gravistimulation changes expression of genes encoding putative carrier proteins of auxin polar transport in etiolated pea epicotyls

    Science.gov (United States)

    Hoshino, T.; Hitotsubashi, R.; Miyamoto, K.; Tanimoto, E.; Ueda, J.

    STS-95 space experiment has showed that auxin polar transport in etiolated epicotyls of pea (Pisum sativum L. cv. Alaska) seedlings is controlled by gravistimulation. In Arabidopsis thaliana auxin polar transport has considered to be regulated by efflux and influx carrier proteins in plasma membranes, AtPIN1 and AtAUX1, respectively. In order to know how gravistimuli control auxin polar transport in etiolated pea epicotyls at molecular levels, strenuous efforts have been made, resulting in successful isolation of full-length cDNAs of a putative auxin efflux and influx carriers, PsPIN2 and PsAUX1, respectively. Significantly high levels in homology were found on nucleotide and deduced amino acid sequences among PsPIN2, PsPIN1 (accession no. AY222857, Chawla and DeMason, 2003) and AtPINs, and also among PsAUX1, AtAUX1 and their related genes. Phylogenetic analyses based on the deduced amino acid sequences revealed that PsPIN2 belonged to a subclade including AtPIN3, AtPIN4 relating to lateral transport of auxin, while PsPIN1 belonged to the same clade as AtPIN1 relating to auxin polar transport. In the present study, we examined the effects of gravistimuli on the expression of PsPINs and PsAUX1 in etiolated pea seedlings by northern blot analysis. Expression of PsPIN1, PsPIN2 and PsAUX1 in hook region of 3.5-d-old etiolated pea seedlings grown under simulated microgravity conditions on a 3-D clinostat increased as compared with that of the seedlings grown under 1 g conditions. On the other hand, that of PsPIN1 and PsAUX1 in the 1st internode region under simulated microgravity conditions on a 3-D clinostat also increased, while that of PsPIN2 was affected little. These results suggest that expression of PsPIN1, PsPIN2 and PsAUX1 regulating polar/lateral transport of auxin is substantially under the control of gravity. A possible role of PsPINs and PsAUX1 of auxin polar transport in etiolated pea seedlings will also be discussed.

  14. Highly polarized Th17 cells induce EAE via a T-bet-independent mechanism

    Science.gov (United States)

    Grifka-Walk, Heather M.; Lalor, Stephen J.; Segal, Benjamin M.

    2013-01-01

    Summary In the MOG35-55 induced EAE model, autoreactive Th17 cells that accumulate in the central nervous system (CNS) acquire Th1 characteristics via a T-bet dependent mechanism. It remains to be determined whether Th17 plasticity and encephalitogenicity are causally related to each other. Here we show that IL-23 polarized Tbet−/− Th17 cells are unimpaired in either activation or proliferation, and induce higher quantities of the chemokines RANTES and CXCL2 than wildtype (WT) Th17 cells. Unlike their WT counterparts, they retain an IL-17hiIFNγneg-lo cytokine profile following adoptive transfer into syngeneic hosts. This population of highly polarized Th17 effectors is capable of mediating EAE, albeit with a milder clinical course. It has previously been reported that the signature Th1 and Th17 effector cytokines, IFNγ and IL-17, are dispensable for the development of autoimmune demyelinating disease. The current study demonstrates that the “master regulator” transcription factor, T-bet, is also not universally required for encephalitogenicity. Our results contribute to a growing body of data showing heterogeneity of myelin-reactive T cells and the independent mechanisms they employ to inflict damage to CNS tissues, complicating the search for therapeutic targets relevant across the spectrum of individuals with multiple sclerosis. PMID:23878008

  15. Effects of cell cycle noise on excitable gene circuits

    CERN Document Server

    Veliz-Cuba, Alan; Bennett, Matthew R; Josić, Krešimir; Ott, William

    2016-01-01

    We assess the impact of cell cycle noise on gene circuit dynamics. For bistable genetic switches and excitable circuits, we find that transitions between metastable states most likely occur just after cell division and that this concentration effect intensifies in the presence of transcriptional delay. We explain this concentration effect with a 3-states stochastic model. For genetic oscillators, we quantify the temporal correlations between daughter cells induced by cell division. Temporal correlations must be captured properly in order to accurately quantify noise sources within gene networks.

  16. Stem Cell Based Gene Therapy in Prostate Cancer

    OpenAIRE

    Jae Heon Kim; Hong Jun Lee; Yun Seob Song

    2014-01-01

    Current prostate cancer treatment, especially hormone refractory cancer, may create profound iatrogenic outcomes because of the adverse effects of cytotoxic agents. Suicide gene therapy has been investigated for the substitute modality for current chemotherapy because it enables the treatment targeting the cancer cells. However the classic suicide gene therapy has several profound side effects, including immune-compromised due to viral vector. Recently, stem cells have been regarded as a new ...

  17. Large Animal Models of Hematopoietic Stem Cell Gene Therapy

    OpenAIRE

    Trobridge, Grant D.; Kiem, Hans-Peter

    2010-01-01

    Large animal models have been instrumental in advancing hematopoietic stem cell (HSC) gene therapy. Here we review the advantages of large animal models, their contributions to the field of HSC gene therapy, and recent progress in this field. Several properties of human HSCs including their purification, their cell-cycle characteristics, their response to cytokines, and the proliferative demands put on them after transplantation are more similar in large animal models than in mice. Progress i...

  18. Development of gene and stem cell therapy for ocular neurodegeneration

    Institute of Scientific and Technical Information of China (English)

    Jing-Xue; Zhang; Ning-Li; Wang; Qing-Jun; Lu

    2015-01-01

    Retinal degenerative diseases pose a serious threat to eye health, but there is currently no effective treatment available. Recent years have witnessed rapid development of several cutting-edge technologies, such as gene therapy, stem cell therapy, and tissue engineering. Due to the special features of ocular structure, some of these technologies have been translated into ophthalmological clinic practice with fruitful achievements, setting a good example for other fields. This paper reviews the development of the gene and stem cell therapies in ophthalmology.

  19. Atypical Cadherin Fat1 Is Required for Lens Epithelial Cell Polarity and Proliferation but Not for Fiber Differentiation

    OpenAIRE

    Sugiyama, Yuki; Shelley, Elizabeth J.; Badouel, Caroline; McNeill, Helen; McAvoy, John W.

    2015-01-01

    Using knockout mice, we show that atypical cadherin Fat1 is essential for lens epithelial cells to maintain cell junctions, polarity, and proliferation but not for terminal fiber cell differentiation. We also report that Fat4 does not exhibit functional redundancy with Fat1 during lens development.

  20. Hox genes and mesenchymal stem cells

    OpenAIRE

    Ackema, Karin

    2008-01-01

    textabstractWe hypothesised that Hox proteins may play a role in the establishment of regional differences between different MSC isolates. The first step in understanding the potential function and regulation of Hox genes in MSC is to establish their exact expression patterns. We have mapped Hox gene expression in MSC from different sites of the body. The resulting expression profiles could then be compared to their anatomical origin and their differentiation potential. This showed that Hox g...

  1. Stromal mesenchyme cell genes of the human prostate and bladder

    Directory of Open Access Journals (Sweden)

    Pascal Laura E

    2005-12-01

    Full Text Available Abstract Background Stromal mesenchyme cells play an important role in epithelial differentiation and likely in cancer as well. Induction of epithelial differentiation is organ-specific, and the genes responsible could be identified through a comparative genomic analysis of the stromal cells from two different organs. These genes might be aberrantly expressed in cancer since cancer could be viewed as due to a defect in stromal signaling. We propose to identify the prostate stromal genes by analysis of differentially expressed genes between prostate and bladder stromal cells, and to examine their expression in prostate cancer. Methods Immunohistochemistry using antibodies to cluster designation (CD cell surface antigens was first used to characterize the stromas of the prostate and bladder. Stromal cells were prepared from either prostate or bladder tissue for cell culture. RNA was isolated from the cultured cells and analyzed by DNA microarrays. Expression of candidate genes in normal prostate and prostate cancer was examined by RT-PCR. Results The bladder stroma was phenotypically different from that of the prostate. Most notable was the presence of a layer of CD13+ cells adjacent to the urothelium. This structural feature was also seen in the mouse bladder. The prostate stroma was uniformly CD13-. A number of differentially expressed genes between prostate and bladder stromal cells were identified. One prostate gene, proenkephalin (PENK, was of interest because it encodes a hormone. Secreted proteins such as hormones and bioactive peptides are known to mediate cell-cell signaling. Prostate stromal expression of PENK was verified by an antibody raised against a PENK peptide, by RT-PCR analysis of laser-capture microdissected stromal cells, and by database analysis. Gene expression analysis showed that PENK expression was down-regulated in prostate cancer. Conclusion Our findings show that the histologically similar stromas of the prostate and

  2. Periplasmic Acid Stress Increases Cell Division Asymmetry (Polar Aging of Escherichia coli.

    Directory of Open Access Journals (Sweden)

    Michelle W Clark

    Full Text Available Under certain kinds of cytoplasmic stress, Escherichia coli selectively reproduce by distributing the newer cytoplasmic components to new-pole cells while sequestering older, damaged components in cells inheriting the old pole. This phenomenon is termed polar aging or cell division asymmetry. It is unknown whether cell division asymmetry can arise from a periplasmic stress, such as the stress of extracellular acid, which is mediated by the periplasm. We tested the effect of periplasmic acid stress on growth and division of adherent single cells. We tracked individual cell lineages over five or more generations, using fluorescence microscopy with ratiometric pHluorin to measure cytoplasmic pH. Adherent colonies were perfused continually with LBK medium buffered at pH 6.00 or at pH 7.50; the external pH determines periplasmic pH. In each experiment, cell lineages were mapped to correlate division time, pole age and cell generation number. In colonies perfused at pH 6.0, the cells inheriting the oldest pole divided significantly more slowly than the cells inheriting the newest pole. In colonies perfused at pH 7.50 (near or above cytoplasmic pH, no significant cell division asymmetry was observed. Under both conditions (periplasmic pH 6.0 or pH 7.5 the cells maintained cytoplasmic pH values at 7.2-7.3. No evidence of cytoplasmic protein aggregation was seen. Thus, periplasmic acid stress leads to cell division asymmetry with minimal cytoplasmic stress.

  3. The Wnt Frizzled Receptor MOM-5 Regulates the UNC-5 Netrin Receptor through Small GTPase-Dependent Signaling to Determine the Polarity of Migrating Cells.

    Directory of Open Access Journals (Sweden)

    Naomi Levy-Strumpf

    2015-08-01

    Full Text Available Wnt and Netrin signaling regulate diverse essential functions. Using a genetic approach combined with temporal gene expression analysis, we found a regulatory link between the Wnt receptor MOM-5/Frizzled and the UNC-6/Netrin receptor UNC-5. These two receptors play key roles in guiding cell and axon migrations, including the migration of the C. elegans Distal Tip Cells (DTCs. DTCs migrate post-embryonically in three sequential phases: in the first phase along the Antero-Posterior (A/P axis, in the second, along the Dorso-Ventral (D/V axis, and in the third, along the A/P axis. Loss of MOM-5/Frizzled function causes third phase A/P polarity reversals of the migrating DTCs. We show that an over-expression of UNC-5 causes similar DTC A/P polarity reversals and that unc-5 deficits markedly suppress the A/P polarity reversals caused by mutations in mom-5/frizzled. This implicates MOM-5/Frizzled as a negative regulator of unc-5. We provide further evidence that small GTPases mediate MOM-5's regulation of unc-5 such that one outcome of impaired function of small GTPases like CED-10/Rac and MIG-2/RhoG is an increase in unc-5 function. The work presented here demonstrates the existence of cross talk between components of the Netrin and Wnt signaling pathways and provides further insights into the way guidance signaling mechanisms are integrated to orchestrate directed cell migration.

  4. New circular polarization selective surface concepts based on the Pierrot cell using printed circuit technology

    Science.gov (United States)

    Lopez, Humberto Israel

    This M.A.Sc. thesis focuses on finding an alternative method of constructing a circular polarization selective surface (CPSS) based on the Pierrot cell using the standard printed circuit technology. This technique uses a folded flexible substrate, which enables the implementation of the 3D Pierrot cells on a single metal layer defined with precision printed circuit board techniques, without the need for metalized via holes. Different topologies of the CPSS are analyzed in order to make the CPSS more efficient in terms of bandwidth and independence on the direction of propagation of the incident wave. A left-hand CPSS is designed to illustrate the benefits of the proposed approach. The first approach is a simple Pierrot unit cell CPSS which is optimized to have good reflection and transmission coefficients. A prototype is built and then characterized in a test bench operating in the K-band. For the fabricated prototype, the transmission coefficients of plane waves at normal incidence in the right-hand and the left-hand circular polarizations are --0.48 dB and --24 dB respectively. The bandwidth for which the transmission coefficient of the incident left-handed incident wave is greater than --3 dB was of 17.6%. These results are in good agreement with simulations results obtained with HFSS. A second variant considered is a Pierrot cell with a series load in the middle segment. With this cell it is possible to equalize the frequencies giving a better operation in the right- and left-handed circular polarized waves. There is an improvement for the co-pol to cross-pol ratio for the RHCP waves of 10 dB at 20 GHz. The added load does not affect the performance for the left-hand circular polarization, as expected. The third modification is a Pierrot cell at 90 degrees. This cell is designed to allow the combination of two Pierrot cells working at different frequencies on the same substrate in order to increase the frequency bandwidth of the CPSS. Unfortunately, the axial

  5. Cell and gene therapy in Australia.

    Science.gov (United States)

    Martiniello-Wilks, R; Rasko, J E J

    2007-01-01

    The expansion of human cells to produce cell therapeutic products for the treatment of disease is, with few exceptions, an experimental therapy. Because cell therapies involve a biological product, often with some genetic or other modification, they require extensive pre-clinical research and development. Cell therapy production processes and premises require licensing by the Therapeutic Goods Administration. In this review, timed to coincide with the international meetings of the ISCT and ISSCR in Australia, we describe some promising cell therapies currently under development. PMID:17464751

  6. Impact of methoxyacetic acid on mouse Leydig cell gene expression

    Directory of Open Access Journals (Sweden)

    Waxman David J

    2010-06-01

    Full Text Available Abstract Background Methoxyacetic acid (MAA is the active metabolite of the widely used industrial chemical ethylene glycol monomethyl ether, which is associated with various developmental and reproductive toxicities, including neural toxicity, blood and immune disorders, limb degeneration and testicular toxicity. Testicular toxicity is caused by degeneration of germ cells in association with changes in gene expression in both germ cells and Sertoli cells of the testis. This study investigates the impact of MAA on gene expression in testicular Leydig cells, which play a critical role in germ cell survival and male reproductive function. Methods Cultured mouse TM3 Leydig cells were treated with MAA for 3, 8, and 24 h and changes in gene expression were monitored by genome-wide transcriptional profiling. Results A total of 3,912 MAA-responsive genes were identified. Ingenuity Pathway analysis identified reproductive system disease, inflammatory disease and connective tissue disorder as the top biological functions affected by MAA. The MAA-responsive genes were classified into 1,366 early responders, 1,387 mid-responders, and 1,138 late responders, based on the time required for MAA to elicit a response. Analysis of enriched functional clusters for each subgroup identified 106 MAA early response genes involved in transcription regulation, including 32 genes associated with developmental processes. 60 DNA-binding proteins responded to MAA rapidly but transiently, and may contribute to the downstream effects of MAA seen for many mid and late response genes. Genes within the phosphatidylinositol/phospholipase C/calcium signaling pathway, whose activity is required for potentiation of nuclear receptor signaling by MAA, were also enriched in the set of early MAA response genes. In contrast, many of the genes responding to MAA at later time points encode membrane proteins that contribute to cell adhesion and membrane signaling. Conclusions These findings

  7. Engineering Synthetic Gene Circuits in Living Cells with CRISPR Technology.

    Science.gov (United States)

    Jusiak, Barbara; Cleto, Sara; Perez-Piñera, Pablo; Lu, Timothy K

    2016-07-01

    One of the goals of synthetic biology is to build regulatory circuits that control cell behavior, for both basic research purposes and biomedical applications. The ability to build transcriptional regulatory devices depends on the availability of programmable, sequence-specific, and effective synthetic transcription factors (TFs). The prokaryotic clustered regularly interspaced short palindromic repeat (CRISPR) system, recently harnessed for transcriptional regulation in various heterologous host cells, offers unprecedented ease in designing synthetic TFs. We review how CRISPR can be used to build synthetic gene circuits and discuss recent advances in CRISPR-mediated gene regulation that offer the potential to build increasingly complex, programmable, and efficient gene circuits in the future. PMID:26809780

  8. Screening for the Most Suitable Reference Genes for Gene Expression Studies in Equine Milk Somatic Cells.

    Directory of Open Access Journals (Sweden)

    Jakub Cieslak

    Full Text Available Apart from the well-known role of somatic cell count as a parameter reflecting the inflammatory status of the mammary gland, the composition of cells isolated from milk is considered as a valuable material for gene expression studies in mammals. Due to its unique composition, in recent years an increasing interest in mare's milk consumption has been observed. Thus, investigating the genetic background of horse's milk variability presents and interesting study model. Relying on 39 milk samples collected from mares representing three breeds (Polish Primitive Horse, Polish Cold-blooded Horse, Polish Warmblood Horse we aimed to investigate the utility of equine milk somatic cells as a source of mRNA and to screen the best reference genes for RT-qPCR using geNorm and NormFinder algorithms. The results showed that despite relatively low somatic cell counts in mare's milk, the amount and the quality of the extracted RNA are sufficient for gene expression studies. The analysis of the utility of 7 potential reference genes for RT-qPCR experiments for the normalization of equine milk somatic cells revealed some differences between the outcomes of the applied algorithms, although in both cases the KRT8 and TOP2B genes were pointed as the most stable. Analysis by geNorm showed that the combination of 4 reference genes (ACTB, GAPDH, TOP2B and KRT8 is required for apropriate RT-qPCR experiments normalization, whereas NormFinder algorithm pointed the combination of KRT8 and RPS9 genes as the most suitable. The trial study of the relative transcript abundance of the beta-casein gene with the use of various types and numbers of internal control genes confirmed once again that the selection of proper reference gene combinations is crucial for the final results of each real-time PCR experiment.

  9. Flat leaf formation realized by cell-division control and mutual recessive gene regulation.

    Science.gov (United States)

    Hayakawa, Yoshinori; Tachikawa, Masashi; Mochizuki, Atsushi

    2016-09-01

    Most of the land plants generally have dorsoventrally flat leaves, maximizing the surface area of both upper (adaxial) side and lower (abaxial) side. The former is specialized for light capturing for photosynthesis and the latter is specialized for gas exchange. From findings of molecular genetics, it has been considered that the coupled dynamics between tissue morphogenesis and gene regulation for cell identity is responsible for making flat leaves. The hypothesis claims that a flat leaf is generated under two assumptions, (i) two mutually recessive groups of genes specify adaxial and abaxial sides of a leaf, (ii) cell divisions are induced at the limited region in the leaf margin where both of two groups are expressed. We examined the plausibility and possibility of this hypothesis from the dynamical point of view. We studied a mathematical model where two processes are coupled, tissue morphogenesis induced by cell division and deformation, and dynamics of gene regulations. From the analysis of the model we found that the classically believed hypothesis is not sufficient to generate flat leaves with high probability. We examined several different modifications and revision of the model. Then we found that a simple additional rule of polarized cell division facilitates flat leaf formation. The result of our analysis gives prediction of possible mechanism, which can be easily verified in experiments. PMID:27287339

  10. Influence of polar solvents on photovoltaic performance of Monascusred dye-sensitized solar cell

    Science.gov (United States)

    Lee, Jae Wook; Kim, Tae Young; Ko, Hyun Seok; Han, Shin; Lee, Suk-Ho; Park, Kyung Hee

    Dye-sensitized solar cells (DSSCs) were assembled using natural dyes extracted from Monascus red pigment as a sensitizer. In this work, we studied the adsorption characteristics for harvesting sunlight and the electrochemical behavior for electron transfer in Monascus red DSSC using different solvents. The effect of polar aprotic and protic solvents including water, ethanol, and dimethylsulfoxide (DMSO) used in the sensitization process was investigated for the improvement in conversion efficiency of a cell. As for the Monascus red dye-sensitized electrode in DMSO solvent, the solar cell yields a short-circuit current density (Jsc) of 1.23 mA/cm2, a photovoltage (Voc) of 0.75 V, and a fill factor of 0.72, corresponding to an energy conversion efficiency (η) of 0.66%.

  11. Polarization of calcium signaling and fluid secretion in salivary gland cells.

    Science.gov (United States)

    Ambudkar, I S

    2012-01-01

    The secretion of fluid, electrolytes, and protein by exocrine gland acinar cells is a vectorial process that requires the coordinated regulation of multiple channel and transporter proteins, signaling components, as well as mechanisms involved in vesicular fusion and water transport. Most critical in this is the regulation of cytosolic free [Ca(2+)] ([Ca(2+)](i)) in response to neurotransmitter stimulation. Control of [Ca(2+)](i) increase in specific regions of the cell is the main determinant of fluid and electrolyte secretion in salivary gland acinar cells as it regulates several major ion flux mechanisms as well as the water channel that are required for this process. Polarized [Ca(2+)](i) signals are also essential for protein secretion in pancreatic acinar cells. Thus, the mechanisms that generate and modulate these compartmentalized [Ca(2+)](i) signals are central to the regulation of exocrine secretion. These mechanisms include membrane receptors for neurotransmitters, intracellular Ca(2+) release channels, Ca(2+) entry channels, as well Ca(2+) as pumps and mitochondria. The spatial arrangement of proteins involved in Ca(2+) signaling is of primary significance in the generation of specific compartmentalized [Ca(2+)](i) signals. Within these domains, both local and global [Ca(2+)](i) changes are tightly controlled. Control of secretion is also dependent on the targeting of ion channels and transporters to specific domains in the cell where their regulation by [Ca(2+)](i) signals is facilitated. Together, the polarized localization of Ca(2+) signaling and secretory components drive vectorial secretion of fluid, electrolytes, and proteins in the exocrine salivary glands and pancreas. This review will discuss recent findings which have led to resolution of the molecular components underlying the spatio-temporal control of [Ca(2+)](i) signals in exocrine gland cells and their role in secretion. PMID:23061636

  12. The PDZ Protein Na+/H+ Exchanger Regulatory Factor-1 (NHERF1) Regulates Planar Cell Polarity and Motile Cilia Organization

    Science.gov (United States)

    Stolz, Donna B.; Tsang, Michael; Friedman, Peter A.; Romero, Guillermo

    2016-01-01

    Directional flow of the cerebrospinal fluid requires coordinated movement of the motile cilia of the ependymal epithelium that lines the cerebral ventricles. Here we report that mice lacking the Na+/H+ Exchanger Regulatory Factor 1 (NHERF1/Slc9a3r1, also known as EBP50) develop profound communicating hydrocephalus associated with fewer and disorganized ependymal cilia. Knockdown of NHERF1/slc9a3r1 in zebrafish embryos also causes severe hydrocephalus of the hindbrain and impaired ciliogenesis in the otic vesicle. Ultrastructural analysis did not reveal defects in the shape or organization of individual cilia. Similar phenotypes have been described in animals with deficiencies in Wnt signaling and the Planar Cell Polarity (PCP) pathway. We show that NHERF1 binds the PCP core genes Frizzled (Fzd) and Vangl. We further show that NHERF1 assembles a ternary complex with Fzd4 and Vangl2 and promotes translocation of Vangl2 to the plasma membrane, in particular to the apical surface of ependymal cells. Taken together, these results strongly support an important role for NHERF1 in the regulation of PCP signaling and the development of functional motile cilia. PMID:27055101

  13. Transcriptional Wiring of Cell Wall-Related Genes in Arabidopsis

    Institute of Scientific and Technical Information of China (English)

    Marek Mutwil; Colin Ruprecht; Federico M. Giorgi; Martin Bringmann; Bj(o)rn Usadel; Staffan Persson

    2009-01-01

    Transcriptional coordination, or co-expression, of genes may signify functional relatedness of the correspond-ing proteins. For example, several genes involved in secondary cell wall cellulose biosynthesis are co-expressed with genes engaged in the synthesis of xylan, which is a major component of the secondary cell wall. To extend these types of anal-yses, we investigated the co-expression relationships of all Carbohydrate-Active enZYmes (CAZy)-related genes for Arabidopsis thaliana. Thus, the intention was to transcriptionally link different cell wall-related processes to each other, and also to other biological functions. To facilitate easy manual inspection, we have displayed these interactions as networks and matrices, and created a web-based interface (http://aranet.mpimp-golm.mpg.de/corecarb) containing downloadable files for all the transcriptional associations.

  14. Planar Cell Polarity Protein Localization in the Secretory Ameloblasts of Rat Incisors

    OpenAIRE

    Nishikawa, Sumio; Kawamoto, Tadafumi

    2012-01-01

    The localization of the planar cell polarity proteins Vang12, frizzled-3, Vang11, and Celsr1 in the rat incisors was examined using immunocytochemistry. The results showed that Vang12 was localized at two regions of the Tomes’ processes of inner enamel–secretory ameloblasts in rat incisors: a proximal and a distal region. In contrast, frizzled-3 was localized at adherens junctions of the proximal and distal areas of inner enamel– and outer enamel–secretory ameloblasts, where N-cadherin and β-...

  15. Neurohypophysial Receptor Gene Expression by Thymic T Cell Subsets and Thymic T Cell Lymphoma Cell Lines

    Directory of Open Access Journals (Sweden)

    I. Hansenne

    2004-01-01

    transcribed in thymic epithelium, while immature T lymphocytes express functional neurohypophysial receptors. Neurohypophysial receptors belong to the G protein-linked seven-transmembrane receptor superfamily and are encoded by four distinct genes, OTR, V1R, V2R and V3R. The objective of this study was to identify the nature of neurohypophysial receptor in thymic T cell subsets purified by immunomagnetic selection, as well as in murine thymic lymphoma cell lines RL12-NP and BW5147. OTR is transcribed in all thymic T cell subsets and T cell lines, while V3R transcription is restricted to CD4+ CD8+ and CD8+ thymic cells. Neither V1R nor V2R transcripts are detected in any kind of T cells. The OTR protein was identified by immunocytochemistry on thymocytes freshly isolated from C57BL/6 mice. In murine fetal thymic organ cultures, a specific OTR antagonist does not modify the percentage of T cell subsets, but increases late T cell apoptosis further evidencing the involvement of OT/OTR signaling in the control of T cell proliferation and survival. According to these data, OTR and V3R are differentially expressed during T cell ontogeny. Moreover, the restriction of OTR transcription to T cell lines derived from thymic lymphomas may be important in the context of T cell leukemia pathogenesis and treatment.

  16. Potential of HSV-TK gene in gene-radiotherapy for human bladder carcinoma cells

    International Nuclear Information System (INIS)

    Objective: To explore the killing effect of retrovirus-mediated HSV-TK/GCV system combined with radiotherapy on human bladder carcinoma cells. Methods: By using retrovirus-mediated gene transfer technique, hygromycin phosphotransferase-thymidine kinase fusion gene (HyTK) was transferred into EJ bladder carcinoma cells. Antitumor effects were observed after irradiation with 60Co γ-rays as well as gancyclovir treatment. Results: EJ bladder carcinoma cells transduced with HyTK gene and treated with low concentrations of gancyclovir, which alone showed little cytotoxicity, were highly sensitive to radiation. The sensitizer enhancement ratio (SER) was 1.36. Conclusion: The combination of retrovirus-mediated HyTK/GCV gene therapy with standard radiation therapy might improve the therapeutic effectiveness for bladder carcinoma in clinical practice

  17. Experiments on Gene Transferring to Primary Hematopoietic Cells by Liposome

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    Liposomes have showed many advantages in mediating exogenous gene into many cell types in vitro and in vivo. But few data are available concerning gene transfer into hematopoietic cells. In this report, we described two-marker genes (Neo R and Lac Z) co-transferred into hematopoietic cells of human and mouse by using liposome in vitro. The efficiency of gene transfer was tested by Xgal staining and observation of colony formation. The X-gal blue staining rate of transduced cells was about (13.33±2. 68) % in human and about (16. 28±2.95) % in mouse without G418 selection. After G418 selection, the blue cell rate was (46. 06±3.47)%in human and (43. 45±4. 1) % in mouse, which were markedly higher than those before selection, suggesting that high-efficiency gene transfer and expression could be attained in primary hematopoietic cells using this easy and harmless transduction protocol. At the same time, this protocol provided experimental data for clinicians to investigate the biology of marrow reconstitution and trace the origin of relapse after autologous bone marrow transplantation for the patients with leukemia.

  18. Isolating human DNA repair genes using rodent-cell mutants

    International Nuclear Information System (INIS)

    The DNA repair systems of rodent and human cells appear to be at least as complex genetically as those in lower eukaryotes and bacteria. The use of mutant lines of rodent cells as a means of identifying human repair genes by functional complementation offers a new approach toward studying the role of repair in mutagenesis and carcinogenesis. In each of six cases examined using hybrid cells, specific human chromosomes have been identified that correct CHO cell mutations affecting repair of damage from uv or ionizing radiations. This finding suggests that both the repair genes and proteins may be virtually interchangeable between rodent and human cells. Using cosmid vectors, human repair genes that map to chromosome 19 have cloned as functional sequences: ERCC2 and XRCC1. ERCC1 was found to have homology with the yeast excision repair gene RAD10. Transformants of repair-deficient cell lines carrying the corresponding human gene show efficient correction of repair capacity by all criteria examined. 39 refs., 1 fig., 1 tab

  19. Exine dehiscing induces rape microspore polarity, which results in different daughter cell fate and fixes the apical–basal axis of the embryo

    Science.gov (United States)

    Tang, Xingchun; Liu, Yuan; Sun, Meng-xiang

    2013-01-01

    The roles of cell polarity and the first asymmetric cell division during early embryogenesis in apical–basal cell fate determination remain unclear. Previously, a novel Brassica napus microspore embryogenesis system was established, by which rape exine-dehisced microspores were induced by physical stress. Unlike traditional microspore culture, cell polarity and subsequent asymmetric division appeared in the exine-dehisced microspore, which finally developed into a typical embryo with a suspensor. Further studies indicated that polarity is critical for apical–basal cell fate determination and suspensor formation. However, the pattern of the first division was not only determined by cell polarity but was also regulated by the position of the ruptured exine. The first division could be equal or unequal, with its orientation essentially perpendicular to the polar axis. In both types of cell division, the two daughter cells could have different cell fates and give rise to an embryo with a suspensor, similar to zygotic apical–basal cell differentiation. The alignment of the two daughter cells is consistent with the orientation of the apical–basal axis of future embryonic development. Thus, the results revealed that exine dehiscing induces rape microspore polarization, and this polarity results in a different cell fate and fixes the apical–basal axis of embryogenesis, but is uncoupled from cell asymmetric division. The present study demonstrated the relationships among cell polarity, asymmetric cell division, and cell fate determination in early embryogenesis. PMID:23162119

  20. Exine dehiscing induces rape microspore polarity, which results in different daughter cell fate and fixes the apical-basal axis of the embryo.

    Science.gov (United States)

    Tang, Xingchun; Liu, Yuan; He, Yuqing; Ma, Ligang; Sun, Meng-Xiang

    2013-01-01

    The roles of cell polarity and the first asymmetric cell division during early embryogenesis in apical-basal cell fate determination remain unclear. Previously, a novel Brassica napus microspore embryogenesis system was established, by which rape exine-dehisced microspores were induced by physical stress. Unlike traditional microspore culture, cell polarity and subsequent asymmetric division appeared in the exine-dehisced microspore, which finally developed into a typical embryo with a suspensor. Further studies indicated that polarity is critical for apical-basal cell fate determination and suspensor formation. However, the pattern of the first division was not only determined by cell polarity but was also regulated by the position of the ruptured exine. The first division could be equal or unequal, with its orientation essentially perpendicular to the polar axis. In both types of cell division, the two daughter cells could have different cell fates and give rise to an embryo with a suspensor, similar to zygotic apical-basal cell differentiation. The alignment of the two daughter cells is consistent with the orientation of the apical-basal axis of future embryonic development. Thus, the results revealed that exine dehiscing induces rape microspore polarization, and this polarity results in a different cell fate and fixes the apical-basal axis of embryogenesis, but is uncoupled from cell asymmetric division. The present study demonstrated the relationships among cell polarity, asymmetric cell division, and cell fate determination in early embryogenesis. PMID:23162119

  1. Sds22, a PP1 phosphatase regulatory subunit, regulates epithelial cell polarity and shape [Sds22 in epithelial morphology

    OpenAIRE

    Sung Hsin-Ho; Fletcher Georgina; Hidalgo Cristina; Grusche Felix A; Sahai Erik; Thompson Barry J

    2009-01-01

    Abstract Background How epithelial cells adopt their particular polarised forms is poorly understood. In a screen for genes regulating epithelial morphology in Drosophila, we identified sds22, a conserved gene previously characterised in yeast. Results In the columnar epithelia of imaginal discs or follicle cells, mutation of sds22 causes contraction of cells along their apical-basal axis, resulting in a more cuboidal morphology. In addition, the mutant cells can also display altered cell pol...

  2. Expression of segment polarity genes in brachiopods supports a non-segmental ancestral role of engrailed for bilaterians.

    Science.gov (United States)

    Vellutini, Bruno C; Hejnol, Andreas

    2016-01-01

    The diverse and complex developmental mechanisms of segmentation have been more thoroughly studied in arthropods, vertebrates and annelids-distantly related animals considered to be segmented. Far less is known about the role of "segmentation genes" in organisms that lack a segmented body. Here we investigate the expression of the arthropod segment polarity genes engrailed, wnt1 and hedgehog in the development of brachiopods-marine invertebrates without a subdivided trunk but closely related to the segmented annelids. We found that a stripe of engrailed expression demarcates the ectodermal boundary that delimits the anterior region of Terebratalia transversa and Novocrania anomala embryos. In T. transversa, this engrailed domain is abutted by a stripe of wnt1 expression in a pattern similar to the parasegment boundaries of insects-except for the expression of hedgehog, which is restricted to endodermal tissues of the brachiopod embryos. We found that pax6 and pax2/5/8, putative regulators of engrailed, also demarcate the anterior boundary in the two species, indicating these genes might be involved in the anterior patterning of brachiopod larvae. In a comparative phylogenetic context, these findings suggest that bilaterians might share an ancestral, non-segmental domain of engrailed expression during early embryogenesis. PMID:27561213

  3. Poldip2 controls vascular smooth muscle cell migration by regulating focal adhesion turnover and force polarization

    Science.gov (United States)

    Datla, Srinivasa Raju; McGrail, Daniel J.; Vukelic, Sasa; Huff, Lauren P.; Lyle, Alicia N.; Pounkova, Lily; Lee, Minyoung; Seidel-Rogol, Bonnie; Khalil, Mazen K.; Hilenski, Lula L.; Terada, Lance S.; Dawson, Michelle R.; Lassègue, Bernard

    2014-01-01

    Polymerase-δ-interacting protein 2 (Poldip2) interacts with NADPH oxidase 4 (Nox4) and regulates migration; however, the precise underlying mechanisms are unclear. Here, we investigated the role of Poldip2 in focal adhesion turnover, as well as traction force generation and polarization. Poldip2 overexpression (AdPoldip2) in vascular smooth muscle cells (VSMCs) impairs PDGF-induced migration and induces a characteristic phenotype of long cytoplasmic extensions. AdPoldip2 also prevents the decrease in spreading and increased aspect ratio observed in response to PDGF and slightly impairs cell contraction. Moreover, AdPoldip2 blocks focal adhesion dissolution and sustains H2O2 levels in focal adhesions, whereas Poldip2 knockdown (siPoldip2) significantly decreases the number of focal adhesions. RhoA activity is unchanged when focal adhesion dissolution is stimulated in control cells but increases in AdPoldip2-treated cells. Inhibition of RhoA blocks Poldip2-mediated attenuation of focal adhesion dissolution, and overexpression of RhoA or focal adhesion kinase (FAK) reverses the loss of focal adhesions induced by siPoldip2, indicating that RhoA and FAK mediate the effect of Poldip2 on focal adhesions. Nox4 silencing prevents focal adhesion stabilization by AdPoldip2 and induces a phenotype similar to siPoldip2, suggesting a role for Nox4 in Poldip2-induced focal adhesion stability. As a consequence of impaired focal adhesion turnover, PDGF-treated AdPoldip2 cells are unable to reduce and polarize traction forces, a necessary first step in migration. These results implicate Poldip2 in VSMC migration via regulation of focal adhesion turnover and traction force generation in a Nox4/RhoA/FAK-dependent manner. PMID:25063792

  4. Normal and tumor-derived myoepithelial cells differ in their ability to interact with luminal breast epithelial cells for polarity and basement membrane deposition

    Energy Technology Data Exchange (ETDEWEB)

    Gudjonsson, Thorarinn; Ronnov-Jessen, Lone; Villadsen, Rene; Rank, Fritz; Bissell, Mina J.; Petersen, Ole William

    2001-10-04

    The signals that determine the correct polarity of breast epithelial structures in vivo are not understood. We have shown previously that luminal epithelial cells can be polarized when cultured within a reconstituted basement membrane gel. We reasoned that such cues in vivo may be given by myoepithelial cells. Accordingly, we used an assay where luminal epithelial cells are incorrectly polarized to test this hypothesis. We show that culturing human primary luminal epithelial cells within collagen-I gels leads to formation of structures with no lumina and with reverse polarity as judged by dual stainings for sialomucin, epithelial specific antigen or occludin. No basement membrane is deposited, and {beta}4-integrin staining is negative. Addition of purified human myoepithelial cells isolated from normal glands corrects the inverse polarity, and leads to formation of double-layered acini with central lumina. Among the laminins present in the human breast basement membrane (laminin-1, -5 and -10/11), laminin-1 was unique in its ability to substitute for myoepithelial cells in polarity reversal. Myoepithelial cells were purified also from four different breast cancer sources including a biphasic cell line. Three out of four samples either totally lacked the ability to interact with luminal epithelial cells, or conveyed only correction of polarity in a fraction of acini. This behavior was directly related to the ability of the tumor myoepithelial cells to produce {alpha}-1 chain of laminin. In vivo, breast carcinomas were either negative for laminin-1 (7/12 biopsies) or showed a focal, fragmented deposition of a less intensely stained basement membrane (5/12 biopsies). Dual staining with myoepithelial markers revealed that tumorassociated myoepithelial cells were either negative or weakly positive for expression of laminin-1, establishing a strong correlation between loss of laminin-1 and breast cancer. We conclude that the double-layered breast acinus may be

  5. Gene function in early mouse embryonic stem cell differentiation

    Directory of Open Access Journals (Sweden)

    Campbell Pearl A

    2007-03-01

    Full Text Available Abstract Background Little is known about the genes that drive embryonic stem cell differentiation. However, such knowledge is necessary if we are to exploit the therapeutic potential of stem cells. To uncover the genetic determinants of mouse embryonic stem cell (mESC differentiation, we have generated and analyzed 11-point time-series of DNA microarray data for three biologically equivalent but genetically distinct mESC lines (R1, J1, and V6.5 undergoing undirected differentiation into embryoid bodies (EBs over a period of two weeks. Results We identified the initial 12 hour period as reflecting the early stages of mESC differentiation and studied probe sets showing consistent changes of gene expression in that period. Gene function analysis indicated significant up-regulation of genes related to regulation of transcription and mRNA splicing, and down-regulation of genes related to intracellular signaling. Phylogenetic analysis indicated that the genes showing the largest expression changes were more likely to have originated in metazoans. The probe sets with the most consistent gene changes in the three cell lines represented 24 down-regulated and 12 up-regulated genes, all with closely related human homologues. Whereas some of these genes are known to be involved in embryonic developmental processes (e.g. Klf4, Otx2, Smn1, Socs3, Tagln, Tdgf1, our analysis points to others (such as transcription factor Phf21a, extracellular matrix related Lama1 and Cyr61, or endoplasmic reticulum related Sc4mol and Scd2 that have not been previously related to mESC function. The majority of identified functions were related to transcriptional regulation, intracellular signaling, and cytoskeleton. Genes involved in other cellular functions important in ESC differentiation such as chromatin remodeling and transmembrane receptors were not observed in this set. Conclusion Our analysis profiles for the first time gene expression at a very early stage of m

  6. Gene array identification of Ipf1/Pdx1-/- regulated genes in pancreatic progenitor cells

    Directory of Open Access Journals (Sweden)

    Rydén Patrik

    2007-11-01

    Full Text Available Abstract Background The homeodomain transcription factor IPF1/PDX1 exerts a dual role in the pancreas; Ipf1/Pdx1 global null mutants fail to develop a pancreas whereas conditional inactivation of Ipf1/Pdx1 in β-cells leads to impaired β-cell function and diabetes. Although several putative target genes have been linked to the β-cell function of Ipf1/Pdx1, relatively little is known with respect to genes regulated by IPF1/PDX1 in early pancreatic progenitor cells. Results Microarray analyses identified a total of 111 genes that were differentially expressed in e10.5 pancreatic buds of Ipf1/Pdx1-/- embryos. The expression of one of these, Spondin 1, which encodes an extracellular matrix protein, has not previously been described in the pancreas. Quantitative real-time RT-PCR analyses and immunohistochemical analyses also revealed that the expression of FgfR2IIIb, that encodes the receptor for FGF10, was down-regulated in Ipf1/Pdx1-/- pancreatic progenitor cells. Conclusion This microarray analysis has identified a number of candidate genes that are differentially expressed in Ipf1/Pdx1-/- pancreatic buds. Several of the differentially expressed genes were known to be important for pancreatic progenitor cell proliferation and differentiation whereas others have not previously been associated with pancreatic development.

  7. Rapid paracellular transmigration of Campylobacter jejuni across polarized epithelial cells without affecting TER: role of proteolytic-active HtrA cleaving E-cadherin but not fibronectin

    Directory of Open Access Journals (Sweden)

    Boehm Manja

    2012-04-01

    Full Text Available Abstract Background Campylobacter jejuni is one of the most important bacterial pathogens causing food-borne illness worldwide. Crossing the intestinal epithelial barrier and host cell entry by C. jejuni is considered the primary reason of damage to the intestinal tissue, but the molecular mechanisms as well as major bacterial and host cell factors involved in this process are still widely unclear. Results In the present study, we characterized the serine protease HtrA (high-temperature requirement A of C. jejuni as a secreted virulence factor with important proteolytic functions. Infection studies and in vitro cleavage assays showed that C. jejuni’s HtrA triggers shedding of the extracellular E-cadherin NTF domain (90 kDa of non-polarised INT-407 and polarized MKN-28 epithelial cells, but fibronectin was not cleaved as seen for H. pylori’s HtrA. Deletion of the htrA gene in C. jejuni or expression of a protease-deficient S197A point mutant did not lead to loss of flagella or reduced bacterial motility, but led to severe defects in E-cadherin cleavage and transmigration of the bacteria across polarized MKN-28 cell layers. Unlike other highly invasive pathogens, transmigration across polarized cells by wild-type C. jejuni is highly efficient and is achieved within a few minutes of infection. Interestingly, E-cadherin cleavage by C. jejuni occurs in a limited fashion and transmigration required the intact flagella as well as HtrA protease activity, but does not reduce transepithelial electrical resistance (TER as seen with Salmonella, Shigella, Listeria or Neisseria. Conclusion These results suggest that HtrA-mediated E-cadherin cleavage is involved in rapid crossing of the epithelial barrier by C. jejuni via a very specific mechanism using the paracellular route to reach basolateral surfaces, but does not cleave the fibronectin receptor which is necessary for cell entry.

  8. Rapid paracellular transmigration of Campylobacter jejuni across polarized epithelial cells without affecting TER: role of proteolytic-active HtrA cleaving E-cadherin but not fibronectin

    LENUS (Irish Health Repository)

    Boehm, Manja

    2012-04-25

    AbstractBackgroundCampylobacter jejuni is one of the most important bacterial pathogens causing food-borne illness worldwide. Crossing the intestinal epithelial barrier and host cell entry by C. jejuni is considered the primary reason of damage to the intestinal tissue, but the molecular mechanisms as well as major bacterial and host cell factors involved in this process are still widely unclear.ResultsIn the present study, we characterized the serine protease HtrA (high-temperature requirement A) of C. jejuni as a secreted virulence factor with important proteolytic functions. Infection studies and in vitro cleavage assays showed that C. jejuni’s HtrA triggers shedding of the extracellular E-cadherin NTF domain (90 kDa) of non-polarised INT-407 and polarized MKN-28 epithelial cells, but fibronectin was not cleaved as seen for H. pylori’s HtrA. Deletion of the htrA gene in C. jejuni or expression of a protease-deficient S197A point mutant did not lead to loss of flagella or reduced bacterial motility, but led to severe defects in E-cadherin cleavage and transmigration of the bacteria across polarized MKN-28 cell layers. Unlike other highly invasive pathogens, transmigration across polarized cells by wild-type C. jejuni is highly efficient and is achieved within a few minutes of infection. Interestingly, E-cadherin cleavage by C. jejuni occurs in a limited fashion and transmigration required the intact flagella as well as HtrA protease activity, but does not reduce transepithelial electrical resistance (TER) as seen with Salmonella, Shigella, Listeria or Neisseria.ConclusionThese results suggest that HtrA-mediated E-cadherin cleavage is involved in rapid crossing of the epithelial barrier by C. jejuni via a very specific mechanism using the paracellular route to reach basolateral surfaces, but does not cleave the fibronectin receptor which is necessary for cell entry.

  9. Classification of dendritic cell phenotypes from gene expression data

    Directory of Open Access Journals (Sweden)

    Zolezzi Francesca

    2011-08-01

    Full Text Available Abstract Background The selection of relevant genes for sample classification is a common task in many gene expression studies. Although a number of tools have been developed to identify optimal gene expression signatures, they often generate gene lists that are too long to be exploited clinically. Consequently, researchers in the field try to identify the smallest set of genes that provide good sample classification. We investigated the genome-wide expression of the inflammatory phenotype in dendritic cells. Dendritic cells are a complex group of cells that play a critical role in vertebrate immunity. Therefore, the prediction of the inflammatory phenotype in these cells may help with the selection of immune-modulating compounds. Results A data mining protocol was applied to microarray data for murine cell lines treated with various inflammatory stimuli. The learning and validation data sets consisted of 155 and 49 samples, respectively. The data mining protocol reduced the number of probe sets from 5,802 to 10, then from 10 to 6 and finally from 6 to 3. The performances of a set of supervised classification models were compared. The best accuracy, when using the six following genes --Il12b, Cd40, Socs3, Irgm1, Plin2 and Lgals3bp-- was obtained by Tree Augmented Naïve Bayes and Nearest Neighbour (91.8%. Using the smallest set of three genes --Il12b, Cd40 and Socs3-- the performance remained satisfactory and the best accuracy was with Support Vector Machine (95.9%. These data mining models, using data for the genes Il12b, Cd40 and Socs3, were validated with a human data set consisting of 27 samples. Support Vector Machines (71.4% and Nearest Neighbour (92.6% gave the worst performances, but the remaining models correctly classified all the 27 samples. Conclusions The genes selected by the data mining protocol proposed were shown to be informative for discriminating between inflammatory and steady-state phenotypes in dendritic cells. The

  10. Identification of astrocytoma associated genes including cell surface markers

    International Nuclear Information System (INIS)

    Despite intense effort the treatment options for the invasive astrocytic tumors are still limited to surgery and radiation therapy, with chemotherapy showing little or no increase in survival. The generation of Serial Analysis of Gene Expression (SAGE) profiles is expected to aid in the identification of astrocytoma-associated genes and highly expressed cell surface genes as molecular therapeutic targets. SAGE tag counts can be easily added to public expression databases and quickly disseminated to research efforts worldwide. We generated and analyzed the SAGE transcription profiles of 25 primary grade II, III and IV astrocytomas [1]. These profiles were produced as part of the Cancer Genome Anatomy Project's SAGE Genie [2], and were used in an in silico search for candidate therapeutic targets by comparing astrocytoma to normal brain transcription. Real-time PCR and immunohistochemistry were used for the validation of selected candidate target genes in 2 independent sets of primary tumors. A restricted set of tumor-associated genes was identified for each grade that included genes not previously associated with astrocytomas (e.g. VCAM1, SMOC1, and thymidylate synthetase), with a high percentage of cell surface genes. Two genes with available antibodies, Aquaporin 1 and Topoisomerase 2A, showed protein expression consistent with transcript level predictions. This survey of transcription in malignant and normal brain tissues reveals a small subset of human genes that are activated in malignant astrocytomas. In addition to providing insights into pathway biology, we have revealed and quantified expression for a significant portion of cell surface and extra-cellular astrocytoma genes

  11. Identification of astrocytoma associated genes including cell surface markers

    Directory of Open Access Journals (Sweden)

    Eberhart Charles G

    2004-07-01

    Full Text Available Abstract Background Despite intense effort the treatment options for the invasive astrocytic tumors are still limited to surgery and radiation therapy, with chemotherapy showing little or no increase in survival. The generation of Serial Analysis of Gene Expression (SAGE profiles is expected to aid in the identification of astrocytoma-associated genes and highly expressed cell surface genes as molecular therapeutic targets. SAGE tag counts can be easily added to public expression databases and quickly disseminated to research efforts worldwide. Methods We generated and analyzed the SAGE transcription profiles of 25 primary grade II, III and IV astrocytomas 1. These profiles were produced as part of the Cancer Genome Anatomy Project's SAGE Genie 2, and were used in an in silico search for candidate therapeutic targets by comparing astrocytoma to normal brain transcription. Real-time PCR and immunohistochemistry were used for the validation of selected candidate target genes in 2 independent sets of primary tumors. Results A restricted set of tumor-associated genes was identified for each grade that included genes not previously associated with astrocytomas (e.g. VCAM1, SMOC1, and thymidylate synthetase, with a high percentage of cell surface genes. Two genes with available antibodies, Aquaporin 1 and Topoisomerase 2A, showed protein expression consistent with transcript level predictions. Conclusions This survey of transcription in malignant and normal brain tissues reveals a small subset of human genes that are activated in malignant astrocytomas. In addition to providing insights into pathway biology, we have revealed and quantified expression for a significant portion of cell surface and extra-cellular astrocytoma genes.

  12. Dkk-1 Inhibits Intestinal Epithelial Cell Migration by Attenuating Directional Polarization of Leading Edge Cells

    OpenAIRE

    Koch, Stefan; Capaldo, Christopher T.; Samarin, Stanislav; Nava, Porfirio; Neumaier, Irmgard; Skerra, Arne; Sacks, David B; Parkos, Charles A.; Nusrat, Asma

    2009-01-01

    Wnt signaling pathways regulate proliferation, motility, and survival in a variety of human cell types. Dickkopf-1 (Dkk-1) is a secreted Wnt antagonist that has been proposed to regulate tissue homeostasis in the intestine. In this report, we show that Dkk-1 is secreted by intestinal epithelial cells after wounding and that it inhibits cell migration by attenuating the directional orientation of migrating epithelial cells. Dkk-1 exposure induced mislocalized activation of Cdc42 in migrating c...

  13. In search of suitable reference genes for gene expression studies of human renal cell carcinoma by real-time PCR

    Directory of Open Access Journals (Sweden)

    Kristiansen Glen

    2007-06-01

    Full Text Available Abstract Background Housekeeping genes are commonly used as endogenous reference genes for the relative quantification of target genes in gene expression studies. No conclusive systematic study comparing the suitability of different candidate reference genes in clear cell renal cell carcinoma has been published to date. To remedy this situation, 10 housekeeping genes for normalizing purposes of RT-PCR measurements already recommended in various studies were examined with regard to their usefulness as reference genes. Results The expression of the potential reference genes was examined in matched malignant and non-malignant tissue specimens from 25 patients with clear cell renal cell carcinoma. Quality assessment of isolated RNA performed with a 2100 Agilent Bioanalyzer showed a mean RNA integrity number of 8.7 for all samples. The between-run variations related to the crossing points of PCR reactions of a control material ranged from 0.17% to 0.38%. The expression of all genes did not depend on age, sex, and tumour stage. Except the genes TATA box binding protein (TBP and peptidylprolyl isomerase A (PPIA, all genes showed significant differences in expression between malignant and non-malignant pairs. The expression stability of the candidate reference genes was additionally controlled using the software programs geNorm and NormFinder. TBP and PPIA were validated as suitable reference genes by normalizing the target gene ADAM9 using these two most stably expressed genes in comparison with up- and down-regulated housekeeping genes of the panel. Conclusion Our study demonstrated the suitability of the two housekeeping genes PPIA and TBP as endogenous reference genes when comparing malignant tissue samples with adjacent normal tissue samples from clear cell renal cell carcinoma. Both genes are recommended as reference genes for relative gene quantification in gene profiling studies either as single gene or preferably in combination.

  14. Absence of transepithelial anion exchange by rabbit OMCD: Evidence against reversal of cell polarity

    International Nuclear Information System (INIS)

    In the rabbit cortical collecting duct (CCD), Cl tracer crosses the epithelium predominantly via an anion exchange system that operates in either a Cl-Cl or Cl-HCO3 exchange mode. In the present study, the authors used the 36Cl lumen-to-bath rate coefficient (KCl, nm/s), a sensitive measurement of CCD transepithelial anion transport, to investigate the nature of Cl transport in the medullary collecting duct dissected from inner stripe, outer medulla (OMCD). The KCl in OMCD perfused and bathed in HCO3-Ringer solution was low and similar to that value observed in the CCD when anion exchange is inhibited and Cl permeates the epithelium by diffusion. To test the hypothesis that metabolic alkalosis could reverse the polarity of intercalated cells and thus induce an apical Cl-HCO3 exchanger in H+-secreting OMCD cells, they measured KCl in OMCD from rabbits make alkalotic by deoxycorticosterone and furosemide. Although the base-line KCl was slightly higher than in OMCD from control rabbits, the value was still far lower than the KCl under comparable conditions in CCD. They conclude (1) Cl transport across the MCD by anion exchange is immeasurably low or nonexistent; (2) unlike the CCD, Cl transport in OMCD is not responsive to cAMP; and (3) metabolic alkalosis does not induce an apical anion exchanger in OMCD, i.e., does not cause epithelial polarity reversal

  15. 10.6% Certified Colloidal Quantum Dot Solar Cells via Solvent-Polarity-Engineered Halide Passivation.

    Science.gov (United States)

    Lan, Xinzheng; Voznyy, Oleksandr; García de Arquer, F Pelayo; Liu, Mengxia; Xu, Jixian; Proppe, Andrew H; Walters, Grant; Fan, Fengjia; Tan, Hairen; Liu, Min; Yang, Zhenyu; Hoogland, Sjoerd; Sargent, Edward H

    2016-07-13

    Colloidal quantum dot (CQD) solar cells are solution-processed photovoltaics with broad spectral absorption tunability. Major advances in their efficiency have been made via improved CQD surface passivation and device architectures with enhanced charge carrier collection. Herein, we demonstrate a new strategy to improve further the passivation of CQDs starting from the solution phase. A cosolvent system is employed to tune the solvent polarity in order to achieve the solvation of methylammonium iodide (MAI) and the dispersion of hydrophobic PbS CQDs simultaneously in a homogeneous phase, otherwise not achieved in a single solvent. This process enables MAI to access the CQDs to confer improved passivation. This, in turn, allows for efficient charge extraction from a thicker photoactive layer device, leading to a certified solar cell power conversion efficiency of 10.6%, a new certified record in CQD photovoltaics. PMID:27351104

  16. Gene profiling of growth factor independence 1B gene (Gfi-1B) in leukemic cells.

    Science.gov (United States)

    Koldehoff, Michael; Zakrzewski, Johannes L; Klein-Hitpass, Ludger; Beelen, Dietrich W; Elmaagacli, Ahmet H

    2008-01-01

    To investigate the molecular effects of growth factor independence 1B (Gfi-1B), a transcription factor essential for the development of hematopoietic cells and differentiation of erythroid and megakaryocytic lineages, the naturally Gfi-1B overexpressing cell line K562 was cultured in the presence of Gfi-1B target-specific small interfering RNA (siRNA). SiRNA treatment significantly knocked down Gfi-1B expression with an efficiency of nearly 90%. Analysis of the siRNA silencing protocol by colony-forming units ensured that it was not cytotoxic. Samples from Gfi-1B overexpressing cells and cells with knocked-down Gfi-1B were analyzed by oligonucleotide microarray technology and based upon rigorous statistical analysis of the data; relevant genes were chosen for confirmation by reserve transcriptase-polymerase chain reaction, including MYC/MYCBP and CDKN1A. Interestingly, transcripts within components of the signalling cascade of immune cells (PLD1, LAMP1, HSP90, IL6ST), of the tyrosine kinase pathway (TPR, RAC3) and of the transcription factors (RAC3, CEP290, JEM-1, ATR, MYC, SMC3, RARA, RBBP6) were found to be differentially expressed in Gfi-1B overexpressing cells compared to controls. Individual genes such as ZDHHC17, DMXL1, ZNF292 were found to be upregulated in Gfi-1B overexpressing cells. In addition, down-regulated transcripts showed cell signaling transcripts for several chemokine gene members including GNAL, CXCL5, GNL3L, GPR65, TMEM30, BCL11B and transcription factors (GTF2H3, ATXN3). In conclusion, several essential cell signalling factors, as well as transcriptional and post-translational regulation genes were differentially expressed in cells that overexpressed Gfi-1B compared to control cells with knocked-down Gfi-1B. Our data indicate that Gfi-1B signalling is important for commitment and maturation of hematopoietic cell populations. PMID:18224412

  17. MHV-A59 enters polarized murine epithelial cells through the apical surface but is released basolaterally

    NARCIS (Netherlands)

    Rossen, J W; Voorhout, W F; Horzinek, M C; van der Ende, A; Strous, G J; Rottier, P J

    1995-01-01

    Coronaviruses have a marked tropism for epithelial cells. Entry and release of the porcine transmissible gastroenteritis virus (TGEV) is restricted to apical surfaces of polarized epithelial cells, as we have recently shown (J. W. A. Rossen, C. P. J. Bekker, W. F. Voorhout, G. J. A. M. Strous, A. va

  18. UV-induced changes in cell cycle and gene expression within rabbit lens epithelial cells

    Energy Technology Data Exchange (ETDEWEB)

    Sidjanin, D. [Northern Illinois Univ., De Kalb, IL (United States). Dept. of Biological Sciences; Grdina, D. [Argonne National Lab., IL (United States); Woloschak, G.E. [Northern Illinois Univ., De Kalb, IL (United States). Dept. of Biological Sciences

    1994-11-01

    Damage to lens epithelial cells is a probable initiation process in cataract formation induced by ultraviolet radiation. These experiments investigated the ability of 254 nm radiation on cell cycle progression and gene expression in rabbit lens epithelial cell line N/N1003A. No changes in expression of c-fos, c-jun, alpha- tubulin, or vimentin was observed following UV exposure. Using flow cytometry, an accumulation of cells in G1/S phase of the cell cycle 1 hr following exposure. The observed changes in gene expression, especially the decreased histone transcripts reported here may play a role in UV induced inhibition of cell cycle progression.

  19. NOD2 gene expression in Paneth cells and monocytes.

    OpenAIRE

    Lala, S. G.

    2006-01-01

    Introduction: Mutations in the NOD2 gene are associated with the development of Crohn's disease, an inflammatory disorder of the gastrointestinal tract. The NOD2 protein induces cellular activation in response to the bacterial antigen muramyl dipeptide (MDP). The NOD2 gene is mainly expressed by circulating blood monocytes although NOD2-associated Crohn's disease involves mainly the terminal ileum. Paneth cells, which are most numerous in the terminal ileum, are specialised intestinal epithel...

  20. Optimising gene therapy of hypoparathyroidism with hematopoietic stem cells

    Institute of Scientific and Technical Information of China (English)

    ZHOU Yi; L(U) Bing-jie; XU Ping; SONG Chun-fang

    2005-01-01

    Background The treatment of hypoparathyroidism (HPT) is still a difficult clinical problem, which necessitates a new therapy. Gene therapy of HPT has been valuable, but how to improve the gene transfer efficiency and expression stability is a problem. This study was designed to optimize the gene therapy of HPT with hematopoietic stem cells (HSCs) recombined with the parathyroid hormone (PTH) gene. Methods The human PTH gene was amplified by polymerase chain reaction (PCR) from pcDNA3.1-PTH vectors and inserted into murine stem cell virus (MSCV) vectors with double enzyme digestion (EcoRI and XhoI). The recombinant vectors were transfected into PA317 packaging cell lines by the lipofectin method and screened by G418 selective medium. The condensed recombinant retroviruses were extracted and used to infect HSCs, which were injected into mice suffering from HPT. The change of symptoms and serum levels of PTH and calcium in each group of mice were investigated. Results The human PTH gene was inserted into MSCV vectors successfully and the titres were up to 2×107 colony forming unit (CFU)/ml in condensed retroviral solution. The secretion of PTH reached 15 ng·10-6·cell-1 per 48 hours. The wild type viruses were not detected via PCR amplification, so they were safe for use. The mice suffering from HPT recovered quickly and the serum levels of calcium and PTH remained normal for about three months after the HSCs recombined with PTH were injected into them. The therapeutic effect of this method was better than simple recombinant retroviruses injection.Conclusions The recombinant retroviral vectors MSCV-PTH and the high-titre condensed retroviral solution recombined with the PTH gene are obtained. The recombinant retroviral solution could infect HSCs at a high rate of efficiency. The infected HSCs could cure HPT in mice. This method has provided theoretical evidence for the clinical gene therapy of HPT.

  1. The evolutionary emergence of cell type-specific genes inferred from the gene expression analysis of Hydra

    Science.gov (United States)

    Hwang, Jung Shan; Ohyanagi, Hajime; Hayakawa, Shiho; Osato, Naoki; Nishimiya-Fujisawa, Chiemi; Ikeo, Kazuho; David, Charles N.; Fujisawa, Toshitaka; Gojobori, Takashi

    2007-01-01

    Cell lineages of cnidarians including Hydra represent the fundamental cell types of metazoans and provides us a unique opportunity to study the evolutionary diversification of cell type in the animal kingdom. Hydra contains epithelial cells as well as a multipotent interstitial cell (I-cell) that gives rise to nematocytes, nerve cells, gland cells, and germ-line cells. We used cDNA microarrays to identify cell type-specific genes by comparing gene expression in normal Hydra with animals lacking the I-cell lineage, so-called epithelial Hydra. We then performed in situ hybridization to localize expression to specific cell types. Eighty-six genes were shown to be expressed in specific cell types of the I-cell lineage. An additional 29 genes were expressed in epithelial cells and were down-regulated in epithelial animals lacking I-cells. Based on the above information, we constructed a database (http://hydra.lab.nig.ac.jp/hydra/), which describes the expression patterns of cell type-specific genes in Hydra. Most genes expressed specifically in either I-cells or epithelial cells have homologues in higher metazoans. By comparison, most nematocyte-specific genes and approximately half of the gland cell- and nerve cell-specific genes are unique to the cnidarian lineage. Because nematocytes, gland cells, and nerve cells appeared along with the emergence of cnidarians, this suggests that lineage-specific genes arose in cnidarians in conjunction with the evolution of new cell types required by the cnidarians. PMID:17766437

  2. Thiazolidinediones inhibit REG Iα gene transcription in gastrointestinal cancer cells

    International Nuclear Information System (INIS)

    REG (Regenerating gene) Iα protein functions as a growth factor for gastrointestinal cancer cells, and its mRNA expression is strongly associated with a poor prognosis in gastrointestinal cancer patients. We here demonstrated that PPARγ-agonist thiazolidinediones (TZDs) inhibited cell proliferation and REG Iα protein/mRNA expression in gastrointestinal cancer cells. TZDs inhibited the REG Iα gene promoter activity, via its cis-acting element which lacked PPAR response element and could not bind to PPARγ, in PPARγ-expressing gastrointestinal cancer cells. The inhibition was reversed by co-treatment with a specific PPARγ-antagonist GW9662. Although TZDs did not inhibit the REG Iα gene promoter activity in PPARγ-non-expressing cells, PPARγ overexpression in the cells recovered their inhibitory effect. Taken together, TZDs inhibit REG Iα gene transcription through a PPARγ-dependent pathway. The TZD-induced REG Iα mRNA reduction was abolished by cycloheximide, indicating the necessity of novel protein(s) synthesis. TZDs may therefore be a candidate for novel anti-cancer drugs for patients with gastrointestinal cancer expressing both REG Iα and PPARγ.

  3. How actin/myosin crosstalks guide the adhesion, locomotion and polarization of cells.

    Science.gov (United States)

    Sackmann, Erich

    2015-11-01

    Cell-tissue-tissue interaction is determined by specific short range forces between cell adhesion molecules (CAMs) and ligands of the tissue, long range repulsion forces mediated by cell surface grafted macromolecules and adhesion-induced elastic stresses in the cell envelope. This interplay of forces triggers the rapid random clustering of tightly coupled linkers. By coupling of actin gel patches to the intracellular domains of the CAMs, these clusters can grow in a secondary process resulting in the formation of functional adhesion microdomains (ADs). The ADs can act as biochemical steering centers by recruiting and activating functional proteins, such as GTPases and associated regulating proteins, through electrostatic-hydrophobic forces with cationic lipid domains that act as attractive centers. First, I summarize physical concepts of cell adhesion revealed by studies of biomimetic systems. Then I describe the role of the adhesion domains as biochemical signaling platforms and force transmission centers promoting cellular protrusions, in terms of a shell string model of cells. Protrusion forces are generated by actin gelation triggered by molecular machines (focal adhesion kinase (FAK), Src-kinases and associated adaptors) which assemble around newly formed integrin clusters. They recruit and activate the GTPases Rac-1 and actin gelation promoters to charged membrane domains via electrostatic-hydrophobic forces. The cell front is pushed forward in a cyclic and stepwise manner and the step-width is determined by the dynamics antagonistic interplay between Rac-1 and RhoA. The global cell polarization in the direction of motion is mediated by the actin-microtubule (MT) crosstalk at adhesion domains. Supramolecular actin-MT assemblies at the front help to promote actin polymerization. At the rear they regulate the dismantling of the ADs through the Ca(++)-mediated activation of the protease calpain and trigger their disruption by RhoA mediated contraction via

  4. Impact of the cell division cycle on gene circuits

    Science.gov (United States)

    Bierbaum, Veronika; Klumpp, Stefan

    2015-12-01

    In growing cells, protein synthesis and cell growth are typically not synchronous, and, thus, protein concentrations vary over the cell division cycle. We have developed a theoretical description of genetic regulatory systems in bacteria that explicitly considers the cell division cycle to investigate its impact on gene expression. We calculate the cell-to-cell variations arising from cells being at different stages in the division cycle for unregulated genes and for basic regulatory mechanisms. These variations contribute to the extrinsic noise observed in single-cell experiments, and are most significant for proteins with short lifetimes. Negative autoregulation buffers against variation of protein concentration over the division cycle, but the effect is found to be relatively weak. Stronger buffering is achieved by an increased protein lifetime. Positive autoregulation can strongly amplify such variation if the parameters are set to values that lead to resonance-like behaviour. For cooperative positive autoregulation, the concentration variation over the division cycle diminishes the parameter region of bistability and modulates the switching times between the two stable states. The same effects are seen for a two-gene mutual-repression toggle switch. By contrast, an oscillatory circuit, the repressilator, is only weakly affected by the division cycle.

  5. Gene expression profiles identify inflammatory signatures in dendritic cells.

    Directory of Open Access Journals (Sweden)

    Anna Torri

    Full Text Available Dendritic cells (DCs constitute a heterogeneous group of antigen-presenting leukocytes important in activation of both innate and adaptive immunity. We studied the gene expression patterns of DCs incubated with reagents inducing their activation or inhibition. Total RNA was isolated from DCs and gene expression profiling was performed with oligonucleotide microarrays. Using a supervised learning algorithm based on Random Forest, we generated a molecular signature of inflammation from a training set of 77 samples. We then validated this molecular signature in a testing set of 38 samples. Supervised analysis identified a set of 44 genes that distinguished very accurately between inflammatory and non inflammatory samples. The diagnostic performance of the signature genes was assessed against an independent set of samples, by qRT-PCR. Our findings suggest that the gene expression signature of DCs can provide a molecular classification for use in the selection of anti-inflammatory or adjuvant molecules with specific effects on DC activity.

  6. Identification and Characterization of Renal Cell Carcinoma Gene Markers

    Directory of Open Access Journals (Sweden)

    Louis S. Liou

    2007-01-01

    Full Text Available 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 identifi ed 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 categorie at a signifi cance 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.

  7. Escherichia coli isolated from a Crohn's disease patient adheres, invades, and induces inflammatory responses in polarized intestinal epithelial cells.

    Science.gov (United States)

    Eaves-Pyles, Tonyia; Allen, Christopher A; Taormina, Joanna; Swidsinski, Alexander; Tutt, Christopher B; Jezek, G Eric; Islas-Islas, Martha; Torres, Alfredo G

    2008-07-01

    Inflammatory diseases of the intestinal tract are a major health concern both in the United States and around the world. Evidence now suggests that a new category of Escherichia coli, designated Adherent Invasive E. coli (AIEC) is highly prevalent in Crohn's Disease (CD) patients. AIEC strains have been shown to colonize and adhere to intestinal epithelial cells (IEC). However, the role AIEC strains play in the induction of an inflammatory response is not known. Therefore, we examined several E. coli strains (designated LF82, O83:H1, 6604 and 6655) that were isolated from CD patients for their ability to induce inflammation in two IEC, Caco-2BBe and T-84 cells. Results showed that each strain had varying abilities to adhere to and invade IEC as well as induced cytokine secretion from polarized IEC. However, E. coli O83:H1 displayed the best characteristics of AIEC strains as compared to the prototype AIEC strain LF82, inducing cytokine secretion from IEC and promoting immune cell migration through IEC. Upon further analysis, E. coli O83:H1 did not harbor virulence genes present in known pathogenic intestinal organisms. Further characterization of E. coli O83:H1 virulence determinants showed that a non-flagellated O83:H1 strain significantly decreased the organism's ability to adhere to and invade both IEC and elicit IEC cytokine secretion compared to the wild type and complemented strains. These findings demonstrate that E. coli O83:H1 possesses the characteristics of the AIEC LF82 strain that may contribute to the low-grade, chronic inflammation observed in Crohn's disease. PMID:17900983

  8. From single-cell to cell-pool transcriptomes: stochasticity in gene expression and RNA splicing.

    Science.gov (United States)

    Marinov, Georgi K; Williams, Brian A; McCue, Ken; Schroth, Gary P; Gertz, Jason; Myers, Richard M; Wold, Barbara J

    2014-03-01

    Single-cell RNA-seq mammalian transcriptome studies are at an early stage in uncovering cell-to-cell variation in gene expression, transcript processing and editing, and regulatory module activity. Despite great progress recently, substantial challenges remain, including discriminating biological variation from technical noise. Here we apply the SMART-seq single-cell RNA-seq protocol to study the reference lymphoblastoid cell line GM12878. By using spike-in quantification standards, we estimate the absolute number of RNA molecules per cell for each gene and find significant variation in total mRNA content: between 50,000 and 300,000 transcripts per cell. We directly measure technical stochasticity by a pool/split design and find that there are significant differences in expression between individual cells, over and above technical variation. Specific gene coexpression modules were preferentially expressed in subsets of individual cells, including one enriched for mRNA processing and splicing factors. We assess cell-to-cell variation in alternative splicing and allelic bias and report evidence of significant differences in splice site usage that exceed splice variation in the pool/split comparison. Finally, we show that transcriptomes from small pools of 30-100 cells approach the information content and reproducibility of contemporary RNA-seq from large amounts of input material. Together, our results define an experimental and computational path forward for analyzing gene expression in rare cell types and cell states. PMID:24299736

  9. Highly parallel identification of essential genes in cancer cells.

    Science.gov (United States)

    Luo, Biao; Cheung, Hiu Wing; Subramanian, Aravind; Sharifnia, Tanaz; Okamoto, Michael; Yang, Xiaoping; Hinkle, Greg; Boehm, Jesse S; Beroukhim, Rameen; Weir, Barbara A; Mermel, Craig; Barbie, David A; Awad, Tarif; Zhou, Xiaochuan; Nguyen, Tuyen; Piqani, Bruno; Li, Cheng; Golub, Todd R; Meyerson, Matthew; Hacohen, Nir; Hahn, William C; Lander, Eric S; Sabatini, David M; Root, David E

    2008-12-23

    More complete knowledge of the molecular mechanisms underlying cancer will improve prevention, diagnosis and treatment. Efforts such as The Cancer Genome Atlas are systematically characterizing the structural basis of cancer, by identifying the genomic mutations associated with each cancer type. A powerful complementary approach is to systematically characterize the functional basis of cancer, by identifying the genes essential for growth and related phenotypes in different cancer cells. Such information would be particularly valuable for identifying potential drug targets. Here, we report the development of an efficient, robust approach to perform genome-scale pooled shRNA screens for both positive and negative selection and its application to systematically identify cell essential genes in 12 cancer cell lines. By integrating these functional data with comprehensive genetic analyses of primary human tumors, we identified known and putative oncogenes such as EGFR, KRAS, MYC, BCR-ABL, MYB, CRKL, and CDK4 that are essential for cancer cell proliferation and also altered in human cancers. We further used this approach to identify genes involved in the response of cancer cells to tumoricidal agents and found 4 genes required for the response of CML cells to imatinib treatment: PTPN1, NF1, SMARCB1, and SMARCE1, and 5 regulators of the response to FAS activation, FAS, FADD, CASP8, ARID1A and CBX1. Broad application of this highly parallel genetic screening strategy will not only facilitate the rapid identification of genes that drive the malignant state and its response to therapeutics but will also enable the discovery of genes that participate in any biological process. PMID:19091943

  10. Efficient retrovirus-mediated transfer of cell-cycle control genes to transformed cells

    Directory of Open Access Journals (Sweden)

    B.E. Strauss

    1999-07-01

    Full Text Available The use of gene therapy continues to be a promising, yet elusive, alternative for the treatment of cancer. The origins of cancer must be well understood so that the therapeutic gene can be chosen with the highest chance of successful tumor regression. The gene delivery system must be tailored for optimum transfer of the therapeutic gene to the target tissue. In order to accomplish this, we study models of G1 cell-cycle control in both normal and transformed cells in order to understand the reasons for uncontrolled cellular proliferation. We then use this information to choose the gene to be delivered to the cells. We have chosen to study p16, p21, p53 and pRb gene transfer using the pCL-retrovirus. Described here are some general concepts and specific results of our work that indicate continued hope for the development of genetically based cancer treatments.

  11. Polar flagellar biosynthesis and a regulator of flagellar number influence spatial parameters of cell division in Campylobacter jejuni.

    Directory of Open Access Journals (Sweden)

    Murat Balaban

    2011-12-01

    Full Text Available Spatial and numerical regulation of flagellar biosynthesis results in different flagellation patterns specific for each bacterial species. Campylobacter jejuni produces amphitrichous (bipolar flagella to result in a single flagellum at both poles. These flagella confer swimming motility and a distinctive darting motility necessary for infection of humans to cause diarrheal disease and animals to promote commensalism. In addition to flagellation, symmetrical cell division is spatially regulated so that the divisome forms near the cellular midpoint. We have identified an unprecedented system for spatially regulating cell division in C. jejuni composed by FlhG, a regulator of flagellar number in polar flagellates, and components of amphitrichous flagella. Similar to its role in other polarly-flagellated bacteria, we found that FlhG regulates flagellar biosynthesis to limit poles of C. jejuni to one flagellum. Furthermore, we discovered that FlhG negatively influences the ability of FtsZ to initiate cell division. Through analysis of specific flagellar mutants, we discovered that components of the motor and switch complex of amphitrichous flagella are required with FlhG to specifically inhibit division at poles. Without FlhG or specific motor and switch complex proteins, cell division occurs more often at polar regions to form minicells. Our findings suggest a new understanding for the biological requirement of the amphitrichous flagellation pattern in bacteria that extend beyond motility, virulence, and colonization. We propose that amphitrichous bacteria such as Campylobacter species advantageously exploit placement of flagella at both poles to spatially regulate an FlhG-dependent mechanism to inhibit polar cell division, thereby encouraging symmetrical cell division to generate the greatest number of viable offspring. Furthermore, we found that other polarly-flagellated bacteria produce FlhG proteins that influence cell division, suggesting that

  12. Gene Profiling Technique to Accelerate Stem Cell Therapies for Eye Diseases

    Science.gov (United States)

    ... to accelerate stem cell therapies for eye diseases Gene profiling technique to accelerate stem cell therapies for ... The method simultaneously measures the expression of multiple genes, allowing scientists to quickly characterize cells according to ...

  13. Estrogen-responsive genes overlap with triiodothyronine-responsive genes in a breast carcinoma cell line.

    Science.gov (United States)

    Figueiredo, Nancy Bueno; Cestari, Sílvia Helena; Conde, Sandro José; Luvizotto, Renata Azevedo Melo; De Sibio, Maria Teresa; Perone, Denise; Katayama, Maria Lúcia Hirata; Carraro, Dirce Maria; Brentani, Helena Paula; Brentani, Maria Mitzi; Nogueira, Célia Regina

    2014-01-01

    It has been well established that estrogen plays an important role in the progression and treatment of breast cancer. However, the role of triiodothyronine (T₃) remains controversial. We have previously shown its capacity to stimulate the development of positive estrogen receptor breast carcinoma, induce the expression of genes (PR, TGF-alpha) normally stimulated by estradiol (E₂), and suppress genes (TGF-beta) normally inhibited by E₂. Since T₃ regulates growth hormones, metabolism, and differentiation, it is important to verify its action on other genes normally induced by E₂. Therefore, we used DNA microarrays to compare gene expression patterns in MCF-7 breast adenocarcinoma cells treated with E₂ and T₃. Several genes were modulated by both E₂ and T₃ in MCF-7 cells (Student's t-test, P 2.0, pFDR < 0.05). We confirmed our microarray results by real-time PCR. Our findings reveal that certain genes in MCF-7 cells can be regulated by both E₂ and T₃. PMID:24587767

  14. Specifically targeted gene therapy for small-cell lung cancer

    DEFF Research Database (Denmark)

    Christensen, C.L.; Zandi, R.; Gjetting, T.;

    2009-01-01

    DNA into malignant cells causing them to die. Since SCLC is a highly disseminated malignancy, the gene therapeutic agent must be administered systemically, obligating a high level of targeting of tumor tissue and the use of delivery vehicles designed for systemic circulation of the therapeutic DNA......Small-cell lung cancer (SCLC) is a highly malignant disease with poor prognosis. Hence, there is great demand for new therapies that can replace or supplement the current available treatment regimes. Gene therapy constitutes a promising strategy and relies on the principle of introducing exogenous...

  15. Mn bioavailability by polarized Caco-2 cells: comparison between Mn gluconate and Mn oxyprolinate

    Directory of Open Access Journals (Sweden)

    Fulgenzi Alessandro

    2011-07-01

    Full Text Available Abstract Background Micronutrient inadequate intake is responsible of pathological deficiencies and there is a need of assessing the effectiveness of metal supplementation, frequently proposed to rebalance poor diets. Manganese (Mn is present in many enzymatic intracellular systems crucial for the regulation of cell metabolism, and is contained in commercially available metal supplements. Methods We compared the effects of two different commercial Mn forms, gluconate (MnGluc and oxyprolinate (MnOxP. For this purpose we used the polarized Caco-2 cells cultured on transwell filters, an established in vitro model of intestinal epithelium. Since micronutrient deficiency may accelerate mitochondrial efficiency, the mitochondrial response of these cells, in the presence of MnGluc and MnOxP, by microscopy methods and by ATP luminescence assay was used. Results In the presence of both MnOxP and MnGluc a sustained mitochondrial activity was shown by mitoTraker labeling (indicative of mitochondrial respiration, but ATP intracellular content remained comparable to untreated cells only in the presence of MnOxP. In addition MnOxP transiently up-regulated the antioxidant enzyme Mn superoxide dismutase more efficiently than MnGluc. Both metal treatments preserved NADH and βNADPH diaphorase oxidative activity, avoided mitochondrial dysfunction, as assessed by the absence of a sustained phosphoERK activation, and were able to maintain cell viability. Conclusions Collectively, our data indicate that MnOxP and MnGluc, and primarily the former, produce a moderate and safe modification of Caco-2 cell metabolism, by activating positive enzymatic mechanisms, thus could contribute to long-term maintenance of cell homeostasis.

  16. The dual effects of polar methanolic extract of Hypericum perforatum L. in bladder cancer cells

    Science.gov (United States)

    Nseyo, U. O.; Nseyo, O. U.; Shiverick, K. T.; Medrano, T.; Mejia, M.; Stavropoulos, N.; Tsimaris, I.; Skalkos, D.

    2007-02-01

    Introduction and background: We have reported on the polar methanolic fraction (PMF) of Hypericum Perforatum L as a novel photosensitizing agent for photodynamic therapy (PDT) and photodynamic diagnosis (PDD). PMF has been tested in human leukemic cells, HL-60 cells, cord blood hemopoietic progenitor cells, bladder cancers derived from metastatic lymph node (T-24) and primary papillary bladder lesion (RT-4). However, the mechanisms of the effects of PMF on these human cell lines have not been elucidated. We have investigated mechanisms of PMF + light versus PMF-alone (dark experiment) in T-24 human bladder cancer cells. Methods: PMF was prepared from an aerial herb of HPL which was brewed in methanol and extracted with ether and methanol. Stock solutions of PMF were made in DSMO and stored in dark conditions. PMF contains 0.57% hypericin and 2.52% hyperforin. The T24 cell line was obtained from American Type Culture Collection (ATCC). In PDT treatment, PMF (60μg/ml) was incubated with cells, which were excited with laser light (630nm) 24 hours later. Apoptosis was determined by DNA fragmentation/laddering assay. DNA isolation was performed according to the manufacture's instructions with the Kit (Oncogene Kit#AM41). Isolated DNA samples were separated by electrophoresis in 1.5% in agarose gels and bands were visualized by ethidium bromide labeling. The initial cell cycle analysis and phase distribution was by flow cytometry. DNA synthesis was measured by [3H] thymidine incorporation, and cell cycle regulatory proteins were assayed by Western immunoblot. Results: The results of the flow cytometry showed PMF +light induced significant (40%) apoptosis in T24 cells, whereas Light or PMF alone produced little apoptosis. The percentage of cells in G 0/G I phase was decreased by 25% and in G2/M phase by 38%. The main impact was observed on the S phase which was blocked by 78% from the specific photocytotoxic process. DNA laddering analysis showed that PMF (60

  17. Clusters of conserved beta cell marker genes for assessment of beta cell phenotype.

    Directory of Open Access Journals (Sweden)

    Geert A Martens

    Full Text Available BACKGROUND AND METHODOLOGY: The aim of this study was to establish a gene expression blueprint of pancreatic beta cells conserved from rodents to humans and to evaluate its applicability to assess shifts in the beta cell differentiated state. Genome-wide mRNA expression profiles of isolated beta cells were compared to those of a large panel of other tissue and cell types, and transcripts with beta cell-abundant and -selective expression were identified. Iteration of this analysis in mouse, rat and human tissues generated a panel of conserved beta cell biomarkers. This panel was then used to compare isolated versus laser capture microdissected beta cells, monitor adaptations of the beta cell phenotype to fasting, and retrieve possible conserved transcriptional regulators. PRINCIPAL FINDINGS: A panel of 332 conserved beta cell biomarker genes was found to discriminate both isolated and laser capture microdissected beta cells from all other examined cell types. Of all conserved beta cell-markers, 15% were strongly beta cell-selective and functionally associated to hormone processing, 15% were shared with neuronal cells and associated to regulated synaptic vesicle transport and 30% with immune plus gut mucosal tissues reflecting active protein synthesis. Fasting specifically down-regulated the latter cluster, but preserved the neuronal and strongly beta cell-selective traits, indicating preserved differentiated state. Analysis of consensus binding site enrichment indicated major roles of CREB/ATF and various nutrient- or redox-regulated transcription factors in maintenance of differentiated beta cell phenotype. CONCLUSIONS: Conserved beta cell marker genes contain major gene clusters defined by their beta cell selectivity or by their additional abundance in either neural cells or in immune plus gut mucosal cells. This panel can be used as a template to identify changes in the differentiated state of beta cells.

  18. Prediction of epigenetically regulated genes in breast cancer cell lines

    Energy Technology Data Exchange (ETDEWEB)

    Loss, Leandro A; Sadanandam, Anguraj; Durinck, Steffen; Nautiyal, Shivani; Flaucher, Diane; Carlton, Victoria EH; Moorhead, Martin; Lu, Yontao; Gray, Joe W; Faham, Malek; Spellman, Paul; Parvin, Bahram

    2010-05-04

    Methylation of CpG islands within the DNA promoter regions is one mechanism that leads to aberrant gene expression in cancer. In particular, the abnormal methylation of CpG islands may silence associated genes. Therefore, using high-throughput microarrays to measure CpG island methylation will lead to better understanding of tumor pathobiology and progression, while revealing potentially new biomarkers. We have examined a recently developed high-throughput technology for measuring genome-wide methylation patterns called mTACL. Here, we propose a computational pipeline for integrating gene expression and CpG island methylation profles to identify epigenetically regulated genes for a panel of 45 breast cancer cell lines, which is widely used in the Integrative Cancer Biology Program (ICBP). The pipeline (i) reduces the dimensionality of the methylation data, (ii) associates the reduced methylation data with gene expression data, and (iii) ranks methylation-expression associations according to their epigenetic regulation. Dimensionality reduction is performed in two steps: (i) methylation sites are grouped across the genome to identify regions of interest, and (ii) methylation profles are clustered within each region. Associations between the clustered methylation and the gene expression data sets generate candidate matches within a fxed neighborhood around each gene. Finally, the methylation-expression associations are ranked through a logistic regression, and their significance is quantified through permutation analysis. Our two-step dimensionality reduction compressed 90% of the original data, reducing 137,688 methylation sites to 14,505 clusters. Methylation-expression associations produced 18,312 correspondences, which were used to further analyze epigenetic regulation. Logistic regression was used to identify 58 genes from these correspondences that showed a statistically signifcant negative correlation between methylation profles and gene expression in the

  19. Improved retroviral suicide gene transfer in colon cancer cell lines after cell synchronization with methotrexate

    Directory of Open Access Journals (Sweden)

    Nordlinger Bernard

    2011-10-01

    Full Text Available Abstract Background Cancer gene therapy by retroviral vectors is mainly limited by the level of transduction. Retroviral gene transfer requires target cell division. Cell synchronization, obtained by drugs inducing a reversible inhibition of DNA synthesis, could therefore be proposed to precondition target cells to retroviral gene transfer. We tested whether drug-mediated cell synchronization could enhance the transfer efficiency of a retroviral-mediated gene encoding herpes simplex virus thymidine kinase (HSV-tk in two colon cancer cell lines, DHDK12 and HT29. Methods Synchronization was induced by methotrexate (MTX, aracytin (ara-C or aphidicolin. Gene transfer efficiency was assessed by the level of HSV-TK expression. Transduced cells were driven by ganciclovir (GCV towards apoptosis that was assessed using annexin V labeling by quantitative flow cytometry. Results DHDK12 and HT29 cells were synchronized in S phase with MTX but not ara-C or aphidicolin. In synchronized DHDK12 and HT29 cells, the HSV-TK transduction rates were 2 and 1.5-fold higher than those obtained in control cells, respectively. Furthermore, the rate of apoptosis was increased two-fold in MTX-treated DHDK12 cells after treatment with GCV. Conclusions Our findings indicate that MTX-mediated synchronization of target cells allowed a significant improvement of retroviral HSV-tk gene transfer, resulting in an increased cell apoptosis in response to GCV. Pharmacological control of cell cycle may thus be a useful strategy to optimize the efficiency of retroviral-mediated cancer gene therapy.

  20. Expression of a human placental alkaline phosphatase gene in transfected cells: Use as a reporter for studies of gene expression

    International Nuclear Information System (INIS)

    The human placental alkaline phosphatase gene has been cloned and reintroduced into mammalian cells. When a plasmid carrying the gene under control of the simian virus 40 early promoter (pSV2Apap) is transfected into a variety of different cell types, placental alkaline phosphatase activity can readily be detected by using whole cell suspensions or cell lysates. Alkaline phosphatase activity can also be visualized directly in individual transfected cells by histochemical staining. The gene is appropriate for use as a reporter in studies of gene regulation since its expression is dependent on the presence of exogenous transcription control elements. The overall assay to detect the expression of the gene is quantitative, very rapid, and inexpensive. Cotransfections of cells with pSV2Apap and a related plasmid carrying the bacterial chloramphenicol acetyltransferase gene (pSV2Acat) indicate that transcription of these two genes is detected with roughly the same sensitivity

  1. Gene Expression Music Algorithm-Based Characterization of the Ewing Sarcoma Stem Cell Signature

    OpenAIRE

    Staege, Martin Sebastian

    2016-01-01

    Gene Expression Music Algorithm (GEMusicA) is a method for the transformation of DNA microarray data into melodies that can be used for the characterization of differentially expressed genes. Using this method we compared gene expression profiles from endothelial cells (EC), hematopoietic stem cells, neuronal stem cells, embryonic stem cells (ESC), and mesenchymal stem cells (MSC) and defined a set of genes that can discriminate between the different stem cell types. We analyzed the behavior ...

  2. Analysis of polarization methods for elimination of power overshoot in microbial fuel cells

    KAUST Repository

    Watson, Valerie J.

    2011-01-01

    Polarization curves from microbial fuel cells (MFCs) often show an unexpectedly large drop in voltage with increased current densities, leading to a phenomenon in the power density curve referred to as "power overshoot". Linear sweep voltammetry (LSV, 1 mV s- 1) and variable external resistances (at fixed intervals of 20 min) over a single fed-batch cycle in an MFC both resulted in power overshoot in power density curves due to anode potentials. Increasing the anode enrichment time from 30 days to 100 days did not eliminate overshoot, suggesting that insufficient enrichment of the anode biofilm was not the primary cause. Running the reactor at a fixed resistance for a full fed-batch cycle (~ 1 to 2 days), however, completely eliminated the overshoot in the power density curve. These results show that long times at a fixed resistance are needed to stabilize current generation by bacteria in MFCs, and that even relatively slow LSV scan rates and long times between switching circuit loads during a fed-batch cycle may produce inaccurate polarization and power density results for these biological systems. © 2010 Elsevier B.V. All rights reserved.

  3. Down-Regulation of Cell Surface Receptors Is Modulated by Polar Residues within the Transmembrane Domain

    Science.gov (United States)

    Zaliauskiene, Lolita; Kang, Sunghyun; Brouillette, Christie G.; Lebowitz, Jacob; Arani, Ramin B.; Collawn, James F.

    2000-01-01

    How recycling receptors are segregated from down-regulated receptors in the endosome is unknown. In previous studies, we demonstrated that substitutions in the transferrin receptor (TR) transmembrane domain (TM) convert the protein from an efficiently recycling receptor to one that is rapidly down regulated. In this study, we demonstrate that the “signal” within the TM necessary and sufficient for down-regulation is Thr11Gln17Thr19 (numbering in TM). Transplantation of these polar residues into the wild-type TR promotes receptor down-regulation that can be demonstrated by changes in protein half-life and in receptor recycling. Surprisingly, this modification dramatically increases the TR internalization rate as well (∼79% increase). Sucrose gradient centrifugation and cross-linking studies reveal that propensity of the receptors to self-associate correlates with down-regulation. Interestingly, a number of cell surface proteins that contain TM polar residues are known to be efficiently down-regulated, whereas recycling receptors for low-density lipoprotein and transferrin conspicuously lack these residues. Our data, therefore, suggest a simple model in which specific residues within the TM sequences dramatically influence the fate of membrane proteins after endocytosis, providing an alternative signal for down-regulation of receptor complexes to the well-characterized cytoplasmic tail targeting signals. PMID:10930460

  4. High performance single step co-fired solid oxide fuel cells (SOFC): Polarization measurements and analysis

    Science.gov (United States)

    Yoon, Kyung Joong

    At present, one of the major obstacles for the commercialization of solid oxide fuel cell (SOFC) power systems is their high manufacturing costs expressed in terms of SOFC system cost per unit power ($/kW). In this work, anode-supported planar SOFCs were fabricated by a cost-competitive single step co-firing process. The cells were comprised of a porous Ni + yittria-stabilized zirconia (YSZ) anode support, a porous-fine-grained Ni + YSZ anode active layer for some experiments, a dense YSZ electrolyte, a porous-fine-grained Ca-doped LaMnO3 (LCM) + YSZ cathode active layer, and a porous LCM cathode current collector layer. The fabrication process involved tape casting or high shear compaction (HSC) of the anode support followed by screen printing of the remaining component layers. The cells were then co-fired at 1300˜1340°C for 2 hours. The performance of the cell fabricated with the tape casting anode was improved by minimizing various polarization losses through experimental and theoretical modeling approaches, and the maximum power density of 1.5 W/cm 2 was obtained at 800°C with humidified hydrogen (3% H2O) and air. The cells were also tested with various compositions of humidified hydrogen (3˜70% H2O) to simulate the effect of practical fuel utilization on the cell performance. Based on these measurements, an analytical model describing anodic reactions was developed to understand reaction kinetics and rate limiting steps. The cell performance at high fuel utilization was significantly improved by increasing the number of the reaction sites near the anode-electrolyte interface. For anode substrate fabrication, the HSC process offers many advantages such as low fabrication costs, high production throughput, and good control of shrinkage and thickness over the conventional tape casting process. HSC process was successfully employed in single step co-firing process, and SOFCs fabricated with HSC anodes showed adequate performance both at low and high fuel

  5. Gene expression analysis of cell death induction by Taurolidine in different malignant cell lines

    International Nuclear Information System (INIS)

    The anti-infective agent Taurolidine (TRD) has been shown to have cell death inducing properties, but the mechanism of its action is largely unknown. The aim of this study was to identify potential common target genes modulated at the transcriptional level following TRD treatment in tumour cell lines originating from different cancer types. Five different malignant cell lines (HT29, Chang Liver, HT1080, AsPC-1 and BxPC-3) were incubated with TRD (100 μM, 250 μM and 1000 μM). Proliferation after 8 h and cell viability after 24 h were analyzed by BrdU assay and FACS analysis, respectively. Gene expression analyses were carried out using the Agilent -microarray platform to indentify genes which displayed conjoint regulation following the addition of TRD in all cell lines. Candidate genes were subjected to Ingenuity Pathways Analysis and selected genes were validated by qRT-PCR and Western Blot. TRD 250 μM caused a significant inhibition of proliferation as well as apoptotic cell death in all cell lines. Among cell death associated genes with the strongest regulation in gene expression, we identified pro-apoptotic transcription factors (EGR1, ATF3) as well as genes involved in the ER stress response (PPP1R15A), in ubiquitination (TRAF6) and mitochondrial apoptotic pathways (PMAIP1). This is the first conjoint analysis of potential target genes of TRD which was performed simultaneously in different malignant cell lines. The results indicate that TRD might be involved in different signal transduction pathways leading to apoptosis

  6. Gene, Stem Cell, and Alternative Therapies for SCA 1

    Science.gov (United States)

    Wagner, Jacob L.; O'Connor, Deirdre M.; Donsante, Anthony; Boulis, Nicholas M.

    2016-01-01

    Spinocerebellar ataxia 1 is an autosomal dominant disease characterized by neurodegeneration and motor dysfunction. In disease pathogenesis, polyglutamine expansion within Ataxin-1, a gene involved in transcriptional repression, causes protein nuclear inclusions to form. Most notably, neuronal dysfunction presents in Purkinje cells. However, the effect of mutant Ataxin-1 is not entirely understood. Two mouse models are employed to represent spinocerebellar ataxia 1, a B05 transgenic model that specifically expresses mutant Ataxin-1 in Purkinje cells, and a Sca1 154Q/2Q model that inserts the polyglutamine expansion into the mouse Ataxin-1 locus so that the mutant Ataxin-1 is expressed in all cells that express Ataxin-1. This review aims to summarize and evaluate the wide variety of therapies proposed for spinocerebellar ataxia 1, specifically gene and stem cell therapies.

  7. Influence of viral genes on the cell-to-cell spread of RNA silencing

    OpenAIRE

    Zhou, Yu; Ryabov, Eugene; Zhang, Xuemei; Hong, Yiguo

    2008-01-01

    The turnip crinkle virus-based vector TCV–GFPΔCP had been devised previously to study cell-to-cell and long-distance spread of virus-induced RNA silencing. TCV–GFPΔCP, which had been constructed by replacing the coat protein (CP) gene with a green fluorescent protein (GFP) coding sequence, was able to induce RNA silencing in single epidermal cells, from which RNA silencing spread from cell-to-cell. Using this unique local silencing assay together with mutagenesis analysis, two TCV genes, p8 a...

  8. Identification of novel Notch target genes in T cell leukaemia

    Directory of Open Access Journals (Sweden)

    Warrander Fiona

    2009-06-01

    Full Text Available Abstract Background Dysregulated Notch signalling is believed to play an important role in the development and maintenance of T cell leukaemia. At a cellular level, Notch signalling promotes proliferation and inhibits apoptosis of T cell acute lymphoblastic leukaemia (T-ALL cells. In this study we aimed to identify novel transcriptional targets of Notch signalling in the T-ALL cell line, Jurkat. Results RNA was prepared from Jurkat cells retrovirally transduced with an empty vector (GFP-alone or vectors containing constitutively active forms of Notch (N1ΔE or N3ΔE, and used for Affymetrix microarray analysis. A subset of genes found to be regulated by Notch was chosen for real-time PCR validation and in some cases, validation at the protein level, using several Notch-transduced T-ALL and non-T-ALL leukaemic cell lines. As expected, several known transcriptional target of Notch, such as HES1 and Deltex, were found to be overexpressed in Notch-transduced cells, however, many novel transcriptional targets of Notch signalling were identified using this approach. These included the T cell costimulatory molecule CD28, the anti-apoptotic protein GIMAP5, and inhibitor of DNA binding 1 (1D1. Conclusion The identification of such downstream Notch target genes provides insights into the mechanisms of Notch function in T cell leukaemia, and may help identify novel therapeutic targets in this disease.

  9. Using a GFP-gene fusion technique to study the cell cycle-dependent distribution of calmodulin in living cells

    Institute of Scientific and Technical Information of China (English)

    李朝军; 吕品; 张东才

    1999-01-01

    In this study, a green fluorescent protein (GFP)-calmodulin (CaM) fusion gene method was used to examine the distribution of calmodulin during various stages of cell cycle. First, it was found that the distribution of CaM in living cells changes with the cell cycle. CaM was found mainly in the cytoplasm during G1 phase. It began to move into the nucleus when the cell entered S phase. At G2 phase, CaM became more concentrated in the nucleus than in cytoplasm. Second, the accumulation of CaM in the nucleus during G2 phase appeared to be related to the onset of mitosis, since inhibiting the activation of CaM at this stage resulted in blocking the nuclear membrane breakdown and chromatin condensation. Finally, after the cell entered mitosis, a high concentration of CaM was found at the polar regions of the mitotic spindle. At this time, inhibiting the activity of CaM would cause a disruption of the spindle structure. The relationship between the stage-specific distribution of CaM and its function in regulat

  10. The reduction half cell in biomaterials corrosion: oxygen diffusion profiles near and cell response to polarized titanium surfaces.

    Science.gov (United States)

    Gilbert, J L; Zarka, L; Chang, E; Thomas, C H

    1998-11-01

    Mechanically assisted corrosion processes can greatly increase the oxidation currents generated in passivating alloy systems like Co-Cr and titanium due to oxide film disruption. When oxide films are abraded, repassivation and ionic dissolution both occur at rates that are orders of magnitude higher than undisrupted surfaces. The excess electrons generated by these anodic processes must be consumed in corresponding reduction reactions that include the reduction of oxygen. If large enough, these reduction reactions may locally deplete the concentration of solution-dissolved oxygen and, in turn, affect cell behavior in the vicinity of the implant surface. To date, this hypothesis has not been tested. In the present study, a scanning electrochemical microscope was used to measure oxygen concentration profiles in vitro near a planar titanium electrode polarized to different voltages representative of those attainable by titanium undergoing mechanically assisted corrosion. The potentials investigated ranged from 0 mV to -1000 mV (AgCl). The oxygen concentration as a function of distance from the titanium surface was measured using a platinum-iridium microelectrode and an amperometric technique. Also, preliminary experiments were performed to assess the response of rat calvarial osteoblast-rich cells cultured for 2 h on titanium samples polarized to two different potentials (0 mV and -1000 mV versus AgCl). The results of this study indicate that oxygen concentrations near titanium surfaces are affected by sample potentials out to probe-sample distances as great as 500 microm. Within 2 microm of the surface, oxygen concentrations decreased by 15 to 25% for sample potentials between -100 and -500 mV. At potentials more negative than -600 mV, the oxygen concentration dropped rapidly to near zero by -900 mV. The cell experiments showed a statistically significant difference in the amount of cell spreading, as measured by projected cell area, between the two groups (p < 0

  11. Euphorbia tirucalli modulates gene expression in larynx squamous cell carcinoma

    OpenAIRE

    Franco-Salla, Gabriela Bueno; Prates, Janesly; Cardin, Laila Toniol; dos Santos, Anemari Ramos Dinarte; Silva Jr, Wilson Araújo da; da Cunha, Bianca Rodrigues; Tajara, Eloiza Helena; Oliani, Sonia Maria; Rodrigues-Lisoni, Flávia Cristina

    2016-01-01

    Background Some plants had been used in the treatment of cancer and one of these has attracted scientific interest, the Euphorbia tirucalli (E. tirucalli), used in the treatment of asthma, ulcers, warts has active components with activities scientifically proven as antimutagenic, anti-inflammatory and anticancer. Methods We evaluate the influence of the antitumoral fraction of the E. tirucalli latex in the larynx squamous cell carcinoma (Hep-2), on the morphology, cell proliferation and gene ...

  12. The effect of adenovirus-mediated gene expression of FHIT in small cell lung cancer cells

    DEFF Research Database (Denmark)

    Zandi, Roza; Xu, Kai; Poulsen, Hans S;

    2011-01-01

    The candidate tumor suppressor fragile histidine traid (FHIT) is frequently inactivated in small cell lung cancer (SCLC). Mutations in the p53 gene also occur in the majority of SCLC leading to the accumulation of the mutant protein. Here we evaluated the effect of FHIT gene therapy alone or in...... combination with the mutant p53-reactivating molecule, PRIMA-1(Met)/APR-246, in SCLC. Overexpression of FHIT by recombinant adenoviral vector (Ad-FHIT)-mediated gene transfer in SCLC cells inhibited their growth by inducing apoptosis and when combined with PRIMA-1(Met)/APR-246, a synergistic cell growth...

  13. Susceptible genes and molecular pathways related to heavy ion irradiation in oral squamous cell carcinoma cells

    International Nuclear Information System (INIS)

    Background and purpose: Heavy ion beams are high linear energy transfer (LET) radiation characterized by a higher relative biologic effectiveness than low LET radiation. The aim of the current study was to determine the difference of gene expression between heavy ion beams and X-rays in oral squamous cell carcinoma (OSCC)-derived cells. Materials and methods: The OSCC cells were irradiated with accelerated carbon or neon ion irradiation or X-rays using three different doses. We sought to identify genes the expression of which is affected by carbon and neon ion irradiation using Affymetrix GeneChip analysis. The identified genes were analyzed using the Ingenuity Pathway Analysis Tool to investigate the functional network and gene ontology. Changes in mRNA expression in the genes were assessed by real-time quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR). Results: The microarray analysis identified 84 genes that were modulated by carbon and neon ion irradiation at all doses in OSCC cells. Among the genes, three genes (TGFBR2, SMURF2, and BMP7) and two genes (CCND1 and E2F3), respectively, were found to be involved in the transforming growth factor β-signaling pathway and cell cycle:G1/S checkpoint regulation pathway. The qRT-PCR data from the five genes after heavy ion irradiation were consistent with the microarray data (P < 0.01). Conclusion: Our findings should serve as a basis for global characterization of radiation-regulated genes and pathways in heavy ion-irradiated OSCC

  14. A Review of Gene Delivery and Stem Cell Based Therapies for Regenerating Inner Ear Hair Cells

    Directory of Open Access Journals (Sweden)

    Michael S. Detamore

    2011-09-01

    Full Text Available Sensory neural hearing loss and vestibular dysfunction have become the most common forms of sensory defects, affecting millions of people worldwide. Developing effective therapies to restore hearing loss is challenging, owing to the limited regenerative capacity of the inner ear hair cells. With recent advances in understanding the developmental biology of mammalian and non-mammalian hair cells a variety of strategies have emerged to restore lost hair cells are being developed. Two predominant strategies have developed to restore hair cells: transfer of genes responsible for hair cell genesis and replacement of missing cells via transfer of stem cells. In this review article, we evaluate the use of several genes involved in hair cell regeneration, the advantages and disadvantages of the different viral vectors employed in inner ear gene delivery and the insights gained from the use of embryonic, adult and induced pluripotent stem cells in generating inner ear hair cells. Understanding the role of genes, vectors and stem cells in therapeutic strategies led us to explore potential solutions to overcome the limitations associated with their use in hair cell regeneration.

  15. CHD7 targets active gene enhancer elements to modulate ES cell-specific gene expression.

    Directory of Open Access Journals (Sweden)

    Michael P Schnetz

    2010-07-01

    Full Text Available CHD7 is one of nine members of the chromodomain helicase DNA-binding domain family of ATP-dependent chromatin remodeling enzymes found in mammalian cells. De novo mutation of CHD7 is a major cause of CHARGE syndrome, a genetic condition characterized by multiple congenital anomalies. To gain insights to the function of CHD7, we used the technique of chromatin immunoprecipitation followed by massively parallel DNA sequencing (ChIP-Seq to map CHD7 sites in mouse ES cells. We identified 10,483 sites on chromatin bound by CHD7 at high confidence. Most of the CHD7 sites show features of gene enhancer elements. Specifically, CHD7 sites are predominantly located distal to transcription start sites, contain high levels of H3K4 mono-methylation, found within open chromatin that is hypersensitive to DNase I digestion, and correlate with ES cell-specific gene expression. Moreover, CHD7 co-localizes with P300, a known enhancer-binding protein and strong predictor of enhancer activity. Correlations with 18 other factors mapped by ChIP-seq in mouse ES cells indicate that CHD7 also co-localizes with ES cell master regulators OCT4, SOX2, and NANOG. Correlations between CHD7 sites and global gene expression profiles obtained from Chd7(+/+, Chd7(+/-, and Chd7(-/- ES cells indicate that CHD7 functions at enhancers as a transcriptional rheostat to modulate, or fine-tune the expression levels of ES-specific genes. CHD7 can modulate genes in either the positive or negative direction, although negative regulation appears to be the more direct effect of CHD7 binding. These data indicate that enhancer-binding proteins can limit gene expression and are not necessarily co-activators. Although ES cells are not likely to be affected in CHARGE syndrome, we propose that enhancer-mediated gene dysregulation contributes to disease pathogenesis and that the critical CHD7 target genes may be subject to positive or negative regulation.

  16. Dynamics of Cdc42 network embodies a Turing-type mechanism of yeast cell polarity.

    Science.gov (United States)

    Goryachev, Andrew B; Pokhilko, Alexandra V

    2008-04-30

    Complex biochemical networks can be understood by identifying their principal regulatory motifs and mode of action. We model the early phase of budding yeast cellular polarization and show that the biochemical processes in the presumptive bud site comprise a Turing-type mechanism. The roles of the prototypical activator and substrate are played by GTPase Cdc42 in its active and inactive states, respectively. We demonstrate that the nucleotide cycling of Cdc42 converts cellular energy into a stable cluster of activated Cdc42. This energy drives a continuous membrane-cytoplasmic exchange of the cluster components to counteract diffusive spread of the cluster. This exchange explains why only one bud forms per cell cycle, because the winner-takes-all competition of candidate sites inevitably selects a single site. PMID:18381072

  17. Production of p53 gene knockout rats by homologous recombination in embryonic stem cells

    OpenAIRE

    Tong, Chang; Li, Ping; Wu, Nancy L; Yan, Youzhen; Ying, Qi-Long

    2010-01-01

    The use of homologous recombination to modify genes in embryonic stem (ES) cells provides a powerful means to elucidate gene function and create disease models1. Application of this technology to engineer genes in rats has previously been impossible in the absence of germline competent ES cells in this species. We have recently established authentic rat ES cells2, 3. Here we report the generation of the first gene knockout rats using the ES cell-based gene targeting technology. We designed a ...

  18. Double suicide genes selectively kill human umbilical vein endothelial cells

    Directory of Open Access Journals (Sweden)

    Liu Lunxu

    2011-02-01

    Full Text Available Abstract Background To construct a recombinant adenovirus containing CDglyTK double suicide genes and evaluate the killing effect of the double suicide genes driven by kinase domain insert containing receptor (KDR promoter on human umbilical vein endothelial cells. Methods Human KDR promoter, Escherichia coli (E. coli cytosine deaminase (CD gene and the herpes simplex virus-thymidine kinase (TK gene were cloned using polymerase chain reaction (PCR. Plasmid pKDR-CDglyTK was constructed with the KDR promoter and CDglyTK genes. A recombinant adenoviral plasmid AdKDR-CDglyTK was then constructed and transfected into 293 packaging cells to grow and harvest adenoviruses. KDR-expressing human umbilical vein endothelial cells (ECV304 and KDR-negative liver cancer cell line (HepG2 were infected with the recombinant adenoviruses at different multiplicity of infection (MOI. The infection rate was measured by green fluorescent protein (GFP expression. The infected cells were cultured in culture media containing different concentrations of prodrugs ganciclovir (GCV and/or 5-fluorocytosine (5-FC. The killing effects were measured using two different methods, i.e. annexin V-FITC staining and terminal transferase-mediated dUTP nick end-labeling (TUNEL staining. Results Recombinant adenoviruses AdKDR-CDglyTK were successfully constructed and they infected ECV304 and HepG2 cells efficiently. The infection rate was dependent on MOI of recombinant adenoviruses. ECV304 cells infected with AdKDR-CDglyTK were highly sensitive to GCV and 5-FC. The cell survival rate was dependent on both the concentration of the prodrugs and the MOI of recombinant adenoviruses. In contrast, there were no killing effects in the HepG2 cells. The combination of two prodrugs was much more effective in killing ECV304 cells than GCV or 5-FC alone. The growth of transgenic ECV304 cells was suppressed in the presence of prodrugs. Conclusion AdKDR-CDglyTK/double prodrog system may be a useful

  19. Activation and polar sequestration of PopA, a c-di-GMP effector protein involved in Caulobacter crescentus cell cycle control

    DEFF Research Database (Denmark)

    Ozaki, Shogo; Schalch-Moser, Annina; Zumthor, Ludwig; Manfredi, Pablo; Ebbensgaard, Anna Elisabeth; Schirmer, Tilman; Jenal, Urs

    2014-01-01

    When Caulobacter crescentus enters S-phase the replication initiation inhibitor CtrA dynamically positions to the old cell pole to be degraded by the polar ClpXP protease. Polar delivery of CtrA requires PopA and the diguanylate cyclase PleD that positions to the same pole. Here we present evidence...... that PopA originated through gene duplication from its paralogue response regulator PleD and subsequent co-option as c-di-GMP effector protein. While the C-terminal catalytic domain (GGDEF) of PleD is activated by phosphorylation of the N-terminal receiver domain, functional adaptation has reversed...... PopA to the cell pole in response to c-di-GMP binding. In agreement with the divergent activation and targeting mechanisms, distinct markers sequester PleD and PopA to the old cell pole upon S-phase entry. Together these data indicate that PopA adopted a novel role as topology specificity factor to...

  20. Gene targeting in embryonic stem cells, II: conditional technologies

    Science.gov (United States)

    Genome modification via transgenesis has allowed researchers to link genotype and phenotype as an alternative approach to the characterization of random mutations through evolution. The synergy of technologies from the fields of embryonic stem (ES) cells, gene knockouts, and protein-mediated recombi...

  1. Lentiviral hematopoietic stem cell gene therapy in inherited metabolic disorders

    NARCIS (Netherlands)

    G. Wagemaker (Gerard)

    2014-01-01

    textabstractAfter more than 20 years of development, lentiviral hematopoietic stem cell gene therapy has entered the stage of initial clinical implementation for immune deficiencies and storage disorders. This brief review summarizes the development and applications, focusing on the lysosomal enzyme

  2. Polarizing T and B cell responses by APC-targeted subunit vaccines.

    Directory of Open Access Journals (Sweden)

    Gunnveig eGrødeland

    2015-07-01

    Full Text Available Current influenza vaccines mostly aim at the induction of specific neutralizing antibodies. While antibodies are important for protection against a particular virus strain, T cells can recognize epitopes that will offer broader protection against influenza. We have previously developed a DNA vaccine format by which protein antigens can be targeted specifically to receptors on antigen presenting cells (APCs. The DNA-encoded vaccine proteins are homodimers, each chain consisting of a targeting unit, a dimerization unit, and an antigen. The strategy of targeting antigen to APCs greatly enhances immune responses as compared to non-targeted controls. Furthermore, targeting of antigen to different receptors on APCs can polarize the immune response to different arms of immunity. Here, we discuss how targeting of hemagglutinin (HA to MHC class II molecules increases Th2 and IgG1 antibody responses, whereas targeting to chemokine receptors XCR1 or CCR1/3/5 increases Th1 and IgG2a responses, in addition to CD8+ T cell responses. We also discuss these results in relation to work published by others on APC-targeting. Differential targeting of APC surface molecules may allow the induction of tailor-made phenotypes of adaptive immune responses that are optimal for protection against various infectious agents, including influenza virus.

  3. Isolation of genes predominantly expressed in guard cells and epidermal cells of Nicotiana glauca.

    Science.gov (United States)

    Smart, L B; Cameron, K D; Bennett, A B

    2000-04-01

    Guard cells are specialized and metabolically active cells which arise during the differentiation of the epidermis. Using Nicotiana glauca epidermal peels as a source of purified guard cells, we have constructed a cDNA library from guard cell RNA. In order to isolate genes that are predominantly expressed in guard cells, we performed a differential screen of this library, comparing the hybridization of a radiolabeled cDNA probe synthesized from guard cell RNA to that from a mesophyll cell cDNA probe. Sixteen clones were isolated based on their greater level of hybridization with the guard cell probe. Of these, eight had high homology to lipid transfer protein (LTP), two were similar to glycine-rich protein (GRP), and one displayed high homology to proline-rich proteins from Arabidopsis thaliana (AtPRP2, AtPRP4) and from potato guard cells (GPP). Northern analysis confirmed that one or more NgLTP genes, NgGRP1, and NgGPP1 are all differentially expressed, with highest levels in guard cells, and low or undetectable levels in mesophyll cells and in roots. In addition, all are induced to some degree in drought-stressed guard cells. NgLTP and NgGRP1 expression was localized by in situ hybridization to the guard cells and pavement cells in the epidermis. NgGRP1 expression was also detected in cells of the vasculature. Genomic Southern analysis indicated that LTP is encoded by a family of highly similar genes in N. glauca. This work has identified members of a subset of epidermis- and guard cell-predominant genes, whose protein products are likely to contribute to the unique properties acquired by guard cells and pavement cells during differentiation. PMID:10890533

  4. RNA Interference Targeting Leptin Gene Effect on Hepatic Stellate Cells

    Institute of Scientific and Technical Information of China (English)

    XUE Xiulan; LIN Jusheng; SONG Yuhu; SUN Xuemei; ZHOU Hejun

    2005-01-01

    To construct the specific siRNA expression vectors and investigate their effect on leptin and collagen I in HSC, which provide a new approach to the prevent and treat hepatic fibrosis. The five siRNAs against leptin gene were transcript synthesized intracellularly by expression templates of plasmid vector psiRNA-hH1neo. The recombinant leptin siRNA plasmid vectors could express in eukaryocyte , and then to evaluate them by using enzyme cutting and sequencing. The recombinant plasmids were transfected into HSCs using Lipofectamine methods respectively. The cells were selected after growing in DMEM containing 300 μg/mL G418 for about 4 weeks. Gene expression of leptin and collagen I were showed by Western blot analysis and reverse transcription polymerase chain reaction (RT-PCR). Identification by enzyme cutting and sequencing showed that the leptin siRNA expression vectors were constructed successfully, and leptin siRNA could inhibit the leptin and collagen I gene expression effectively. It was concluded that RNA interference-mediated silencing of leptin gene diminished leptin and collagen I gene expression in HSCs. Furthermore, attenuated the extracellular matrix over-deposition at the same time. Leptin gene is ideal targets of gene therapy for liver fibrosis.

  5. Signaling at the inhibitory natural killer cell immune synapse regulates lipid raft polarization but not class I MHC clustering.

    Science.gov (United States)

    Fassett, M S; Davis, D M; Valter, M M; Cohen, G B; Strominger, J L

    2001-12-01

    Natural killer (NK) cell cytotoxicity is determined by a balance of positive and negative signals. Negative signals are transmitted by NK inhibitory receptors (killer immunoglobulin-like receptors, KIR) at the site of membrane apposition between an NK cell and a target cell, where inhibitory receptors become clustered with class I MHC ligands in an organized structure known as an inhibitory NK immune synapse. Immune synapse formation in NK cells is poorly understood. Because signaling by NK inhibitory receptors could be involved in this process, the human NK tumor line YTS was transfected with signal-competent and signal-incompetent KIR2DL1. The latter were generated by truncating the KIR2DL1 cytoplasmic tail or by introducing mutations in the immunoreceptor tyrosine-based inhibition motifs. The KIR2DL1 mutants retained their ability to cluster class I MHC ligands on NK cell interaction with appropriate target cells. Therefore, receptor-ligand clustering at the inhibitory NK immune synapse occurs independently of KIR2DL1 signal transduction. However, parallel examination of NK cell membrane lipid rafts revealed that KIR2DL1 signaling is critical for blocking lipid raft polarization and NK cell cytotoxicity. Moreover, raft polarization was inhibited by reagents that disrupt microtubules and actin filaments, whereas synapse formation was not. Thus, NK lipid raft polarization and inhibitory NK immune synapse formation occur by fundamentally distinct mechanisms. PMID:11724921

  6. Reprogramming resistant genes: in-depth comparison of gene expressions among iPS, ES and somatic cells.

    Directory of Open Access Journals (Sweden)

    Natalia ePolouliakh

    2013-01-01

    Full Text Available Transcription factor based reprogramming reverts adult cells to an embryonic state, yielding potential for generating different tissue types. However, recent reports indicated the substantial differences in pattern of gene expression between induced pluripotent stem (iPS cells and embryonic stem (ES cells. In this study we compare gene expression signatures of different iPS and ES cell lines and relate expression profiles of differently expressed genes to their expression status in somatic cells. As a result, we discovered that genes resistant to reprogramming comprise two major clusters, which are reprogramming dependent ‘Induced Genes’ and somatic origin ‘Inherited Genes’, both exhibiting preferences in methylation marks. Closer look into the Induced Genes by means of the transcription regulation analysis predicted several groups of genes with various roles in reprogramming and transgene DNA binding model. We believe that our results are a helpful source for biologists for further improvement of iPS cell technology.

  7. Gene expression profiling of circulating tumor cells and peripheral blood mononuclear cells from breast cancer patients

    Czech Academy of Sciences Publication Activity Database

    Hensler, M.; Vancurova, I.; Becht, E.; Palata, O.; Strnad, P.; Tesarova, P.; Cabinakova, M.; Švec, David; Kubista, Mikael; Bartunkova, J.; Spisek, R.; Sojka, L.

    2016-01-01

    Roč. 5, č. 4 (2016), e1102827. ISSN 2162-402X Institutional support: RVO:86652036 Keywords : Breast cancer * gene expression profiling * circulating tumor cells Subject RIV: FD - Oncology ; Hematology

  8. The effect of adenovirus-mediated gene expression of FHIT in small cell lung cancer cells

    DEFF Research Database (Denmark)

    Zandi, Roza; Xu, Kai; Poulsen, Hans S;

    2011-01-01

    The candidate tumor suppressor fragile histidine traid (FHIT) is frequently inactivated in small cell lung cancer (SCLC). Mutations in the p53 gene also occur in the majority of SCLC leading to the accumulation of the mutant protein. Here we evaluated the effect of FHIT gene therapy alone or in...

  9. Microbubble-Enhanced Ultrasound Gene Transfer into Fibroblast Cells

    Science.gov (United States)

    Hirayama, Kota; Kaneko, Yukio; Tei, Yuichi; Matsumoto, Yoichiro

    2007-05-01

    Ultrasound finds many applications in the medical field, including ultrasound imaging, non-invasive treatment of tumors and lithotripsy. Ultrasound also has a potential to deliver some therapeutic materials, such as genes, drugs or proteins into cells. It is known that microbubbles can improve the delivery efficiency. It is believed that therapeutic materials can pass through the cell membrane whose permeability is increased by microbubble destruction or the ultrasound pressure. In this study, we investigated the delivery of GFP plasmid gene into the fibroblast cells. Ultrasound (frequency = 2.1 MHz, duty cycle = 10%) was used to irradiate the cultured cells through a medium that contains microbubbles and GFP plasmid. GFP plasmid transfection could be easily observed by fluorescence microscopy. Ultrasound irradiation under a variety of conditions resulted in successful GFP plasmid delivery. Microbubbles enhanced GFP transfection, and conclusions were drawn as to the relationship between gene transfection and various ultrasound exposure parameters. We also investigated the effect of ultrasound intensity on cell viability.

  10. Gene Expression Profiling Predicts Survival in Conventional Renal Cell Carcinoma.

    Directory of Open Access Journals (Sweden)

    2005-12-01

    Full Text Available BACKGROUND: Conventional renal cell carcinoma (cRCC accounts for most of the deaths due to kidney cancer. Tumor stage, grade, and patient performance status are used currently to predict survival after surgery. Our goal was to identify gene expression features, using comprehensive gene expression profiling, that correlate with survival. METHODS AND FINDINGS: Gene expression profiles were determined in 177 primary cRCCs using DNA microarrays. Unsupervised hierarchical clustering analysis segregated cRCC into five gene expression subgroups. Expression subgroup was correlated with survival in long-term follow-up and was independent of grade, stage, and performance status. The tumors were then divided evenly into training and test sets that were balanced for grade, stage, performance status, and length of follow-up. A semisupervised learning algorithm (supervised principal components analysis was applied to identify transcripts whose expression was associated with survival in the training set, and the performance of this gene expression-based survival predictor was assessed using the test set. With this method, we identified 259 genes that accurately predicted disease-specific survival among patients in the independent validation group (p < 0.001. In multivariate analysis, the gene expression predictor was a strong predictor of survival independent of tumor stage, grade, and performance status (p < 0.001. CONCLUSIONS: cRCC displays molecular heterogeneity and can be separated into gene expression subgroups that correlate with survival after surgery. We have identified a set of 259 genes that predict survival after surgery independent of clinical prognostic factors.

  11. WT1-specific T cell receptor gene therapy: improving TCR function in transduced T cells.

    Science.gov (United States)

    Stauss, Hans J; Thomas, Sharyn; Cesco-Gaspere, Michela; Hart, Daniel P; Xue, Shao-An; Holler, Angelika; King, Judy; Wright, Graham; Perro, Mario; Pospori, Constantina; Morris, Emma

    2008-01-01

    Adoptive transfer of antigen-specific T lymphocytes is an attractive form of immunotherapy for haematological malignancies and cancer. The difficulty of isolating antigen-specific T lymphocytes for individual patients limits the more widespread use of adoptive T cell therapy. The demonstration that cloned T cell receptor (TCR) genes can be used to produce T lymphocyte populations of desired specificity offers new opportunities for antigen-specific T cell therapy. The first trial in humans demonstrated that TCR gene-modified T cells persisted for an extended time period and reduced tumor burden in some patients. The WT1 protein is an attractive target for immunotherapy of leukemia and solid cancer since elevated expression has been demonstrated in AML, CML, MDS and in breast, colon and ovarian cancer. In the past, we have isolated high avidity CTL specific for a WT1-derived peptide presented by HLA-A2 and cloned the TCR alpha and beta genes of a WT1-specific CTL line. The genes were inserted into retroviral vectors for transduction of human peripheral blood T lymphocytes of leukemia patients and normal donors. The treatment of leukemia-bearing NOD/SCID mice with T cells transduced with the WT1-specific TCR eliminated leukemia cells in the bone marrow of most mice, while treatment with T cells transduced with a TCR of irrelevant specificity did not diminish the leukemia burden. In order to improve the safety and efficacy of TCR gene therapy, we have developed lentiviral TCR gene transfer. In addition, we employed strategies to enhance TCR expression while avoiding TCR mis-pairing. It may be possible to generate dominant TCR constructs that can suppress the expression of the endogenous TCR on the surface of transduced T cells. The development of new TCR gene constructs holds great promise for the safe and effective delivery of TCR gene therapy for the treatment of malignancies. PMID:17855129

  12. Automatic Control of Gene Expression in Mammalian Cells.

    Science.gov (United States)

    Fracassi, Chiara; Postiglione, Lorena; Fiore, Gianfranco; di Bernardo, Diego

    2016-04-15

    Automatic control of gene expression in living cells is paramount importance to characterize both endogenous gene regulatory networks and synthetic circuits. In addition, such a technology can be used to maintain the expression of synthetic circuit components in an optimal range in order to ensure reliable performance. Here we present a microfluidics-based method to automatically control gene expression from the tetracycline-inducible promoter in mammalian cells in real time. Our approach is based on the negative-feedback control engineering paradigm. We validated our method in a monoclonal population of cells constitutively expressing a fluorescent reporter protein (d2EYFP) downstream of a minimal CMV promoter with seven tet-responsive operator motifs (CMV-TET). These cells also constitutively express the tetracycline transactivator protein (tTA). In cells grown in standard growth medium, tTA is able to bind the CMV-TET promoter, causing d2EYFP to be maximally expressed. Upon addition of tetracycline to the culture medium, tTA detaches from the CMV-TET promoter, thus preventing d2EYFP expression. We tested two different model-independent control algorithms (relay and proportional-integral (PI)) to force a monoclonal population of cells to express an intermediate level of d2EYFP equal to 50% of its maximum expression level for up to 3500 min. The control input is either tetracycline-rich or standard growth medium. We demonstrated that both the relay and PI controllers can regulate gene expression at the desired level, despite oscillations (dampened in the case of the PI controller) around the chosen set point. PMID:26414746

  13. Identifying genes that mediate anthracyline toxicity in immune cells

    Directory of Open Access Journals (Sweden)

    Amber eFrick

    2015-04-01

    Full Text Available The role of the immune system in response to chemotherapeutic agents remains elusive. The interpatient variability observed in immune and chemotherapeutic cytotoxic responses is likely, at least in part, due to complex genetic differences. Through the use of a panel of genetically diverse mouse inbred strains, we developed a drug screening platform aimed at identifying genes underlying these chemotherapeutic cytotoxic effects on immune cells. Using genome-wide association studies (GWAS, we identified four genome-wide significant quantitative trait loci (QTL that contributed to the sensitivity of doxorubicin and idarubicin in immune cells. Of particular interest, a locus on chromosome 16 was significantly associated with cell viability following idarubicin administration (p = 5.01x10-8. Within this QTL lies App, which encodes amyloid beta precursor protein. Comparison of dose-response curves verified that T-cells in App knockout mice were more sensitive to idarubicin than those of C57BL/6J control mice (p < 0.05.In conclusion, the cellular screening approach coupled with GWAS led to the identification and subsequent validation of a gene involved in T-cell viability after idarubicin treatment. Previous studies have suggested a role for App in in vitro and in vivo cytotoxicity to anticancer agents; the overexpression of App enhances resistance, while the knockdown of this gene is deleterious to cell viability. Thus, further investigations should include performing mechanistic studies, validating additional genes from the GWAS, including Ppfia1 and Ppfibp1, and ultimately translating the findings to in vivo and human studies.

  14. Regulation of histone gene expression during the cell cycle.

    Science.gov (United States)

    Meshi, T; Taoka, K I; Iwabuchi, M

    2000-08-01

    The steady-state level of histone mRNAs fluctuates coordinately with chromosomal DNA synthesis during the cell cycle. Such an S phase-specific expression pattern results from transcriptional activation of histone genes coupled with the onset of replication and from transcriptional repression of the genes as well as specific destabilization of histone mRNAs around the end of the S phase. Proliferation-coupled and S phase-specific expression of histone genes is primarily achieved by the activities of the proximal promoter regions, where several conserved cis-acting elements have been identified. Among them, three kinds of Oct-containing composite elements (OCEs) play a pivotal role in S phase-specific transcriptional activation. Other ones, such as Nona, solo-Oct, and CCGTC motifs, appear to modulate the functions of OCEs to enhance or repress the transcriptional level, possibly depending on the state of the cells. Here, we review the growing evidence concerning the regulatory mechanisms by which plant histone genes are expressed S phase-specifically in proliferating cells. PMID:11089867

  15. Early gene regulation of osteogenesis in embryonic stem cells

    KAUST Repository

    Kirkham, Glen R.

    2012-01-01

    The early gene regulatory networks (GRNs) that mediate stem cell differentiation are complex, and the underlying regulatory associations can be difficult to map accurately. In this study, the expression profiles of the genes Dlx5, Msx2 and Runx2 in mouse embryonic stem cells were monitored over a 48 hour period after exposure to the growth factors BMP2 and TGFβ1. Candidate GRNs of early osteogenesis were constructed based on published experimental findings and simulation results of Boolean and ordinary differential equation models were compared with our experimental data in order to test the validity of these models. Three gene regulatory networks were found to be consistent with the data, one of these networks exhibited sustained oscillation, a behaviour which is consistent with the general view of embryonic stem cell plasticity. The work cycle presented in this paper illustrates how mathematical modelling can be used to elucidate from gene expression profiles GRNs that are consistent with experimental data. © 2012 The Royal Society of Chemistry.

  16. Screening radiosensitizing-related genes mediated by elemene in lung adenocarcinoma A549 cells by using gene chip

    International Nuclear Information System (INIS)

    Objective: To screen radiosensitizing-related genes mediated by elemene in lung adenocarcinoma A549 cells by using gene chip. Methods: MTT test was used to calculate the IC50 of elemene. (1) The effect of radiosensitivity was detected by colony forming assay. A549 cells were divided into 2 groups: radiation group and radiation + elemene group. Oligonucleotide chip was used to screen the gene expression changes of A549 cells from these 2 groups. The up-regulated gene Egr-1 and the down-regulated gene CyclinD1 were selected to undergo RT-PCR so as to confirm the reliability of the result. Results: MTT test showed the elemene inhibited the proliferation of the A549 cells dose-dependently. The IC50 value of elemene on the A549 cells was 120 mg/L. (2) 10 mg/L elemene had radiosensitising effect on A549 cells.The values of SERD0 and SERDq obtained from the survival curve were (1.54±0. 20) and (1.43±0.15) respectively. Gene chip screened 122 differentially-expressed genes, including 89 up-regulated genes and 33 down-regulated genes. (3) These altered genes could be related to cell structure, substance metabolism,cell proliferation, cell differentiation, signal transduction, material transport, DNA repair, apoptosis, immune response and so forth. The RT-PCR results of Egr-1 and Cyclin D1 were consistent with the gene chip analysis. Conclusions: The mechanism of elemene enhancing the radiosensitivity of lung adenocarcinoma A549 cells is the result of participation and collaboration of multiple genes. Further study of the newly-discovered differentially-expressed gene helps find out new radiosensitizational targets of elemene. (authors)

  17. Radiation improves gene transfer into human ovarian carcinoma cells

    International Nuclear Information System (INIS)

    Purpose/Objective: Poor gene transfer is the major stumbling block to successful gene therapy today. We hypothesized that ionizing radiation might activate cellular recombination, and so improve stable gene transfer. During studies to quantitate radiation activated recombination, we also found that both plasmid and adenoviral vector transduction could be increased by irradiation. The studies presented here describe the effects of irradiation on gene transduction efficiency (both transient and stable transduction) in several human ovarian carcinoma lines, as a prelude to in vivo animal studies. Materials and Methods: The effect of irradiation on stable gene transfer efficiency was determined in human ovarian carcinoma cell lines (SKOV3, CAOV3 and PA1). Either irradiated or unirradiated cells were transfected with pRSVZ plasmid (containing a LacZ expression cassette) in either the supercoiled and linearized (XmnI) forms and β-galactosidase expression followed with time. Transfection efficiency was measured by flow cytometry following FDG staining at 0, 48, and 96 hours after irradiation. FDG is converted to a fluorescent metabolite by LacZ, and thus reflects the transfection efficiency of the LacZ containing vector. Vector quantitation was also performed by southern hybridization. Stable transduction efficiency was measured 14 -35 days after irradiation. Optimization of the time of irradiation with respect to transfection was performed. Since cells demonstrated increased stable recombination for as long as 96 hours after irradiation, continuous low dose rate and multiple radiation fractions were also tested. These experiments were repeated using the Ad5CMVlacZ. Dividing cells were exposed to Ad5CMVlacZ at an MOI of 0.1,1,5,10 and 100 to determine optimum transfection concentration. Transduction efficiency was again measured at various intervals to determine the radiation dose and interval post transfection which provides the maximum increase in transfection

  18. Identification of genes associated with cisplatin resistance in human oral squamous cell carcinoma cell line

    Directory of Open Access Journals (Sweden)

    Zhang Ping

    2006-09-01

    Full Text Available Abstract Background Cisplatin is widely used for chemotherapy of head and neck squamous cell carcinoma. However, details of the molecular mechanism responsible for cisplatin resistance are still unclear. The aim of this study was to identify the expression of genes related to cisplatin resistance in oral squamous cell carcinoma cells. Methods A cisplatin-resistant cell line, Tca/cisplatin, was established from a cisplatin-sensitive cell line, Tca8113, which was derived from moderately-differentiated tongue squamous cell carcinoma. Global gene expression in this resistant cell line and its sensitive parent cell line was analyzed using Affymetrix HG-U95Av2 microarrays. Candidate genes involved in DNA repair, the MAP pathway and cell cycle regulation were chosen to validate the microarray analysis results. Cell cycle distribution and apoptosis following cisplatin exposure were also investigated. Results Cisplatin resistance in Tca/cisplatin cells was stable for two years in cisplatin-free culture medium. The IC50 for cisplatin in Tca/cisplatin was 6.5-fold higher than that in Tca8113. Microarray analysis identified 38 genes that were up-regulated and 25 that were down-regulated in this cell line. Some were novel candidates, while others are involved in well-characterized mechanisms that could be relevant to cisplatin resistance, such as RECQL for DNA repair and MAP2K6 in the MAP pathway; all the genes were further validated by Real-time PCR. The cell cycle-regulated genes CCND1 and CCND3 were involved in cisplatin resistance; 24-hour exposure to 10 μM cisplatin induced a marked S phase block in Tca/cisplatin cells but not in Tca8113 cells. Conclusion The Tca8113 cell line and its stable drug-resistant variant Tca/cisplatin provided a useful model for identifying candidate genes responsible for the mechanism of cisplatin resistance in oral squamous cell carcinoma. Our data provide a useful basis for screening candidate targets for early diagnosis

  19. Identification of genes associated with cisplatin resistance in human oral squamous cell carcinoma cell line

    International Nuclear Information System (INIS)

    Cisplatin is widely used for chemotherapy of head and neck squamous cell carcinoma. However, details of the molecular mechanism responsible for cisplatin resistance are still unclear. The aim of this study was to identify the expression of genes related to cisplatin resistance in oral squamous cell carcinoma cells. A cisplatin-resistant cell line, Tca/cisplatin, was established from a cisplatin-sensitive cell line, Tca8113, which was derived from moderately-differentiated tongue squamous cell carcinoma. Global gene expression in this resistant cell line and its sensitive parent cell line was analyzed using Affymetrix HG-U95Av2 microarrays. Candidate genes involved in DNA repair, the MAP pathway and cell cycle regulation were chosen to validate the microarray analysis results. Cell cycle distribution and apoptosis following cisplatin exposure were also investigated. Cisplatin resistance in Tca/cisplatin cells was stable for two years in cisplatin-free culture medium. The IC50 for cisplatin in Tca/cisplatin was 6.5-fold higher than that in Tca8113. Microarray analysis identified 38 genes that were up-regulated and 25 that were down-regulated in this cell line. Some were novel candidates, while others are involved in well-characterized mechanisms that could be relevant to cisplatin resistance, such as RECQL for DNA repair and MAP2K6 in the MAP pathway; all the genes were further validated by Real-time PCR. The cell cycle-regulated genes CCND1 and CCND3 were involved in cisplatin resistance; 24-hour exposure to 10 μM cisplatin induced a marked S phase block in Tca/cisplatin cells but not in Tca8113 cells. The Tca8113 cell line and its stable drug-resistant variant Tca/cisplatin provided a useful model for identifying candidate genes responsible for the mechanism of cisplatin resistance in oral squamous cell carcinoma. Our data provide a useful basis for screening candidate targets for early diagnosis and further intervention in cisplatin resistance

  20. Recombinant cells that highly express chromosomally-integrated heterologous gene

    Science.gov (United States)

    Ingram, Lonnie O.; Ohta, Kazuyoshi; Wood, Brent E.

    2007-03-20

    Recombinant host cells are obtained that comprise (A) a heterologous, polypeptide-encoding polynucleotide segment, stably integrated into a chromosome, which is under transcriptional control of an endogenous promoter and (B) a mutation that effects increased expression of the heterologous segment, resulting in enhanced production by the host cells of each polypeptide encoded by that segment, relative to production of each polypeptide by the host cells in the absence of the mutation. The increased expression thus achieved is retained in the absence of conditions that select for cells displaying such increased expression. When the integrated segment comprises, for example, ethanol-production genes from an efficient ethanol producer like Zymomonas mobilis, recombinant Escherichia coli and other enteric bacterial cells within the present invention are capable of converting a wide range of biomass-derived sugars efficiently to ethanol.

  1. Gene analysis and dynamics of tumor stem cell in human glioblastoma cells after radiation

    International Nuclear Information System (INIS)

    Because glioblastoma is the most malignant central nervous system (CNS) tumor, it is very difficult to cure despite surgery and adjuvant therapy. At present, surgery, radiotherapy, and chemotherapy are combined in the treatment of each patient. However, glioblastoma have radiotherapy and chemotherapy resistance, and this is not a radical treatment. We suspect that the tumor stem cell affects the recurrence, radiotherapy resistance and chemotherapy resistance of the tumor. Many studies suggest that tumor stem cells play an important role in tumorgenesis and tumor progression. Using human glioblastoma cell lines (T98G, A172), irradiated (0 Gy, 30 Gy, 60 Gy) glioblastoma cells were prepared under the same conditions as clinical therapy. We performed the analysis of cell proliferation rate, side population analysis by fluorescence-activated cell sorter (FACS), isolation of CD133+ cells and genetic analysis (human stem cell), using these cells. In the results of this study, the stem cell-related genes were highly expressed in the CD133+ cells compared with the CD133- cells. Therefore, it suggested that the CD133+ cells may contain tumor stem cells. In T98G, when compared to unirradiated cells and 60 Gy-irradiated cells, the cell proliferation rate for 30 Gy-irradiated cells tended to be higher, and stem cell-related genes were highly expressed in 30 Gy-irradiated CD133+ cells. In other words, in T98G, from the viewpoint of antitumor effects, the results suggest that chemotherapy may show more effect in 30 Gy-irradiated. In this genetic analysis, we suggest that CD133+ cells strongly affect tumor proliferation. In addition, CD133+ cells affect the resistance and the effect of treatments because some kind of changes occur in CD133+ cells after radiation. (author)

  2. Gene expression profile differences in high and low metastatic human ovarian cancer cell lines by gene chip

    Institute of Scientific and Technical Information of China (English)

    许沈华; 牟瀚舟; 吕桂泉; 朱赤红; 羊正炎; 高永良; 楼洪坤; 刘祥麟; 程勇; 杨文

    2002-01-01

    Objectives To study the difference between gene expressions of high (H0-8910PM) and low (HO-8910) metastatic human ovarian carcinoma cell lines and screen novel associated genes by cDNA microarray. Methods cDNA retro-transcribed from equal quantities of mRNA derived from high and low metastatic tumor cells or normal ovarian tissues were labeled with Cy5 and Cy3 fluorescein as probes. The mixed probe was hybridized with two pieces of BioDoor 4096 double dot human whole gene chip and scanned with a ScanArray 3000 laser scanner. The acquired image was analyzed by ImaGene 3.0 software. Results A total of 355 genes with expression levels more than 3 times larger were found by comparing the HO-8910 cell with normal ovarian epithelial cells. A total of 323 genes with expression levels more than 3 times larger in HO-8910PM cells compared to normal ovarian epithelium cells were also detected. A total of 165 genes whose expression levels were more than two times those of HO-8910PM cells compared to their mother cell line (HO-8910) were detected. Twenty-one genes with expression levels >3 times were found from comparison of these two tumor cell lines.Conclusions cDNA microarray techniques are effective in screening differential gene expression between two human ovarian cancer cell lines (H0-8910PM; HO-8910) and normal ovarian epithelial cells. These genes may be related to the genesis and development of ovarian carcinoma. Analysis of the human ovarian cancer gene expression profile with cDNA microarray may help in gene diagnosis, treatment and prevention.

  3. Effects of space flight exposure on cell growth, tumorigenicity and gene expression in cancer cells

    Science.gov (United States)

    Yang, Cheng; Li, Yuehui; Zhang, Zhijie; Luo, Chen; Tong, Yongqing; Zhou, Guohua; Xie, Pingli; Hu, Jinyue; Li, Guancheng

    2008-12-01

    It is well recognized that harsh outer space environment, consisting of microgravity and radiation, poses significant health risks for human cells. To investigate potential effects of the space environment exposure on cancer cells we examined the biological changes in Caski cells carried by the "Shen Zhou IV" spaceship. After exposure for 7 days in spaceflight, 1440 survival subclonal cell lines were established and 4 cell lines were screened. 44F10 and 17E3 were selected because of their increased cell proliferation and tumorigenesis, while 48A9 and 31F2 had slower cytological events. Experiments with cell proliferation assay, flow cytometry, soft agar assay, tumorigenesis assay and DNA microarray analysis have shown that selected cell lines presented multiple biological changes in cell morphology, cell growth, tumorigenicity and gene expression. These results suggest that space environment exposure can make significant biological impact on cancer cells and provide an entry point to find the immunological target of tumorigenesis.

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

    OpenAIRE

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

    1998-01-01

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

  5. Generation of sphingosine-1-phosphate by apoptotic cells and its impact on macrophage polarization in cancer development

    OpenAIRE

    Weigert, Andreas

    2008-01-01

    The removal of apoptotic cells (AC) can be regarded as an integral component of the program to terminate inflammation. Clearance of AC by professional phagocytes such as macrophages induces an anti-inflammatory phenotype in the latter ones. Anti-inflammatory or M2 polarization is also observed in macrophages infiltrating certain human tumors. These tumor-associated macrophages (TAM) contribute actively to tumor progression by promoting immune evasion, angiogenesis and tumor cell survival. The...

  6. T Cells and Gene Regulation: The Switching On and Turning Up of Genes after T Cell Receptor Stimulation in CD8 T Cells

    Science.gov (United States)

    Conley, James M.; Gallagher, Michael P.; Berg, Leslie J.

    2016-01-01

    Signaling downstream of the T cell receptor (TCR) is directly regulated by the dose and affinity of peptide antigen. The strength of TCR signaling drives a multitude of T cell functions from development to differentiation. CD8 T cells differentiate into a diverse pool of effector and memory cells after activation, a process that is critical for pathogen clearance and is highly regulated by TCR signal strength. T cells rapidly alter their gene expression upon activation. Multiple signaling pathways downstream of the TCR activate transcription factors, which are critical for this process. The dynamics between proximal TCR signaling, transcription factor activation and CD8 T cell function are discussed here. We propose that inducible T cell kinase (ITK) acts as a rheostat for gene expression. This unique regulation of TCR signaling by ITK provides a possible signaling mechanism for the promotion of a diverse T cell repertoire in response to pathogen. PMID:26973653

  7. Repression of the albumin gene in Novikoff hepatoma cells

    International Nuclear Information System (INIS)

    Novikoff hepatoma cells have lost their capacity to synthesize albumin. As a first approach to study the mechanisms underlying this event, in vitro translation in a reticulocyte system was performed using total polyadenylated mRNA from rat liver and Novikoff hepatoma cells. Immunoprecipitation of the in vitro translation products with albumin-specific antibody revealed a total lack of albumin synthesis in Novikoff hepatoma, suggesting the absence of functional albumin mRNA in these cells. Titration experiments using as probe albumin cDNA cloned in pBR322 plasmid demonstrated the absence of albumin-specific sequences in both polysomal and nuclear polyadenylated and total RNA from Novikoff cells. This albumin recombinant plasmid was obtained by screening a rat liver cDNA library with albumin [/sup 32/P]cDNA reverse transcribed from immuno-precipitated mRNA. The presence of an albumin-specific gene insert was documented with translation assays as well as by restriction mapping. Repression of the albumin gene at the transcriptional level was further demonstrated by RNA blotting experiments using the cloned albumin cDNA probe. Genomic DNA blots using the cloned albumin cDNA as probe did not reveal any large-scale deletions, insertions, or rearrangements in the albumin gene, suggesting that the processes involved in the suppression of albumin mRNA synthesis do not involve extensive genomic rearrangements

  8. Expression of caspase-3 gene in apoptotic HL-60 cell and different human tumor cell lines

    International Nuclear Information System (INIS)

    Objective: To research the expression of caspase-3 gene in the apoptotic and the control HL-60 cells and in the different human tumor cell lines. Methods: Caspase-3 mRNA in the control and γ-radiation-induced apoptotic HL-60 cells, and in the 6 types of human tumor cell lines, was analysed by Northern blot. Results: The caspase-3 gene transcript was more highly expressed in leukemia cells HL-60, CEM, K562 and neuroblastoma SH-SY5Y than in cervical adenocarcinoma HeLa and breast carcinoma MCF7, and more highly in the radiation-induced apoptotic HL-60 than in the control HL-60 cells. Conclusion: The high level of expression of caspase-3 may aid the efforts to understand the tumor cell sensitivity to radiation, apoptosis and its inherent ability to survive

  9. Reference gene selection for head and neck squamous cell carcinoma gene expression studies

    Directory of Open Access Journals (Sweden)

    Raynal Caroline

    2009-08-01

    Full Text Available Abstract Background It is no longer adequate to choose reference genes blindly. We present the first study that defines the suitability of 12 reference genes commonly used in cancer studies (ACT, ALAS, B2M, GAPDH, HMBS, HPRT, KALPHA, RPS18, RPL27, RPS29, SHAD and TBP for the normalization of quantitative expression data in the field of head and neck squamous cell carcinoma (HNSCC. Results Raw expression levels were measured by RT-qPCR in HNSCC and normal matched mucosa of 46 patients. We analyzed the expression stability using geNorm and NormFinder and compared the expression levels between subgroups. In HNSCC and/or normal mucosa, the four best normalization genes were ALAS, GAPDH, RPS18 and SHAD and the most stable combination of two genes was GAPDH-SHAD. We recommend using KALPHA-TBP for the study of T1-T2 tumors, RPL27-SHAD for T3-T4 tumors, KALPHA-SHAD for N0 tumors, and ALAS-TBP for N+ tumors. ACT, B2M, GAPDH, HMBS, HPRT, KALPHA, RPS18, RPS29, SHAD and TBP were slightly misregulated ( Conclusion In the field of HNSCC, this study will guide researchers in selecting the most appropriate reference genes from among 12 potentially suitable reference genes, depending on the specific setting of their experiments.

  10. Gene Profiling of Aortic Valve Interstitial Cells under Elevated Pressure Conditions: Modulation of Inflammatory Gene Networks

    Directory of Open Access Journals (Sweden)

    James N. Warnock

    2011-01-01

    Full Text Available The study aimed to identify mechanosensitive pathways and gene networks that are stimulated by elevated cyclic pressure in aortic valve interstitial cells (VICs and lead to detrimental tissue remodeling and/or pathogenesis. Porcine aortic valve leaflets were exposed to cyclic pressures of 80 or 120 mmHg, corresponding to diastolic transvalvular pressure in normal and hypertensive conditions, respectively. Linear, two-cycle amplification of total RNA, followed by microarray was performed for transcriptome analysis (with qRT-PCR validation. A combination of systems biology modeling and pathway analysis identified novel genes and molecular mechanisms underlying the biological response of VICs to elevated pressure. 56 gene transcripts related to inflammatory response mechanisms were differentially expressed. TNF-α, IL-1α, and IL-1β were key cytokines identified from the gene network model. Also of interest was the discovery that pentraxin 3 (PTX3 was significantly upregulated under elevated pressure conditions (41-fold change. In conclusion, a gene network model showing differentially expressed inflammatory genes and their interactions in VICs exposed to elevated pressure has been developed. This system overview has detected key molecules that could be targeted for pharmacotherapy of aortic stenosis in hypertensive patients.

  11. Regulators of gene expression in Enteric Neural Crest Cells are putative Hirschsprung disease genes.

    Science.gov (United States)

    Schriemer, Duco; Sribudiani, Yunia; IJpma, Arne; Natarajan, Dipa; MacKenzie, Katherine C; Metzger, Marco; Binder, Ellen; Burns, Alan J; Thapar, Nikhil; Hofstra, Robert M W; Eggen, Bart J L

    2016-08-01

    The enteric nervous system (ENS) is required for peristalsis of the gut and is derived from Enteric Neural Crest Cells (ENCCs). During ENS development, the RET receptor tyrosine kinase plays a critical role in the proliferation and survival of ENCCs, their migration along the developing gut, and differentiation into enteric neurons. Mutations in RET and its ligand GDNF cause Hirschsprung disease (HSCR), a complex genetic disorder in which ENCCs fail to colonize variable lengths of the distal bowel. To identify key regulators of ENCCs and the pathways underlying RET signaling, gene expression profiles of untreated and GDNF-treated ENCCs from E14.5 mouse embryos were generated. ENCCs express genes that are involved in both early and late neuronal development, whereas GDNF treatment induced neuronal maturation. Predicted regulators of gene expression in ENCCs include the known HSCR genes Ret and Sox10, as well as Bdnf, App and Mapk10. The regulatory overlap and functional interactions between these genes were used to construct a regulatory network that is underlying ENS development and connects to known HSCR genes. In addition, the adenosine receptor A2a (Adora2a) and neuropeptide Y receptor Y2 (Npy2r) were identified as possible regulators of terminal neuronal differentiation in GDNF-treated ENCCs. The human orthologue of Npy2r maps to the HSCR susceptibility locus 4q31.3-q32.3, suggesting a role for NPY2R both in ENS development and in HSCR. PMID:27266404

  12. Selective and site-specific mobilization of dermal dendritic cells and Langerhans cells by Th1- and Th2-polarizing adjuvants

    OpenAIRE

    Sen, Debasish; Forrest, Luette; Kepler, Thomas B.; Parker, Ian; Cahalan, Michael D.

    2010-01-01

    Dendritic cells (DCs) initiate and polarize adaptive immune responses toward varying functional outcomes. By means of intravital two-photon microscopy, we report that dermal dendritic cells (DDCs) and Langerhans cells (LCs) are differentially mobilized during contact sensitization and by adjuvants such as unmethylated CpG oligonucleotide (CpG) and LPS that induce T helper type 1 (Th1) responses, or papain that induces T helper type 2 (Th2) responses. In ear pinna, contact sensitization, CpG, ...

  13. In vitro Th1 and Th2 cell polarization is severely influenced by the initial ratio of naïve and memory CD4+ T cells

    DEFF Research Database (Denmark)

    Blom, Lars; Poulsen, Lars K.

    even small percentages (99% naïve CD4+ T cells resulted in better Th1 and Th2 polarization with significant reduced fractions of IL-4+ and IFN-γ+ CD4+ T cells, respectively. Moreover, the Th2 primed >99% naïve CD4+ T cells showed significantly higher ratio of IL-4+:IFN-γ+ (>4 fold) and GATA-3:+T...

  14. Par-3 partitioning defective 3 homolog (C. elegans) and androgen-induced prostate proliferative shutoff associated protein genes are mutationally inactivated in prostate cancer cells

    International Nuclear Information System (INIS)

    Gene identification by nonsense-mediated mRNA decay inhibition (GINI) has proven its usefulness in identifying mutant genes in cancer cell lines. An increase in transcription in response to NMD inhibition of a subset of genes is a major cause of false positives when genes are selected for sequencing analysis. To distinguish between mRNA accumulations caused by stress response-induced transcription and nonsense-containing mRNA stabilizations is a challenge in identifying mutant genes using GINI. To identify potential tumor-suppressor genes mutated in prostate cancer cell lines, we applied a version of GINI that involves inhibition of NMD in two steps. In the first step, NMD is inhibited in duplicate tissue-culture plates. During this step, both the substrate for NMD and stress-response mRNA transcripts are accumulated in cells. In the second step, transcription is inhibited in both plates and NMD is inhibited in one plate and released in the second plate. Microarray analysis of gene-expression profiles in both plates after the second step detects only the differences in mRNA degradation but not in mRNA accumulation. Analyzing gene expression profile alterations in 22RV1 and LNCaP prostate cancer cells following NMD inhibition we selected candidates for sequencing analysis in both cell lines. Sequencing identified inactivating mutations in both alleles of the PARD3 and AS3 genes in the LNCaP and 22RV1 cells, respectively. Introduction of a wild-type PARD3 cDNA into the LNCaP cells resulted in a higher proliferation rate in tissue culture, a higher adhesion of LNCaP cells to the components of extracellular matrix and impaired the growth of the LNCaP cells in soft agar and in a three-dimensional cell-culture. The mutational inactivation in a prostate cancer cell line of the PARD3 gene involved in asymmetric cell division and maintenance of cell-polarity suggests that the loss of cell-polarity contributes to prostate carcinogenesis

  15. Discrimination of meniscal cell phenotypes using gene expression profiles

    Directory of Open Access Journals (Sweden)

    M Son

    2012-03-01

    Full Text Available The lack of quantitative and objective metrics to assess cartilage and meniscus cell phenotypes contributes to the challenges in fibrocartilage tissue engineering. Although functional assessment of the final resulting tissue is essential, initial characterization of cell sources and quantitative description of their progression towards the natural, desired cell phenotype would provide an effective tool in optimizing cell-based tissue engineering strategies. The purpose of this study was to identify quantifiable characteristics of meniscal cells and thereby find phenotypical markers that could effectively categorize cells based on their tissue of origin (cartilage, inner, middle, and outer meniscus. The combination of gene expression ratios collagen VI/collagen II, ADAMTS-5/collagen II, and collagen I/collagen II was the most effective indicator of variation among different tissue regions. We additionally demonstrate a possible application of these quantifiable metrics in evaluating the use of serially passaged chondrocytes as a possible cell source in fibrocartilage engineering. Comparing the ratios of the passaged chondrocytes and the native meniscal cells may provide direction to optimize towards the desired cell phenotype. We have thus shown that measurable markers defining the characteristics of the native meniscus can establish a standard by which different tissue engineering strategies can be objectively assessed. Such metrics could additionally be useful in exploring the different stages of meniscal degradation in osteoarthritis and provide some insight in the disease progression.

  16. The FRIABLE1 gene product affects cell adhesion in Arabidopsis.

    Directory of Open Access Journals (Sweden)

    Lutz Neumetzler

    Full Text Available Cell adhesion in plants is mediated predominantly by pectins, a group of complex cell wall associated polysaccharides. An Arabidopsis mutant, friable1 (frb1, was identified through a screen of T-DNA insertion lines that exhibited defective cell adhesion. Interestingly, the frb1 plants displayed both cell and organ dissociations and also ectopic defects in organ separation. The FRB1 gene encodes a Golgi-localized, plant specific protein with only weak sequence similarities to known proteins (DUF246. Unlike other cell adhesion deficient mutants, frb1 mutants do not have reduced levels of adhesion related cell wall polymers, such as pectins. Instead, FRB1 affects the abundance of galactose- and arabinose-containing oligosaccharides in the Golgi. Furthermore, frb1 mutants displayed alteration in pectin methylesterification, cell wall associated extensins and xyloglucan microstructure. We propose that abnormal FRB1 action has pleiotropic consequences on wall architecture, affecting both the extensin and pectin matrices, with consequent changes to the biomechanical properties of the wall and middle lamella, thereby influencing cell-cell adhesion.

  17. Altered epigenetic regulation of homeobox genes in human oral squamous cell carcinoma cells

    Energy Technology Data Exchange (ETDEWEB)

    Marcinkiewicz, Katarzyna M.; Gudas, Lorraine J., E-mail: ljgudas@med.cornell.edu

    2014-01-01

    To gain insight into oral squamous cell carcinogenesis, we performed deep sequencing (RNAseq) of non-tumorigenic human OKF6-TERT1R and tumorigenic SCC-9 cells. Numerous homeobox genes are differentially expressed between OKF6-TERT1R and SCC-9 cells. Data from Oncomine, a cancer microarray database, also show that homeobox (HOX) genes are dysregulated in oral SCC patients. The activity of Polycomb repressive complexes (PRC), which causes epigenetic modifications, and retinoic acid (RA) signaling can control HOX gene transcription. HOXB7, HOXC10, HOXC13, and HOXD8 transcripts are higher in SCC-9 than in OKF6-TERT1R cells; using ChIP (chromatin immunoprecipitation) we detected PRC2 protein SUZ12 and the epigenetic H3K27me3 mark on histone H3 at these genes in OKF6-TERT1R, but not in SCC-9 cells. In contrast, IRX1, IRX4, SIX2 and TSHZ3 transcripts are lower in SCC-9 than in OKF6-TERT1R cells. We detected SUZ12 and the H3K27me3 mark at these genes in SCC-9, but not in OKF6-TERT1R cells. SUZ12 depletion increased HOXB7, HOXC10, HOXC13, and HOXD8 transcript levels and decreased the proliferation of OKF6-TERT1R cells. Transcriptional responses to RA are attenuated in SCC-9 versus OKF6-TERT1R cells. SUZ12 and H3K27me3 levels were not altered by RA at these HOX genes in SCC-9 and OKF6-TERT1R cells. We conclude that altered activity of PRC2 is associated with dysregulation of homeobox gene expression in human SCC cells, and that this dysregulation potentially plays a role in the neoplastic transformation of oral keratinocytes. - Highlights: • RNAseq elucidates differences between non-tumorigenic and tumorigenic oral keratinocytes. • Changes in HOX mRNA in SCC-9 vs. OKF6-TERT1R cells are a result of altered epigenetic regulation. • RNAseq shows that retinoic acid (RA) influences gene expression in both OKF6-TERT1R and SCC-9 cells.

  18. Altered epigenetic regulation of homeobox genes in human oral squamous cell carcinoma cells

    International Nuclear Information System (INIS)

    To gain insight into oral squamous cell carcinogenesis, we performed deep sequencing (RNAseq) of non-tumorigenic human OKF6-TERT1R and tumorigenic SCC-9 cells. Numerous homeobox genes are differentially expressed between OKF6-TERT1R and SCC-9 cells. Data from Oncomine, a cancer microarray database, also show that homeobox (HOX) genes are dysregulated in oral SCC patients. The activity of Polycomb repressive complexes (PRC), which causes epigenetic modifications, and retinoic acid (RA) signaling can control HOX gene transcription. HOXB7, HOXC10, HOXC13, and HOXD8 transcripts are higher in SCC-9 than in OKF6-TERT1R cells; using ChIP (chromatin immunoprecipitation) we detected PRC2 protein SUZ12 and the epigenetic H3K27me3 mark on histone H3 at these genes in OKF6-TERT1R, but not in SCC-9 cells. In contrast, IRX1, IRX4, SIX2 and TSHZ3 transcripts are lower in SCC-9 than in OKF6-TERT1R cells. We detected SUZ12 and the H3K27me3 mark at these genes in SCC-9, but not in OKF6-TERT1R cells. SUZ12 depletion increased HOXB7, HOXC10, HOXC13, and HOXD8 transcript levels and decreased the proliferation of OKF6-TERT1R cells. Transcriptional responses to RA are attenuated in SCC-9 versus OKF6-TERT1R cells. SUZ12 and H3K27me3 levels were not altered by RA at these HOX genes in SCC-9 and OKF6-TERT1R cells. We conclude that altered activity of PRC2 is associated with dysregulation of homeobox gene expression in human SCC cells, and that this dysregulation potentially plays a role in the neoplastic transformation of oral keratinocytes. - Highlights: • RNAseq elucidates differences between non-tumorigenic and tumorigenic oral keratinocytes. • Changes in HOX mRNA in SCC-9 vs. OKF6-TERT1R cells are a result of altered epigenetic regulation. • RNAseq shows that retinoic acid (RA) influences gene expression in both OKF6-TERT1R and SCC-9 cells

  19. Exine dehiscing induces rape microspore polarity, which results in different daughter cell fate and fixes the apical–basal axis of the embryo

    OpenAIRE

    Tang, Xingchun; Liu, Yuan; He, Yuqing; Ma, Ligang; Sun, Meng-xiang

    2012-01-01

    The roles of cell polarity and the first asymmetric cell division during early embryogenesis in apical–basal cell fate determination remain unclear. Previously, a novel Brassica napus microspore embryogenesis system was established, by which rape exine-dehisced microspores were induced by physical stress. Unlike traditional microspore culture, cell polarity and subsequent asymmetric division appeared in the exine-dehisced microspore, which finally developed into a typical embryo with a suspen...

  20. The influence of non polar and polar molecules in mouse motile cells membranes and pure lipid bilayers.

    Directory of Open Access Journals (Sweden)

    Francisco J Sierra-Valdez

    Full Text Available We report an experimental study of mouse sperm motility that shows chief aspects characteristic of neurons: the anesthetic (produced by tetracaine and excitatory (produced by either caffeine or calcium effects and their antagonic action. While tetracaine inhibits sperm motility and caffeine has an excitatory action, the combination of these two substances balance the effects, producing a motility quite similar to that of control cells. We also study the effects of these agents (anesthetic and excitatory on the melting points of pure lipid liposomes constituted by 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC and dipalmitoyl phosphatidic acid (DPPA. Tetracaine induces a large fluidization of the membrane, shifting the liposomes melting transition temperature to much lower values. The effect of caffeine is null, but its addition to tetracaine-doped liposomes greatly screen the fluidization effect. A high calcium concentration stiffens pure lipid membranes and strongly reduces the effect of tetracaine. Molecular Dynamics Simulations are performed to further understand our experimental findings at the molecular level. We find a strong correlation between the effect of antagonic molecules that could explain how the mechanical properties suitable for normal cell functioning are affected and recovered.

  1. Perinatal stem-cell and gene therapy for hemoglobinopathies.

    Science.gov (United States)

    Surbek, Daniel; Schoeberlein, Andreina; Wagner, Anna

    2008-08-01

    Most genetic diseases of the lymphohematopoietic system, including hemoglobinopathies, can now be diagnosed early in gestation. However, as yet, prenatal treatment is not available. Postnatal therapy by hematopoietic stem cell (HSC) transplantation from bone marrow, mobilized peripheral blood, or umbilical cord blood is possible for several of these diseases, in particular for the hemoglobinopathies, but is often limited by a lack of histocompatible donors, severe treatment-associated morbidity, and preexisting organ damage that developed before birth. In-utero transplantation of allogeneic HSC has been performed successfully in various animal models and recently in humans. However, the clinical success of this novel treatment is limited to diseases in which the fetus is affected by severe immunodeficiency. The lack of donor cell engraftment in nonimmunocompromised hosts is thought to be due to immunologic barriers, as well as to competitive fetal marrow population by host HSCs. Among the possible strategies to circumvent allogeneic HLA barriers, the use of gene therapy by genetically corrected autologous HSCs in the fetus is one of the most promising approaches. The recent development of strategies to overcome failure of efficient transduction of quiescent hematopoietic cells using new vector constructs and transduction protocols opens new perspectives for gene therapy in general, as well as for prenatal gene transfer in particular. The fetus might be especially susceptible for successful gene therapy approaches because of the developing, expanding hematopoietic system during gestation and the immunologic naiveté early in gestation, precluding immune reaction towards the transgene by inducing tolerance. Ethical issues, in particular regarding treatment safety, must be addressed more closely before clinical trials with fetal gene therapy in human pregnancies can be initiated. PMID:18420474

  2. Current status of gene transfer into haemopoietic progenitor cells: application to Langerhans cell histiocytosis.

    OpenAIRE

    M. Brenner

    1994-01-01

    A number of recent studies have shown that it is possible to obtain significant levels of gene transfer and expression in marrow progenitor cells and their progeny by using retroviral vectors. The data obtained from these studies and the possible applications to Langerhans cell histiocytosis (LCH) are reviewed.

  3. The water-born protein pheromones of the polar protozoan ciliate, Euplotes nobilii: Coding genes and molecular structures

    Science.gov (United States)

    Vallesi, Adriana; Alimenti, Claudio; Di Giuseppe, Graziano; Dini, Fernando; Pedrini, Bill; Wüthrich, Kurt; Luporini, Pierangelo

    2010-08-01

    The protozoan ciliate Euplotes nobilii found in Antarctic and Arctic coastal waters relies on secretion of water-soluble cell type-specific signal proteins (pheromones) to regulate its vegetative growth and sexual mating. For three of these psychrophilic pheromones we previously determined the three-dimensional structures by nuclear magnetic resonance (NMR) spectroscopy with protein solutions purified from the natural sources, which led to evidence that their adaptation to cold is primarily achieved by increased flexibility through an extension of regions free of regular secondary structures, and by increased exposure of negative charges on the protein surface. Then we cloned the coding genes of these E. nobilii pheromones from the transcriptionally active cell somatic nucleus (macronucleus) and characterized the full-length sequences. These sequences all contain an open reading frame of 252-285 nucleotides, which is specific for a cytoplasmic pheromone precursor that requires two proteolytic cleavages to remove a signal peptide and a pro segment before release of the mature protein into the extracellular environment. The 5‧ and 3‧ non-coding regions are two- to three-fold longer than the coding region and appear to be tightly conserved, probably in relation to the inclusion of intron sequences destined to be alternatively removed to play key regulatory roles in the mechanism of the pheromone gene expression.

  4. MGMT enrichment and second gene co-expression in hematopoietic progenitor cells using separate or dual-gene lentiviral vectors.

    Science.gov (United States)

    Roth, Justin C; Alberti, Michael O; Ismail, Mourad; Lingas, Karen T; Reese, Jane S; Gerson, Stanton L

    2015-01-22

    The DNA repair gene O(6)-methylguanine-DNA methyltransferase (MGMT) allows efficient in vivo enrichment of transduced hematopoietic stem cells (HSC). Thus, linking this selection strategy to therapeutic gene expression offers the potential to reconstitute diseased hematopoietic tissue with gene-corrected cells. However, different dual-gene expression vector strategies are limited by poor expression of one or both transgenes. To evaluate different co-expression strategies in the context of MGMT-mediated HSC enrichment, we compared selection and expression efficacies in cells cotransduced with separate single-gene MGMT and GFP lentivectors to those obtained with dual-gene vectors employing either encephalomyocarditis virus (EMCV) internal ribosome entry site (IRES) or foot and mouth disease virus (FMDV) 2A elements for co-expression strategies. Each strategy was evaluated in vitro and in vivo using equivalent multiplicities of infection (MOI) to transduce 5-fluorouracil (5-FU) or Lin(-)Sca-1(+)c-kit(+) (LSK)-enriched murine bone marrow cells (BMCs). The highest dual-gene expression (MGMT(+)GFP(+)) percentages were obtained with the FMDV-2A dual-gene vector, but half of the resulting gene products existed as fusion proteins. Following selection, dual-gene expression percentages in single-gene vector cotransduced and dual-gene vector transduced populations were similar. Equivalent MGMT expression levels were obtained with each strategy, but GFP expression levels derived from the IRES dual-gene vector were significantly lower. In mice, vector-insertion averages were similar among cells enriched after dual-gene vectors and those cotransduced with single-gene vectors. These data demonstrate the limitations and advantages of each strategy in the context of MGMT-mediated selection, and may provide insights into vector design with respect to a particular therapeutic gene or hematologic defect. PMID:25479595

  5. EVALUATION OF CYTOKINE GENE POLYMORPHISM IN B CELL LYMPHOID MALIGNANCIES

    Directory of Open Access Journals (Sweden)

    E. L. Nazarova

    2014-01-01

    Full Text Available Previous studies with some solid tumors has shown that polymorphisms of certain cytokine genes may be used as predictors of clinical outcome in the patients. It seemed important to evaluate potential correlations between production of certain pro- and anti-inflammatory cytokines and co-receptor molecules, and promoter polymorphism of the cytokine genes involved into regulation of cell proliferation, differentiation, apoptosis, lipid metabolism and blood clotting in the patients with hematological malignancies. The article contains our results concerning associations between of IL-1β, -2, -4, -10, -17, TNFα, and allelic polymorphisms of their genes in 62 patients with B cell lymphoid malignancies in an ethnically homogenous group (self-identified as Russians. We have shown that the GА and AA genotypes of the G-308A polymorphism in TNFα gene are significantly associated with increased production of this cytokine, being more common in aggressive non-Hodgkin lymphomas, more rare in multiple myeloma and in indolent non-Hodgkin lymphomas.

  6. Gene expression pattern of functional neuronal cells derived from human bone marrow mesenchymal stromal cells

    Directory of Open Access Journals (Sweden)

    Bron Dominique

    2008-04-01

    Full Text Available Abstract Background Neuronal tissue has limited potential to self-renew or repair after neurological diseases. Cellular therapies using stem cells are promising approaches for the treatment of neurological diseases. However, the clinical use of embryonic stem cells or foetal tissues is limited by ethical considerations and other scientific problems. Thus, bone marrow mesenchymal stomal cells (BM-MSC could represent an alternative source of stem cells for cell replacement therapies. Indeed, many studies have demonstrated that MSC can give rise to neuronal cells as well as many tissue-specific cell phenotypes. Methods BM-MSC were differentiated in neuron-like cells under specific induction (NPBM + cAMP + IBMX + NGF + Insulin. By day ten, differentiated cells presented an expression profile of real neurons. Functionality of these differentiated cells was evaluated by calcium influx through glutamate receptor AMPA3. Results Using microarray analysis, we compared gene expression profile of these different samples, before and after neurogenic differentiation. Among the 1943 genes differentially expressed, genes down-regulated are involved in osteogenesis, chondrogenesis, adipogenesis, myogenesis and extracellular matrix component (tuftelin, AGC1, FADS3, tropomyosin, fibronectin, ECM2, HAPLN1, vimentin. Interestingly, genes implicated in neurogenesis are increased. Most of them are involved in the synaptic transmission and long term potentialisation as cortactin, CASK, SYNCRIP, SYNTL4 and STX1. Other genes are involved in neurite outgrowth, early neuronal cell development, neuropeptide signaling/synthesis and neuronal receptor (FK506, ARHGAP6, CDKRAP2, PMCH, GFPT2, GRIA3, MCT6, BDNF, PENK, amphiregulin, neurofilament 3, Epha4, synaptotagmin. Using real time RT-PCR, we confirmed the expression of selected neuronal genes: NEGR1, GRIA3 (AMPA3, NEF3, PENK and Epha4. Functionality of these neuron-like cells was demonstrated by Ca2+ influx through glutamate

  7. Screening of differentially expressed genes related to differentiation and proliferation by gene expression profiling of different grade astrocytoma cell lines

    Institute of Scientific and Technical Information of China (English)

    Yi Zeng; Zhong Yang; Yangyun Han; Chao You

    2008-01-01

    BACKGROUND: The detection of differential gene expression in brain is possible by cDNA microarray technology, and the screening of differentially expressed genes might provide a biological basis for gene-targeted therapy for tumors. OBJECTIVE: To detect the differential expression of genes among astrocytoma SHG-44 (WHO grade IV), CHG-5 (WHO grade II), and ATRA-treated SHG-44 cell lines by cDNA microarray. DESIGN: Laboratory experiments in vitro.SETTING: Department of Neurobiology, the Third Military Medical University. MATERIALS: The experiment was performed at the Department of Neurobiology in the Third Military Medical University of the Chinese PLA from January to October 2007. The SHG-44 cell line (WHO grade Ⅳ) was established by Prof. Ziwei Du, and the CHG-5 cell line (WHO grade II) was set up by Prof. Xiuwu Bian from the Third Military Medical University of the Chinese PLA. The cDNA microarray containing 9182 known genes was prepared and provided by Dr. Yang Zhong at the City University of Hong Kong. MAIN OUTCOME MEASURES: The identification of genes that were similarly regulated (overlapping) during tumor progression and differentiation, by comparison of gene expression profiles between CHG-5 and SHG-44 cells, and between SHG-44 cells with or without treatment with ATRA. RESULTS: Thirty-one overlapping genes were found to have similar regulatory effects on astrocytomas; among them, twenty genes were up-regulated and eleven were down-regulated in both comparisons between CHG-5 and SHG-44 cells, and between SHG-44 cells with or without treatment with ATRA. The four reported genes, SERPINF1, MAPK11, HIF1A and SOD2, were up-regulated in this study.CONCLUSION: The differentially expressed genes in different grade astrocytoma cell lines were revealed primarily by cDNA microarray; among them, five identified overlapping genes, SERPINF1, MAPK11, DCTN2, HIF1A and SOD2, were related to the malignant progression of astrocytoma cells.

  8. Improvement the equation of polarization curve of a proton exchange membrane fuel cell at different channel geometry

    Science.gov (United States)

    Khazaee, I.

    2015-12-01

    The polarization curve of a proton exchange membrane fuel cell is an important parameter which is expressed by the change of voltage and current of it that indicates the performance of the cell. The voltage of the cell is a function of temperature that is expressed by the Nernst equation and the equation of voltage losses such as activation loss, ohmic loss and concentration loss. In this study a new correlation for polarization curve is obtained that it in addition to temperature, a new parameter is involved in it that shows the effect of the geometry of cross-section area of channels. For this purpose three PEM fuel cells with different channels geometry of rectangular, elliptical and triangular have constructed. The active area of each cell is 25 cm2 that its weight is 1300 g. The material of the gas diffusion layer is carbon clothes, the membrane is nafion 117 and the catalyst layer is a plane with 0.004 g/cm2 platinum. Also a test bench designed and constructed for testing the cell and a series of experiments are carried out to investigate the influence of the geometry of the cell on performance of the cell. The results show that when the geometry of channel is rectangular the performance of the cell is better than the triangular and elliptical channel.

  9. Novel genes underlying beta cell survival in metabolic stress

    OpenAIRE

    Singh, Himadri; Farouk, Mohammed; Bose, Barish Baran; Singh, Prabhakar

    2013-01-01

    Relative insulin deficiency, in response to increased metabolic demand (obesity, genetic insulin resistance, pregnancy and aging) lead to Type2 diabetes. Susceptibility of the type 2 diabetes has a genetic basis, as a subset of people with risk factors (obesity, Insulin Resistance, pregnancy), develop Type2 Diabetes. We aimed to identify ‘cluster’ of overexpressed genes, underlying increased beta cell survival in diabetes resistant C57BL/6J ob/ob mice (compared to diabetes susceptible BTBR ob...

  10. Development of gene and stem cell therapy for ocular neurodegeneration

    OpenAIRE

    Zhang, Jing-Xue; Wang, Ning-Li; Lu, Qing-Jun

    2015-01-01

    Retinal degenerative diseases pose a serious threat to eye health, but there is currently no effective treatment available. Recent years have witnessed rapid development of several cutting-edge technologies, such as gene therapy, stem cell therapy, and tissue engineering. Due to the special features of ocular structure, some of these technologies have been translated into ophthalmological clinic practice with fruitful achievements, setting a good example for other fields. This paper reviews t...

  11. Highly parallel identification of essential genes in cancer cells

    OpenAIRE

    Luo, Biao; Cheung, Hiu Wing; Subramanian, Aravind; Sharifnia, Tanaz; Okamoto, Michael; Yang, Xiaoping; Hinkle, Greg; Boehm, Jesse S.; Beroukhim, Rameen; Weir, Barbara A.; Mermel, Craig; Barbie, David A; Awad, Tarif; Zhou, Xiaochuan; Nguyen, Tuyen Van

    2008-01-01

    More complete knowledge of the molecular mechanisms underlying cancer will improve prevention, diagnosis and treatment. Efforts such as The Cancer Genome Atlas are systematically characterizing the structural basis of cancer, by identifying the genomic mutations associated with each cancer type. A powerful complementary approach is to systematically characterize the functional basis of cancer, by identifying the genes essential for growth and related phenotypes in different cancer cells. Such...

  12. Cu₂ZnSnS(4x)Se(4(1-x)) solar cells from polar nanocrystal inks.

    Science.gov (United States)

    van Embden, Joel; Chesman, Anthony S R; Della Gaspera, Enrico; Duffy, Noel W; Watkins, Scott E; Jasieniak, Jacek J

    2014-04-01

    A facile ligand exchange method for dispersing Cu2ZnSnS4 (CZTS) nanocrystals (NCs) in environmentally benign polar solvents, such as ethanol or n-propanol, at high concentrations (up to 200 mg/mL) is demonstrated. This approach has been applied to CZTS nanocrystals synthesized via scalable, noninjection methods to formulate colloidally stable inks that are suitable for the solution processing of solar cell devices. Unlike other inks currently used to fabricate NC solar cells, the CZTS nanocrystal ink developed here circumvents the need for hydrazine, pyridine, or thiol coordinating solvents. By combining our polar CZTS inks with optimized selenization procedures, substrate CZTSSe solar cells have been successfully fabricated with device efficiencies of 7.7%. PMID:24690032

  13. Synchrotron radiation X-ray microfluorescence reveals polarized distribution of atomic elements during differentiation of pluripotent stem cells.

    Directory of Open Access Journals (Sweden)

    Simone C Cardoso

    Full Text Available The mechanisms underlying pluripotency and differentiation in embryonic and reprogrammed stem cells are unclear. In this work, we characterized the pluripotent state towards neural differentiated state through analysis of trace elements distribution using the Synchrotron Radiation X-ray Fluorescence Spectroscopy. Naive and neural-stimulated embryoid bodies (EB derived from embryonic and induced pluripotent stem (ES and iPS cells were irradiated with a spatial resolution of 20 µm to make elemental maps and qualitative chemical analyses. Results show that these embryo-like aggregates exhibit self-organization at the atomic level. Metallic elements content rises and consistent elemental polarization pattern of P and S in both mouse and human pluripotent stem cells were observed, indicating that neural differentiation and elemental polarization are strongly correlated.

  14. Methylation of Gene CHFR Promoter in Acute Leukemia Cells

    Institute of Scientific and Technical Information of China (English)

    GONG Hui; LIU Wengli; ZHOU Jianfeng; XU Huizhen

    2005-01-01

    Summary: In order to explore whether gene CHFR was inactivated by methylation in leukemia cells, the expression of CHFR was examined before and after treatment with demethylation agent in Molt-4, Jurkat and U937 leukemia cell lines by means of RT-PCR. The methylation of promoter in Molt-4, Jurkat and U937 cells as well as 41 acute leukemia patients was analyzed by MS-PCR. The results showed that methylation of CHFR promoter was inactivated and could be reversed by treatment with a demethylating agent in Molt-4, Jurkat and U937. CHFR promoter methylation was detected in 39 % of acute leukemia patients. There was no difference in incidence of CHFR promoter methylation between acute myelocytic leukemia and acute lymphocytic leukemia. In conclusion, CHFR is frequently inactivated in acute leukemia and is a good candidate for the leukemia supper gene. By affecting mitotic checkpoint function, CHFR inactivation likely plays a key role in tumorigenesis in acute leukemia. Moreover, the methylation of gene CHFR appears to be a good index with which to predict the sensitivity of acute leukemia to microtubule inhibitors.

  15. Autofocus Correction of Azimuth Phase Error and Residual Range Cell Migration in Spotlight SAR Polar Format Imagery

    CERN Document Server

    Mao, Xinhua; Zhu, Zhaoda

    2012-01-01

    Synthetic aperture radar (SAR) images are often blurred by phase perturbations induced by uncompensated sensor motion and /or unknown propagation effects caused by turbulent media. To get refocused images, autofocus proves to be useful post-processing technique applied to estimate and compensate the unknown phase errors. However, a severe drawback of the conventional autofocus algorithms is that they are only capable of removing one-dimensional azimuth phase errors (APE). As the resolution becomes finer, residual range cell migration (RCM), which makes the defocus inherently two-dimensional, becomes a new challenge. In this paper, correction of APE and residual RCM are presented in the framework of polar format algorithm (PFA). First, an insight into the underlying mathematical mechanism of polar reformatting is presented. Then based on this new formulation, the effect of polar reformatting on the uncompensated APE and residual RCM is investigated in detail. By using the derived analytical relationship betwee...

  16. Intracellular photoreceptive site for blue light-induced cell division in protonemata of the fern Adiantum [Pteridophyta]: Further analyses by polarized light irradiation and cell centrifugation

    International Nuclear Information System (INIS)

    The intracellular localization of the photoreceptive site for blue light-induced cell division in single-celled protonemata of Adiantum capillus-veneris L. was investigated using polarized light irradiation and protonemal cell centrifugation. The response to irradiation with polarized blue light showed no dependence on the direction of light polarization. However, centrifugation of the protonemata followed by microbeam irradiation showed that the site of blue light perception could be displaced together with the nucleus. Centrifugal treatment changed the distribution of intracellular organelles at the time of light exposure and basipetally displaced the nucleus about 90μm. This treatment had no effect on the induction of cell division with blue light if the protonemata were centrifuged again acropetally after the light treatment. Microbeam (30×30 μm2) irradiation with blue light of the apical 45–75 βm region, the receptive site of blue light in non-centrifuged cell, did not induce cell division. However, cell division was induced by irradiation of the nucleus-containing region, indicating that the photoreceptive site was displaced together with the nucleus by the centrifugation. These results suggest that the blue light receptor regulating cell division in Adiantum protonemata is not likely to be located on the plasma membrane. (author)

  17. Reporter gene expression in dendritic cells after gene gun administration of plasmid DNA

    OpenAIRE

    Watkins, Craig; Hopkins, John; Harkiss, Gordon

    2005-01-01

    Dendritic cells (DC) play an integral role in plasmid DNA vaccination. However, the interaction between plasmid DNA and DC in vivo is incompletely understood. In this report, we utilise the sheep pseudoafferent cannulation model to examine the interaction between plasmid DNA encoding enhanced green fluorescent protein (pEGFP) and afferent lymph DC (ALDC) following gene gun administration. The results show that peaks of fluorescent ALDC tended to appear around days 1-4 and 9-13, then erratical...

  18. Expression changes of cell-cell adhesion-related genes in colorectal tumors

    OpenAIRE

    Bujko, Mateusz; KOBER, PAULINA; Mikula, Michal; Ligaj, Marcin; Ostrowski, Jerzy; Siedlecki, Janusz Aleksander

    2015-01-01

    Epithelial tissues achieve a highly organized structure due to cell-cell junction complexes. Carcinogenesis is accompanied by changes in cell interactions and tissue morphology, which appear in the early stages of benign tumors and progress along with invasive potential. The aim of the present study was to analyze the changes in expression levels of genes encoding intercellular junction proteins that have been previously identified to be differentially expressed in colorectal tumors compared ...

  19. Polyfluorene Electrolytes Interfacial Layer for Efficient Polymer Solar Cells: Controllably Interfacial Dipoles by Regulation of Polar Groups.

    Science.gov (United States)

    Liu, Huimin; Hu, Lin; Wu, Feiyan; Chen, Lie; Chen, Yiwang

    2016-04-20

    The polar groups in the conjugated polyelectrolytes (CPEs) can create the favorable dipoles at the electrode/active layer interface, which is critical for the CPEs to minimize the interfacial energy barrier in polymer solar cells (PSCs). Herein, a series of CPEs based on poly [(9,9-bis(3'-(N,N-dimethylamino)propyl)-2,7-fluorene)-co-2,7-(9,9-dioctylfluorene)] derivates (PFNs) (PFN30, PFN50, PFN70, and PFN100) with different mole ratio of polar groups (-N(C2H5)2) were designed and synthesized to investigate the effect of the numbers of polar groups on the interfacial dipoles. Controllably interfacial dipoles could be readily achieved by only tuning the numbers of -N(C2H5)2 in PFNs, as revealed by the work function of the PFNs modified ITO gradually reduced as the loadings of the -N(C2H5)2 increased. In addition, increasing the numbers of -N(C2H5)2 in PFNs were also favorable for developing the smooth and homogeneous morphology of the active layer. As a result, the content of the polar amine in the PFNs exerted great influence on the performance of polymer solar cells. Increasing the numbers of the pendent -N(C2H5)2 could effectively improve the power conversion efficiency (PCE) of the devices. Among these PFNs, PFN100 with the highest content of -N(C2H5)2 polar groups delivered the device with the best PCE of 3.27%. It indicates tailoring the content of the polar groups in the CPEs interlayer is a facial and promising approach for interfacial engineering to developing high performance PSCs. PMID:27028166

  20. Global gene expression analyses of hematopoietic stem cell-like cell lines with inducible Lhx2 expression

    Directory of Open Access Journals (Sweden)

    Lundeberg Joakim

    2006-04-01

    Full Text Available Abstract Background Expression of the LIM-homeobox gene Lhx2 in murine hematopoietic cells allows for the generation of hematopoietic stem cell (HSC-like cell lines. To address the molecular basis of Lhx2 function, we generated HSC-like cell lines where Lhx2 expression is regulated by a tet-on system and hence dependent on the presence of doxycyclin (dox. These cell lines efficiently down-regulate Lhx2 expression upon dox withdrawal leading to a rapid differentiation into various myeloid cell types. Results Global gene expression of these cell lines cultured in dox was compared to different time points after dox withdrawal using microarray technology. We identified 267 differentially expressed genes. The majority of the genes overlapping with HSC-specific databases were those down-regulated after turning off Lhx2 expression and a majority of the genes overlapping with those defined as late progenitor-specific genes were the up-regulated genes, suggesting that these cell lines represent a relevant model system for normal HSCs also at the level of global gene expression. Moreover, in situ hybridisations of several genes down-regulated after dox withdrawal showed overlapping expression patterns with Lhx2 in various tissues during embryonic development. Conclusion Global gene expression analysis of HSC-like cell lines with inducible Lhx2 expression has identified genes putatively linked to self-renewal / differentiation of HSCs, and function of Lhx2 in organ development and stem / progenitor cells of non-hematopoietic origin.

  1. Development a new equation of polarization curve for a proton exchange membrane fuel cell at different channel geometry

    Directory of Open Access Journals (Sweden)

    I. Khazaee

    2014-01-01

    Full Text Available The polarization curve of a proton exchange membrane fuel cell is an important parameter that is used to investigate the performance of it that is expressed with the Nernst equation with the equation of losses the voltage such as activation loss, ohmic loss and concentration loss that they are a function of temperature of the cell and the current density. In this study a new correlation for polarization curve is obtained that it is a function of temperature, current density and a new parameter of cross-section geometry of channels. For this purpose three PEM fuel cells with different channels geometry of rectangular, elliptical and triangular have constructed. The active area of each cell is that its weight is 1300gr. The material of the gas diffusion layer is Carbon clothes, the membrane is nafion112 and the catalyst layer is a plane with 0.004 gr/cm2 Platinum. Also a test bench designed and constructed for testing the cell and a series of experiments are carried out to investigate the influence of the geometry of the cell on performance of the cell. The results show that when the geometry of channel is rectangular the performance of the cell is better than the triangular and elliptical channel.

  2. Bipolar switching polarity reversal by electrolyte layer sequence in electrochemical metallization cells with dual-layer solid electrolytes.

    Science.gov (United States)

    Soni, Rohit; Meuffels, Paul; Petraru, Adrian; Hansen, Mirko; Ziegler, Martin; Vavra, Ondrej; Kohlstedt, Hermann; Jeong, Doo Seok

    2013-12-21

    Bipolar switching behaviours of electrochemical metallization (ECM) cells with dual-layer solid electrolytes (SiOx-Ge0.3Se0.7) were analyzed. Type 1 ECM cell, Pt (bottom electrode)/SiOx/Ge0.3Se0.7/Cu (top electrode), exhibited typical eightwise current-voltage (I-V) hysteresis of ECM cells whereas Type 2 ECM cell, Pt (bottom electrode)/Ge0.3Se0.7/SiOx/Cu(top electrode), showed counter-eightwise hysteresis. In addition, absolute off-switching voltage in Type 2 cell is lower than that in Type 1 cell while on-switching voltage in both cells is almost the same. An attempt to understand this electrolyte-stack-sequence-depending switching polarity reversal was made in terms of the ECM cell potential change upon the electrolyte stack sequence and the consequent change in Cu filament growth direction. Relevant experimental evidence for the hypothesis was obtained regarding the switching behaviours. Furthermore, given the switching polarity reversal, feasibility of serial complementary resistive switches was also demonstrated. PMID:24177268

  3. ET-67SUICIDE GENE THERAPY FOR GLIOMA USING MULTILINEAGE-DEFFERENTIATING STRESS ENDURING (MUSE) CELLS

    OpenAIRE

    Yamasaki, Tomohiro; Wakao, Shohei; KAWAJI, Hiroshi; Suzuki, Tomo; Kamio, Yoshinobu; AMANO, SHINJI; Sameshima, Tetsuro; Sakai, Naoto; TOKUYAMA, TSUTOMU; Dezawa, Mari; NAMBA, HIROKI

    2014-01-01

    INTRODUCTION: We have been investigating cell-based glioma gene therapy using various kinds of stem cells transduced with the herpes simplex virus thymidine kinase gene (HSVtk). In our previous study, we used SSEA3/CD105 double-positive multilineage-differentiating stress-enduring (Muse) cells transduced with HSVtk (Muse-tk cells) as the vehicle for HSVtk/ganciclovir (GCV) gene therapy. We demonstrated a potent in vitro tumoricidal bystander effect for various glioma cells. In the present stu...

  4. Treating hearing disorders with cell and gene therapy

    Science.gov (United States)

    Gillespie, Lisa N.; Richardson, Rachael T.; Nayagam, Bryony A.; Wise, Andrew K.

    2014-12-01

    Hearing loss is an increasing problem for a substantial number of people and, with an aging population, the incidence and severity of hearing loss will become more significant over time. There are very few therapies currently available to treat hearing loss, and so the development of new therapeutic strategies for hearing impaired individuals is of paramount importance to address this unmet clinical need. Most forms of hearing loss are progressive in nature and therefore an opportunity exists to develop novel therapeutic approaches to slow or halt hearing loss progression, or even repair or replace lost hearing function. Numerous emerging technologies have potential as therapeutic options. This paper details the potential of cell- and gene-based therapies to provide therapeutic agents to protect sensory and neural cells from various insults known to cause hearing loss; explores the potential of replacing lost sensory and nerve cells using gene and stem cell therapy; and describes the considerations for clinical translation and the challenges that need to be overcome.

  5. Dependence of InGaN solar cell performance on polarization-induced electric field and carrier lifetime

    International Nuclear Information System (INIS)

    The effects of Mg-induced net acceptor doping concentration and carrier lifetime on the performance of a p—i—n InGaN solar cell are investigated. It is found that the electric field induced by spontaneous and piezoelectric polarization in the i-region could be totally shielded when the Mg-induced net acceptor doping concentration is sufficiently high. The polarization-induced potential barriers are reduced and the short circuit current density is remarkably increased from 0.21 mA/cm2 to 0.95 mA/cm2 by elevating the Mg doping concentration. The carrier lifetime determined by defect density of i-InGaN also plays an important role in determining the photovoltaic properties of solar cell. The short circuit current density severely degrades, and the performance of InGaN solar cell becomes more sensitive to the polarization when carrier lifetime is lower than the transit time. This study demonstrates that the crystal quality of InGaN absorption layer is one of the most important challenges in realizing high efficiency InGaN solar cells. (interdisciplinary physics and related areas of science and technology)

  6. Heat shock genes – integrating cell survival and death

    Indian Academy of Sciences (India)

    Richa Arya; Moushami Mallik; Subhash C Lakhotia

    2007-04-01

    Heat shock induced gene expression and other cellular responses help limit the damage caused by stress and thus facilitate cellular recovery. Cellular damage also triggers apoptotic cell death through several pathways. This paper briefly reviews interactions of the major heat shock proteins with components of the apoptotic pathways. Hsp90, which acts as a chaperone for unstable signal transducers to keep them poised for activation, interacts with RIP and Akt and promotes NF-B mediated inhibition of apoptosis; in addition it also blocks some steps in the apoptotic pathways. Hsp70 is mostly anti-apoptotic and acts at several levels like inhibition of translocation of Bax into mitochondria, release of cytochrome c from mitochondria, formation of apoptosome and inhibition of activation of initiator caspases. Hsp70 also modulates JNK, NF-B and Akt signaling pathways in the apoptotic cascade. In contrast, Hsp60 has both anti- and pro-apoptotic roles. Cytosolic Hsp60 prevents translocation of the pro-apoptotic protein Bax into mitochondria and thus promotes cell survival but it also promotes maturation of procaspase-3, essential for caspase mediated cell death. Our recent in vivo studies show that RNAi for the Hsp60D in Drosophila melanogaster prevents induced apoptosis. Hsp27 exerts its anti-apoptotic influence by inhibiting cytochrome c and TNF-mediated cell death. crystallin suppresses caspase-8 and cytochrome c mediated activation of caspase-3. Studies in our laboratory also reveal that absence or reduced levels of the developmentally active as well as stress induced non-coding hsr transcripts, which are known to sequester diverse hnRNPs and related nuclear RNA-binding proteins, block induced apoptosis in Drosophila. Modulation of the apoptotic pathways by Hsps reflects their roles as ``weak links” between various ``hubs” in cellular networks. On the other hand, non-coding RNAs, by virtue of their potential to bind with multiple proteins, can act as ``hubs” in

  7. Bioelectric modulation of macrophage polarization

    Science.gov (United States)

    Li, Chunmei; Levin, Michael; Kaplan, David L.

    2016-02-01

    Macrophages play a critical role in regulating wound healing and tissue regeneration by changing their polarization state in response to local microenvironmental stimuli. The native roles of polarized macrophages encompass biomaterials and tissue remodeling needs, yet harnessing or directing the polarization response has been largely absent as a potential strategy to exploit in regenerative medicine to date. Recent data have revealed that specific alteration of cells’ resting potential (Vmem) is a powerful tool to direct proliferation and differentiation in a number of complex tissues, such as limb regeneration, craniofacial patterning and tumorigenesis. In this study, we explored the bioelectric modulation of macrophage polarization by targeting ATP sensitive potassium channels (KATP). Glibenclamide (KATP blocker) and pinacidil (KATP opener) treatment not only affect macrophage polarization, but also influence the phenotype of prepolarized macrophages. Furthermore, modulation of cell membrane electrical properties can fine-tune macrophage plasticity. Glibenclamide decreased the secretion and gene expression of selected M1 markers, while pinacidil augmented M1 markers. More interestingly, glibencalmide promoted macrophage alternative activation by enhancing certain M2 markers during M2 polarization. These findings suggest that control of bioelectric properties of macrophages could offer a promising approach to regulate macrophage phenotype as a useful tool in regenerative medicine.

  8. Blazed vector grating liquid crystal cells with photocrosslinkable polymeric alignment films fabricated by one-step polarizer rotation method

    Science.gov (United States)

    Kawai, Kotaro; Kuzuwata, Mitsuru; Sasaki, Tomoyuki; Noda, Kohei; Kawatsuki, Nobuhiro; Ono, Hiroshi

    2014-12-01

    Blazed vector grating liquid crystal (LC) cells, in which the directors of low-molar-mass LCs are antisymmetrically distributed, were fabricated by one-step exposure of an empty glass cell inner-coated with a photocrosslinkable polymer LC (PCLC) to UV light. By adopting a LC cell structure, twisted nematic (TN) and homogeneous (HOMO) alignments were obtained in the blazed vector grating LC cells. Moreover, the diffraction efficiency of the blazed vector grating LC cells was greatly improved by increasing the thickness of the device in comparison with that of a blazed vector grating with a thin film structure obtained in our previous study. In addition, the diffraction efficiency and polarization states of ±1st-order diffracted beams from the resultant blazed vector grating LC cells were controlled by designing a blazed pattern in the alignment films, and these diffraction properties were well explained on the basis of Jones calculus and the elastic continuum theory of nematic LCs.

  9. Mutation and gene transfer of neutral amino acid transport System L genes in mammalian cells

    International Nuclear Information System (INIS)

    The authors are attempting to clone the genes coding for amino acid transport System L. Chinese hamster ovary (CHO) cell mutants that are temperature sensitive in their leucyl-tRNA synthetase show temperature-dependent regulation of System L. Temperature resistant mutants isolated from these cells have constitutively derepressed System L activity. Somatic cell fusion studies using these mutants have suggested that a trans-acting element controls regulation of System L. Mutants with reduced transport activity were isolated by a 3H-suicide selection. The growth of these mutant cells is limited by the transport defect. CHO mutants were transformed with a human cosmid library, followed by selection at high temperatures and low leucine concentrations. Some transformants have increased levels of System L activity, suggesting that human genes coding for leucine transport have been incorporated into the CHO genome. Human sequences were rescued by a lambda in vitro packaging system. These sequences hybridize to vector and total human DNA. Experiments are being done to confirm that these sequences indeed code for transport System L. They are also attempting to label membrane components of amino acid transporters by group-specific modifying reagents

  10. Clusters of conserved beta cell marker genes for assessment of beta cell phenotype

    DEFF Research Database (Denmark)

    Martens, Geert A; Jiang, Lei; Hellemans, Karine H;

    2011-01-01

    of a large panel of other tissue and cell types, and transcripts with beta cell-abundant and -selective expression were identified. Iteration of this analysis in mouse, rat and human tissues generated a panel of conserved beta cell biomarkers. This panel was then used to compare isolated versus laser......The aim of this study was to establish a gene expression blueprint of pancreatic beta cells conserved from rodents to humans and to evaluate its applicability to assess shifts in the beta cell differentiated state. Genome-wide mRNA expression profiles of isolated beta cells were compared to those...... capture microdissected beta cells, monitor adaptations of the beta cell phenotype to fasting, and retrieve possible conserved transcriptional regulators....

  11. Differential regulation of NAB corepressor genes in Schwann cells

    Directory of Open Access Journals (Sweden)

    Sachdev Shrikesh

    2007-12-01

    Full Text Available Abstract Background Myelination of peripheral nerves by Schwann cells requires not only the Egr2/Krox-20 transactivator, but also the NGFI-A/Egr-binding (NAB corepressors, which modulate activity of Egr2. Previous work has shown that axon-dependent expression of Egr2 is mediated by neuregulin stimulation, and NAB corepressors are co-regulated with Egr2 expression in peripheral nerve development. NAB corepressors have also been implicated in macrophage development, cardiac hypertrophy, prostate carcinogenesis, and feedback regulation involved in hindbrain development. Results To test the mechanism of NAB regulation in Schwann cells, transfection assays revealed that both Nab1 and Nab2 promoters are activated by Egr2 expression. Furthermore, direct binding of Egr2 at these promoters was demonstrated in vivo by chromatin immunoprecipitation analysis of myelinating sciatic nerve, and binding of Egr2 to the Nab2 promoter was stimulated by neuregulin in primary Schwann cells. Although Egr2 expression activates the Nab2 promoter more highly than Nab1, we surprisingly found that only Nab1 – but not Nab2 – expression levels were reduced in sciatic nerve from Egr2 null mice. Analysis of the Nab2 promoter showed that it is also activated by ETS proteins (Ets2 and Etv1/ER81 and is bound by Ets2 in vivo. Conclusion Overall, these results indicate that induction of Nab2 expression in Schwann cells involves not only Egr2, but also ETS proteins that are activated by neuregulin stimulation. Although Nab1 and Nab2 play partially redundant roles, regulation of Nab2 expression by ETS factors explains several observations regarding regulation of NAB genes. Finally, these data suggest that NAB proteins are not only feedback inhibitors of Egr2, but rather that co-induction of Egr2 and NAB genes is involved in forming an Egr2/NAB complex that is crucial for regulation of gene expression.

  12. CHD7, the gene mutated in CHARGE syndrome, regulates genes involved in neural crest cell guidance.

    Science.gov (United States)

    Schulz, Yvonne; Wehner, Peter; Opitz, Lennart; Salinas-Riester, Gabriela; Bongers, Ernie M H F; van Ravenswaaij-Arts, Conny M A; Wincent, Josephine; Schoumans, Jacqueline; Kohlhase, Jürgen; Borchers, Annette; Pauli, Silke

    2014-08-01

    Heterozygous loss of function mutations in CHD7 (chromodomain helicase DNA-binding protein 7) lead to CHARGE syndrome, a complex developmental disorder affecting craniofacial structures, cranial nerves and several organ systems. Recently, it was demonstrated that CHD7 is essential for the formation of multipotent migratory neural crest cells, which migrate from the neural tube to many regions of the embryo, where they differentiate into various tissues including craniofacial and heart structures. So far, only few CHD7 target genes involved in neural crest cell development have been identified and the role of CHD7 in neural crest cell guidance and the regulation of mesenchymal-epithelial transition are unknown. Therefore, we undertook a genome-wide microarray expression analysis on wild-type and CHD7 deficient (Chd7 (Whi/+) and Chd7 (Whi/Whi)) mouse embryos at day 9.5, a time point of neural crest cell migration. We identified 98 differentially expressed genes between wild-type and Chd7 (Whi/Whi) embryos. Interestingly, many misregulated genes are involved in neural crest cell and axon guidance such as semaphorins and ephrin receptors. By performing knockdown experiments for Chd7 in Xenopus laevis embryos, we found abnormalities in the expression pattern of Sema3a, a protein involved in the pathogenesis of Kallmann syndrome, in vivo. In addition, we detected non-synonymous SEMA3A variations in 3 out of 45 CHD7-negative CHARGE patients. In summary, we discovered for the first time that Chd7 regulates genes involved in neural crest cell guidance, demonstrating a new aspect in the pathogenesis of CHARGE syndrome. Furthermore, we showed for Sema3a a conserved regulatory mechanism across different species, highlighting its significance during development. Although we postulated that the non-synonymous SEMA3A variants which we found in CHD7-negative CHARGE patients alone are not sufficient to produce the phenotype, we suggest an important modifier role for SEMA3A in the

  13. Increased malignancy of oral squamous cell carcinomas (oscc) is associated with macrophage polarization in regional lymph nodes – an immunohistochemical study

    OpenAIRE

    Wehrhan, Falk; Büttner-Herold, Maike; Hyckel, Peter; Moebius, Patrick; Preidl, Raimund; Distel, Luitpold; Ries, Jutta; Amann, Kerstin; Schmitt, Christian; Neukam, Friedrich W.; Weber, Manuel

    2014-01-01

    Background It is largely accepted that specific immunological parameters in solid malignancies are associated with patient’s prognosis. Recently a correlation of macrophage polarization with histomorphological parameters could also be shown in oral squamous cell carcinoma (oscc). The observed tumor derived peripheral immune tolerance could be associated with the macrophage polarization in regional tumor draining lymph nodes. So far there are no studies analyzing the macrophage polarization in...

  14. Evidence for involvement of Wnt signalling in body polarities, cell proliferation, and the neuro-sensory system in an adult ctenophore.

    Directory of Open Access Journals (Sweden)

    Muriel Jager

    Full Text Available Signalling through the Wnt family of secreted proteins originated in a common metazoan ancestor and greatly influenced the evolution of animal body plans. In bilaterians, Wnt signalling plays multiple fundamental roles during embryonic development and in adult tissues, notably in axial patterning, neural development and stem cell regulation. Studies in various cnidarian species have particularly highlighted the evolutionarily conserved role of the Wnt/β-catenin pathway in specification and patterning of the primary embryonic axis. However in another key non-bilaterian phylum, Ctenophora, Wnts are not involved in early establishment of the body axis during embryogenesis. We analysed the expression in the adult of the ctenophore Pleurobrachia pileus of 11 orthologues of Wnt signalling genes including all ctenophore Wnt ligands and Fz receptors and several members of the intracellular β-catenin pathway machinery. All genes are strongly expressed around the mouth margin at the oral pole, evoking the Wnt oral centre of cnidarians. This observation is consistent with primary axis polarisation by the Wnts being a universal metazoan feature, secondarily lost in ctenophores during early development but retained in the adult. In addition, local expression of Wnt signalling genes was seen in various anatomical structures of the body including in the locomotory comb rows, where their complex deployment suggests control by the Wnts of local comb polarity. Other important contexts of Wnt involvement which probably evolved before the ctenophore/cnidarian/bilaterian split include proliferating stem cells and progenitors irrespective of cell types, and developing as well as differentiated neuro-sensory structures.

  15. Evidence for involvement of Wnt signalling in body polarities, cell proliferation, and the neuro-sensory system in an adult ctenophore.

    Science.gov (United States)

    Jager, Muriel; Dayraud, Cyrielle; Mialot, Antoine; Quéinnec, Eric; le Guyader, Hervé; Manuel, Michaël

    2013-01-01

    Signalling through the Wnt family of secreted proteins originated in a common metazoan ancestor and greatly influenced the evolution of animal body plans. In bilaterians, Wnt signalling plays multiple fundamental roles during embryonic development and in adult tissues, notably in axial patterning, neural development and stem cell regulation. Studies in various cnidarian species have particularly highlighted the evolutionarily conserved role of the Wnt/β-catenin pathway in specification and patterning of the primary embryonic axis. However in another key non-bilaterian phylum, Ctenophora, Wnts are not involved in early establishment of the body axis during embryogenesis. We analysed the expression in the adult of the ctenophore Pleurobrachia pileus of 11 orthologues of Wnt signalling genes including all ctenophore Wnt ligands and Fz receptors and several members of the intracellular β-catenin pathway machinery. All genes are strongly expressed around the mouth margin at the oral pole, evoking the Wnt oral centre of cnidarians. This observation is consistent with primary axis polarisation by the Wnts being a universal metazoan feature, secondarily lost in ctenophores during early development but retained in the adult. In addition, local expression of Wnt signalling genes was seen in various anatomical structures of the body including in the locomotory comb rows, where their complex deployment suggests control by the Wnts of local comb polarity. Other important contexts of Wnt involvement which probably evolved before the ctenophore/cnidarian/bilaterian split include proliferating stem cells and progenitors irrespective of cell types, and developing as well as differentiated neuro-sensory structures. PMID:24391946

  16. A systematic study on drug-response associated genes using baseline gene expressions of the Cancer Cell Line Encyclopedia

    Science.gov (United States)

    Liu, Xiaoming; Yang, Jiasheng; Zhang, Yi; Fang, Yun; Wang, Fayou; Wang, Jun; Zheng, Xiaoqi; Yang, Jialiang

    2016-03-01

    We have studied drug-response associated (DRA) gene expressions by applying a systems biology framework to the Cancer Cell Line Encyclopedia data. More than 4,000 genes are inferred to be DRA for at least one drug, while the number of DRA genes for each drug varies dramatically from almost 0 to 1,226. Functional enrichment analysis shows that the DRA genes are significantly enriched in genes associated with cell cycle and plasma membrane. Moreover, there might be two patterns of DRA genes between genders. There are significantly shared DRA genes between male and female for most drugs, while very little DRA genes tend to be shared between the two genders for a few drugs targeting sex-specific cancers (e.g., PD-0332991 for breast cancer and ovarian cancer). Our analyses also show substantial difference for DRA genes between young and old samples, suggesting the necessity of considering the age effects for personalized medicine in cancers. Lastly, differential module and key driver analyses confirm cell cycle related modules as top differential ones for drug sensitivity. The analyses also reveal the role of TSPO, TP53, and many other immune or cell cycle related genes as important key drivers for DRA network modules. These key drivers provide new drug targets to improve the sensitivity of cancer therapy.

  17. Benzyl isothiocyanate alters the gene expression with cell cycle regulation and cell death in human brain glioblastoma GBM 8401 cells.

    Science.gov (United States)

    Tang, Nou-Ying; Chueh, Fu-Shin; Yu, Chien-Chih; Liao, Ching-Lung; Lin, Jen-Jyh; Hsia, Te-Chun; Wu, King-Chuen; Liu, Hsin-Chung; Lu, Kung-Wen; Chung, Jing-Gung

    2016-04-01

    Glioblastoma multiforme (GBM) is a highly malignant devastating brain tumor in adults. Benzyl isothiocyanate (BITC) is one of the isothiocyanates that have been shown to induce human cancer cell apoptosis and cell cycle arrest. Herein, the effect of BITC on cell viability and apoptotic cell death and the genetic levels of human brain glioblastoma GBM 8401 cells in vitro were investigated. We found that BITC induced cell morphological changes, decreased cell viability and the induction of cell apoptosis in GBM 8401 cells was time-dependent. cDNA microarray was used to examine the effects of BITC on GBM 8401 cells and we found that numerous genes associated with cell death and cell cycle regulation in GBM 8401 cells were altered after BITC treatment. The results show that expression of 317 genes was upregulated, and two genes were associated with DNA damage, the DNA-damage-inducible transcript 3 (DDIT3) was increased 3.66-fold and the growth arrest and DNA-damage-inducible α (GADD45A) was increased 2.34-fold. We also found that expression of 182 genes was downregulated and two genes were associated with receptor for cell responses to stimuli, the EGF containing fibulin-like extracellular matrix protein 1 (EFEMP1) was inhibited 2.01-fold and the TNF receptor-associated protein 1 (TRAP1) was inhibited 2.08-fold. BITC inhibited seven mitochondria ribosomal genes, the mitochondrial ribosomal protein; tumor protein D52 (MRPS28) was inhibited 2.06-fold, the mitochondria ribosomal protein S2 (MRPS2) decreased 2.07-fold, the mitochondria ribosomal protein L23 (MRPL23) decreased 2.08-fold, the mitochondria ribosomal protein S2 (MRPS2) decreased 2.07-fold, the mitochondria ribosomal protein S12 (MRPS12) decreased 2.08-fold, the mitochondria ribosomal protein L12 (MRPL12) decreased 2.25-fold and the mitochondria ribosomal protein S34 (MRPS34) was decreased 2.30-fold in GBM 8401 cells. These changes of gene expression can provide the effects of BITC on the

  18. Cell cycle and anti-estrogen effects synergize to regulate cell proliferation and ER target gene expression.

    Directory of Open Access Journals (Sweden)

    Mathieu Dalvai

    Full Text Available Antiestrogens are designed to antagonize hormone induced proliferation and ERalpha target gene expression in mammary tumor cells. Commonly used drugs such as OH-Tamoxifen and ICI 182780 (Fulvestrant block cell cycle progression in G0/G1. Inversely, the effect of cell cycle stage on ER regulated gene expression has not been tested directly. We show that in ERalpha-positive breast cancer cells (MCF-7 the estrogen receptor gene and downstream target genes are cell cycle regulated with expression levels varying as much as three-fold between phases of the cell cycle. Steroid free culture conditions commonly used to assess the effect of hormones or antiestrogens on gene expression also block MCF-7 cells in G1-phase when several ERalpha target genes are overexpressed. Thus, cell cycle effects have to be taken into account when analyzing the impact of hormonal treatments on gene transcription. We found that antiestrogens repress transcription of several ERalpha target genes specifically in S phase. This observation corroborates the more rapid and strong impact of antiestrogen treatments on cell proliferation in thymidine, hydroxyurea or aphidicolin arrested cells and correlates with an increase of apoptosis compared to similar treatments in lovastatin or nocodazol treated cells. Hence, cell cycle effects synergize with the action of antiestrogens. An interesting therapeutic perspective could be to enhance the action of anti-estrogens by associating hormone-therapy with specific cell cycle drugs.

  19. Gene transcriptional profiles in human lymphoblastoid cells with low and high doses of irradiation

    International Nuclear Information System (INIS)

    Objective: To compare the gene expression difference between 0.1 and 5 Gy X-ray irradiated cells,and to explore its possible mechanism. Methods: A cDNA microarray corresponding to 45033 human genes was used to analyze the transcriptional profiles of normal human lymphoblastoid AHH-1 cells at 4 h after 0.1 or 5 Gy irradiation. The genes with a fold change ≥ 2.0 were identified as the differentially expressed genes. real-lime PCR and Western blot were used to confirm the expression of PERP. Results: The microarray assay showed that there were 760 up-regulated genes and 1222 down-regulated genes in the cells at 0.1 Gy, while there were 744 genes down-regulated and 457 genes up-regulated in the cells at 5 Gy. In addition, 55 genes were commonly up-regulated and 339 genes commonly down-regulated at 0.1 and 5 Gy. The predominant biological processes of the differential genes responding to low-dose radiation include cell-cell signaling transduction and DNA damage response, and the altered genes after 5 Gy irradiation were related to cell proliferation, differentiation, and apoptosis. Moreover, the expression of PERP gene was down regulated, which was consistent with the data of microarray assay. Conclusions: The quantitative and qualitative differences in the gene expressions may contribute to the diverse biological effects induced by low or high doses of ionizing radiation. (authors)

  20. Analysis of Gene Expression in the K562-n High Tumorigenitic Human Leukemia Cell Line

    Institute of Scientific and Technical Information of China (English)

    Shuqing Lü; Xiaoping Xu; Fang Xia; JianMin Wang

    2005-01-01

    OBJECTIVE The human leukemia K562-n cell line displays much higher tumorigenic actively in nude mice compared with its parental K562 cell line. The molecular mechanism of the differences in tumorigenicity between K562-n and K562 in nude mice was examined.METHODS The differences in gene expression between K562 and K562-n cells were analyzed by using cDNA microarrays.RESULTS Among the12,800 genes examined, there was a significant difference in expression of 139 genes between K562-n and K562 cells.Eighty-five of these genes have been registered in the GeneBank and 54are unknown. The genes accessible from the GeneBank include:1)oncogenes and tumor-supressor genes; 2) genes related to transcription regulation, the cell cycle and apoptosis; 3) genes related to the cytoskeleton and cytokinetics; 4) genes related to metabolism and transport; 5) genes related to immune function. There were also some differently expressed genes with mixed functions.CONCLUSION There are many genes differentially expressed between K562-n and K562 cells .The high tumorigenicity of the human leukemia K562-n cell line in nude mice might be related to its specific geneexpression profile.

  1. Expression and subcellular localization of aquaporin water channels in the polarized hepatocyte cell line, WIF-B

    Directory of Open Access Journals (Sweden)

    Marinelli Raúl A

    2005-08-01

    Full Text Available Abstract Background Recent data suggest that canalicular bile secretion involves selective expression and coordinated regulation of aquaporins (AQPs, a family of water channels proteins. In order to further characterize the role of AQPs in this process, an in vitro cell system with retained polarity and expression of AQPs and relevant solute transporters involved in bile formation is highly desirable. The WIF-B cell line is a highly differentiated and polarized rat hepatoma/human fibroblast hybrid, which forms abundant bile canalicular structures. This cell line has been reported to be a good in vitro model for studying hepatocyte polarity. Results Using RT-PCR, immunoblotting and confocal immunofluorescence, we showed that WIF-B cells express the aquaporin water channels that facilitate the osmotically driven water movements in the liver, i.e. AQP8, AQP9, and AQP0; as well as the key solute transporters involved in the generation of canalicular osmotic gradients, i.e., the bile salt export pump Bsep, the organic anion transporter Mrp2 and the chloride bicarbonate exchanger AE2. The subcellular localization of the AQPs and the solute transporters in WIF-B cells was similar to that in freshly isolated rat hepatocytes and in intact liver. Immunofluorescent costaining studies showed intracellular colocalization of AQP8 and AE2, suggesting the possibility that these transporters are expressed in the same population of pericanalicular vesicles. Conclusion The hepatocyte cell line WIF-B retains the expression and subcellular localization of aquaporin water channels as well as key solute transporters for canalicular bile secretion. Thus, these cells can work as a valuable tool for regulatory and mechanistic studies of the biology of bile formation.

  2. Photon harvesting, coloring and polarizing in photovoltaic cell integrated color filters: efficient energy routing strategies for power-saving displays

    Science.gov (United States)

    Wen, Long; Chen, Qin; Song, Shichao; Yu, Yan; Jin, Lin; Hu, Xin

    2015-07-01

    We describe the integral electro-optical strategies that combine the functionalities of photovoltaic (PV) electricity generation and color filtering as well as polarizing to realize more efficient energy routing in display technology. Unlike the conventional pigment-based filters and polarizers, which absorb substantial amounts of unwanted spectral components and dissipate them in the form of heat, we propose converting the energy of those photons into electricity by constructing PV cell-integrated color filters based on a selectively transmitting aluminum (Al) rear electrode perforated with nanoholes (NHs). Combining with a dielectric-metal-dielectric (DMD) front electrode, the devices were optimized to enable efficient cavity-enhanced photon recycling in the PV functional layers. We perform a comprehensive theoretical and numerical analysis to explore the extraordinary optical transmission (EOT) through the Al NHs and identify basic design rules for achieving structural coloring or polarizing in our PV color filters. We show that the addition of thin photoactive polymer layers on the symmetrically configured Al NH electrode narrows the bandwidth of the EOT-assisted high-pass light filtering due to the strongly damped anti-symmetric coupling of the surface modes excited on the front and rear surface of the Al NHs, which facilitates the whole visible coloring with relatively high purity for the devices. By engineering the cut-off characteristics of the plasmonic waveguide mode supported by the circular or ellipsoidal Al NHs, beyond the photon recycling capacity, PV color filters and PV polarizing color filters that allow polarization-insensitive and strong polarization-anisotropic color filtering were demonstrated. The findings presented here may shed some light on expanding the utilization of PV electricity generation across new-generation energy-saving electrical display devices.

  3. Gene expression in epithelial cells in response to pneumovirus infection

    Directory of Open Access Journals (Sweden)

    Rosenberg Helene F

    2001-05-01

    Full Text Available Abstract Respiratory syncytial virus (RSV and pneumonia virus of mice (PVM are viruses of the family Paramyxoviridae, subfamily pneumovirus, which cause clinically important respiratory infections in humans and rodents, respectively. The respiratory epithelial target cells respond to viral infection with specific alterations in gene expression, including production of chemoattractant cytokines, adhesion molecules, elements that are related to the apoptosis response, and others that remain incompletely understood. Here we review our current understanding of these mucosal responses and discuss several genomic approaches, including differential display reverse transcription-polymerase chain reaction (PCR and gene array strategies, that will permit us to unravel the nature of these responses in a more complete and systematic manner.

  4. HBV X Gene Transfection Upregulates IL-1β and IL-6 Gene Expression and Induces Rat Glomerular Mesangial Cell Proliferation

    Institute of Scientific and Technical Information of China (English)

    Hongzhu LU; Jianhua ZHOU

    2008-01-01

    The X gene of HBV encodes a 17-KD protein, termed HBx, which has been shown to function as a transcriptional trans-activator of a variety of viral and cellular promoter/enhancer elements. The aim of this study was to investigate the effect of HBx on gene expression of interleukin (IL)-1β and IL-6, and proliferation of rat mesangial cells in vitro. The X gene of HBV was amplified by PCR assay, and inserted into the eukaryotic expression vector pCI-neo. The structure of recombinant pCI-neo-X plasmid was proved by restrict endonuclease digestion and sequencing analysis. pCI-neo-X was transfected into cultured rat mesangial cell line in vitro via liposome. HBx expression in transfected mesangial cells was detected by Western blot. The IL-1β and IL-6 mRNA expression in those cells was assayed by semiquantitative RT-PCR. Mesangial cell proliferation was tested by MTT. The results showed that HBx was obviously expressed in cultured mesangial cell line at 36th and 48th h after transfection. The expression of IL-1β and IL-6 mRNA was simultaneously increased. The cell proliferation was also obvious at the same time. It was concluded that HBx gene transfection could induce IL-1β and IL-6 gene expression and mesangial cell proliferation. HBx may play a critical role in mesangial cell proliferation through upregulation of the IL-1β and IL-6 gene expression.

  5. Wdpcp, a PCP protein required for ciliogenesis, regulates directional cell migration and cell polarity by direct modulation of the actin cytoskeleton.

    Directory of Open Access Journals (Sweden)

    Cheng Cui

    2013-11-01

    Full Text Available Planar cell polarity (PCP regulates cell alignment required for collective cell movement during embryonic development. This requires PCP/PCP effector proteins, some of which also play essential roles in ciliogenesis, highlighting the long-standing question of the role of the cilium in PCP. Wdpcp, a PCP effector, was recently shown to regulate both ciliogenesis and collective cell movement, but the underlying mechanism is unknown. Here we show Wdpcp can regulate PCP by direct modulation of the actin cytoskeleton. These studies were made possible by recovery of a Wdpcp mutant mouse model. Wdpcp-deficient mice exhibit phenotypes reminiscent of Bardet-Biedl/Meckel-Gruber ciliopathy syndromes, including cardiac outflow tract and cochlea defects associated with PCP perturbation. We observed Wdpcp is localized to the transition zone, and in Wdpcp-deficient cells, Sept2, Nphp1, and Mks1 were lost from the transition zone, indicating Wdpcp is required for recruitment of proteins essential for ciliogenesis. Wdpcp is also found in the cytoplasm, where it is localized in the actin cytoskeleton and in focal adhesions. Wdpcp interacts with Sept2 and is colocalized with Sept2 in actin filaments, but in Wdpcp-deficient cells, Sept2 was lost from the actin cytoskeleton, suggesting Wdpcp is required for Sept2 recruitment to actin filaments. Significantly, organization of the actin filaments and focal contacts were markedly changed in Wdpcp-deficient cells. This was associated with decreased membrane ruffling, failure to establish cell polarity, and loss of directional cell migration. These results suggest the PCP defects in Wdpcp mutants are not caused by loss of cilia, but by direct disruption of the actin cytoskeleton. Consistent with this, Wdpcp mutant cochlea has normal kinocilia and yet exhibits PCP defects. Together, these findings provide the first evidence, to our knowledge, that a PCP component required for ciliogenesis can directly modulate the actin

  6. Study in static mode of a photovoltaic cell bi facial to crystalline silicon under electric polarization and constant multispectral illumination

    International Nuclear Information System (INIS)

    The theoretical study in static mode of a photovoltaic cell bi facial to silicon under electric polarization and multispectral illumination is presented. Through this study, various expressions of the parameters of recombination have been established as well for an illumination by the face before an illumination by the back face. Curves of variation of the densities of carriers, densities of photocurrent, speeds of recombinations and photo tensions have been traced for the two modes of illumination

  7. Quantitative analysis of cell-type specific gene expression in the green alga Volvox carteri

    Directory of Open Access Journals (Sweden)

    Hallmann Armin

    2006-12-01

    Full Text Available Abstract Background The multicellular alga Volvox carteri possesses only two cell types: mortal, motile somatic cells and potentially immortal, immotile reproductive cells. It is therefore an attractive model system for studying how cell-autonomous cytodifferentiation is programmed within a genome. Moreover, there are ongoing genome projects both in Volvox carteri and in the closely related unicellular alga Chlamydomonas reinhardtii. However, gene sequencing is only the beginning. To identify cell-type specific expression and to determine relative expression rates, we evaluate the potential of real-time RT-PCR for quantifying gene transcript levels. Results Here we analyze a diversified pool of 39 target genes by real-time RT-PCR for each cell type. This gene pool contains previously known genes with unknown localization of cellular expression, 28 novel genes which are described in this study for the first time, and a few known, cell-type specific genes as a control. The respective gene products are, for instance, part of photosynthesis, cellular regulation, stress response, or transport processes. We provide expression data for all these genes. Conclusion The results show that quantitative real-time RT-PCR is a favorable approach to analyze cell-type specific gene expression in Volvox, which can be extended to a much larger number of genes or to developmental or metabolic mutants. Our expression data also provide a basis for a detailed analysis of individual, previously unknown, cell-type specifically expressed genes.

  8. Improved polarization of mesoporous electrodes of a proton exchange membrane fuel cell using N-methyl-2-pyrrolidinone

    International Nuclear Information System (INIS)

    Highlights: • Enhancement of decal-transfer rate by controlling hydrophilicity of the CL. • Well-distributed CL microstructures by using NMP-containing ink. • Improved cell polarization by achieving highly connected Pt/C agglomerates. • Effect of different solvents on the electrochemical performance of the CL. -- Abstract: Both the catalytic and ohmic polarization of a proton exchange membrane fuel cell must be improved to achieve commercialization. Herein, high-performance membrane electrode assemblies (MEAs) were prepared using an amide-type chemical, i.e., N-methyl-2-pyrrolidinone (NMP) as a solvent for the catalyst ink. Influence of different solvent on the catalyst-layer (CL) surface microstructure was clarified to achieve high fuel-cell performance. The electrochemical performance of the MEAs made from different ink formulation was examined in detail using electrochemical impedance spectroscopy and cyclic voltammetry. As a result, the NMP–glycerol ink with a 10 wt.% glycerol was used to form a dense and well-connected Pt/C–Nafion agglomerated CL, followed by enhanced catalytic and ohmic polarizations relative to the glycerol and NMP CLs

  9. Polarization Imaging Apparatus for Cell and Tissue Imaging and Diagnostics Project

    Data.gov (United States)

    National Aeronautics and Space Administration — In recent years there has been an increasing interest in the propagation of polarized light in randomly scattering media. The investigation of backscattered light...

  10. Polarization Imaging Apparatus for Cell and Tissue Imaging and Diagnostics Project

    Data.gov (United States)

    National Aeronautics and Space Administration — This work proposes to capitalize on our Phase I success in a novel visible-near infrared Stokes polarization imaging technology based on high performance fast...

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

    International Nuclear Information System (INIS)

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

  12. Gene expression profiling in multipotent DFAT cells derived from mature adipocytes

    International Nuclear Information System (INIS)

    Highlights: → Adipocyte dedifferentiation is evident in a significant decrease in typical genes. → Cell proliferation is strongly related to adipocyte dedifferentiation. → Dedifferentiated adipocytes express several lineage-specific genes. → Comparative analyses using publicly available datasets boost the interpretation. -- Abstract: Cellular dedifferentiation signifies the withdrawal of cells from a specific differentiated state to a stem cell-like undifferentiated state. However, the mechanism of dedifferentiation remains obscure. Here we performed comparative transcriptome analyses during dedifferentiation in mature adipocytes (MAs) to identify the transcriptional signatures of multipotent dedifferentiated fat (DFAT) cells derived from MAs. Using microarray systems, we explored similarly expressed as well as significantly differentially expressed genes in MAs during dedifferentiation. This analysis revealed significant changes in gene expression during this process, including a significant reduction in expression of genes for lipid metabolism concomitantly with a significant increase in expression of genes for cell movement, cell migration, tissue developmental processes, cell growth, cell proliferation, cell morphogenesis, altered cell shape, and cell differentiation. Our observations indicate that the transcriptional signatures of DFAT cells derived from MAs are summarized in terms of a significant decrease in functional phenotype-related genes and a parallel increase in cell proliferation, altered cell morphology, and regulation of the differentiation of related genes. A better understanding of the mechanisms involved in dedifferentiation may enable scientists to control and possibly alter the plasticity of the differentiated state, which may lead to benefits not only in stem cell research but also in regenerative medicine.

  13. Gene expression profiling in multipotent DFAT cells derived from mature adipocytes

    Energy Technology Data Exchange (ETDEWEB)

    Ono, Hiromasa [Laboratory of Cell and Tissue Biology, College of Bioresource Sciences, Nihon University, 1866 Kameino, Fujisawa, Kanagawa 252-8510 (Japan); Database Center for Life Science (DBCLS), Research Organization of Information and Systems (ROIS), Faculty of Engineering Bldg.12 2-11-16 Yayoi, Bunkyo-ku, Tokyo 113-0032 (Japan); Oki, Yoshinao [Laboratory of Cell and Tissue Biology, College of Bioresource Sciences, Nihon University, 1866 Kameino, Fujisawa, Kanagawa 252-8510 (Japan); Bono, Hidemasa [Database Center for Life Science (DBCLS), Research Organization of Information and Systems (ROIS), Faculty of Engineering Bldg.12 2-11-16 Yayoi, Bunkyo-ku, Tokyo 113-0032 (Japan); Kano, Koichiro, E-mail: kkano@brs.nihon-u.ac.jp [Laboratory of Cell and Tissue Biology, College of Bioresource Sciences, Nihon University, 1866 Kameino, Fujisawa, Kanagawa 252-8510 (Japan)

    2011-04-15

    Highlights: {yields} Adipocyte dedifferentiation is evident in a significant decrease in typical genes. {yields} Cell proliferation is strongly related to adipocyte dedifferentiation. {yields} Dedifferentiated adipocytes express several lineage-specific genes. {yields} Comparative analyses using publicly available datasets boost the interpretation. -- Abstract: Cellular dedifferentiation signifies the withdrawal of cells from a specific differentiated state to a stem cell-like undifferentiated state. However, the mechanism of dedifferentiation remains obscure. Here we performed comparative transcriptome analyses during dedifferentiation in mature adipocytes (MAs) to identify the transcriptional signatures of multipotent dedifferentiated fat (DFAT) cells derived from MAs. Using microarray systems, we explored similarly expressed as well as significantly differentially expressed genes in MAs during dedifferentiation. This analysis revealed significant changes in gene expression during this process, including a significant reduction in expression of genes for lipid metabolism concomitantly with a significant increase in expression of genes for cell movement, cell migration, tissue developmental processes, cell growth, cell proliferation, cell morphogenesis, altered cell shape, and cell differentiation. Our observations indicate that the transcriptional signatures of DFAT cells derived from MAs are summarized in terms of a significant decrease in functional phenotype-related genes and a parallel increase in cell proliferation, altered cell morphology, and regulation of the differentiation of related genes. A better understanding of the mechanisms involved in dedifferentiation may enable scientists to control and possibly alter the plasticity of the differentiated state, which may lead to benefits not only in stem cell research but also in regenerative medicine.

  14. Differential expression of the klf6 tumor suppressor gene upon cell damaging treatments in cancer cells

    Energy Technology Data Exchange (ETDEWEB)

    Gehrau, Ricardo C.; D' Astolfo, Diego S.; Andreoli, Veronica [Centro de Investigaciones en Bioquimica Clinica e Inmunologia (CIBICI-CONICET), Departamento de Bioquimica Clinica, Facultad de Ciencias Quimicas, Universidad Nacional de Cordoba, Haya de la Torre y Medina Allende, Ciudad Universitaria, 5000 Cordoba (Argentina); Bocco, Jose L., E-mail: jbocco@fcq.unc.edu.ar [Centro de Investigaciones en Bioquimica Clinica e Inmunologia (CIBICI-CONICET), Departamento de Bioquimica Clinica, Facultad de Ciencias Quimicas, Universidad Nacional de Cordoba, Haya de la Torre y Medina Allende, Ciudad Universitaria, 5000 Cordoba (Argentina); Koritschoner, Nicolas P. [Centro de Investigaciones en Bioquimica Clinica e Inmunologia (CIBICI-CONICET), Departamento de Bioquimica Clinica, Facultad de Ciencias Quimicas, Universidad Nacional de Cordoba, Haya de la Torre y Medina Allende, Ciudad Universitaria, 5000 Cordoba (Argentina)

    2011-02-10

    The mammalian Krueppel-like factor 6 (KLF6) is involved in critical roles such as growth-related signal transduction, cell proliferation and differentiation, development, apoptosis and angiogenesis. Also, KLF6 appears to be an emerging key factor during cancer development and progression. Its expression is thoroughly regulated by several cell-damaging stimuli. DNA damaging agents at lethal concentrations induce a p53-independent down-regulation of the klf6 gene. To investigate the impact of external stimuli on human klf6 gene expression, its mRNA level was analyzed using a cancer cell line profiling array system, consisting in an assortment of immobilized cDNAs from multiple cell lines treated with several cell-damaging agents at growth inhibitory concentrations (IC{sub 50}). Cell-damaging agents affected the klf6 expression in 62% of the cDNA samples, though the expression pattern was not dependent on the cell origin type. Interestingly, significant differences (p < 0.0001) in KLF6 mRNA levels were observed depending on the cellular p53 status upon cell damage. KLF6 expression was significantly increased in 63% of p53-deficient cells (122/195). Conversely, KLF6 mRNA level decreased nearly 4 fold in more than 70% of p53+/+ cells. In addition, klf6 gene promoter activity was down-regulated by DNA damaging agents in cells expressing the functional p53 protein whereas it was moderately increased in the absence of functional p53. Consistent results were obtained for the endogenous KLF6 protein level. Results indicate that human klf6 gene expression is responsive to external cell damage mediated by IC{sub 50} concentrations of physical and chemical stimuli in a p53-dependent manner. Most of these agents are frequently used in cancer therapy. Induction of klf6 expression in the absence of functional p53 directly correlates with cell death triggered by these compounds, whereas it is down-regulated in p53+/+ cells. Hence, klf6 expression level could represent a valuable

  15. Cell and gene therapy in Duchenne muscular dystrophy.

    Science.gov (United States)

    Morgan, J E

    1994-02-01

    Experiments in mice have supported the idea of treating Duchenne muscular dystrophy (DMD) by implanting normal muscle precursor cells into dystrophin-deficient muscles. However, similar experiments on DMD patients have had little success. Gene therapy for DMD, by introducing dystrophin constructs via retroviral or adenoviral vectors, has been shown to be possible in the mouse, but the efficiency and safety aspects of this technique will have to be carefully examined before similar experiments can be attempted in man. Direct injection of dystrophin cDNA constructs into mdx muscles has given rise to very low levels of dystrophin and this may be a possibility for the treatment of heart muscle. PMID:7514447

  16. A gold nanoparticle pentapeptide: gene fusion to induce therapeutic gene expression in mesenchymal stem cells.

    Science.gov (United States)

    Muroski, Megan E; Morgan, Thomas J; Levenson, Cathy W; Strouse, Geoffrey F

    2014-10-22

    Mesenchymal stem cells (MSC) have been identified as having great potential as autologous cell therapeutics to treat traumatic brain injury and spinal injury as well as neuronal and cardiac ischemic events. All future clinical applications of MSC cell therapies must allow the MSC to be harvested, transfected, and induced to express a desired protein or selection of proteins to have medical benefit. For the full potential of MSC cell therapy to be realized, it is desirable to systematically alter the protein expression of therapeutically beneficial biomolecules in harvested MSC cells with high fidelity in a single transfection event. We have developed a delivery platform on the basis of the use of a solid gold nanoparticle that has been surface modified to produce a fusion containing a zwitterionic, pentapeptide designed from Bax inhibiting peptide (Ku70) to enhance cellular uptake and a linearized expression vector to induce enhanced expression of brain-derived neurotrophic factor (BDNF) in rat-derived MSCs. Ku70 is observed to effect >80% transfection following a single treatment of femur bone marrow isolated rat MSCs with efficiencies for the delivery of a 6.6 kbp gene on either a Au nanoparticle (NP) or CdSe/ZnS quantum dot (QD). Gene expression is observed within 4 d by optical measurements, and secretion is observed within 10 d by Western Blot analysis. The combination of being able to selectively engineer the NP, to colocalize biological agents, and to enhance the stability of those agents has provided the strong impetus to utilize this novel class of materials to engineer primary MSCs. PMID:25198921

  17. A polarization-independent liquid crystal phase modulation using polymer-network liquid crystals in a 90° twisted cell

    Science.gov (United States)

    Lin, Yi-Hsin; Chen, Ming-Syuan; Lin, Wei-Chih; Tsou, Yu-Shih

    2012-07-01

    A polarization-independent liquid crystal phase modulation using polymer-network liquid crystals in a 90° twisted cell (T-PNLC) is demonstrated. T-PNLC consists of three layers. Liquid crystal (LC) directors in the two layers near glass substrates are orthogonal to each other and those two layers modulate two eigen-polarizations of an incident light. As a result, two eigen-polarizations of an incident light experience the same phase shift. In the middle layer, LC directors are perpendicular to the glass substrate and contribute no phase shift. The phase shift of T-PNLC is electrically tunable and polarization-independent. T-PNLC does not require any bias voltage for operation. The phase shift is 0.28 π rad for the voltage of 30 Vrms. By measuring and analyzing the optical phase shift of T-PNLC at the oblique incidence of transverse magnetic wave, the pretilt angle of LC directors and the effective thickness of three layers are obtained and discussed. The potential applications are spatial light modulators, laser beam steering, and micro-lens arrays.

  18. Extracellular enzymatic activities of cold-adapted bacteria from polar oceans and effect of temperature and salinity on cell growth

    Institute of Scientific and Technical Information of China (English)

    Zeng Yinxin; Yu Yong; Chen Bo; Li Huirong

    2004-01-01

    The potential of 324 bacteria isolated from different habitats in polar oceans to produce a variety of extracellular enzymatic activities at low temperature was investigated. By plate assay, lipase, protease, amylase, gelatinase, agarase, chitinase or cellulase were detected. Lipases were generally present by bacteria living in polar oceans. Protease-producing bacteria held the second highest proportion in culturable isolates. Strains producing amylase kept a relative stable proportion of around 30% in different polar marine habitats. All 50 Arctic sea-ice bacteria producing proteases were cold-adapted strains, however, only 20% were psychrophilic. 98% of them could grow at 3% NaCl, and 56% could grow without NaCl. On the other hand, 98% of these sea-ice bacteria produced extracellular proteases with optimum temperature at or higher than 35℃, well above the upper temperature limit of cell growth. Extracellular enzymes including amylase, agarase, cellulase and lipase released by bacteria from seawater or sediment in polar oceans, most expressed maximum activities between 25 and 35℃. Among extracellular enzymes released by bacterial strain BSw20308, protease expressed maximum activity at 40℃, higher than 35℃ of polysaccharide hydrolases and 25℃ of lipase.

  19. Differential gene expression in human hepatocellular carcinoma Hep3B cells induced by apoptosis-related gene BNIPL-2

    Institute of Scientific and Technical Information of China (English)

    Li Xie; Wen-Xin Qin; Xiang-Huo He; Hui-Qun Shu; Gen-Fu Yao; Da-Fang Wan; Jian-Ren Gu

    2004-01-01

    AIM: Bcl-2/adenovirus E1B 19 ku interacting protein 2-like (BNIPL-2) is a novel protein recently identified in our laboratory. BNIPL-2 is homologous to human BNIP-2, a potentially proapoptotic protein, and can interact with Bcl-2 and Cdc42GAP and promote apoptosis in BEL-7402 cells.Here we report the gene-expression profile regulated by BNIPL-2 in human hepatocarcinoma Hep3B cells and the analysis of its potential roles in cell apoptosis.METHODS: BNIPL-2 was overexpressed in Hep3B cells using tetracycline inducible or Tet-on system. Screened by Western blot, the cells with low background and high induction fold of BNIPL-2 were obtained. We performed Atlas human cDNA expression array hybridization on these cells and analyzed the data with Quantarray software to identify BNIPL-2-regulated genes and their expression profile. RT-PCR was used to confirm the altered expression level of part of genes identified by the Atlas array hybridization.RESULTS: Fifteen of 588 genes spotted on the Atlas membrane showed altered expression levels in BNIPL-2-transfected Hep3B-Tet-on cells, in which 8 genes involved in cell apoptosis or growth inhibition were up-regulated and 7 genes involved in cellular proliferation were downregulated following overexpression of BNIPL-2.CONCLUSION: cDNA array is a powerful tool to explore gene expression profiles under inducible conditions. The data obtained using the cDNA expression microarray technology indicates that BNIPL-2 may play its roles in apoptosis through regulating the expression of genes associated with cell apoptosis, growth inhibition and cell proliferation.

  20. Inhibitory Effect of Isoflavones on Prostate Cancer Cells and PTEN Gene

    Institute of Scientific and Technical Information of China (English)

    2006-01-01

    Objective To explore the mechanisms by which genistein and daidzein inhibit the growth of prostate cancer cells. Methods LNCaP and PC-3 cells were exposed to genistein and daidzein and cell viability was determined by MTT assay and cytotoxicity of the drugs by LDH test. Flow cytometry (FCM) was used to assess the cell cycle in LNCaP and PC-3 cells.Reverse transcription-polymerase chain reaction (RT-PCR) was applied to examine the expression of PTEN gene (a tumor suppressor gene), estrogen receptor alpha gene (Erα), estrogen receptor beta gene (Erβ), androgen receptor gene (AR) and vascular endothelial growth factor gene (VEGF). Results The viability of PC-3 and LNCaP cells decreased with increasing concentrations and exposure time of genistein and daidzein. Genistein increased G2/M phase cells in PC-3 cells while decreased S phase cells in LNCaP cells in a dose-dependent manner. Daidzein exerted no influence on the cell cycle of LNCaP and PC-3 cells, but the apoptosis percentage of LNCaP cells was elevated significantly by daidzein. Genistein induced the expression of PTEN gene in PC-3 and LNCaP cells. Daidzein induced the expression of PTEN gene in LNCaP but not in PC-3 cells. The expression of VEGF, Erα and Erβ genes decreased and AR gene was not expressed after incubation with genistein and daidzein in PC-3 cells. In LNCaP cells, the expression of VEGF and AR gene decreased but there was no change in the expression of Erα and Erβ gene after incubation with genistein and daidzein. Conclusion Genistein and daidzein exert a time- and dose-dependent inhibitory effect on PC-3 and LNCaP cells. The down-regulation of ER gene by daidzein influences the growth of PC-3 cells directly. The inhibition of PC-3 cells by genistein and that of LNCaP cells by genistein and daidzein may be via Akt pathway that is repressed by PTEN gene, which subsequently down-regulates the expression of AR and VEGF genes. Our results suggest that the expression of PTEN gene plays a key

  1. Parathyroid hormone-related protein regulates tumor-relevant genes in breast cancer cells.

    NARCIS (Netherlands)

    Dittmer, A.; Vetter, M.; Schunke, D.; Span, P.N.; Sweep, C.G.J.; Thomssen, C.; Dittmer, J.

    2006-01-01

    The effect of endogenous parathyroid hormone-related protein (PTHrP) on gene expression in breast cancer cells was studied. We suppressed PTHrP expression in MDA-MB-231 cells by RNA interference and analyzed changes in gene expression by microarray analysis. More than 200 genes showed altered expres

  2. Compact Circularly Polarized Patch Antenna Using a Composite Right/Left-Handed Transmission Line Unit-Cell

    OpenAIRE

    Geng, L.; Wang, G. M.; Zhang, C. X.; Gao, X. J.; Zong, B. F.

    2013-01-01

    A compact circularly polarized (CP) patch antenna using a composite right/left-handed (CRLH) transmission line (TL) unit-cell is proposed. The CRLH TL unit-cell includes a complementary split ring resonator (CSRR) for shunt inductance and a gap loaded with a circular-shaped slot for series capacitance. The CSRR can decrease the TM10 mode resonance frequency, thus reducing the electrical size of the proposed antenna. In addition, the asymmetry of the CSRR brings about the TM01 mode, which can ...

  3. Musashi1 modulates cell proliferation genes in the medulloblastoma cell line Daoy

    International Nuclear Information System (INIS)

    Musashi1 (Msi1) is an RNA binding protein with a central role during nervous system development and stem cell maintenance. High levels of Msi1 have been reported in several malignancies including brain tumors thereby associating Msi1 and cancer. We used the human medulloblastoma cell line Daoy as model system in this study to knock down the expression of Msi1 and determine the effects upon soft agar growth and neurophere formation. Quantitative RT-PCR was conducted to evaluate the expression of cell proliferation, differentiation and survival genes in Msi1 depleted Daoy cells. We observed that MSI1 expression was elevated in Daoy cells cultured as neurospheres compared to those grown as monolayer. These data indicated that Msi1 might be involved in regulating proliferation in cancer cells. Here we show that shRNA mediated Msi1 depletion in Daoy cells notably impaired their ability to form colonies in soft agar and to grow as neurospheres in culture. Moreover, differential expression of a group of Notch, Hedgehog and Wnt pathway related genes including MYCN, FOS, NOTCH2, SMO, CDKN1A, CCND2, CCND1, and DKK1, was also found in the Msi1 knockdown, demonstrating that Msi1 modulated the expression of a subset of cell proliferation, differentiation and survival genes in Daoy. Our data suggested that Msi1 may promote cancer cell proliferation and survival as its loss seems to have a detrimental effect in the maintenance of medulloblastoma cancer cells. In this regard, Msi1 might be a positive regulator of tumor progression and a potential target for therapy

  4. Musashi1 modulates cell proliferation genes in the medulloblastoma cell line Daoy

    Directory of Open Access Journals (Sweden)

    Hung Jaclyn Y

    2008-09-01

    Full Text Available Abstract Background Musashi1 (Msi1 is an RNA binding protein with a central role during nervous system development and stem cell maintenance. High levels of Msi1 have been reported in several malignancies including brain tumors thereby associating Msi1 and cancer. Methods We used the human medulloblastoma cell line Daoy as model system in this study to knock down the expression of Msi1 and determine the effects upon soft agar growth and neurophere formation. Quantitative RT-PCR was conducted to evaluate the expression of cell proliferation, differentiation and survival genes in Msi1 depleted Daoy cells. Results We observed that MSI1 expression was elevated in Daoy cells cultured as neurospheres compared to those grown as monolayer. These data indicated that Msi1 might be involved in regulating proliferation in cancer cells. Here we show that shRNA mediated Msi1 depletion in Daoy cells notably impaired their ability to form colonies in soft agar and to grow as neurospheres in culture. Moreover, differential expression of a group of Notch, Hedgehog and Wnt pathway related genes including MYCN, FOS, NOTCH2, SMO, CDKN1A, CCND2, CCND1, and DKK1, was also found in the Msi1 knockdown, demonstrating that Msi1 modulated the expression of a subset of cell proliferation, differentiation and survival genes in Daoy. Conclusion Our data suggested that Msi1 may promote cancer cell proliferation and survival as its loss seems to have a detrimental effect in the maintenance of medulloblastoma cancer cells. In this regard, Msi1 might be a positive regulator of tumor progression and a potential target for therapy.

  5. Distinct gene expression signatures in human embryonic stem cells differentiated towards definitive endoderm at single-cell level

    DEFF Research Database (Denmark)

    Norrman, Karin; Strömbeck, Anna; Semb, Henrik; Ståhlberg, Anders

    2013-01-01

    of anterior definitive endoderm (DE). Here, we differentiated human embryonic stem cells towards DE using three different activin A based treatments. Differentiation efficiencies were evaluated by gene expression profiling over time at cell population level. A panel of key markers was used to study...... were useful to monitor the temporal expression of genes involved in primitive streak formation and endoderm formation, while single-cell analysis allowed us to study cell culture heterogeneity and fingerprint individual cells. In addition, single-cell analysis revealed distinct gene expression patterns...

  6. CIRCADIAN CLOCK AND CELL CYCLE GENE EXPRESSION: IN MOUSE MAMMARY EPITHELIAL CELLS AND IN THE DEVELOPING MOUSE MAMMARY GLAND

    OpenAIRE

    Metz, Richard P.; Qu, Xiaoyu; Laffin, Brian; Earnest, David; Porter, Weston W.

    2006-01-01

    Mouse mammary epithelial cells (HC-11) and mammary tissues were analyzed for developmental changes in circadian clock, cellular proliferation and differentiation marker genes. Expression of the clock genes, Per1 and Bmal1, were elevated in differentiated HC-11 cells whereas Per2 mRNA levels were higher in undifferentiated cells. This differentiation-dependent profile of clock gene expression was consistent with that observed in mouse mammary glands as Per1 and Bmal1 mRNA levels were elevated ...

  7. Water-polysaccharide interactions in the primary cell wall of Arabidopsis thaliana from polarization transfer solid-state NMR.

    Science.gov (United States)

    White, Paul B; Wang, Tuo; Park, Yong Bum; Cosgrove, Daniel J; Hong, Mei

    2014-07-23

    Polysaccharide-rich plant cell walls are hydrated under functional conditions, but the molecular interactions between water and polysaccharides in the wall have not been investigated. In this work, we employ polarization transfer solid-state NMR techniques to study the hydration of primary-wall polysaccharides of the model plant, Arabidopsis thaliana. By transferring water (1)H polarization to polysaccharides through distance- and mobility-dependent (1)H-(1)H dipolar couplings and detecting it through polysaccharide (13)C signals, we obtain information about water proximity to cellulose, hemicellulose, and pectins as well as water mobility. Both intact and partially extracted cell wall samples are studied. Our results show that water-pectin polarization transfer is much faster than water-cellulose polarization transfer in all samples, but the extent of extraction has a profound impact on the water-polysaccharide spin diffusion. Removal of calcium ions and the consequent extraction of homogalacturonan (HG) significantly slowed down spin diffusion, while further extraction of matrix polysaccharides restored the spin diffusion rate. These trends are observed in cell walls with similar water content, thus they reflect inherent differences in the mobility and spatial distribution of water. Combined with quantitative analysis of the polysaccharide contents, our results indicate that calcium ions and HG gelation increase the amount of bound water, which facilitates spin diffusion, while calcium removal disrupts the gel and gives rise to highly dynamic water, which slows down spin diffusion. The recovery of spin diffusion rates after more extensive extraction is attributed to increased water-exposed surface areas of the polysaccharides. Water-pectin spin diffusion precedes water-cellulose spin diffusion, lending support to the single-network model of plant primary walls in which a substantial fraction of the cellulose surface is surrounded by pectins. PMID:24984197

  8. Comparison of gene expression profiles in chromate transformed BEAS-2B cells.

    Directory of Open Access Journals (Sweden)

    Hong Sun

    Full Text Available BACKGROUND: Hexavalent chromium [Cr(VI] is a potent human carcinogen. Occupational exposure has been associated with increased risk of respiratory cancer. Multiple mechanisms have been shown to contribute to Cr(VI induced carcinogenesis, including DNA damage, genomic instability, and epigenetic modulation, however, the molecular mechanism and downstream genes mediating chromium's carcinogenicity remain to be elucidated. METHODS/RESULTS: We established chromate transformed cell lines by chronic exposure of normal human bronchial epithelial BEAS-2B cells to low doses of Cr(VI followed by anchorage-independent growth. These transformed cell lines not only exhibited consistent morphological changes but also acquired altered and distinct gene expression patterns compared with normal BEAS-2B cells and control cell lines (untreated that arose spontaneously in soft agar. Interestingly, the gene expression profiles of six Cr(VI transformed cell lines were remarkably similar to each other yet differed significantly from that of either control cell lines or normal BEAS-2B cells. A total of 409 differentially expressed genes were identified in Cr(VI transformed cells compared to control cells. Genes related to cell-to-cell junction were upregulated in all Cr(VI transformed cells, while genes associated with the interaction between cells and their extracellular matrices were down-regulated. Additionally, expression of genes involved in cell proliferation and apoptosis were also changed. CONCLUSION: This study is the first to report gene expression profiling of Cr(VI transformed cells. The gene expression changes across individual chromate exposed clones were remarkably similar to each other but differed significantly from the gene expression found in anchorage-independent clones that arose spontaneously. Our analysis identified many novel gene expression changes that may contribute to chromate induced cell transformation, and collectively this type of

  9. Cell-specific regulation of a Brassica napus CMS-associated gene by a nuclear restorer with related effects on a floral homeotic gene promoter.

    Science.gov (United States)

    Geddy, Rachel; Mahé, Laetitia; Brown, Gregory G

    2005-02-01

    Cytoplasmic male sterility (CMS) is a maternally inherited defect in pollen production specified by novel mitochondrial genes. It can be suppressed by nuclear restorer (Rf) genes which normally downregulate expression of a CMS-associated novel mitochondrial gene. Two forms of Brassica napus CMS, nap and pol, are associated with related chimeric genes, orf222 and orf224, respectively. We show that in pol and nap CMS, anther locule development is asynchronous and asymmetric, that one or more locules within each anther may fail to develop entirely and that CMS anthers display polarity in locule development. We show, by in situ hybridization, that orf222 transcripts accumulate in sterile anthers prior to development of morphological differences between CMS and restored stamens, and remain preferentially localized to microsporangia. In fertility-restored anthers, however, orf222 transcript levels remain low throughout development. Some sporogenous and meiotic cells differentiate within CMS anthers and form functional pollen despite retaining high orf222 transcript levels, suggesting that the effect of orf222 expression in blocking pollen development is limited to an early and specific stage. Transcripts of other mitochondrial genes, exemplified by atp6 and cob, and of the nuclear-encoded ATP synthase gamma subunit, accumulate preferentially in the microsporangia of both sterile and fertile anthers. Thus nuclear fertility restoration reduces orf222 transcript levels in a gene and tissue-specific manner. We observe differences between CMS and fertile plants in the timing and patterning of APETALA3 promoter activity that suggest a possible basis for the developmental abnormalities of CMS flowers. PMID:15659093

  10. Simvastatin modulates mesenchymal stromal cell proliferation and gene expression.

    Science.gov (United States)

    Zanette, Dalila Lucíola; Lorenzi, Julio Cesar Cetrulo; Panepucci, Rodrigo Alexandre; Palma, Patricia Vianna Bonini; Dos Santos, Daiane Fernanda; Prata, Karen Lima; Silva, Wilson Araújo

    2015-01-01

    Statins are widely used hypocholesterolemic drugs that block the mevalonate pathway, responsible for the biosysnthesis of cholesterol. However, statins also have pleiotropic effects that interfere with several signaling pathways. Mesenchymal stromal cells (MSC) are a heterogeneous mixture of cells that can be isolated from a variety of tissues and are identified by the expression of a panel of surface markers and by their ability to differentiate in vitro into osteocytes, adipocytes and chondrocytes. MSC were isolated from amniotic membranes and bone marrows and characterized based on ISCT (International Society for Cell Therapy) minimal criteria. Simvastatin-treated cells and controls were directly assayed by CFSE (Carboxyfluorescein diacetate succinimidyl ester) staining to assess their cell proliferation and their RNA was used for microarray analyses and quantitative PCR (qPCR). These MSC were also evaluated for their ability to inhibit PBMC (peripheral blood mononuclear cells) proliferation. We show here that simvastatin negatively modulates MSC proliferation in a dose-dependent way and regulates the expression of proliferation-related genes. Importantly, we observed that simvastatin increased the percentage of a subset of smaller MSC, which also were actively proliferating. The association of MSC decreased size with increased pluripotency and the accumulating evidence that statins may prevent cellular senescence led us to hypothesize that simvastatin induces a smaller subpopulation that may have increased ability to maintain the entire pool of MSC and also to protect them from cellular senescence induced by long-term cultures/passages in vitro. These results may be important to better understand the pleiotropic effects of statins and its effects on the biology of cells with regenerative potential. PMID:25874574

  11. Simvastatin modulates mesenchymal stromal cell proliferation and gene expression.

    Directory of Open Access Journals (Sweden)

    Dalila Lucíola Zanette

    Full Text Available Statins are widely used hypocholesterolemic drugs that block the mevalonate pathway, responsible for the biosysnthesis of cholesterol. However, statins also have pleiotropic effects that interfere with several signaling pathways. Mesenchymal stromal cells (MSC are a heterogeneous mixture of cells that can be isolated from a variety of tissues and are identified by the expression of a panel of surface markers and by their ability to differentiate in vitro into osteocytes, adipocytes and chondrocytes. MSC were isolated from amniotic membranes and bone marrows and characterized based on ISCT (International Society for Cell Therapy minimal criteria. Simvastatin-treated cells and controls were directly assayed by CFSE (Carboxyfluorescein diacetate succinimidyl ester staining to assess their cell proliferation and their RNA was used for microarray analyses and quantitative PCR (qPCR. These MSC were also evaluated for their ability to inhibit PBMC (peripheral blood mononuclear cells proliferation. We show here that simvastatin negatively modulates MSC proliferation in a dose-dependent way and regulates the expression of proliferation-related genes. Importantly, we observed that simvastatin increased the percentage of a subset of smaller MSC, which also were actively proliferating. The association of MSC decreased size with increased pluripotency and the accumulating evidence that statins may prevent cellular senescence led us to hypothesize that simvastatin induces a smaller subpopulation that may have increased ability to maintain the entire pool of MSC and also to protect them from cellular senescence induced by long-term cultures/passages in vitro. These results may be important to better understand the pleiotropic effects of statins and its effects on the biology of cells with regenerative potential.

  12. Localization of Core Planar Cell Polarity Proteins, PRICKLEs, in Ameloblasts of Rat Incisors: Possible Regulation of Enamel Rod Decussation

    International Nuclear Information System (INIS)

    To confirm the possible involvement of planar cell polarity proteins in odontogenesis, one group of core proteins, PRICKLE1, PRICKLE2, PRICKLE3, and PRICKLE4, was examined in enamel epithelial cells and ameloblasts by immunofluorescence microscopy. PRICKLE1 and PRICKLE2 showed similar localization in the proliferation and secretory zones of the incisor. Immunoreactive dots and short rods in ameloblasts and stratum intermedium cells were evident in the proliferation to differentiation zone, but in the secretion zone, cytoplasmic dots decreased and the distal terminal web was positive for PRICKLE1 and PRICKLE2. PRICKLE3 and PRICKLE4 showed cytoplasmic labeling in ameloblasts and other enamel epithelial cells. Double labeling of PRICKLE2 with VANGL1, which is another planar cell polarity protein, showed partial co-localization. To examine the transport route of PRICKLE proteins, PRICKLE1 localization was examined after injection of a microtubule-disrupting reagent, colchicine, and was compared with CX43, which is a membrane protein transported as vesicles via microtubules. The results confirmed the retention of immunoreactive dots for PRICKLE1 in the cytoplasm of secretory ameloblasts of colchicine-injected animals, but fewer dots were observed in control animals. These results suggest that PRICKLE1 and PRICKLE2 are transported as vesicles to the junctional area, and are involved in pattern formation of distal junctional complexes and terminal webs of ameloblasts, further implying a role in the formed enamel rod arrangement

  13. Alterations in radioresistance of eucaryotic cells after the transfer of genomic wildtype DNA and metallothionein genes

    International Nuclear Information System (INIS)

    The presented paper describes experiments concerning the alteration of radiosensitivity of eucaryotic cells after gene transfer. Ionizing radiation (γ- or X-ray) induces DNA single- or double strand breaks, which are religated by an unknown repair system. Repair deficient cells are highly sensitive to ionizing radiation. In the experiments described, cells from a patient with the heritable disease Ataxia telangiectasia were used as well as two X-ray sensitive CHO mutant cell lines. After gene transfer of an intact human DNA repair gene or a metallothionein gene the cells should regain radioresistance. (orig.)

  14. CD133-targeted gene transfer into long-term repopulating hematopoietic stem cells.

    Science.gov (United States)

    Brendel, Christian; Goebel, Benjamin; Daniela, Abriss; Brugman, Martijn; Kneissl, Sabrina; Schwäble, Joachim; Kaufmann, Kerstin B; Müller-Kuller, Uta; Kunkel, Hana; Chen-Wichmann, Linping; Abel, Tobias; Serve, Hubert; Bystrykh, Leonid; Buchholz, Christian J; Grez, Manuel

    2015-01-01

    Gene therapy for hematological disorders relies on the genetic modification of CD34(+) cells, a heterogeneous cell population containing about 0.01% long-term repopulating cells. Here, we show that the lentiviral vector CD133-LV, which uses a surface marker on human primitive hematopoietic stem cells (HSCs) as entry receptor, transfers genes preferentially into cells with high engraftment capability. Transduction of unstimulated CD34(+) cells with CD133-LV resulted in gene marking of cells with competitive proliferative advantage in vitro and in immunodeficient mice. The CD133-LV-transduced population contained significantly more cells with repopulating capacity than cells transduced with vesicular stomatitis virus (VSV)-LV, a lentiviral vector pseudotyped with the vesicular stomatitis virus G protein. Upon transfer of a barcode library, CD133-LV-transduced cells sustained gene marking in vivo for a prolonged period of time with a 6.7-fold higher recovery of barcodes compared to transduced control cells. Moreover, CD133-LV-transduced cells were capable of repopulating secondary recipients. Lastly, we show that this targeting strategy can be used for transfer of a therapeutic gene into CD34(+) cells obtained from patients suffering of X-linked chronic granulomatous disease. In conclusion, direct gene transfer into CD133(+) cells allows for sustained long-term engraftment of gene corrected cells. PMID:25189742

  15. Multi-gene epigenetic silencing of tumor suppressor genes in T-cell lymphoma cells; delayed expression of the p16 protein upon reversal of the silencing

    DEFF Research Database (Denmark)

    Nagasawa, T; Zhang, Q; Raghunath, P N; Wong, H Y; El-Salem, M; Szallasi, A; Marzec, M; Gimotty, P; Rook, A H; Vonderheid, E C; Odum, Niels; Wasik, M A

    2006-01-01

    To understand better T-cell lymphomagenesis, we examined promoter CpG methylation and mRNA expression of closely related genes encoding p16, p15, and p14 tumor suppressor genes in cultured malignant T-cells that were derived from cutaneous, adult type, and anaplastic lymphoma kinase (ALK)-express......To understand better T-cell lymphomagenesis, we examined promoter CpG methylation and mRNA expression of closely related genes encoding p16, p15, and p14 tumor suppressor genes in cultured malignant T-cells that were derived from cutaneous, adult type, and anaplastic lymphoma kinase (ALK......)-expressing T-cell lymphomas. p16 gene was epigenetically silenced in all but one of the 10 malignant T-cell lines examined, p15 gene silenced in roughly half of the lines, and p14 was the least frequently affected. Extensive methylation of the p16 promoter was seen in six out of 10 cutaneous T-cell lymphoma...... patient samples and corresponded with lack of p16 protein expression in the cases examined. Treatment of cultured T-cells with the DNA methyltransferase inhibitor, 5-aza-2-deoxy-cytidine, resulted in reversal of the p16 gene silencing. However, expression of p16 protein was delayed in relationship to p16...

  16. A multimodality reporter gene for monitoring transplanted stem cells

    International Nuclear Information System (INIS)

    Introduction: The aim of this study is to explore the feasibility of a triple-fused reporter gene, termed TGF [herpes simplex virus type 1 thymidine kinase (HSV1-tk), enhanced green fluorescent protein (eGFP) and firefly luciferase (Fluc)], to monitor stem cells using multimodality molecular imaging. Methods: A recombinant adenovirus vector carrying the triple-fused reporter gene (Ad5-TGF) was constructed. Bone marrow mesenchymal stem cells (BMSCs) were transfected with different virus titers of Ad5-TGF [multiplicities of infection (MOIs) were 0, 50, 100, 150, 200 and 250]. The mRNA and protein expressions of HSV1-tk, eGFP and Fluc in the transfected BMSCs were evaluated using polymerase chain reaction and Western blot. After the transfection of the BMSCs with different virus titers of Ad5-TGF (MOIs were 25, 50, 75, 100 and 125), their uptake rates of 131I-FIAU were measured. Whole-body fluorescence, bioluminescence and micro-positron emission tomography (PET) images were acquired 1 day after the transfected BMSCs were injected into the left forelimb of rats. Results: After the transfection with different titers of Ad5-TGF, the positive transfection rate reached a peak (70%) when the MOI was 100. HSV1-tk, eGFP and Fluc mRNA and protein were detected in the Ad5-TGF-transfected BMSCs, which implies their successful transfection and expression. The BMSCs uptake of 131I-FIAU increased with the adenovirus titer and incubation time and reached a plateau (approximately 5.3%) after 3 h. Strong signals were observed in the injected left forearms in the fluorescence, bioluminescence and micro-PET images. Conclusions: A triple-fused reporter gene, TGF, can be used as a multifunctional molecular probe for multimodality imaging.

  17. Influence of carbon support microstructure on the polarization behavior of a polymer electrolyte membrane fuel cell membrane electrode assemblies

    Energy Technology Data Exchange (ETDEWEB)

    Guha, Abhishek; Schiraldi, David A. [Department of Macromolecular Science and Engineering and Case Advanced Power Institute, Case Western Reserve University, 10900 Euclid Avenue, 2100 Adelbert Rd, Cleveland, OH 44106-7202 (United States); Zawodzinski, Thomas A. Jr. [Department of Chemical Engineering and Case Advanced Power Institute, Case Western Reserve University, 10900 Euclid Avenue, Cleveland, OH 44106 (United States)

    2010-08-15

    The influence of carbon support morphology on the polarization behavior of a PEM fuel cell membrane electrode assembly has been investigated in this communication. Nanometer sized platinum electrocatalyst particles were deposited on lower surface area fibrous (carbon nanofibers) and particulate carbon supports (carbon blacks) by the well-documented ethylene glycol route for supported electrocatalyst synthesis. These supported catalyst systems were subsequently utilized to prepare catalyst inks and membrane electrode assemblies (MEA) in conjunction with a perflurosulfonated ionomeric membrane-Nafion {sup registered}. Level of liquid Nafion binder in the supported catalyst inks was varied and the ramifications of such a variation on polarization behavior of the MEA determined. The trend in polarization performance was found to be independent of the carbon support morphology in the various ink compositions. The two varieties of carbon supports were also mixed together in various weight ratios and platinum was deposited by the glycol method. Key parameters such as the platinum content on carbon and platinum particle size were determined to be independent of the nature of the supports on which the particles had been deposited. The results indicate that lower surface area carbon supports of vastly contrasting morphologies can be interchangeably employed as catalyst support materials in a PEM fuel cell MEA. (author)

  18. Multimodality imaging of reporter gene expression using a novel fusion vector in living cells and animals

    Science.gov (United States)

    Gambhir; Sanjiv , Pritha; Ray

    2009-04-28

    Novel double and triple fusion reporter gene constructs harboring distinct imageable reporter genes are provided, as well as applications for the use of such double and triple fusion constructs in living cells and in living animals using distinct imaging technologies.

  19. Differential gene expression in CD8+ cells exhibiting noncytotoxic anti-HIV activity

    International Nuclear Information System (INIS)

    Suppressive subtractive hybridization with polymerase chain reaction was used to identify the gene(s) associated with the CD8+ cell noncytotoxic anti-HIV response. The differences in gene expression profiles of CD8+ cells from a pair of discordant HIV-positive identical twins were studied. Forty-nine genes were identified as expressed at higher levels in the CD8+ cells from the infected twin that inhibited viral replication. The differential expression of these genes was then evaluated using Q-PCR to determine if this gene expression pattern is evident in CD8+ cells from other HIV-positive subjects showing this antiviral activity. Three genes, including one unknown, were found to have significantly increased expression in antiviral CD8+ cells

  20. Distribution specificity of polarized populations of T helper cells in patients with chronic hepatitis B virus infection

    Institute of Scientific and Technical Information of China (English)

    JIANG Rong-long; FENG Xiao-rong; LU Qiao-sheng; LUO Kang-xian; FU Ning

    2001-01-01

    Objective: To investigate the roles of the polarized populations of T helper cells isolated from the peripheral blood mononuclear cells (PBMCs) of individuals with chronic hepatitis B virus (HBV) infection. Methods: PBMCs from patients with chronic HBV infection were separated routinely, stimulated by PMA, ionomycin and monensin, and the production of IL-4, IFN-γ and TGF-β by CD4+ T cells was observed by flow cytometry(FACS). Results: The percentages of the T cells producing IFN-γ, IL-4 or TGF-β ranged from 2.3% to 18.6%, 1.1% to 8.7% and 0.7% to 7.1% respectively among CD4+ cells from non-infected individuals. The majority of CD4+ T cells in PBMCs from individuals with chronic HBV infection were Th0 cells. The proportion of Th1 cells in patients with active chronic hepatitis B was higher than that in patients at inactive stage of the disease (P<0.05), indicating a significant elevation of Thl cells with the hepatic inflammation activity. The percentage of Th2 cells in individuals with HBV infection was higher than that in controls (P<0.05),but showed no difference between different patients (P>0.05). The percentage of Th3 cells was higher in asymptomatic HBV carriers than that in patients with chronic hepatitis B and in healthy controls (P<0.05). Conclusions: Th1-type cytokines are related with hepatic inflammation activity of chronic hepatitis B, and Th2 cells may be associated w